14 Appendix

Appendix
List of Publications and GenBank Submissions
Research papers
1. Dutta D and Ghosh K (2015) Screening of extracellular enzyme-producing and

pathogen inhibitory gut bacteria as putative probiotics in mrigal, Cirrhinus
mrigala (Hamilton, 1822). International Journal of Fisheries and Aquatic
studies 2: 310–318 (Impact factor- 0.55)
2. Banerjee S, Mukherjee A, Dutta D and Ghosh K (2015) Evaluation of Chitinolytic
Gut Microbiota in Some Carps and Optimization of Culture Conditions for
Chitinase Production by the Selected Bacteria. Journal of Microbiology,
Biotechnology and Food Sciences 5: 12-19 (Impact factor- 0.98)
3. Dutta D, Banerjee S, Mukherjee A and Ghosh K (2015) Selection and Probiotic
Characterization of Exoenzyme-Producing Bacteria Isolated From The Gut of
Catla Catla (Actinopterygii: Cypriniformes:Cyprinidae). Acta Ichthyologica et
Piscatoria, 45: 373-384 (Impact factor- 0.62)
4. Banerjee S, Mukherjee A, Dutta D and Ghosh K (2016) Non-Starch Polysaccharide
Degrading Gut Bacteria in Indian Major Carps and Exotic Carps. Jordan Journal
of Biological Sciences 9: 69-78
5. Mukherjee A, Dutta D, Banerjee S, Ringø E, Breines EM, Hareide E, Chandra G and
Ghosh K (2016) Potential Probiotics from Indian Major Carp, Cirrhinus mrigala.
Characterization, Pathogen Inhibitory Activity, Partial Characterization of
Bacteriocin and Production of Exoenzymes. Research in Veterinary Science 108:
76-84 (Impact factor- 1.50)
6. Mukherjee A, Dutta D, Banerjee S, Ringø E, Breines EM, Hareide E, Chandra G and
Ghosh K (2017) Culturable autochthonous gut bacteria in rohu, Labeo rohita. In
vitro growth inhibition against pathogenic Aeromonas spp., stability in gut, bio-
safety and identification by 16S rRNA gene sequencing. Symbiosis
doi:10.1007/s13199-017-0474-7 (Impact factor- 1.28)
NCBI GenBank Submission

1. Bacillus subtilis subsp. spizizenii (KF623286)
2. Bacillus tequilensis (KF623287)
3. Bacillus aerius (KF623288)
4. Bacillus sonorensis (KF623289)
5. Bacillus amyloliquefaciens (KF623290)
6. Bacillus sonorensis (KF623291)
i
Bacillus subtilis subsp. spizizenii strain LR3H1A 16S ribosomal RNA
gene, partial sequence
GenBank: KF623286.1
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LOCUS KF623286 1564 bp DNA linear BCT 03-NOV-2013
DEFINITION Bacillus subtilis subsp. spizizenii strain LR3H1A 16S ribosomal RNA
gene, partial sequence.
ACCESSION KF623286
VERSION KF623286.1
KEYWORDS .
SOURCE Bacillus subtilis subsp. spizizenii
ORGANISM Bacillus subtilis subsp. spizizenii
Bacteria; Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus.
REFERENCE 1 (bases 1 to 1564)
AUTHORS Dutta,D. and Ghosh,K.
TITLE Exo-enzyme producing bacteria
JOURNAL Unpublished
TITLE Direct Submission
JOURNAL Submitted (04-SEP-2013) Department of Zoology, The University of
Burdwan, Golapbag, Burdwan, West Bengal 713104, India
COMMENT ##Assembly-Data-START##
Sequencing Technology :: Sanger dideoxy sequencing
##Assembly-Data-END##
FEATURES Location/Qualifiers
source 1..1564
/organism="Bacillus subtilis subsp. spizizenii"
/mol_type="genomic DNA"
/strain="LR3H1A"
/isolation_source="gut"
/host="fish"
/sub_species="spizizenii"
/db_xref="taxon:96241"
/country="India"
/altitude="40 m"
/collection_date="16-Jun-2011"
/collected_by="Dipanjan Dutta"
rRNA <1..>1564
/product="16S ribosomal RNA"
ORIGIN
1 ggtaattatt agatatggga agaatttttt gggttgtttt aaacgctcag gacgaacgct
61 ggcggcgtgc ctaatacatg caagtcgagc ggacagatgg gagcttgctc cctgatgtta
121 gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat aactccggga
181 aaccggggct aataccggat ggttgtttga accgcatggt tcaaacataa aaggtggctt
241 cggctaccac ttacagatgg acccgcggcg cattagctag ttggtgaggt aacggctcac
301 caaggcaacg atgcgtagcc gacctgagag ggtgatcggc cacactggga ctgagacacg
361 gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga aagtctgacg
421 gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa agctctgttg ttagggaaga
481 acaagtaccg ttcgaatagg gcggtacctt gacggtacct aaccagaaag ccacggctaa
541 ctacgtgcca gcagccgcgg taatacgtag ggggcaaacg ttgtccgcaa ttattgggcg
601 taaagggctc gcaggcggtt tcttaagtct gatgtgaaag cccccggctc aaccggggag
661 ggtcattgga aactggggaa cttgagtgca gaagaggaga gtggaattcc acgtgtagcg
721 gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag gcgactctct ggtctgtaac
781 tgacgctgag gagcgaaaac acgcccagcg aacaggatta gataccctgg tagtccacgc
841 cgtaaacgat gagtgctaag tgttaggggg tttccgcccc ttagtgctgc agctaacgca
901 ttaagcactc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa ttgacggggg
961 cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt
1021 cttgacatcc tctgacaatc ctagagatag gacgtcccct tcgggggcag agtgacaggt
1081 ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc
1141 aacccttgat cttagttgcc agcattcagt tgggcactct aaggtgactg ccggtgacaa
1201 accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac
1261 gtgctacaat ggacagaaca aagggcagcg aaaccgcgag gttaagccaa tcccacaaat
1321 ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa tcgctagtaa
1381 tcgcggatca gcatgccgcg gttgaatacg ttcccgggcc ttgtacacac cgcccgtcac
1441 accacgagag tttgtaacac ccgaagtcgg tgaggtaacc ttttaggagc cagccgccga
1501 aggtgggaca gatgattggg tgatctacag ggaggggcgt ccccaatata aaaaaaaggc
1561 cttt
ii
Bacillus tequilensis strain LR3F3P 16S ribosomal RNA gene, partial
sequence
GenBank: KF623287.1
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DEFINITION Bacillus tequilensis strain LR3F3P 16S ribosomal RNA gene, partial
sequence.
ACCESSION KF623287
VERSION KF623287.1
KEYWORDS .
SOURCE Bacillus tequilensis
ORGANISM Bacillus tequilensis
TITLE Exo-enzyme producing microorganism
JOURNAL Unpublished
source 1..1563
/organism="Bacillus tequilensis"
/strain="LR3F3P"
/host="fish"
/country="India"
/altitude="40 m"
/collection_date="20-Aug-2011"
rRNA <1..>1563
ORIGIN
1 ggaggggggg ggggggtttt ttaggttttt tttccagctc aggacgaacg ctggcggcgt
61 gcctaataca tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga
121 cgggtgagta acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg
181 ctaataccgg atggttgttt gaaccgcatg gttcaaacat aaaaggtggc ttcggctacc
241 acttacagat ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcga
301 cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac
361 tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg
421 ccgcgtgagt gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtac
481 cgttcgaata gggcggtacc ttgacggtac ctaaccagaa agccacggct aactacgtgc
541 cagcagccgc ggtaatacgt acggggaaag cgttgtccgg aattattggg cgtaaagggc
601 tcgcaggcgg tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg
661 gaaactgggg aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg
721 cgtagagatg tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg
781 aggagcgaaa acgaggccag cgaacaggat tagataccct ggtagtccac gccgtaaacg
841 atgagtgcta agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac
901 tccgcctggg gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca
961 agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat
1021 cctctgacaa tcctagagat aggacgtccc cttcgggggc agagtgacag gtggtgcatg
1081 gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg
1141 atcttagttg ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg
1201 aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca
1261 atggacagaa caaagggcag cgaaaccgcg aggttaagcc aatcccacaa atctgttctc
1321 agttcggatc gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat
1381 cagcatgccg cggttgaata cgttcccggg ccttgtacac accgcccgtc acaccacgag
1441 agtttgtaac acccgaagtc ggtgaggtaa ccttttagga gccagccgcc gaaggtggga
1501 cagatgattg ggtgactaca gggggggagg ggggaggaga agagagaaaa gggccaaaag
1561 acc
iii
Bacillus aerius strain CCH1A 16S ribosomal RNA gene, partial
sequence
GenBank: KF623288.1
FASTA Graphics
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DEFINITION Bacillus aerius strain CCH1A 16S ribosomal RNA gene, partial
sequence.
ACCESSION KF623288
VERSION KF623288.1
KEYWORDS .
SOURCE Bacillus aerius
ORGANISM Bacillus aerius
TITLE Putative probiotic strain isolated from Catla
JOURNAL Unpublished
source 1..1585
/organism="Bacillus aerius"
/strain="CCH1A"
/host="fish"
/country="India"
/altitude="40 m"
/collection_date="28-Dec-2011"
rRNA <1..>1585
ORIGIN
1 ctttttgttt gcccggtttg ggggttgggg gggttttata tatatataaa gtttttttcc
61 gcaggacgaa cgctggcggc gtgcctaata catgcaagtc gagcggacag atgggagctt
121 gctccctgat gttagcggcg gacgggtgag taacacgtgg gtaacctgcc tgtaagactg
181 ggataactcc gggaaaccgg ggctaatacc ggatgcttga ttgaaccgca tggttcaatt
241 ataaaaggtg gcttttagct accacttaca gatggacccg cggcgcatta gctagttggt
301 gaggtaacgg ctcaccaagg caacgatgcg tagccgacct gagagggtga tcggccacac
361 tgggactgag acacggccca gactcctacg ggaggcagca gtagggaatc ttccgcaatg
421 gacgaaagtc tgacggagca acgccgcgtg agtgatgaag gttttcggat cgtaaaactc
481 tgttgttagg gaagaacaag taccgttcga atagggcggt accttgacgg tacctaacca
541 gaaagccacg gctaactacg tgccagcagc cgcggtaata cgtagagggc aagcgttgtc
601 cgcaattatt gggcgtaaag cgcgcgcagg cggtttctta agtctgatgt gaaagccccc
661 ggctcaaccg gggagggtca ttggaaactg gggaacttga gtgcagaaga ggagagtgga
721 attccacgtg tagcggtgaa atgcgtagag atgtggagga acaccagtgg cgaaggcgac
781 tctctggtct gtaactgacg ctgaggcgcg aaagagcgcg gagcgaacag gattagatac
841 cctggtagtc cacgccgtaa acgatgagtg ctaagtgtta gagggtttcc gccctttagt
901 gctgcagcaa acgcattaag cactccgcct ggggagtacg gtcgcaagac tgaaactcaa
961 aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga
1021 agaaccttac caggtcttga catcctctga caaccctaga gatagggctt ccccttcggg
1081 ggcagagtga caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag
1141 tcccgcaacg agcgcaaccc ttgatcttag ttgccagcat tcagttgggc actctaaggt
1201 gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg
1261 acctgggcta cacacgtgct acaatgggca gaacaaaggg cagcgaagcc gcgaggctaa
1321 gccaatccca caaatctgtt ctcagttcgg atcgcagtct gcaactcgac tgcgtgaagc
1381 tggaatcgct agtaatcgcg gatcagcatg ccgcgggtga atacgttccc gggccttgta
1441 cacaccgccc gtcacaccac gagagtttgt aacacccgaa gtcggtgagg taaccttttg
1501 gagccagccg ccgaaggtgg gacagatgat tggggactaa gccaagggag ggggggaaaa
1561 aaaaaaaaaa aagaggaaaa aaaga
iv
Bacillus sonorensis strain CCH1Ph 16S ribosomal RNA gene, partial
sequence
GenBank: KF623289.1
FASTA Graphics
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DEFINITION Bacillus sonorensis strain CCH1Ph 16S ribosomal RNA gene, partial
sequence.
ACCESSION KF623289
VERSION KF623289.1
KEYWORDS .
SOURCE Bacillus sonorensis
ORGANISM Bacillus sonorensis
TITLE Enzyme producing gut bacteria
JOURNAL Unpublished
source 1..1586
/organism="Bacillus sonorensis"
/strain="CCH1Ph"
/host="fish"
/country="India"
/altitude="40 m"
/collection_date="03-Feb-2012"
rRNA <1..>1586
ORIGIN
1 gggttttttg gggtttgggg ggggggggct tttttttttt ttttggtttt ttttttcagg
61 cgaacgctgg cggcgtgcct aatacatgca agtcgagcgg acagatggga gcttgctccc
121 tgatgttagc ggcggacggg tgagtaacac gtgggtaacc tgcctgtaag actgggataa
181 ctccgggaaa ccggggctaa taccggatgc ttgattgaac cgcatggttc aattataaaa
241 ggtggctttt agctaccact tacagatgga cccgcggcgc attagctagt tggtgaggta
301 acggctcacc aaggcgacga tgcgtagccg acctgagagg gtgatcggcc acactgggac
361 tgagacacgg cccagactcc tacgggaggc agcagtaggg aatcttccgc aatggacgaa
421 agtctgacgg agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaa actctgttgt
481 tagggaagaa caagtaccgt tcgaataggg cggtaccttg acggtaccta accagaaagc
541 cacggctaac tacgtgccag cagccgcggt aatacgtagg gggcaaacgt tgtccggaat
601 aatgcggcgt aaagcgcgcg caggcggttt cttaagtctg atgtgaaagc ccccggctca
661 accggggagg gtcattggaa actggggaac ttgagtgcag aagaggagag tggaattcca
721 cgtgtagcgg tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg cgactctctg
781 gtctgtaact gacgctgagg cgcgaaaaga gcgcggagcg aacaggatta gataccctgg
841 tagtccacgc cgtaaacgat gagtgctaag tgttagaggg tttccgccct ttagtgctgc
901 agcaaacgca ttaagcactc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa
961 ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac
1021 cttaccaggt cttgacatcc tctgacaacc ctagagatag ggcttcccct tcgggggcag
1081 agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg
1141 caacgagcgc aacccttgat cttagttgcc agcattcagt tgggcactct aaggtgactg
1201 ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg
1261 ggctacacac gtgctacaat gggcagaaca aagggcagcg aagccgcgag gctaagccaa
1321 tcccacaaat ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa
1381 tcgctagtaa tcgcggatca gcatgccgcg gttgaatacg ttcccgggcc ttgtacacac
1441 cgcccgtcac accacggaga gtttgtaaca cccgaagtcc ggtgaggtaa cctttttgga
1501 gccagccgcc gaaggtggga cagatgattg ggtgactaag cagttggggg gggggagaaa
1561 aaaaaataaa aaggaaatta ttacca
v
Bacillus amyloliquefaciens strain CMH1X 16S ribosomal RNA gene,
partial sequence
GenBank: KF623290.1
FASTA Graphics
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DEFINITION Bacillus amyloliquefaciens strain CMH1X 16S ribosomal RNA gene,
partial sequence.
ACCESSION KF623290
VERSION KF623290.1
KEYWORDS .
SOURCE Bacillus amyloliquefaciens
ORGANISM Bacillus amyloliquefaciens
Bacteria; Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus;
Bacillus amyloliquefaciens group.
TITLE Putative probiotic strain isolated from Mrigal
JOURNAL Unpublished
source 1..1573
/organism="Bacillus amyloliquefaciens"
/strain="CMH1X"
/host="fish"
/country="India"
/altitude="40 m"
/collection_date="04-Mar-2012"
rRNA <1..>1573
ORIGIN
1 gccggcctcc cgcctccggc gcgggctcta tataatgttt ttacacgctc aggacgaacg
61 ctggcggcgt gcctaataca tgcaagtcga gcggacagat gggagcttgc tccctgatgt
121 tagcggcgga cgggtgagta acacgtgggt aacctgcctg taagactggg ataactccgg
181 gaaaccgggg ctaataccgg atgcttgttt gaaccgcatg gttcagacat aaaaggtggc
241 ttcggctacc acttacagat ggacccgcgg cgcattagct agttggtgag gtaacggctc
301 accaaggcga cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca
361 cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga
421 cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa
481 gaacaagtgc cgttcaaata gggcggcacc ttgacggtac ctaaccagaa agccacggct
541 aactacgtgc cagcagccgc ggtaatacgt agggggcaaa cggtgtccgc aattattggg
601 cgtaaagggc tcgcaggcgg tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg
661 agggtcattg gaaactgggg aacttgagtg cagaagagga gagtggaatt ccacgtgtag
721 cggtgaaatg cgtagagatg tggaggaaca ccagtggcga aggcgactct ctggtctgta
781 actgacgctg aggagcgaaa gcacgcccag cgaacaggat tagataccct ggtagtccac
841 gccgtaaacg atgagtgcta agtgttaggg ggtttccgcc ccttagtgct gcagctaacg
901 cattaagcac tccgcctggg gagtacggtc gcaagactga aactcaaagg aattgacggg
961 ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag
1021 gtcttgacat cctctgacaa tcctagagat aggacgtccc cttcgggggc agagtgacag
1081 gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc
1141 gcaacccttg atcttagttg ccagcattca gttgggcact ctaaggtgac tgccggtgac
1201 aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac
1261 acgtgctaca atggacagaa caaagggcag cgaaaccgcg aggttaagcc aatcccacaa
1321 atctgttctc agttcggatc gcagtctgca actcgactgc gtgaagctgg aatcgctagt
1381 aatcgcggat cagcatgccg cggttgaata cgttcccggg ccttgtacac accgcccgtc
1441 acaccacgag agtttgtaac acccgaagtc ggtgaggtaa ccttttagga gccagccgcc
1501 gaaggtggga cagatgattg ggtgactaca gaaagaaaag ggggaaaaaa aaaaaaaaaa
1561 gggaggggga aga
vi
Bacillus sonorensis strain CM2H3L 16S ribosomal RNA gene, partial
sequence
GenBank: KF623291.1
FASTA Graphics
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DEFINITION Bacillus sonorensis strain CM2H3L 16S ribosomal RNA gene, partial
sequence.
ACCESSION KF623291
VERSION KF623291.1
KEYWORDS .
SOURCE Bacillus sonorensis
ORGANISM Bacillus sonorensis
TITLE Enzyme producing bacteria from Mrigal
JOURNAL Unpublished
source 1..1558
/organism="Bacillus sonorensis"
/strain="CM2H3L"
/host="fish"
/country="India"
/altitude="40 m"
/collection_date="19-May-2012"
rRNA <1..>1558
ORIGIN
1 gggttttttt tttttagtgt tttttttcgt aggacgaacg ctggcggcgt gcctaataca
61 tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga cgggtgagta
121 acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg
181 atgcttgatt gaaccgcatg gttcaattat aaaaggtggc ttttagctac cacttacaga
241 tggacccgcg gcgcattagc tagttggtga ggtaacggct caccaaggcg acgatgcgta
301 gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg
361 aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag
421 tgatgaaggt tttcggatcg taaaactctg ttgttaggga agaacaagta ccgttcgaat
481 agggcggtac cttgacggta cctaaccaga aagccacggc taactacgtg ccagcagccg
541 cggtaatacg tagggggcaa gcgttgtccg caattattgg gcgtaaagcg cgcgcaggcg
601 gtttcttaag tctgatgtga aagcccccgg ctcaaccggg gagggtcatt ggaaactggg
661 gaacttgagt gcagaagagg agagtggaat tccacgtgta gcggtgaaat gcgtagagat
721 gtggaggaac accagtggcg aaggcgactc tctggtctgt aactgacgct gaggcgcgaa
781 aacaagccga gcgaacagga ttagataccc tggtagtcca cgccgtaaac gatgagtgct
841 aagtgttaga gggtttccgc cctttagtgc tgcagcaaac gcattaagca ctccgcctgg
901 ggagtacggt cgcaagactg aaactcaaag gaattgacgg gggcccgcac aagcggtgga
961 gcatgtggtt taattcgaag caacgcgaag aaccttacca ggtcttgaca tcctctgaca
1021 accctagaga tagggcttcc ccttcggggg cagagtgaca ggtggtgcat ggttgtcgtc
1081 agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gatcttagtt
1141 gccagcattc agttgggcac tctaaggtga ctgccggtga caaaccggag gaaggtgggg
1201 atgacgtcaa atcatcatgc cccttatgac ctgggctaca cacgtgctac aatgggcaga
1261 acaaagggca gcgaagccgc gaggctaagc caatcccaca aatctgttct cagttcggat
1321 cgcagtctgc aactcgactg cgtgaagctg gaatcgctag taatcgcgga tcagcatgcc
1381 gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccacgag agtttgtaac
1441 acccgaagtc ggtgaggtaa ccttttggag ccagccgccg aaggtgggac agatgattgg
1501 ggtgaatcgt acaggggggg gacacgcata gaaggggttt tttttgcccc ggggtttt
vii
International Journal of Fisheries and Aquatic Studies 2015; 2(4): 310-318
ISSN: 2347-5129
IJFAS 2015; 2(4): 310-318 Screening of extracellular enzyme-producing and pathogen
© 2015 IJFAS
www.fisheriesjournal.com inhibitory gut bacteria as putative probiotics in mrigal,
Received: 04-02-2015
Accepted: 25-02-2015
Cirrhinus mrigala (Hamilton, 1822)
Dipanjan Dutta Dipanjan Dutta, Koushik Ghosh
Aquaculture Laboratory,
Department of Zoology, The
Abstract
University of Burdwan,
Golapbag, Burdwan 713 104,
The present study was intended to screen autochthonous bacteria as novel probionts in mrigal, Cirrhinus
West Bengal, India. mrigala. Altogether 117 extracellular enzyme-producing bacteria were isolated from the proximal (PI)
and distal (DI) regions of the gut, out of which 30 strains were primarily selected through qualitative and
Koushik Ghosh quantitative assay of extracellular enzyme activities (viz., amylase, protease, lipase, cellulase, phytase,
Aquaculture Laboratory, xylanase). Further, study of antagonism against 7 potential fish pathogens revealed 13 strains to be
Department of Zoology, The antagonistic against 1 pathogen(s) by cross-streaking and double-layer method. The strains CMH1X
University of Burdwan, and CM2H3L, isolated from the DI region were capable of producing six studied extracellular enzymes
Golapbag, Burdwan 713 104, and antagonistic to 5 tested fish pathogens. Both the strains were competent to grow in fish gut mucus
West Bengal, India. and could resist fish bile juice (8%). Fingerlings of C. mrigala were intraperitoneally injected with these
strains separately and no mortality or external disease symptoms were noticed. Based on the results, the
strains CMH1X and CM2H3L were selected as putative probiotics and identified as Bacillus
amyloliquefaciens (KF623290) and Bacillus sonorensis (KF623291), respectively, through 16S rRNA
partial gene sequence analysis. Although, the presently reported study depicts enzyme-producing
capacity and antimicrobial potential of the gut bacteria in carps, in vivo studies are essential prior to their
application in commercial aquaculture.
Keywords: Antagonism, autochthonous bacteria, Bacillus, extracellular enzymes, Indian major carp
1. Introduction
The use of beneficial microbes as probiotics has a long tradition in animal husbandry [1]. Since
the last two decades probiotics are also being more frequently used in aquaculture. However,
the use of commercial probiotics in fish is somewhat futile as most of the commercial
formulations contain strains isolated from non-fish sources and might not remain viable at high
cell density in the intestinal micro-environment of fish [2]. Hence, screening and selection of
putative probiotics from the host species seems to be reasonable. Extensive research on the gut
microbiota in marine and freshwater fish has confirmed that the gastrointestinal (GI) tract of
fish is an abode of dense microbial population [3, 4, 5] and wide variety of enzymes produced by
GI bacteria could be a contributing source of enzymes in fish [6]. However, screening and
characterization of efficient probiotic isolates from tropical freshwater species is less studied
and merits further exploration.
Preceding studies have suggested varied bacterial species from the GI tract of Indian major
carps, exotic carps and other cultivable teleosts, and apparent beneficial functions of the gut
microbiota pertaining to nutrition of the host fish have been emphasized [6]. On the other hand,
some of the previous studies have attempted selection of probiotic bacteria based on in vitro
antagonism against pathogens [7], or the adhesion and growth in fish intestinal mucus [8, 9]. To
the authors knowledge, reports on the antibacterial efficiency of the extracellular enzyme
producing gut bacteria isolated from the Indian major carps are scarce [10, 11]. Apart from the
functional role that a putative probiotic bacterium might play, viability within the host gut is
Correspondence often believed to be one of the main selection criteria for prospective probiotics [12, 13].
K. Ghosh Therefore, an appraisal of functional attributes together with reports on growth in fish mucus,
Aquaculture Laboratory, resistance to fish bile juice and safety evaluation for the target fish species might be considered
Department of Zoology, The
University of Burdwan,
as an ideal scheme to screen novel probionts from the fish. The presently reported study
Golapbag, Burdwan 713104, primarily aimed as screening and validation of efficient putative probiotics from an Indian
West Bengal, India. major carp, mrigal, Cirrhinus mrigala.
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International Journal of Fisheries and Aquatic Studies
2. Materials and methods suffering from pale gills, bloated appearance, skin ulcerations
2.1. Collection and processing of sample and hemorrhage. Pathogenicity of the isolated strains was
Specimens of mrigal, Cirrhinus mrigala (Hamilton) were checked experimentally by intravenous injection to C. mrigala
collected from three composite carp culture ponds located and by observing the onset of disease in the fish. The
around Burdwan (23°24 N, 87°86 E), West Bengal, India. pathogenic strains were maintained in the laboratory on TSA
Along with natural feeding, the fish were fed a mixture of fish (HiMedia, Mumbai, India) slants at 4 oC. Stock cultures in
meal, rice bran and different oil cakes as supplementary feed. tryptone soya broth (TSB) were stored at -20 °C in 0.9% NaCl
The pond was free from sewage discharge and other with 20% glycerol to provide stable inoculums throughout the
anthropogenic activities. The ranges of water quality study [21].
parameters during the collection period were: temperature
26.127.5 °C, pH 7.17.5 and dissolved oxygen 5.86.9 mg L- 2.4. Evaluation of probiotic properties
1 2.4.1. Extracellular enzyme production: Qualitative and
. Three specimens were obtained from each collection pond,
and thus altogether nine fish (average live weight 350±11.32 quantitative assay
g) were collected, brought to the laboratory with oxygen Following growth on respective media plates, appearance of
packing and distributed separately over 3 aquaria of 75 L each halo zone by flooding the plates with 1% Lugols iodine or
on the basis of their source. 15% HgCl2 indicated amylase and protease activities,
Prior to sacrifice, the fish were starved for 48 h in order to respectively [22]. Clear zone around colonies grown on TB and
clear their GI tracts and anaesthetized by applying 0.03% MPS media plates represented lipase [23] and phytase [20]
tricaine methanesulfonate (MS-222). Ventral surfaces were activities, respectively. Cellulolytic and xylanolytic activities
sterilized using 70% ethanol and fish were dissected were determined on CMC [24] and [19] plates flooded with
aseptically to remove the intestine [14]. The GI tract was Congo red dye. There were three replicates for each
divided into proximal (PI) and distal (DI) parts and processed experimental set. Qualitative extracellular enzyme activity
according to [15] for isolation of autochthonous observed by the appearance of halo (diameter in mm) around
microorganisms. Gut segments from three specimens of C. the colony was presented as scores [25] as follows; 0, nil (no
mrigala were pooled together region-wise for each replicate, halo); 1, low (6-10 mm halo); 2, moderate (11-20 mm halo); 3,
and thus there were three replicates for the study. Pooled good (21-30 mm halo); 4, high (31-39 mm halo); 5, very high
samples of 3 fish were used for each replicate to avoid ( 40 mm halo).
erroneous conclusions due to individual disparity in gut On the basis of the cumulative scores, efficient extracellular
microbiota as described elsewhere [16]. enzyme producing isolates were selected for quantitative
assay. Respective selective broth media were used as
2.2. Isolation of bacterial strains production media for the enzymes. Quantitative assay for the
Homogenates of the pooled intestinal segments of the two production of amylase, cellulase, protease and lipase were
regions were serially (1:10) diluted [17] and each diluted sample performed following the methods described by [26, 27, 28, 29],
(0.1 mL) was poured aseptically onto sterilized Soyabean respectively. A comprehensive description for measurement of
Casein Digest Agar (Tryptone Soya Agar, TSA; HiMedia, these extracellular enzymes and quantitative enzyme assay has
Mumbai, India) media plates to determine the autochthonous been mentioned elsewhere [18]. Quantitative assay of xylanase
culturable heterotrophic aerobic/facultative anaerobic bacterial and phytase activities were measured after [30, 31], respectively.
population. For isolation and enumeration of different Protein content of the enzyme source was measured after [32]
extracellular enzyme-producing bacteria (e.g., amylase, and specific activity of the respective enzymes was expressed
protease, lipase, cellulase, xylanase and phytase), the diluted as units (U).
gut homogenates were spread onto starch (ST), peptone-
gelatin (PG), tributyrin (TB), carboxymethylcellulose (CMC), 2.4.2. Assay for pathogen inhibitory activity
xylan (XY) and modified phytase screening (MPS) media The pathogen inhibitory activity of the promising enzyme-
plates, respectively, following enrichment culture technique. producing isolates were primarily tested against seven fish
ST, PG, TB and CMC media were prepared following [18]. XY pathogens by co-culture or cross streaking method [33, 34]
and MPS media were prepared following [19] and [20], and the strains that showed antagonism against 1 studied fish
respectively. The culture plates were aerobically incubated at pathogens were further evaluated by the double-layer method
[35]
30 °C for 48h to count bacterial colonies following dilution with minor modification. There were three replicates for
plate count method and expressed as log viable counts g-1 GI each experimental set. A clear zone of inhibition (halo) around
tract (LVC) [15]. Number of colonies reported in the present growth of the selected gut bacteria indicated antibacterial
study was an average of three replicates. The well-separated activity and the halo (diameter in mm) around the colony was
colonies were randomly collected, streaked individually on presented as follows; +, low (6-10 mm); ++, moderate (11-20
respective media plates and re-streaked repeatedly to acquire mm); +++, high (21-25 mm); ++++, very high ( 26 mm).
pure cultures. Pure cultures were maintained on slants in a
refrigerator (4 °C) for further study. 2.4.3. Growth on fish mucus
Fish gut mucus was collected from live C. mrigala and
2.3. Fish pathogens and culture maintenance thereafter processed following [36]. Growth on mucus was
Four fish pathogenic strains Aeromonas salmonicida MTCC- determined at 30 °C by counting the number of bacterial cells
1945 (AS), Pseudomonas fluorescens MTCC-103 (PF), with a Petroff-Hausser counting chamber at 24 h, 48 h and 72
Pseudomonas putida MTCC-1072 (PP) and Bacillus mycoides h intervals. To determine viable bacterial cells in mucus,
MTCC-7538 (BM) were acquired from the Microbial Type diluted (upto 10-5) 24 h, 48 h and 72 h cultures were inoculated
Culture Collection, Chandigarh, India. In addition, Aeromonas (0.1 mL) onto TSA plates, incubated at 30 °C for 24 h and
hydrophila (AH), Aeromonas veronii (AV) and Pseudomonas colony-forming units (CFU mL-1) were counted. Sterilized un-
sp. (P) were isolated from diseased fish. The fishes were inoculated mucus was served as the control.
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2.4.4. Bile tolerance described in [25]. The gene encoding 16S rRNA was amplified
Bile tolerance of the selected gut bacteria was evaluated from the isolates by polymerase chain reaction (PCR) using
through determination of minimum inhibitory concentration universal primers 27f (5´-AGAGTTTGATCCTGGCTCAG-
(MIC). Crude bile juice (pH 5.6) was collected from dissected 3´) and 1492r (5´-GGTTACCTTGTTACGACTT-3´).
gall bladder in aseptic condition, filter sterilized through 0.8 Sequenced data were edited using BioEdit Sequence
m and 0.22 m pore size filter papers (HiMedia, Mumbai, Alignment Editor (Version 7.2.0), aligned and analyzed for
India) and stored at -20 °C until use. Different concentrations finding the closest homolog using National Centre for
of bile (5 - 100%, v/v) were used in the agar-wells to Biotechnology Information (NCBI) GenBank and Ribosomal
determine the lowest concentration (MIC) that completely Database Project (RDP) databases. Sequences were deposited
inhibited macroscopic growth of the organisms. to the NCBI GenBank and accession numbers were obtained.
Phylogenetic tree was constructed incorporating 16S rRNA
2.4.5. Safety evaluation partial gene sequences of the closest type strains using MEGA
Bio-safety evaluation of the two putative probionts was carried 5.1Beta4 software following the Maximum Likelihood
out through in vivo studies conducted in 75L glass aquaria Method.
using 45 healthy C. mrigala fingerlings (16 ± 1.27 g). The fish
were acclimatized in the laboratory condition for 2 weeks and 2.6. Statistical Analysis
divided into three equal groups (two experimental and one Statistical analysis of the quantitative enzyme activity data was
control), each with three replicates. The candidate probionts performed by the one-way analysis of variance (ANOVA),
were grown in TSB at 30 °C for 24 h, centrifuged (2800 × g followed by Tukeys test according to [38] using SPSS Version
for 15 min, at 4 °C) and cell pellets were suspended in sterile 10 software [39].
0.9% saline. Each experimental fish received intra-peritoneal
(IP) injection of 1.0 mL containing 109 cells mL-1 of a 3. Results
candidate probiotic bacterium. The fish in control group were Enumeration of gut-bacteria revealed that autochthonous
injected with sterile 0.9% saline [37]. Fish were fed Ad libitum heterotrophic as well as protease, amylase, lipase, cellulase,
with a diet containing approximately 35% crude protein xylanase and phytase producing bacterial populations were
having fish meal as the chief protein source. All groups were present in the proximal (PI) and distal (DI) segments of the GI
kept under observation for 21 days and health status was tract in C. mrigala (Figure 1). Heterotrophic and diverse
checked every day for development of any disease symptom. extracellular enzyme-producing bacterial populations were
found to be predominantly high in the DI region. While
2.5. Identification of isolates by 16S rRNA gene sequence considering extracellular enzyme-producing bacteria,
analysis occurrence of amylolytic bacteria was found to be the highest
The most promising two putative probiotics were identified (LVC = 5.63) followed by cellulolytic bacteria (LVC = 5.34).
through 16S rRNA partial gene sequence analysis after While, xylan-degrading population was noticed to be the
isolation and PCR amplification following the methods lowest (LVC = 3.87).
Fig 1: Log viable counts (LVC g-1 GI tract) of autochthonous bacteria isolated from the proximal intestine (PI) and distal intestine (DI) of
Cirrhinus mrigala. Each column with error bar represents Mean ± Standard error (n=3).
Altogether 117 bacteria were isolated, out of which 30 assay, maximum and minimum scores being 27 and 10,
extracellular enzyme producing strains (14 from PI and 16 respectively (data not shown). Results of the quantitative
from DI) were primarily selected through qualitative enzyme enzyme assay revealed significant differences in the enzyme
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Table 1: Specific activity of the enzymes (U) produced by the primarily selected bacteria isolated from the gut of Cirrhinus mrigala. Data are
Means ± Standard error (n=3)
Strains Amylase(U)1 Protease(U)2 Lipase(U)3 Cellulase(U)4 Phytase (U)5 Xylanase(U)6

Proximal Intestine
CMF1A 161.32±4.31hi ND 4.21±0.23ab ND 201.37±5.62g 4.91±0.22bc
CMF2A 241.14±5.27lm 31.84±1.14b 5.38±0.26i 58.89±2.15b ND 9.96±0.21l
CMF1L 138.83±3.14g 55.83±2.39f 5.08±0.24h 70.16±2.13d 83.64±2.58c 9.07±0.20k
CMF1Ph 157.84±4.29h 56.81±2.33f 4.30±0.21bc 73.52±2.64de 89.73±2.59d 7.39±0.21h
hi fg b d k
CMF2Ph 163.51±4.84 58.84±2.49 4.29±0.23 71.41±2.73 309.81±6.03 7.36±0.28h
CMF3Ph 131.06±3.64f 35.67±1.54cd 4.27±0.26b 60.06±2.91b 208.71±5.53gh 7.35±0.25h
CM2F1L 168.49±4.23ij 71.88±2.19i 4.47±0.27d 55.26±2.17ab 212.29±5.41h 6.86±0.23de
CM2F2L 160.37±4.47hi 60.37±2.67g 4.30±0.24bc 58.36±2.57b 95.67±2.59ef 7.03±0.25ef
CM2F1Ph 119.76±3.06d 77.53±2.58j 4.18±0.26a 58.71±2.04b 86.63±2.65cd 6.66±0.26d
CM3F3C 98.07±2.33c 38.85±1.63e 4.29±0.24b 64.16±2.44c 69.73±2.49b 7.41±0.22hi
CM3F2A 163.52±4.19hi 29.88±1.41b 4.29±0.21b 61.27±2.27bc 96.67±2.65f 6.89±0.21de
b a b a e
CM3F2X 89.94±2.31 18.03±1.06 4.27±0.22 51.17±2.46 93.26±2.09 10.65±0.74n
CM3F2Ph 143.68±3.27h ND ND 59.96±2.67b 288.75±5.38i 10.09±0.87lm
CM3F3Ph 233.67±5.33l ND ND 63.37±2.44bc 206.53±5.48gh 4.74±0.23ab
Distal Intestine
CMH1L 155.46±4.26h 70.43±2.34i 4.51±0.25e 71.85±2.87d 81.57±2.43c 7.34±0.25h
CMH2L ND 54.85±2.56f 4.19±0.26a 64.96±2.56c ND 11.16±0.23op
CMH1P 258.47±5.32mn 33.68±1.41c 4.32±0.22c 57.43±2.41ab ND 11.53±0.23q
i i ab bc ab
CMH3P 165.05±4.14 71.99±2.38 4.21±0.21 63.16±2.70 66.53±2.61 6.88±0.22de
CMH1Ph 134.21±3.69fg 34.59±1.64c 4.30±0.24bc 70.97±2.39d 199.83±4.59g 7.29±0.23h
CMH1X 262.14±5.34o 82.06±2.67jk 5.60±0.26j 72.28±2.67de 362.41±6.76l 10.89±0.46o
de f h de e
CMH4X 123.63±3.11 56.83±2.64 5.07±0.22 72.09±2.51 93.17±2.26 10.03±0.47lm
CMH5X ND 31.70±1.02b 4.35±0.26c 71.14±2.49d 91.41±2.23de 7.36±0.24h
CM2H2L 135.52±3.64fg 57.46±2.69f 4.31±0.29bc 60.19±2.41b ND 4.97±0.28bc
CM2H3L 320.39±6.83p 72.82±2.35i 4.58±0.26f 66.34±2.28c 370.56±6.94lm 17.82±1.13r
CM2H1P 176.53±4.41k 80.24±2.39jk 4.81±0.24g 73.64±2.26de ND 6.85±0.24de
CM2H2P 134.76±3.43fg 54.86±2.47f 4.28±0.28b 56.69±2.37ab ND 7.11±0.25fg
CM3H1A 250.37±5.37m 19.75±1.53a 4.23±0.20ab 53.37±2.36a 207.16±5.63gh 7.08±0.24ef
c h ab bc a
CM3H4C 100.37±3.03 65.53±2.06 4.25±0.25 61.13±2.65 64.49±2.36 4.88±0.23bc
CM3H1X 81.19±2.64a 16.59±1.11a 4.29±0.22b ND 292.14±5.34ij 4.56±0.22a
CM3H2P ND 73.35±2.37i ND 59.61±2.37b 92.52±2.43de 7.69±0.31j
Values with the same superscripts in the same vertical column are not significantly different (P < 0.05).
1
g of maltose liberated ml-1of enzyme extract mg-1 protein min-1
2
g of tyrosine liberated ml-1of enzyme extract mg-1 protein min-1
3 g of free fatty acid liberated ml-1of enzyme extract mg-1 protein min-1
4 g of glucose liberated ml-1of enzyme extract mg-1 protein min-1
5 g of inorganic phosphate liberated ml-1of enzyme extract mg-1 protein min-1
6 mg of D-xylose liberated ml -1of enzyme extract mg-1 protein min-1
ND= Not detected
activities between different bacterial isolates (Table 1). The extracellular enzyme activities revealed that the strains
highest amylolytic was noticed with the isolate CM2H3L CMH1X and CM2H3L (qualitative activity score being 26 and
(320.39±6.83U). The strain CMH1X exhibited maximum 27, respectively) were the most efficient among the 30
protease activity (82.06±2.67U). Both these strains were primarily selected bacterial strains.
isolated from the DI region. Maximum lipase activity was To verify pathogen inhibitory activity, the primarily selected
recorded with the strain CMH1X (5.60±0.26U) isolated from 30 extracellular enzyme-producing bacterial isolates were
the DI region. Maximum cellulase activity was documented further screened against fish pathogens. Out of the 30 isolates,
with the strain CM2H1P (73.64±2.26U) isolated from the DI 13 strains (4 from PI and 9 from DI regions) were found to
region. Maximum phytase (370.56±6.94U) and xylanase inhibit at least one of the tested fish pathogens through cross-
(17.82±1.13U) activities were noticed with the strain streaking method. Pathogen inhibitory activity of these 14
CM2H3L. Overall examination of the six different isolates were further assessed by double layer method and the
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zone of inhibition (halo) produced by the gut isolates were to decline. Log viable cell count (CFU mL-1) also reached
depicted in Table 2. In consequence of the maximum maximum when TSA plates were inoculated with the 48h
extracellular enzyme-producing capacities, the strains CMH1X growth in mucus. Both the candidate probiotic bacteria,
and CM2H3L were also noticed to be antagonistic against 5 CMH1X and CM2H3L, showed tolerance against diluted bile
out of the 7 tested fish pathogens. Therefore, these promising juice and were capable to grow at or below 8% fish bile juice
strains (CMH1X and CM2H3L) were analyzed further for (MIC=8% bile juice). After 21 days of small-scale in vivo
evaluation of other probiotic properties. Both the strains grew experiment, it was revealed that along with the control set
well in gut mucus of C. mrigala (Figure 2). As evident from intra-peritoneal injection of the candidate probiotics did not
the log total microscopic count (cells mL-1) following induce any pathological signs/disease symptoms or mortalities
incubation in the fish gut mucus, the putative probiotic strains in both treatment groups.
exhibited maximum growth at 48h, after which growth started
Table 2: Inhibition zone* produced by the selected gut bacteria in double layer method against the tested fish pathogens#
Strains AH PP AS BM AV P PF
CMF1L + - - - - - +
CM2F1Ph - - ++ - - + -
CM3F2X - + - - - ++ -
CM3F2A - ++ - - - + -
CMH1X ++ +++ - ++++ + - ++
CMH4X + - - - - - -
CMH1L - + - - - + -
CM2H3L - +++ ++++ ++++ - +++ +
CM2H2P - - - - + - -
CM2H2L ++ - - - - - +
CM2H1P - ++ - + + - -
CM3H2P - ++++ - - - - -
CM3H4C - - - + - - +
#AH=Aeromonas hydrophila; PP=Pseudomonas putida; AS= Aeromonas salmonicida; BM=Bacillus mycoides; AV=Aeromonas veronii;
P=Pseudomonas sp.; PF=Pseudomonas fluorescence.
* +, low (6-10 mm halo diameter); ++, moderate (11-20mm halo diameter); +++, high (21-25 mm halo diameter); ++++, very high ( 26 mm
halo diameter)
Fig 2: Log values of total microscopic count (cells mL-1) and viable count (CFU mL-1) of the selected gut bacteria (a) CMH1X (Bacillus
amyloliquefaciens) and (b) CM2H3L (Bacillus sonorensis) grown in gut mucus of Cirrhinus mrigala. Viable count was done on TSA plates
inoculated with respective bacteria cultures of 24 h, 48 h and 72 h duration in fish gut mucus. Each column with error bar represents Mean ±
Standard error (n=3)
Based on the nucleotide homology and phylogenetic analysis (GenBank Accession no. KF623291) that showed maximum
of the 16S rRNA partial gene sequences by nucleotide blast in similarity with the type strain Bacillus sonorensis
the NCBI GenBank and RDP databases, the putative probiotic (AF302118.1). Phylogenetic relation of the two identified
strain CMH1X was identified as Bacillus amyloliquefaciens bacterial isolates with other closely related type strains
(GenBank Accession no. KF623290), which was closest to the retrieved from the RDP database were presented in the
type strain Bacillus amyloliquefaciens (AB255669.1). The dendrogram (Figure 3).
other isolate, CM2H3L was identified as Bacillus sonorensis
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Fig 3: Dendrogram showing phylogenetic relations of the two potential probiotic bacterial strains, Bacillus amyloliquefaciens CMH1X
(KF623290) and Bacillus sonorensis CM2H3L (KF623291), with other closely related strains retrieved from NCBI GenBank and RDP. The
GenBank accession numbers of the reference strains are shown besides the names. Horizontal bars in the dendrogram represent the branch
length. Similarity and homology of the neighbouring sequences have been shown by bootstrap values. Distance matrix was calculated by
Hasegawa-Kishino-Yano model. The scale bar indicates 0.005 substitutions per nucleotide position. Bacillus altitudinis AJ831842.1 served as an
out group.
4. Discussion observations recorded from the GI tracts of the Indian major

In the present study emphasis has been given on the carps [6]. The Indian major carp, C. mrigala has been described
autochthonous microorganisms, as the native flora are as either omnivore, or feeding on detritus arising out from the
supposed to be well adapted to the intended ecological niche. plant feedstuffs [41]. Therefore, in accordance with the
Prior to isolation of microorganisms, the fish were starved for hypothesis that gut bacteria might contribute to the digestion
48 hours and their GI tracts were thoroughly washed with in fish [6]; amylase, cellulase and xylanase activities by the gut
sterile 0.9% saline. Therefore, it may be assumed that the bacteria noticed in the present study might indicate their ability
isolated microorganisms described in the present study to aid in digestion of plant feedstuffs in C. mrigala.
belonged to the autochthonous microbiota as suggested The ability of a bacterial strain to inhibit the growth of
elsewhere [14, 15]. Screening through in vitro tests are pathogenic bacteria has been proposed as the major criteria for
commonly being used to eliminate the less potential the selection of probiotics in many studies [40]. A general
microorganisms from available large pool to a more acceptable consensus developed that probiotic treatment might lead to
number for further testing [40]. Selection of strong extracellular improved protection in fish against several diseases [36].
enzyme producers was considered as the primary criteria for Therefore, demonstration of antagonistic properties against
the candidate probionts in view of inducing improved nutrient some fish pathogens was considered as the second criteria for
utilization to support growth. This preliminary screening has the selection of candidate probionts in the present study.
resulted in an elimination of 74.4% (87 strains out of 117) of Besides Aeromonas spp. as the well-known fish pathogens,
the total isolates from current study. Results of the present both Pseudomonas spp. [42] and Bacillus mycoides [43] are
study depicted that heterotrophic community within the GI described as opportunistic pathogens in fish and therefore,
tract of C. mrigala were represented by diverse bacteria included in the presently reported study to evaluate
capable of producing digestive (amylase, protease, lipase) and antibacterial efficiency of the putative probiotics. Precisely,
anti-nutritional factor degrading (cellulase, phytase, xylanase) two isolates (CMH1X and CM2H3L) with antagonistic
enzymes, which were compliant with the previous activity against five of the seven pathogenic strains tested were
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categorized as strong antagonists. The inhibition zones represent an exact description of the bacterial diversity in fish
exhibited by Bacillus amyloliquefaciens CMH1X (GenBank gut [63, 48]. However, as the major aim of the present study was
Accession no. KF623290) and Bacillus sonorensis CM2H3L to investigate likely function and identify autochthonous gut
(GenBank Accession no. KF623291) in the cross-streaking bacteria in C. mrigala as putative probiotics, the application of
method were similar to those reported by [44] for Alteromonas culture dependent process seems to be rationale. In this study,
sp. P7 (4.0±0.25 mm) and [45] for Bacillus strain BY-9 (3-6 we isolated putative probionts from fish intestine and screened
mm) against the pathogenic Vibriosis harveyi. Gut microbiota their potentiality through in vitro experiments. Nevertheless,
in the freshwater teleosts were fairly dominated by Bacillus assumptions based on in vitro experiments might not comply
spp. [14, 46, 47, 48], which is in agreement with the present study. exactly with in vivo conditions. Therefore, in vivo studies are
Bacillus spp. have been shown to possess adhesion abilities, warranted with these autochthonous strains to determine their
provide immunostimulation and produce bacteriocins [49, 50, 51]. effects on growth and wellbeing of C. mrigala.
Furthermore, Bacillus spp. are favoured as probiotics as they
can be kept in the spore form and stored indefinitely [52]. 6. Acknowledgments
Probiotic effects of B. amyloliquefaciens have been addressed Sincere thanks to the Head, Department of Zoology, The
in few of the recent observations. Application of probiotic B. University of Burdwan, West Bengal, India; The Department
amyloliquefaciens led to improvement of the growth of Science and Technology (FIST programme), New Delhi,
performance, feed conversion ratio, and immunological India and The University Grants Commission (UGC-SAP-
parameters in Nile tilapia, Oreochromis niloticus [53]. In DRS programme), New Delhi, India for providing research
another study, B. amyloliquefaciens FPTB16 has been reported facilities. The authors are grateful to Dr. P.K. Ghosh, USIC,
as putative probiotic for Catla catla on the basis of systemic The University of Burdwan for rendering technical support
and cutaneous mucosal immune responses and disease and Mr. A. Mukherjee for construction of the dendrogram. The
resistance [54]. Besides, pathogen inhibitory potential of several first author is grateful to The Council of Scientific and
bacilli has been indicated elsewhere [10, 55, 56, 57]. Nevertheless, Industrial Research, New Delhi, India for awarding the Junior
enzyme producing capacity together with anti-pathogenic Research Fellowship.
potential has not been addressed widely. In this context,
extracellular digestive enzyme-producing Bacillus 7. References
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
EVALUATION OF CHITINOLYTIC GUT MICROBIOTA IN SOME CARPS AND OPTIMIZATION OF CULTURE
CONDITIONS FOR CHITINASE PRODUCTION BY THE SELECTED BACTERIA
Sudeshna Banerjee, Anjan Mukherjee, Dipanjan Dutta and Koushik Ghosh*

Address(es): Dr. Koushik Ghosh,
Aquaculture Laboratory, Department of Zoology, The University of Burdwan, Golapbag, Burdwan 713 104, West Bengal, India
*Corresponding author: kghoshbu@gmail.com ; kghosh@zoo.buruniv.ac.in doi: 10.15414/jmbfs.2015.5.1.12-19
ARTICLE INFO ABSTRACT
Received 15. 1. 2015 Present study was aimed at isolation of autochthonous chitinase-producing bacteria from the gastrointestinal tracts of 3 Indian Major
Revised 10. 4. 2015 Carps (Labeo rohita, Catla catla, Cirrhinus mrigala) and 3 exotic carps (Hypophthalmichthys molitrix, Ctenopharyngodon idella,
Accepted 28. 4. 2015 Cyprinus carpio). Altogether, 119 bacteria were isolated from both the proximal and distal intestine and screened for chitinolytic
Published 1. 8. 2015 activity. On the basis of chitin hydrolysis zone, 63 isolates were primarily selected for chitinase production, from which 34 potent
strains were further studied for quantitative enzyme assay. Amongst them, the strains HMH1 and CMF2 exhibited potent chitinolytic
activity and were identified as Bacillus pumilus (KF454036) and Bacillus flexus (KF454035), respectively by 16S rRNA partial gene
Regular article
sequence analysis. Optimization of various fermentation parameters (e.g., temperature, pH, inoculums size, surfactant, colloidal chitin
concentration, incubation time, carbon sources, organic and inorganic nitrogen sources) were carried out in chitinase production
medium. Incubation for 72 h at 350C and initial pH 7.5 revealed optimum chitinase productions by B. pumilus HMH1 in the media
supplemented with colloidal chitin 0.1% (w/v), maltose 2% (w/v), ammonium sulphate 1.0% (w/v) and Tween-80 0.2% (v/v). However,
B. flexus CMF2 required 48 h incubation at 35 d initial pH 8.0 with colloidal chitin 0.15% (w/v), sucrose 1% (w/v), yeast extract
2.0% (w/v) and Tween-20 0.2% (v/v) supplementation for optimum yield. The results indicate that there is ample scope for further
research to appraise fish gut microorganisms for chitinase production or as probiotics to improve feed efficiency in fish.
Keywords: Bacillus pumilus, Bacillus flexus, chitinase, fish gut bacteria
INTRODUCTION microorganisms are scarce (Ray et al., 2012). Studies have indicated that fish
feeding on chitin rich diets have higher chitinase activity (Danulat, 1986;
Chitin (C8H13O5N)n has been estimated as the second most abundant biomass in Gutowska et al., 2004). Apart from such sporadic information, likely occurrence
the world after cellulose forming structural component of many fish food of chitinolytic bacteria in fish gut and their significance in feed utilization of the
organisms, including fungi, crustaceans, coelenterates, protozoan and green algae host species are inconclusive and contradictory.
(Rinaudo, 2006; Khoushab and Yamabhai, 2010). Chitin has been reported to Previous studies conducted with Indian major carps indicated beneficial aspects
make up 3.6% (wet weight) of the stomach contents of juvenile black sea bream, of gut associated enzyme-producing microbiota in the host fish with regard to
Acanthopagrus schlegeli (Om et al., 2003) that indicates feeding of chitin rich nutrition (Ghosh et al., 2002a; 2002b; Ray et al., 2010). Application of
organisms in fish. The ability to degrade chitin is considered to involve autochthonous chitinase-producing gut bacteria as probiotics or supplementation
principally the action of the enzyme chitinase (EC 3.2.11.14) that hydrolyzes of bacterial chitinase as feed additive might be assumed as a strategy for effective
insoluble chitin to its oligo and monomeric components. Chitinases are present in utilization of the chitin rich natural feedstuffs in fish. However, screening and
a wide range of organisms including viruses, bacteria, fungi, insects, higher characterization of chitinase-producing autochthonus fish gut microorganisms
plants and animals playing important physiological and ecological roles (Cody et can be viewed as a prerequisite for their likely application in fish. Microbial
al., 1990). investigation on chitin production of chitinase has drawn global attention not only because of its
degradation by bacteria was made by Benecke (1905), who reported chitinolytic extensive application, but also for the need of effective producer organisms.
Bacillus chitinovorus from the polluted waters of Kiel harbour. Therefore, the present study aimed at (1) isolation and enumeration of chitinase-
Freshwater carps cultured in India mostly feed on plankton, algae, aquatic producing gut microorganisms in 3 Indian Major Carps and 3 exotic carps, (2)
organisms and detritus representing omnivorous feeding aptitude (Jhingran, identification of the most promising chitinase-producing micro-organisms by 16S
1997). The chitin content of various copepods (e.g., Cyclops, Diaptomous etc.) rRNA partial gene sequence analysis, and finally (3) optimization of the various
comprising natural food for the carp fry and fingerlings has been reported to process parameters that influence chitinase production by the promising bacterial
range from 21 to 95 mg g-1 by dry weight , 1986). Being rich in strains, Bacillus pumilus HMH1 (KF454036) and Bacillus flexus CMF2
nutrients, the micro-environment of fish gut confers a favourable niche for the (KF454035).
microorganisms (Kar and Ghosh, 2008), and the gut microbiota in fish is closely
related to the food that they use to consume (Han et al., 2010). These distinct MATERIALS AND METHODS
microbial communities may contribute uniquely to the nutrient cycling in the
system et al., 2012). Therefore, feeding on chitin rich components might Experimental fishes
suggest likely occurrence of the chitinase-producing gut microorganisms in fish.
However, in comparison to the comprehensive work conducted on different Three species of Indian major carps (rohu, Labeo rohita; catla, Catla catla;
enzyme-producing gut microorganisms in fish, information on the chitinolytic gut mrigal, Cirrhinus mrigala) and three species of exotic carps (silver carp,

12

J Microbiol Biotech Food Sci / Banerjee et al. 2015 : 5 (1) 12-19
Hypophthalmichthys molitrix; common carp, Cyprinus carpio; grass carp, are presented in Table 1. The collection ponds were free of sewage release or
Ctenopharyngodon idella) were examined in this study. Three specimens of each other anthropogenic activities. The specimens were sampled by a gill-net and
species were collected from three composite carp culture ponds located transported to the laboratory at Golapbag, Burdwan inside oxygen-packed plastic
bags. Ranges of the water quality parameters during the collection period were:
specimens of each species were used in the present study. Their food habits, dissolved oxygen 6.5 7.8 mg L-1, temperature 26.2 27.8 7.2.
average weight and length, average weight of the gut and average length of gut
Table 1 Food habits, average live weight, average fish length, average gut weight and gut length of the fishes examined
Average fish Average fish Average Gut Gut length
Fish Species Food habits*
live weight (g) length (cm) weight (g) (LG) (cm.)
Catla, Catla catla Planktophagous
Rohu, Labeo rohita Omnivorous, mostly plant matter
Mrigal, Cirrhinus mrigala Detrivorous
Silver carp, Hypophthalmichthys
Planktophagous
molitrix
Grass carp, Ctenopharyngodon
Herbivorous, mostly macrophytes
idella
Common carp,Cyprinus carpio Detrivorous
*adapted from Jhingran, 1997

separately on TSA plates to obtain pure cultures. Isolates were individually
Processing of specimens cultured on the colloidal chitin agar plates at 30 days and appearance of
clear zone (due to chitin degradation) surrounding the colonies indicated positive
The fishes were kept separately in de-chlorinated tap water in 100L fibre-glass result of chitinase production. Isolates (colony size: mm) that produced
aquaria according to their source and species. The fish were starved for 48 h to a halo 25 mm (in excess of microbial colony) were selected for quantitative
clear their gastro-intestinal (GI) tracts before being dissected and to remove most enzyme activity.
of the allochthonous microbiota associated with digesta. The fish were
anaesthetized by applying 0.03% of tricaine methanesulfonate (MS-222). The Evaluation of quantitative chitinase activity
ventral surface of each fish was surface sterilized by scrubbing with 1% iodine
solution (Trust and Sparrow, 1974). The fish were dissected aseptically on ice Quantitative chitinase assay was carried out using colloidal chitin as substrate.
tray and their GI tracts were removed. The GI tracts were divided into PI Growth in colloidal chitin broth was centrifuged at 5,000 g for 5 min at 4
(proximal part of the intestine including intestinal bulb) and DI (distal part of the and the cell-free supernatant was used as the crude enzyme. The assay mixture
intestine) parts of the intestine, cut into pieces and opened by a longitudinal containing 0.5% colloidal chitin (1 mL), 25 mM sodium phosphate buffer (0.5
incision, transferred to sterile Petri-dishes and flushed carefully 3 times with mL, pH7.0) and crude enzyme (0.5 mL) was incubated for 1 h at 40 following
0.9% sterile saline solution using an injection syringe in order to remove non- Waghmare et al. (2010). The reducing sugars produced reacted with di-nitro
adherent (allochthonous) microbiota (Ghosh et al., 2010; Khan and Ghosh, salicylic acid (DNSA) and expressed as N-acetyl- -D-glucosamine standards to
2012). Gut segments from three specimens of a species collected from the same demonstrate the chitinase activity (Miller, 1959). Enzyme activity (U) was
pond were pooled together region-wise for each replicate, and there were three defined as the of N-acetyl- -D-glucosamine liberated mg-1 protein min-1.
replicates for each gut segment. Gut segments were homogenized with sterilized Protein content of the enzyme extract was measured using bovine serum albumin
pre-chilled 0.9% sodium chloride solution (1:10; w/v) (Beveridge et al., 1991). as standard (Lowry et al., 1951).
Pooled samples of 3 fish were utilized for each replicate to avoid erroneous
conclusions due to individual inconsistency in gut microorganisms, as described Identification of Isolates by 16S rRNA gene Sequence Analysis
somewhere else et al., 1995; Spanggaard et al. et al.,
2006). The most promising two chitinase producing strains were identified through 16S
rRNA partial gene sequence analysis after isolation and PCR amplification
Microbial Culture following the methods described in Das et al. (2014). The gene encoding 16S
rRNA was amplified from the isolates by polymerase chain reaction (PCR) using
Homogenate of the pooled gut segments of each of the three replicates for each -AGAGTTTGATCCTGGCTCAG- -
fish species and each region of gut was used separately after serial (1:10; up to GGTTACCTTGTTACGACTT-
10-5) dilutions (Beveridge et al., 1991). Diluted samples (100 L) were spread
aseptically within a laminar airflow on sterilized tryptone soy agar (TSA; primer, 2.5 mM MgCl2 U of Taq DNA polymerase
HiMedia, India) plates and incubated at 30 h to determine culturable (Invitrogen). To extract genomic DNA for obtaining template DNA from it,
heterotrophic autochthonous microbiota. Chitinase producing microorganisms colonies were suspended in sterilized saline, centrifuged and the pellet suspended
were isolated by spreading the diluted homogenate (100 L) on sterilized in InstaGene Matrix (Bio-Rad, USA). The cycle used for PCR reaction was: 35
colloidal chitin agar plates and incubated at 30 h. It was assumed that cycles of denaturation at 94
the microbiota, which had formed colonies on the selective colloidal chitin agar extension at 72 (Lane, 1991). PCR products were purified by using
plates, had chitin degrading activity. Colony forming units (CFU) per unit sample Montage PCR Clean up kit (Millipore, USA). Sequencing of the purified PCR
volume of gut homogenate were determined by multiplying the number of products were performed by using Big Dye terminator cycle sequencing kit
colonies formed on each plate by the reciprocal dilution (Rahmatullah and (Applied BioSystems, USA). Sequencing products were resolved on an
Beveridge, 1993) and data were transformed as log viable counts (LVC). automated DNA sequencing system (Applied BioSystems 3730XL, USA).
Colloidal chitin was prepared from the chitin flakes (Hi Media, India) following Sequenced data were edited using BioEdit Sequence Alignment Editor (Version
the modified method of Roberts and Selitrennikoff (1988). Chitin flakes were 7.2.0), aligned and analyzed for finding the closest homolog using National
ground to powder, 5g of powder was added slowly to 90 mL concentrated HCl Centre for Biotechnology Information (NCBI) GenBank and Ribosomal Database
and stirred vigorously for 2 h. Ice-cold 95% ethanol (500 mL) was added to it Project (RDP) databases. Sequences were deposited to the NCBI GenBank and
under vigorous stirring for 30min, kept overnight at 25 and stored at -20 accession numbers were obtained. Phylogenetic tree was constructed
until use. The precipitate was collected by centrifugation at 10,000 g for 15 min incorporating 16S rRNA partial gene sequences of the closest type strains using
and washed with 0.1 M sodium phosphate buffer (pH 7) until the colloidal chitin MEGA 5.1Beta4 software following the Minimum Evolution Method.
became neutral (pH 7.0) (Ahmadi et al., 2008). The well-separated colonies
appeared on colloidal chitin agar plates were randomly picked and streaked

13

Optimization of enzyme production producing bacteria were noticed in the DI region of silver carp, H. molitrix
(LVC=2.35 g-1 intestinal tissue) followed by the DI region of mrigal, C. mrigala
Submerged fermentation was carried out by both of the strains, CMF2 and (LVC=2.27 g-1 intestinal tissue).
HMH1 to optimize various process parameters influencing chitinase production.
Optimization of various process parameters were carried out in chitinase Table 2 Log values of culturable autochthonous aerobic / facultative anaerobic
production medium containing inorganic salts (g/L): 0.7 g KH2PO4; 0.3 g heterotrophic (grown on TSA plates) and chitinase-producing (grown on
K2HPO4; 4 g NaCl; 0.5 g MgSO4, 7H2O; 1 mg FeSO4, 7H2O; 0.1 mg ZnSO4, colloidal chitin agar plates) bacteria isolated from the GI tracts of 3 Indian Major
7H2O and 0.1 mg MnSO4, 7H2O. The parameters studied were: incubation Carps and 3 exotic carps
temperature (250C - 500C), initial pH of the media (5 - 9), inoculum volume (1% Log viable counts (g-1 intestinal tissue)
5%), surfactant (0.2%, v/v) (Tween 20, Tween 40, Tween 80, DMSO), Fish Species Proximate intestine(PI) Distal intestine(DI)
colloidal chitin (0.5-3.0 gL-1) as substrate and incubation period (24 h 120 h). TSA CCA TSA CCA
Further, the medium was supplemented with different carbon sources (1%, w/v) C. catla 5.84 1.46 6.87 1.95
(glucose, sucrose, lactose, maltose and starch) and organic/inorganic nitrogen L. rohita 5.25 1.92 6.33 2.05
sources (1%, w/v) (ammonium sulfate, ammonium nitrate, peptone, yeast extract, C. mrigala 5.33 2.14 6.17 2.27
ammonium chloride and tyrosine). The selected carbon and nitrogen sources H. molitrix 4.85 1.96 6.44 2.35
were varied within a narrow range (1%-5%) to optimize chitinase production. C. idella 5.11 1.25 6.39 1.39
C. carpio 4.76 1.65 5.94 2.23
Statistical Analysis TSA-Tryptone soy agar; CCA- Colloidal Chitin Agar
Statistical analysis of the quantitative enzyme activity data was performed by the Out of the 119 randomly selected isolates, 63 chitinase-producing bacteria were
analysis of variance (ANOVA) Zar (1999) primarily selected by qualitative enzyme assay, which were further evaluated by
using SPSS Ver10 (Kinnear and Gray, 2000). quantitative enzyme assay. Determination of chitinase activity led to select 34
strains (21 from PI and 13 from DI), results of which are depicted in Table 3. The
RESULTS maximum chitinolytic activity was noticed with the strain HMH1 (11.95 0.34
U) isolated from DI of H. molitrix, followed by the strain CMF2 (10.82 0.31 U)
Enumeration of gut microbial community in the 6 fish species studied revealed isolated from the PI of C. mrigala. Therefore, considering the results of chitinase
that autochthonous culturable heterotrophic and chitinase producing activity, the isolates HMH1 and CMF2 were finally selected for identification
microorganisms were present in both PI and DI regions in all the fish species and studied for chitinase production under submerged fermentation for likely use
studied (Table 2). Population levels of culturable autochthonous heterotrophic in future.
aerobic/facultative anaerobic and chitinase- producing bacteria were highest in
the DI regions of all the fish species studied. Maximum counts of chitinase-
Table 3 Chitinase activity of the gut bacterial strains selected through quantitative enzyme assay
Fish species Strains Chitinase (U) Fish species Strains Chitinase (U)
cd hi
LRF1 HMF1
e h
LRF6 HMF5
e g
Labeo rohita LRF2 HMF7
d Hypophthalmichthys molitrix g
LRH4 HMF2
e hi
LRH8 HMH6
h j
CCF6 HMH1
f bc
CCF7 CtIF6
fg b
Catla catla CCF1 CtIF5
e Ctenopharyngodon idella c
CCF2 CtIH1
e c
CCF4 CtIH2
i a
CMF2 CyCF2
f c
CMF3 CyCF3
f d
CMF4 CyCH6
g d
CMF5 CyCH4
Cirrhinus mrigala f Cyprinus carpio d
CMF6 7.84 CyCH1
g
CMH8
fg b
CMH9 CyCH9
f
CMH11
05).
N-acetyl- -D-glucosamine liberated mg-1 protein min-1
Bacillus pumilus (GenBank Accession no. NR112637), while the isolate CMF2
Nucleotide homology and phylogenetic analysis of the 16S rRNA partial gene showed 99% similarity with Bacillus flexus (GenBank Accession no.
sequences by nucleotide blast in the National Centre for Biotechnology NR024691). Phylogenetic relation of the two identified chitinolytic bacteria with
Information (NCBI) GenBank and Ribosomal Database Project (RDP) databases other closely related type strains retrieved from the RDP database are presented
revealed that the strains HMH1 and CMF2 were Bacillus pumilus (GenBank in the dendogram (Figure 1).
Accession no. KF454036) and Bacillus flexus (GenBank Accession no.
KF454035), respectively. The isolate HMH1 showed 98% similarity with

14

Figure 1 Dendogram showing phylogenetic relations of the two most promising chitinase producing bacterial strains, Bacillus pumilus HMH1 (KF454036) and
Bacillus flexus CMF2 (KF454035) with other closely related type strains retrieved from NCBI GenBank. GenBank accession numbers of the reference strains are
shown in parentheses. Horizontal bars in the dendogram represent branch length. Similarity and homology of the neighbouring sequences are shown by the bootstrap
values. Distance matrix calculated by Tamura 3-parameter following Minimum Evolution Method. Scale bar=0.005 substitutions per nucleotide position. Falsibacillus
pallidus EU36818.1 served as an out-group.
Optimum temperature for chitinolytic activity by the both strains, B. pumilus surfactants on chitinase production was determined by adding different
HMH1 and B. flexus CMF2 were noticed to be and 10.21 surfactants viz. Tween 20, Tween 40, Tween 80, DMSO in the production
0.18 U, respectively) (Figure 2a). Further increase in temperature resulted in medium at fixed volume (0.2%, v/v) and presented in Figure 2.d. The results
decrease in the enzyme yield. Initial pH of the medium required for chitinase evidenced maximum chitinase production by B. pumilus HMH1 with Tween 80
production by the strains was evaluated at various pH levels (5.0-9.0). Within the supplementation (14.55 0.21 U), although, Tween 20 was the best for B. flexus
tested pH range, pH 7.5 was optimum for chitinase production by B. pumilus CMF2 (11.75 0.21 U).
HMH1 (14.08 0.25 U), whereas, chitinase yield was the maximum by B. flexus Colloidal chitin was used in the production media as the substrate, as well as the
CMF2 (11.48 0.21 U) at pH 8 (Figure 2b). carbon source. Among the tested levels, 0.1% and 0.15% of colloidal chitin
Effect of percentage of inoculum on chitinase production has been depicted in supported maximum chitinase production by B. pumilus HMH1 (14.64 0.23 U)
Figure 2.c. Chitinase production gradually increased with increase of inoculum and B. flexus CMF2 (11.96 0.21 U), respectively (Figure 2e). Chitinase
percentage leading to maximum enzyme yield at 3.0% for B. pumilus HMH1 productions at different time intervals are presented in Figure 2f. Enzyme
(14.23 0.26 U) and 2.5% for B. flexus CMF2 (11.87 0.19 U), thereafter production increased gradually with incubation time, and maximum production
declined with further increase in the concentration. Influence of various was obtained after 72 h (15.12 0.26 U) and 48 h (12.25 0.21 U) in B. pumilus
HMH1 and B. flexus CMF2, respectively.
Figure 2 Effect of (a) temperature, (b) pH, (c) inoculum size (%, v/v), (d) surfactants (0.2%, v/v) (e) colloidal chitin (substrate) and (f) incubation period
on chitinase production by Bacillus pumilus HMH1 and Bacillus flexus CMF2.

15

Optimization of various supplemented carbon sources (1%, w/v) revealed that the strains, B. pumilus HMH1 (16.91 0.06 U) and B. flexus CMF2 (14.42 0.06
maltose was the most effective carbon source for chitinase production by B. U), respectively (Figure 3d). Moreover, supplementation of additional
pumilus HMH1 (16.87 0.06 U), while sucrose produced the best result for B. ammonium sulfate (>1%) reduced chitinase production by B. pumilus HMH1.
flexus CMF2 (14.53 0.06 U) (Figure 3a). Although, further increase in the However, supplementation of yeast extract at 2% could maximize chitinase
sucrose level diminished chitinase production by B. flexus CMF2, production by B. flexus CMF2 (14.59 0.11 U) (Figure 3e, f). Finally,
supplementation of 2% maltose was noticed as optimum for B. pumilus HMH1 optimization of the fermentation parameters with B. pumilus HMH1 resulted in
(16.97 0.06 U) (Figure 3b,c). 41.5% increase in chitinase production over the initial value, whereas, chitinase
Amongst the diverse organic and inorganic nitrogen sources (1%, w/v) evaluated, production was increased by 42.9% in B. flexus CMF2.
ammonium sulfate and yeast extract sustained maximum chitinase production by
Figure 3 Effect of carbon sources (a) levels of the selected carbon sources (b, c) nitrogen sources (d) and levels of the selected nitrogen sources (e, f)
on chitinase production by Bacillus pumilus HMH1 and Bacillus flexus CMF2.
DISCUSSION may not rule out the presence of extracellular bacterial chitinases representing
symbiotic relationships (Gutowska et al., 2004).
Chitinolytic enzymes are present in a wide range of organisms such as bacteria, In the present study, chitinase-producing strains were noticed through
fungi, yeasts, plants, actinomycetes, arthropods, and also in vertebrates (Hamid quantitative chitinase assay and the two most promising strains (HMH1 and
et al., 2013). There is a growing interest on chitin hydrolysis in aquaculture as CMF2) were identified as B. pumilus (GenBank Accession no. KF454036) and B.
fish consume green algae, crustaceans, zooplanktons, etc. as their food source flexus (GenBank Accession no. KF454035), respectively, based on the 16S rRNA
that contain considerable amount of chitin. Chitinases in the GI tract of fishes partial gene sequence analysis as suggested elsewhere (Roy et al., 2009; Ghosh
may come from the fish itself, its prey and/or the enteric bacteria. To the et al., 2010; Mondal et al., 2010; Ray et al., 2010). Previous reports have also
knowledge, chitinolytic bacteria in the intestine of fish were recorded for the first shown that Bacillus spp. can produce chitinolytic enzymes (Wen et al., 2002;
time in a marine teleost, Lateolabrax (Okutani, 1966). Since then, occurrences Chen et al., 2004; Driss et al., 2005; Waldeck et al., 2006; Chang et al., 2007),
of chtinolytic bacteria within the GI tracts of marine fish species were well however, present study is the first one reporting chitinolytic bacilli from fish gut.
documented (Ray et al., 2012) in comparison to their freshwater counter parts. In Moreover, diverse strains of extracellular enzyme producing Bacillus spp. have
the present investigation, microbial symbionts were isolated from the GI tracts of been identified from the GI tract of freshwater teleosts (for review see Ray et al.,
6 freshwater carp species and some of the isolates exhibited exogenous chitinase 2012), which are in accordance with the present report. Amongst the teleosts,
activity. It may be mentioned that the fish species examined were starved for 48 h previously, chitinolytic Enterobacter, Vibrio and Pseudomonas were reported
and their GI tracts were thoroughly washed with sterile chilled 0.9% saline prior from gray mullets (Hamid et al., 1979), while, chitinase producing Aeromonas
to isolation of microorganisms. Therefore, it is assumed that the microorganisms and Vibrio were isolated from the GI tract of tilapia (Sakata et al., 1980). In
isolated in the present study belong to the autochthonous microbiota as suggested another study, Sakata and Koreeda (1986) reported chitin degrading gut
elsewhere (Ray et al., 2010; Ghosh et al., 2010). Appreciable quantity of bacteria isolated from intestinal contents of tilapia (Sarotherodon niloticus)
chitinase-producing microflora detected in the PI and DI segments of the GI belonging to Plesiomonas shigelloides and Aeromonas hydrophila. Therefore,
tracts in the fish species studied may signify their probable role in degradation of available literatures suggest that chitinolytic bacteria in the Indian Major Carps
ingested chitin through the food. Previously, the fish gut isolates have been (IMC) or other carp species were not detected/evaluated so far, except in the
demonstrated to break down chitin in vivo to aid in the digestion process common carp, Cyprinus carpio (Sugita et al., 1999).
(Goodrich and Morita, 1977; Danulat and Kausch, 1984; MacDonald et al., Optimization of the important physical, chemical and nutritional parameters were
1986; Kono et al., 1987). Further, it may be mentioned that microbial population carried out under submerged fermentation to evaluate chitinase production
was found highest in DI regions of all the fish species studied when compared to potential of the two most promising chitinase-producing bacteria detected in the
the PI regions, which is in conformity with the earlier reports (Mondal et al., present study. Temperature affects a variety of bioprocesses, therefore, the
2008; Ray et al., 2010; Ghosh et al., 2010). Although, endogenous chitinases growth of microorganisms and enzyme production are also affected with
and chitinase genes have been detected in teleosts (Kurokawa et al., 2004), this alteration in incubation temperature. The highest chitinase activity by both the
strains was recorded at 350C. Previous reports by Narayana et al. (2009) and

16

Sudhakar and Nagarajan (2011) also documented maximum chitinase cases, increased growth has been reported due to chitin supplementation
production at 350C by soil isolates Streptomyces sp. ANU6277 and Trichoderma (Gopalakannan and Arul, 2006). Whether this is an evolutionary adaptation to
harzianum, respectively. In another study, Bacillus laterosporus produced high the natural diets to regulate endogenous chitinase production, or symbiotic
chitinase activity at 35 (Shanmugaiah et al., 2008). Further, considerable relation with the chitinolytic microorganisms that would benefit the host fish
levels of chitinase production at 30 might indicate adaptability of both the remains to be investigated. The present study is the first one reporting chitinase-
strains at the tropical water condition. producing microbiota in the GI tracts of the Indian major carps and exotic carps.
Initial pH of the production media not only helps in the chitinase production, but Microorganisms were isolated in the present study by culture dependant methods,
also plays an important role in cell growth (Saima et al. 2013). The results further study involving the PCR amplification technique for the chiA gene might
revealed that pH 7.5 and 8 were optimum for chitinase production by B. pumilus be useful in detecting chitinolytic bacteria associated with fish GI tract as
HMH1 and B. flexus CMF2, respectively. Previous reports also suggested that B. suggested by Sugita and Ito (2006). Whether the gut microbiota isolated in the
laterosporous (Shanmugaiah et al., 2008), Micrococcus sp. AG84 (Annamalai
et al., 2010), Aeromonas sp. JK1 (Ahmadi et al., 2008) and B. pabuli assessment of their role should be given high precedence in future studies.
(Frandberg and Schnurer, 1994) were capable of producing a high amount of Further, the efficient chitinase-producers detected in the present investigation
chitinase at alkaline condition. Optimum chitinase production at alkaline pH may be useful for treatment of chitinous waste and also for production of
noticed in the present study might be due to the fact that the bacterial symbiont different products of hydrolyzed chitin for various applications.
were isolated from the gut of agastric carps and the bacterium was adapted to the
alkaline pH therein as evidenced for phytase-producing gut bacteria in some carp Acknowledgements: The authors are grateful to the Head, Department of
species (Khan and Ghosh, 2013). A pH beyond the optimum level may alter the Zoology, The University of Burdwan, West Bengal, India; The Department of
amino acid composition of the enzyme and thereby diminishes the enzyme Science and Technology (FIST programme), New Delhi, India; and The
activity (Esakkiraj et al., 2009). Chitinase activity gradually increased with University Grants Commission (UGC-SAP-DRS programme), New Delhi, India
increase in inoculum concentration up to 2.5-3.0, and thereafter declined in for providing the research facilities. The first author is grateful to The UGC for
further concentrations. Reduced enzyme production at higher concentrations of awarding the research fellowship.
inoculum might be due to increased competition for nutrient uptake and
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19


ACTA ICHTHYOLOGICA ET PISCATORIA (2015) 45 (4): 373–384 DOI: 10.3750/AIP2015.45.4.05
SELECTION AND PROBIOTIC CHARACTERIZATION OF EXOENZYME-PRODUCING

BACTERIA ISOLATED FROM THE GUT OF CATLA CATLA
(ACTINOPTERYGII: CYPRINIFORMES: CYPRINIDAE)
Dipanjan DUTTA, Sudeshna BANERJEE, Anjan MUKHERJEE, and Koushik GHOSH*
Aquaculture Laboratory, Department of Zoology, The University of Burdwan, Golapbag, Burdwan, West Bengal, India
Dutta D., Banerjee S., Mukherjee A., Ghosh K. 2015. Selection and probiotic characterization of exoen-
zyme-producing bacteria isolated from the gut of Catla catla (Actinopterygii: Cypriniformes: Cyprin-
idae). Acta Ichthyol. Piscat. 45 (4): 373–384.
Background. Exoenzyme-producing gut microbiota and their likely use as probionts have been widely investiga-
ted in major carps. However, reports on exoenzyme-producing capacity together with inhibition of fish pathogens
are scarce. The presently reported study aimed at characterization of novel probionts from the gastro-intestinal
(GI) tract of an Indian major carp—catla, Catla catla (Hamilton, 1822) depending upon extracellular enzyme-pro-
duction, antagonism against Aeromonas spp., growth in fish mucus, bile tolerance, and bio-safety to the target fish.
Materials and methods. GI tracts were divided into proximal and distal parts, homogenized, and plated onto
selective media plates following enrichment culture technique. Exoenzyme-producing isolates were selected thro-
ugh qualitative and quantitative assay of 6 enzymes. Inhibition of Aeromonas spp. was tested through ‘cross-stre-
aking’ and ‘double-layer’ method. Tolerance to GI condition was determined by growth in sterilized fish mucus
and exposure to bile juice. Bio-safety evaluation was done by intra-peritoneal injection of live candidate pro-
biotics and co-culture with autochthonous gut bacteria. Further, 16S rDNA fragments from the putative probiotics
were sequenced, edited, analysed, identified, and deposited to the GenBank.
Results. Seventeen potent isolates were primarily selected through the ability of producing extracellular enzymes,
viz., amylase, protease, lipase, cellulase, phytase, and xylanase. Further study portraying antagonism against
Aeromonas salmonicida, A. hydrophila, and A. veronii led to select the strains CCH1A and CCH1Ph as putative
probiotics. Both the strains were competent to grow in intestinal mucus and could resist diluted bile juice (9% and
10.5%, respectively). The isolates did not produce any disease symptom or mortality in Catla catla fingerlings
during the 21 day trial and were competent to grow with other autochthonous isolates. On the basis of phenotypic
characteristics and 16S rDNA sequence analysis, the strains CCH1A and CCHIPh were identified as Bacillus
aerius (KF623288) and Bacillus sonorensis (KF623289), respectively.
Conclusion. Exoenzyme-producing gut bacteria may inhibit the growth of pathogenic Aeromonas spp. and tolera-
te gastrointestinal condition. Further research is inevitable to explore their potentialities in aquaculture.
Keywords: Indian major carp, Bacillus, extracellular enzyme, antagonism, pathogen
INTRODUCTION
The last decade has seen a growing interest in the ative factor behind mass mortalities of the Indian ma-
application of probiotics through the use of beneficial jor carps, i.e., catla, Catla catla (Hamilton, 1822); rohu,
microorganisms to improve the nutritional properties of Labeo rohita (Hamilton, 1822); and mrigal, Cirrhinus
feed (Verscheure et al. 2000) and to reduce the incidence mrigala (Hamilton, 1822) (see Karunasagar et al. 1986).
of fish diseases by inhibiting the growth of pathogenic Moreover, Catla has been found to be the most suscep-
microorganisms (Balcázar et al. 2006, Kesarcodi-Wat- tible to Aeromonas infection among the Indian major
son et al. 2008). High stocking density of fish under carps in several previous occasions (Khatri et al. 2009).
intensive or semi-intensive culture conditions leads Aeromonas spp. outbreaks in aquaculture are common
to increased feed ration and waste generation with si- in tropical countries and therefore use of antimicrobial
multaneous increase in organic load and bacterial mul- drugs has been suggested as prophylactic measure in
tiplication (Lio-Po and Lim 2014). Motile aeromonads, some of the previous reports (Karunasagar et al. 1986,
being the major bacterial pathogens among warm-water Mukherjee 1991). However, indiscriminate use of antibi-
freshwater fish species are reported to be the main caus- otics has been blamed for the emergence of antibiotic re-
* Correspondence: Dr. Koushik Ghosh, Aquaculture Laboratory, Department of Zoology, The University of Burdwan, Golapbag, Burdwan 713104, West Bengal,
India, phone: +91 9434251606, e-mail: (KG) kghoshbu@gmail.com, kghosh@zoo.buruniv.ac.in, (DD) dd.csirjrf@gmail.com, (SB) sudeshna.jrf@gmail.com, (AM)
anjanmukherjee84@gmail.com.
374 Dutta et al.
sistant bacteria in aquaculture (Akinbowale et al. 2006). segments from three specimens of Catla catla collected
Several countries have introduced restrictions for antibi- from a single pond were pooled together region-wise for
otic usage in aquaculture production (Kesarcodi-Watson each replicate and thus there were three replicates for the
et al. 2008). Alternatively, probiotics have been proposed study. Gut segments for each replicate was homogenized
as biological control agents to confer benefits to the host independently with sterilized chilled 0.9% phosphate
fish species. Enzymatic properties of aerobic (Bairagi et buffered saline (1 : 10, w/v; pH 7.0). Pooled sample of
al. 2002) and anaerobic (Ramirez and Dixon 2003) in- 3 fish were used for each replicate to avoid erroneous
testinal bacteria isolated from diverse fish species have conclusions due to individual disparity in gut microbiota
been shown as the potential role a probiotic organism (Mandal and Ghosh 2013).
may play. However, enzymatic potential along with an- Microbial culture. Homogenates of the pooled gut seg-
tagonistic properties against the potential fish pathogens ments were serially (1 : 10) diluted in NSS (Beveridge et
have been less studied (Sivasubramanian et al. 2012, Luo al. 1991) and poured aseptically (0.1 mL) onto sterilized
et al. 2014, Marlida et al. 2014, Dutta and Ghosh 2015). Soyabean Casein Digest (Tryptone Soya Agar, TSA; Hi-
A number of essential properties could be tested while Media, Mumbai, India) media plates to determine the au-
searching for an effective probiont. A putative probiont tochthonous culturable heterotrophic aerobic/facultative
should be able to colonize well in the gastrointestinal (GI) anaerobic bacteria population. In order to isolate and enu-
tract of the host fish species in order to reveal its bene- merate diverse extracellular enzyme-producing (e.g., am-
ficial effects. Adherence to mucosal layer of intestinal ylase, protease, lipase, cellulase, xylanase, and phytase)
surface (Nikoskelainen et al. 2001, Balcázar et al. 2008), bacteria, the diluted gut homogenates were plated onto
growth on fish mucus and tolerance to the bile juice are starch (ST), peptone-gelatin (PG), tributyrin (TB), car-
the essential attributes that a candidate probiont should boxymethylcellulose (CMC), xylan (XY), and modified
possess (Mukherjee and Ghosh 2014). Antibiotic suscep- phytase screening (MPS) media plates following enrich-
tibility is another important criterion because of the rapid ment culture technique. The culture plates were incubated
emergence of antibiotic resistance of microorganisms in at 30°C for 48 h, bacteria colonies were determined fol-
aquaculture (Ruban and Gunaseelan 2011). In addition, lowing dilution plate count method and mean values of the
the putative probiotic should not be harmful to the resi- replicates were expressed as log viable count · g–1 GI tract
dent or autochthonous gut bacteria as well as to the host (LVC) (Mandal and Ghosh 2013). The well-separated
(Verscheure et al. 2000). colonies were randomly collected, streaked individually
Hence, the aim of the presently reported study was to on respective media plates and re-streaked repeatedly to
isolate and screen novel probiotic bacteria from an Indi- acquire pure cultures. Pure cultures were maintained on
an major carp, catla, Catla catla, based on their ability to slants in a refrigerator (4°C) for further study.
produce different extracellular enzymes, and also to ver- Media composition. ST media (g · L–1): Yeast extract 5;
ify other probiotic properties, e.g., antimicrobial activity Peptone 5; NaCl 5; soluble starch 2; Agar 20; PG media (g
against Aeromonas spp., growth in fish mucus, resistance ·L–1): Peptone 5; Gelatin 4; Yeast extract 3; Agar 20; CMC
to bile, safety evaluation for the target fish and interaction media (g · L–1): Carboxymethylcellulose 5; Yeast extract 5;
with the normal gut flora. Peptone 5; NaCl 5; Agar 20; TB media (g · L–1): Tributyrin
agar base 23.00 g in 990 mL distilled water; Tributyrin 10
MATERIALS AND METHODS mL; XY media (g · L–1): Peptone 5; Yeast extract 2; Mg-
Collection and processing of sample. Nine specimens of SO4.7H2O 0.5; NaCl 0.5; CaCl2 0.15; Birchwood xylan 20;
catla, Catla catla (mean live weight 375 ± 10.97 g) with Agar 20; MPS media (g · L–1): Glucose 10; (NH4)2SO4 1.0;
no external disease symptoms (Smith et al. 2002) were Urea 10; Citric acid 3.0; Sodium citrate 2.0; MgSO4.7H2O
collected from three composite carp culture ponds located 1.0; Sodium phytate 3.0; FeSO4.7H2O 0.01; Agar 20.
around Burdwan (23°24′N, 87°86′E), West Bengal, India. Pathogenic Aeromonas strains and maintenance of
The specimens were brought to the laboratory with oxy- culture. Aeromonas salmonicida MTCC-1945 (AS) and
gen packing and distributed separately over 3 fibre-rein- Aeromonas hydrophila MTCC-1739 (AH) were acquired
forced plastic (FRP) aquaria of 350 L each on the basis of from the Microbial Type Culture Collection, Chandigarh,
their source. The ranges of water quality parameters dur- India. Aeromonas veronii KT737240 (AV) was isolated
ing the collection period were: temperature 26.2–27.8°C, from diseased fish and established as pathogen for Catla
pH 7.1–7.6, and dissolved oxygen 6.2–7.6 mg·L–1. catla in a previous study (Mukherjee and Ghosh 2014).
The fish were starved for 48 h in order to clear their GI The pathogenic strains were maintained on TSA (HiMe-
tracts and anaesthetized by applying 0.03% tricaine meth- dia) slants at 4°C. Stock cultures raised in tryptone soya
anesulfonate (MS-222) prior to sacrifice. Ventral surfaces broth (TSB) were stored at –70°C in 0.9% NaCl with 20%
were sterilized using 70% ethanol and fish were dissected glycerol to give constant inoculums during the study (Su-
aseptically to remove the GI tract (Ghosh et al. 2010). Gut gita et al. 1998).
samples were processed according to Mandal and Ghosh Determination of extracellular enzyme production:
(2013) for isolation of autochthonous microorganisms. qualitative and quantitative assay. Gut isolates were
The GI tracts were divided into proximal (PI) and distal primarily evaluated for qualitative determination of ex-
(DI) parts, flushed thoroughly with normal saline solution tracellular enzyme-producing capacities following growth
(NSS; 0.9% NaCl, w/v), and cut into small pieces. Gut on the selective media plates containing respective sub-
Probiotic attributes of exoenzyme-producing gut bacteria from Catla catla 375
strates. Amylase and protease-producing capacities were to a concentration of 1 mg · mL–1 for use as growth media.
determined on ST and PG plates through the development Samples were then filter-sterilized through 0.8 and 0.22
of clear halo zone when flooded with 1% Lugol’s iodine or μm pore size filter paper (HiMedia) and stored at –80°C
15% HgCl2, respectively (Jacob and Gerstein 1960). Cel- until use. Filter sterilized mucus samples were separately
lulase production was determined on CMC plates flooded inoculated (107 CFU · mL–1) with the two putative probi-
with Congo red prepared with 0.7% agarose (Teather and otics strains and grown at 30°C for 24 h. To calculate the
Wood 1982). Appearance of halo due to the presence of number of viable bacterial cells in mucus, serial dilutions
hydrolysed CMC indicated cellulase production in the me- (10–3, 10–4, and 10–5) were made from 24 h, 48 h, and 72 h
dium. Lipase producing isolates showed halo surrounding cultures, inoculated (0.1 mL) onto TSA plates, incubated
their colony in TB plates containing 1% tributyrin (San- at 30°C for 24 h and colony-forming units (CFU · mL–1)
giliyandi and Gunasekaran 1996). Positive xylanolytic were counted. Plates inoculated with sterilized mucus
isolates were detected after flooding the XY plates with were served as controls.
0.5% (w/v) Congo red and 5% ethanol followed by re- Bile tolerance. Crude bile juice (pH 5.6) was collected
peated washing with 1M NaCl (Ninawe et al. 2006). Clear from dissected gall bladders of the fish used for mucus
zone around the colonies on MPS media plates indicated collection, filter sterilized through 0.8 μm and 0.22 μm
phytase activity of the isolates (Howson and Davis 1983). pore size filter papers (HiMedia, Mumbai, India) and
Qualitative extracellular enzyme activity observed by the stored at –20°C until use. Bacteria grown in TSB (30°C,
appearance of halo (diameter in mm) around the colony 24 h) were centrifuged at 10 000 g (10 min, 4°C) and
was presented as scores (Das et al. 2014) as follows; 0, nil bacterial suspensions were prepared in PBS. Sterile PBS
(no halo); 1, low (6–10 mm halo); 2, moderate (11–20 mm (control) or sterile PBS supplemented with 2%–12%
halo); 3, good (21–30 mm halo); 4, high (31–39 mm halo); (v/v) fish bile juice was inoculated (107 CFU · mL–1)
5, very high (≥ 40 mm halo). with the bacterial suspension as illustrated elsewhere
On the basis of the cumulative scores, efficient ex- (Nikoskelainen et al. 2001, Balcázar et al. 2008, Muk-
tracellular enzyme producing isolates were selected for herjee and Ghosh 2014). Following incubation (1.5 h,
quantitative assay. Respective selective broth media were 30°C), the bacteria samples were serially diluted in ster-
used as production media for the enzymes, viz., amylase, ile PBS and viable bacteria counts were determined on
protease, cellulase, lipase, xylanase, and phytase. Quanti- TSA media plates.
tative assay for the production of amylase, cellulase, pro- Intra-peritoneal injection test. Evaluation of bio-safety
tease, and lipase were performed following the methods of the two putative probionts was performed by in vivo
described by Bernfeld (1955), Denison and Koehn (1977), studies. A total of 45 healthy Catla catla fingerlings (15 ±
Walter (1984), and Bier (1955), respectively. A compre- 1.31 g) was maintained in 75 L glass aquaria and acclima-
hensive description for measurement of these extracellu- tized in the laboratory condition for 2 weeks. Post accli-
lar enzymes and quantitative enzyme assay has been men- matization, the fingerlings were divided into three equal
tioned elsewhere (Bairagi et al. 2002). Quantitative assay groups (two experimental and one control), each with
of xylanase and phytase activities were measured after three replicates (5 fish × 3 groups × 3 replicates). Over-
Bailey et al. (1992) and Yanke et al. (1999), respectively. night cultures of candidate probionts were centrifuged
Enzyme activities were expressed as units (U). (2800 g, 15 min, 4°C) and cell pellets were suspended in
Assay for pathogen inhibitory activity. Pathogen in- sterile 0.9% saline. All fish from each experimental group
hibitory activity of the promising enzyme-producing iso- received intra-peritoneal (IP) injection of 1.0 mL contain-
lates were primarily tested by ‘co-culture’ (cross streaking 109 cells·mL–1 of a candidate probiotic bacterium. The
ing) method (Alippi and Reynaldi 2006) and the strains fish in control group were injected with sterile 0.9% saline
that showed antagonism against ≥1 studied Aeromonas (Mesalhy et al. 2008). Fish were fed ad libitum with a diet
spp. were further evaluated by the ‘double-layer’ meth- containing approximately 35% crude protein having fish-
od (Dopazo et al. 1988) with minor modification. Briefly, meal as the major protein source. The fish were cultured
putative antagonistic strains grown on TSA plates were for 21 days and observed daily for likely development of
killed with chloroform vapour (15 min), overlaid with the any disease symptom.
cultures containing the pathogenic strains and further in- Co-culture test. The two putative probiotic strains
cubated (48 h at 30°C) for the development of inhibition were co-cultured with the previously isolated eight au-
zone (halo). Antibacterial activity indicated by the halo tochthonous fish gut bacteria, e.g., Bacillus licheni-
(diameter in mm) around the colony was presented as fol- formis (KP940379), Bacillus subtilis subsp. spizizenii
lows; +, low (6–10 mm); ++, moderate (11–20 mm); +++, (KP940380), Bacillus aerophilus (KP940381), Bacillus
high (21–25 mm); ++++, very high (≥ 26 mm). There cereus (KP940382), Bacillus safensis (HP940383), Bacil-
were three replicates for each experimental set. lus methylotrophicus (KF559344), Bacillus subtilis sub-
Growth on fish mucus. Mucus from fish skin (Ross et al. sp. spizizenii (KF559346), and Enterobacter hormaechei
2000) and intestine were collected from live Catla catla (KF559347) by ‘cross-streaking’ method. Disappearance
(mean length 17.6 ± 2.16 cm; mean weight 65.28 ± 3.69 g) of the gap on the culture plates indicated compatibility of
and thereafter processed following Mukherjee and Ghosh the putative probionts with the other autochthonous bac-
(2014). Protein concentration of the mucus preparations terial strains.
were determined after Lowry et al. (1951) and adjusted
376 Dutta et al.
Morphological, physiological, and biochemical char- RESULTS

acterization. Colony morphology was studied visually. Isolation of gut bacteria and determination of extracel-
Gram-staining procedure was performed for the determi- lular enzyme-producing capacity. Diverse extracellular
nation of staining property. Endospore forming capacity enzymes (viz. amylase, protease, cellulase, phytase, and
was determined by staining with 5% aqueous malachite xylanase) producing bacteria were represented in both,
green. Culture characteristics were observed in broth and proximal (PI) and distal (DI) segments of the GI tract of
motility was studied using semi-solid media. Bacterial Catla catla (Fig. 1). Heterotrophic and diverse extracellu-
growth was observed at different temperatures (4–50°C), lar enzyme-producing bacterial populations were found to
pH (5.0–10.0), and under anaerobic conditions to see be predominantly high in the DI region. While consider-
whether the bacteria can grow under anaerobic condi- ing different extracellular enzyme-producing bacteria on a
tion as well. Sodium chloride tolerance was determined comparative scale, occurrence of cellulolytic bacteria was
by using different concentrations of NaCl (5%–15%, found to be the highest (LVC = 6.14) followed by lipolytic
w/v). In addition, pure cultures of the bacterial strains bacteria (LVC = 5.77), both in distal intestine. Whereas,
were subjected to various biochemical tests comprising xylan-degrading population in the proximal intestine was
of acid production from carbohydrates, arginine decar- noticed to be the lowest (LVC = 3.48).
boxylation, arginine dihydrolation, urease, and citrate
utilization. 9
GI tract]
8
Identification of the isolates by 16S rRNA gene se- 7
quence analysis. The most promising two putative -1
Log Viable Cell Count [CFU . g
6
probiotics were studied through 16S rRNA partial gene 5
sequence analysis after isolation and PCR amplification 4
following the methods described in Das et al. (2014). The 3
gene encoding 16S rRNA was amplified from the iso- 2
lates by polymerase chain reaction (PCR) using univer- 1
sal primers (27f, 5´-AGAGTTTGATCCTGGCTCAG-3´ 0

Total Amylolytic Proteolytic Lipolytic Cellulolytic Phytate Xylanolytic
and 1492r, 5´-GGTTACCTTGTTACGACTT-3´). The Heterotrophic

Count
bacteria bacteria bacteria bacteria degrading
bacteria
bacteria
PCR reactions were performed using 20 μL of PCR mix PI DI
and 1 μL of template DNA. To extract genomic DNA for

obtaining template DNA from it, colonies were suspend- Fig. 1. Log viable counts of adherent bacteria isolated from
ed in sterilized saline, centrifuged and the pellet suspend- the proximal intestine (PI) and distal intestine (DI) of
ed in InstaGene Matrix (Bio-Rad, USA). The cycle used Catla catla
for PCR reaction was: 35 cycles of denaturation at 94°C
for 45 s, annealing at 55°C for 1 min, extension at 72°C Altogether 118 bacteria were isolated, out of which
for 1 min (Lane 1991). PCR products were purified by 17 extracellular enzyme producing strains (6 from PI and
using Montage PCR Clean up kit (Millipore, USA). Se- 11 from DI) were primarily selected on account of quali-
quencing of the purified PCR products were performed tative enzyme activity data presented as scores (Table 1),
by using Big Dye terminator cycle sequencing kit (Ap- maximum and minimum scores being 29 and 15, respec-
plied BioSystems, USA). Sequencing products were re- tively. Further, results of the quantitative enzyme assay
solved on an automated DNA sequencing system (Ap- revealed significant differences in the enzyme activities
plied BioSystems 3730XL, USA). Sequenced data were among the primarily selected bacterial isolates (Table 2).
edited using BioEdit Sequence Alignment Editor (Ver- The highest amylolytic activity was noticed with the iso-
sion 7.2.0), aligned and analysed for finding the closest late CCH1A (349.37 ± 6.05 U), while it was lowest in the
homolog using National Centre for Biotechnology Infor- isolate CCH3X (125.44 ± 3.83 U). The strain CCH1A ex-
mation (NCBI) GenBank and Ribosomal Database Pro- hibited maximum protease activity (80.40 ± 2.26 U) and
ject (RDP) databases. Sequences were deposited to the minimum protease activity was evidenced with the strain
NCBI GenBank and accession numbers were obtained. CC2F3L (21.75 ± 1.07 U). All these strains were isolated
Phylogenetic tree was constructed incorporating 16S from the DI regions. Maximum lipase activity was recorded
rRNA partial gene sequences of the closest type strains with the strain CCH1A (5.28 ± 0.26 U) isolated from the DI
using MEGA 5.1Beta4 software following the Minimum region, being minimum with the strain CC2F1Ph (4.11 ±
Evolution Method. 0.19 U) isolated from the PI region. Maximum cellulase ac-
Statistical analyses. Statistical analysis of the quantita- tivity was documented with the strain CCF2P (73.51 ± 2.19
tive enzyme activity data was performed by the one-way U) isolated from the PI region, while CC2H2Ph, isolated
analysis of variance (ANOVA), followed by Tukey’s test from the DI region, exhibited minimum cellulase activity
according to Zar (1999) using SPSS Version 10 software (59.59 ± 2.14 U). Maximum phytase activity was docu-
(Kinnear and Gray 2000). mented with the strain CCH1A (396.45 ± 6.31 U) isolated
Ethical issues. Experimental fish were collected and han- from the DI region, while CC2H2Ph isolated from the DI
dled following the approved guidelines of the Institutional region, exhibited minimum phytase activity (66.27 ± 2.18
Ethical Committee. As fish produced in aquaculture farm U). Maximum xylanase activity was documented with the
was used for the study, official permit was not required. strain CCH1Ph (13.33 ± 0.96 U) isolated from the DI re-
gion, while CCF2P, isolated from the DI region, exhibited activity score being 28 and 29, respectively) were the most
minimum xylanase activity (6.54 ± 0.22 U). Overall exam- efficient among the 30 primarily selected bacterial strains.
ination of the six different extracellular enzyme activities Determination of antagonistic activity against
revealed that the strains CCH1A and CCH1Ph (qualitative Aeromonas spp. Further study concerning antagonism
Table 1
Qualitative determination of extracellular enzyme activity by the bacteria isolated from the gut of Catla catla
Enzyme activity
Strain
Amylase1 Protease2 Lipase3 Cellulase4 Phytase5 Xylanase6 Total score
CCF3L 4 5 4 5 2 2 22
Proximal Intestine
CCF2P 4 5 4 5 2 2 22
CC2F1Ph 3 3 2 3 3 3 17
CC2F1A 2 4 3 3 3 3 18
CC2F3L 5 1 4 3 0 2 15
CC3F4A 2 2 2 4 3 2 15
CCH1X 3 2 3 5 0 3 16
CCH3X 2 3 3 5 0 2 15
CCH4X 4 5 4 5 2 3 23
CCH1A 5 5 5 4 5 4 28
Distal Intestine
CCH3A 4 5 4 4 4 2 23
CCH3L 4 4 4 4 3 2 21
CCH3C 3 5 4 4 2 2 20
CCH4C 2 4 3 4 0 2 15
CCH1Ph 5 5 5 5 5 4 29
CC2H2Ph 2 3 3 3 1 3 15
CC2H8L 3 4 3 4 2 3 19
Enzyme activities (with pure culture of bacterial isolates) were presented as scores as described in the text;1on starch (SA) plate; 2on gelatin-
peptone (GP) plate; 3on Tributyrin-agar (TA) plate; 4on carboxymethylcellulose (CMC) plate; 5on modified phytase screen media (MPSM)
plate; 6on birchwood xylan plate; Scores: 0 = nil (no halo), 1 = low (6–10 mm halo diameter), 2 = moderate (11–20mm halo diameter), 3 =
good (21–30 mm halo diameter), 4 = high (31–39 mm halo diameter), 5 = very high (≥ 40 mm halo diameter).
Table 2
Quantitative determination of extracellular enzyme activity (unit activity, U) by the bacteria isolated from the gut
of Catla catla
Enzyme activity [U]

Strain
Amylase1 Protease2 Lipase3 Cellulase4 Phytase5 Xylanase6
CCF3L 241.13 ± 5.28d
73.54±2.28 de
4.36±0.17 ab
72.57 ± 2.19 c
80.50 ± 2.29 bc
7.58 ± 0.34b
Proximal Intestine
CCF2P 250.44 ± 5.87d 74.58 ± 2.34de 4.47 ± 0.27ab 73.51 ± 2.19c 84.54 ± 2.19 bc
6.54 ± 0.22a
CC2F1Ph 191.80 ± 4.28c 55.34 ± 2.44c 4.11 ± 0.19a 62.48 ± 2.30a 198.71 ± 4.84 e
7.33 ± 0.24b
CC2F1A 129.53 ± 3.83a 70.61 ± 2.29d 4.26 ± 0.18ab 59.67 ± 2.38a 191.23 ± 4.70 de
7.27 ± 0.39b
CC2F3L 275.48 ± 5.92e 21.75 ± 1.07a 4.59 ± 0.13b 61.58 ± 2.11a — 7.27 ± 0.34b
CC3F4A 130.37 ± 3.82a 31.36 ± 1.05b 4.18 ± 0.11a 69.46 ± 2.14bc 186.66 ± 4.68 d
7.14 ± 0.32b
CCH1X 167.19 ± 4.87b 31.53 ± 1.14b 4.27 ± 0.15a 71.34 ± 2.27bc — 7.39 ± 0.28b
CCH3X 125.44 ± 3.83a 52.58 ± 2.19c 4.29 ± 0.21ab 72.30 ± 2.36c — 7.02 ± 0.24b
CCH4X 239.45 ± 5.81d 73.58 ± 2.12de 4.51 ± 0.24ab 71.57 ± 2.13bc 86.48 ± 2.22 c
7.20 ± 0.31b
CCH1A 349.37 ± 6.05f 80.40 ± 2.26f 5.28 ± 0.26c 70.52 ± 2.27bc 396.45 ± 6.31 gh
11.76 ± 0.88c
Distal Intestine
CCH3A 247.51 ± 5.03d 74.33 ± 2.40de 4.56 ± 0.14b 67.37 ± 2.19b 210.42 ± 5.87 f
6.98 ± 0.26ab
CCH3L 244.23 ± 5.99d 73.66 ± 2.37de 4.48 ± 0.20ab 61.60 ± 2.22a 200.40 ± 5.94 ef
7.46 ± 0.38b
CCH3C 171.39 ± 4.82b 79.61 ± 2.35f 4.41 ± 0.25ab 68.53 ± 2.29bc 82.48 ± 2.11bc
7.01 ± 0.29ab
CCH4C 133.70 ± 3.73a 71.65 ± 2.23d 4.30 ± 0.27ab 67.62 ± 2.26b — 7.09 ± 0.30ab
CCH1Ph 281.21 ± 5.64e 77.56 ± 2.31e 4.92 ± 0.13c 72.28 ± 2.31c 387.04 ± 6.08 g
13.33 ± 0.96c
CC2H2Ph 127.47 ± 3.81a 54.45 ± 2.52c 4.24 ± 0.28ab 59.59 ± 2.14a 66.27 ± 2.18 a
7.18 ± 0.34b
CC2H8L 186.46 ± 4.78c 71.56 ± 2.21d 4.29 ± 0.18ab 69.40 ± 2.31bc 82.69 ± 2.22 bc
7.17 ± 0.33b
Values are shown as mean ± standard deviation (n = 3); Values with the same superscripts in the same vertical column are not significantly
different (P < 0.05); 1 μg maltose liberated mL-1 of enzyme extract min–1; 2 μg tyrosine liberated mL–1 of enzyme extract min–1; 3μmole free
fatty acid liberated mL–1 of enzyme extract min–1; 4 μg glucose liberated mL–1 of enzyme extract min–1; 5 μg inorganic phosphate liberated
mL–1 of enzyme extract min–1; 6 mg D-xylose liberated mL–1 of enzyme extract min–1.
378 Dutta et al.
against A. hydrophila, A. salmonicida, and A. veronii Table 3

revealed that out of the 30 primarily selected isolates, 4 Inhibition zone produced by the bacteria isolated from
strains (2 each from PI and DI regions) could inhibit at least the gut of Catla catla in double layer method
one of the tested Aeromonas spp. through cross-streaking
method. Pathogen inhibitory activity of these 4 isolates Score
Strain
were also assessed by double layer method and the zone AH AS AV
of inhibition (halo) produced by the gut isolates were de-
CCF2P ++++ ++++ +
picted in Table 3. In consequence of the maximum extra-
CCH1Ph ++++ +++ ++++
cellular enzyme-producing capacities, the strain CCH1Ph
CCH1A ++++ +++ –
was also noticed to be antagonistic against all 3 of the
CC2F1A + – –
among tested fish pathogens, whereas the strain CCH1A
was found to be antagonistic against A. hydrophila and A. AH = Aeromonas hydrophila, AS = Aeromonas salmonicida, AV
= Aeromonas veronii; Score: + low (6–10 mm halo diameter),
salmonicida among the tested pathogens. Therefore, these
++ moderate (11–20mm halo diameter), +++ high (21–25 mm
promising strains (CCH1A and CCH1Ph) were analysed halo diameter), ++++ very high (≥ 26 mm halo diameter), – no
further for evaluation of the other probiotic properties. antagonism.
Phenotypic characterization of the candidate probi-
otic bacteria. Phenotypic characterization of the strains
revealed that both the strains were long, Gram-positive Table 4
rods, catalase positive and capable of forming irregular Key biochemical characteristics differentiating strains
white colonies. Both were capable of utilizing starch, gel- CCH1A (Bacillus aerius) and CCH1Ph (Bacillus sono-
atin, nitrate, and produced acid from arabinose, dextrose, rensis)
mannitol, and xylose. In addition to these common char-
acteristics, the strains differ in some others. CCH1A could Characteristics CCH1A CCH1Ph
grow at 8°C but could not tolerate higher temperatures, Arginine decarboxylase + –
unlike CCH1Ph. It also gives negative results for arginine Arginine dihydrolase – +
dihydrolase and urease test. On the other hand, CCH1Ph Citrate utilization + –
showed better acid tolerance and can utilize a number of Urease – +
carbon sources like cellobiose, inositol, raffinose, and Tolerance of NaCl 11% + –
rhamnose. However, it could not utilize amino acids like Growth at 8°C + –
alanine and tryptophan (Table 4). 40°C – +
Genotypic identification of the selected isolates. Based 45°C – +
on the nucleotide homology and phylogenetic analysis of pH 5.5 – +
the 16S rRNA partial gene sequences by nucleotide blast in Carbon source utilization N-Acetylglucosamine – +
the NCBI GenBank and RDP databases, the putative probi- D-Cellobiose – +
otic strain CCH1A was identified as Bacillus aerius (Gen- Citric acid + –
Bank Accession No. KF623288), which was closest to the Dulcitol – +
type strain Bacillus aerius (AJ831843.1). The other isolate, myo-Inositol – +
CCH1Ph was identified as Bacillus sonorensis (GenBank D-Raffinose – +
Accession No. KF623289) that showed maximum similar- D-Rhamnose – +
ity with the type strain Bacillus sonorensis (AF302118.1). Sodium succinate + –
Phylogenetic relation of the two identified bacterial isolates L-Sorbose + –
with other closely related type strains retrieved from the Thioglycolate + –
RDP database were presented in the dendrogram (Fig. 2). Xylitol + –
Evaluation of growth in skin and intestinal mucus. The Acid from
+ –
strains, CCH1A and CCH1Ph, grew well in mucus of Cat- D-Maltose
la catla (Fig. 3) collected from both skin and intestine. Amino acid utilization L-Alanine + –
Log viable cell count (CFU · mL–1) for both the candidate L-Threonine – +
probionts indicated that the strains were more potent to L-Tryptophan + –
grow in mucus collected from intestine than that of skin. + Positive, – Negative.
Bile tolerance. Both the candidate probiotic bacteria,
CCH1A and CCH1Ph, showed tolerance against diluted didate probiotics did not induce any pathological signs/
bile juice. The selected bacterial strains survived after disease symptoms or mortalities in both treatment groups
1.5 h exposure to different concentrations of bile juice during the 21 days small-scale in vivo experiment. Test
(0%–12%) ranging pH values 5.5–7 (Table 5). Moreover, of co-culture (in vitro) against eight autochthonous fish
marked changes were not detected in the growth of the se- gut bacteria revealed that the selected bacterial strains
lected bacteria even after exposure to the diluted bile juice did not hinder growth of the normal gut flora. Therefore,
(12%) for 24 h (data not presented). the selected putative probionts were compatible with
Bio-safety of the candidate probiotic bacteria. Along the other commonly occurring autochthonous fish gut
with the control set, intra-peritoneal injection of the can- bacteria.
93 Bacillus aerius AJ831843.1
94 CCH1A KF623288
Bacillus sonorensis AF302118.1

100 95 CCH1Ph KF623289
Bacillus amyloliquefaciens AB255669.1
100 Bacillus subtilis subsp. spizizenii AF074970.1
92 Bacillus tequilensis HQ223107.1
Bacillus humi AJ627210.1

69
Bacillus niabensis AY998119.2
50 Bacillus firmus D16268.1
86 Bacillus niacini AB021194.1
93 Bacillus selenatarsenatis AB262082.1
Bacillus algicola FR775437.1
0.005
Fig. 2. Dendrogram showing phylogenetic relations of the two potential probiotic bacterial strains, Bacillus aerius CCH1A
(KF623288) and Bacillus sonorensis CCHIPh (KF623289), isolated from Catla catla, with other closely related strains
retrieved from NCBI GenBank and RDP; The GenBank accession numbers of the reference strains are shown besides
the names; Horizontal bars in the dendrogram represent the branch length; Similarity and homology of the neighbouring
sequences have been shown by bootstrap values; Distance matrix was calculated by Tamura 3+Gamma model; The
scale bar indicates 0.005 substitutions per nucleotide position; Bacillus algicola FR775437.1 served as an outgroup
9 A Table 5
8 Tolerance of the selected isolates at different concentra-
7 tions of fish bile juice for 1.5 h at 30°C
6
5
Log CFU· mL-1
Bile [%]
4 CCH1A CCH1Ph
3 0 6.91 ± 0.007 6.93±0.008
2 1 6.91 ± 0.006 6.93 ± 0.009
1 2 6.88 ± 0.004 6.92 ± 0.007
0 3 6.88 ± 0.004 6.92 ± 0.008
24 48 72 4 6.87 ± 0.004 6.90 ± 0.007
Incubation Period [h] 5 6.86 ± 0.006 6.90 ± 0.005
INTESTINE SKIN 6 6.86 ± 0.003 6.88 ± 0.005
7 6.86 ± 0.005 6.88 ± 0.003
9 B
8 6.84 ± 0.005 6.87 ± 0.006
8
9 6.84 ± 0.007 6.86 ± 0.002
7
10 6.83 ± 0.001 6.86 ± 0.004
6 11 6.81 ± 0.001 6.83 ± 0.003
5 12 6.80 ± 0.003 6.81 ± 0.004
4
Viable count was determined on TSA plates inoculated with bile
3 exposed bacterial suspension; Values are shown as mean ± standard
2 deviation (n = 3)
1
DISCUSSION
0
The primary step for acquisition of potential probionts
24 48 72
to be used in an aquaculture system is the collection of
Incubation Period [h]
background information on the prospective microorgan-
INTESTINE SKIN
isms and their hosts (Gomez-Gil et al. 2000). The present-
Fig. 3. Log values of viable count of the selected gut bacte- ly reported study evaluated population of heterotrophic as
ria isolated from Catla catla: CCH1A (Bacillus aerius) well as different extracellular enzyme-producing bacteria
(A) and CCHIPh (Bacillus sonorensis) (B) grown in in the PI and DI regions of the GI tract in Catla catla.
skin and intestinal mucus of Catla catla; Viable count Although, gut bacteria in catla were noticed to be dom-
was done on TSA plates inoculated with respective bac- inated by the cellulolytic bacteria, existence of lipolytic,
teria cultures of 24 h, 48 h, and 72 h in fish mucus amylolytic, and proteolytic bacteria clearly advocated
omnivorous feeding aptitude of the species as suggested
elsewhere (Dasgupta 1996, Ghosh et al. 2002a, Khabade
380 Dutta et al.
2015). Occurrence of higher numbers of heterotrophic en- The production of antimicrobial substances by some
zyme-producing bacteria in DI region compared to the PI bacteria seemed to play an important role in antagonizing
region was in agreement with the previous reports (Mon- other bacteria in aquatic ecosystems (Dopazo et al. 1988).
dal et al. 2008, Das et al 2014). This might indicate the In view of this context, the use of non-pathogenic and
possibility of microbial degradation of the food material antimicrobial-producing bacteria as probiotic bio-control
in this part of the GI tract in assistance with the highly agents might have a prospect in disease management.
colonized enzyme-producing bacteria (Ghosh et al. 2010). Therefore, pathogen inhibitory activity of the bacteria has
Therefore, evaluation of beneficial attributes of the gut been considered as the major criterion for the selection of
bacteria with high enzyme-producing capacity to select probiotics in several studies (Vine et al. 2006). However,
putative probiotics for fish may be justified. to the authors’ knowledge, such information is scarce with
In search of the best suited bacterial probiotics for fish, respect to gut bacteria from IMCs with high enzymatic
isolation followed by a series of consecutive screening activities (Mukherjee and Ghosh 2014, Dutta and Ghosh
process have been suggested to cover both functional and 2015). Hence, demonstration of antagonistic properties
safety aspects of the putative probiotics (Vine et al. 2006). against Aeromonas spp., the predominant fish pathogen in
Moreover, the presently reported study emphasized au- the tropical countries, was chosen as the second criterion
tochthonous gut microorganisms as the putative probiot- for the selection of candidate probionts in the presently
ics assuming that the resident flora would be well adapted reported study. Among the promising enzyme producers,
within the gut micro-environment of the host fish (Dutta 4 strains were noticed to be antagonistic against at least
and Ghosh 2015). Previous investigations have suggest- one of the three fish pathogens tested. Although a dif-
ed beneficial attributes of the gut bacteria in the digestive ferent degree of pathogen inhibition was detected in this
processes of fish (Austin 2006, Nayak 2010). Likely use study, the inhibition zones exhibited by the strong antag-
of extracellular enzyme producing bacteria as probiotics onists (CCH1Ph, CCF2P, and CCH1A) through the dou-
have called attention to promote nutritional benefits in the ble layer assay were similar to those reported by Ghosh
Indian major carps and other cultivable fish (Ghosh et al. et al. (2007) for B. subtilis SG4 and Krishnan (2014) for
2002a, 2002b, Askarian et al. 2012). Therefore, in view of Bacillus sp. against different strains of A. hydrophila. The
inducing improved nutrient utilization in fish, exogenous strongest inhibitory activity towards the Aeromonas spp.
enzyme producing ability was considered as the primary in terms of the zone of inhibition was displayed by the
criteria for the candidate probionts. Results of the pres- strain CCH1Ph. Our study revealed 4 out of 17 efficient
ently reported study depicted that heterotrophic commu- extracellular enzyme-producing strains to be antagonistic
nity within the GI tract of C. catla were represented by (23.5%) against Aeromonas spp. Previously, Sugita et al.
amylase, protease, lipase (digestive enzymes), cellulase, (1996) documented 3.2% of the gastrointestinal bacterial
phytase, and xylanase (anti-nutritional factor degrading isolates to be antagonistic against 18 different pathogens
enzymes) producing bacteria. The results pertaining quan- including 12 Aeromonas spp. Nonetheless, it could be
titative assay of extracellular enzymes produced by di- hypothesized from the present investigation that the ex-
verse strains isolated in the presently reported study indi- tracellular enzyme-producing bacteria colonizing within
cated that the capacity of extracellular enzyme production the GI tract of Catla catla might offer protection against
varied among the isolates (Table 2). Thus, the strains that the pathogenic Aeromonas strains. Although, the mech-
exhibited a high capacity for secreting exoenzymes could anism of inhibition was not characterized here, preced-
be selected as putative probiotic candidates to facilitate ing studies suggested that the growth of pathogens (e.g.,
feed utilization in the fish. A total of 118 extracellular en- A. hydrophila) might be inhibited by competitive exclu-
zyme producing isolates were obtained from the proximal sion, competition for essential nutrients (e.g., glucose and
and distal parts of the intestine. Scores were assigned to iron) and siderophore production (Lalloo et al. 2010). The
the strains based on the qualitative analysis of the extra- method used in the presently reported study to assay the
cellular enzyme activity and strains with individual score antagonistic effect of the putative probiotics (agar-over-
≥15 (17 strains out of 118) were considered as strong lay/double-layer) demonstrates the influence of diffus-
enzyme producers, which were further confirmed by the ing antimicrobial products on growth of the pathogenic
quantitative assay. This preliminary screening has resulted bacteria. Thus, besides competitive exclusion, our results
in an elimination of 85.6% (n = 101) of the total isolates might indicate secretion of the antibacterial compounds
from current study. Diverse enzyme producing bacteria by the strains CCH1A and CCH1Ph inhibiting the growth
were recorded from the GI tracts of the Indian major carps of Aeromonas spp. as opined by Geraylou et al. (2014)
in previous studies (for review see Ray et al. 2012). The and Mukherjee and Ghosh (2014).
majority of these studies addressed digestive (amylase, Afterwards, considering extracellular enzyme produc-
protease, lipase) and/or anti-nutritional factors (ANF) de- ing capacity and antagonism against Aeromonas spp. to-
grading enzymes (cellulase, phytase) producing capacity gether, two isolates (CCH1A and CCH1Ph) categorized
by the GI tract bacteria. However, the presently reported as putative probiotics were identified by molecular as well
study represented a combined approach to consider the as conventional methods and further characterized for
ability of both digestive as well as anti-nutritional factors verification of other probiotic attributes. Analysis of the
degrading enzymes producing ability as the selection cri- 16S rRNA partial gene sequences and nucleotide homol-
teria of the putative probiotics. ogy revealed that the strains CCH1A and CCH1Ph, were
Bacillus aerius (GenBank Accession No. KF623288) and criterion for any of the probiotic bacteria under critical
Bacillus sonorensis (GenBank Accession No. KF623289), evaluation (Verschuere et al. 2000). Although, several
respectively. Extracellular enzyme-producing ability of Bacilli were demonstrated as probiotics for fish, some
the diverse Bacillus spp. isolated from the GI tract of other strains of B. cereus or B. subtilis were established
different freshwater fish species has been widely docu- as fish pathogens (Pychyński et al. 1981). In the pres-
mented (for review see, Ray et al. 2012). In comparison, ently reported study, small scale in vivo study with the
pathogen inhibitory potential of the bacilli has been rarely two selected putative probiotic strains did not induce any
indicated. B. subtilis SG4 isolated from the gut of mrigal, pathological signs or mortalities in Catla catla and there-
C. mrigala (see Ghosh et al. 2007), B. methylotrophicus fore, considered as safe. The extent of the antagonism by
isolated from channel catfish intestine (Ran et al. 2012), the adherent gut bacteria might also represent possibility
and B. cereus and B. circulans isolated from the gut of of negative interaction against the other resident flora.
some other fish species (Lalloo et al. 2010, Geraylou et al. A co-culture test with the previously isolated autoch-
2014) were reported to be antagonistic against different thonous gut bacteria of catla revealed that the putative
strains of A. hydrophila. In addition, enzyme-producing probiotic strains did not affect their growth. Therefore,
B. thuringiensis isolated from the GI tract of the Atlantic likely co-existence of the diverse autochthonous bacteria
salmon, Salmo salar Linnaeus, 1758, was shown to inhib- and putative probiotics selected in the presently reported
it A. salmonicida subsp. salmonicida and 3 other patho- study could be substantiated within the fish species un-
genic bacteria (Askarian et al. 2012). Extracellular diges- der observation.
tive enzyme-producing B. subtilis BHI344 isolated from
the GI tract of the channel catfish, Ictalurus punctatus CONCLUSIONS AND PERSPECTIVES FOR
(Rafinesque, 1818), was shown to inhibit (in vitro) the THE FUTURE
growth of pathogenic A. hydrophila, A. sobria, and A. cav- As evident from the observations made during the last
iae (see Luo et al. 2014). In the presently reported study, few decades, gut microbiota within the freshwater fishes
B. sonorensis CCH1Ph exhibited inhibitory effects against were somewhat dominated by the Bacillus spp. (Ghosh et
A. hydrophila, A. veronii, and A. salmonicida. Unlike to al. 2010, Ray et al. 2010), which were in conformity with
the present observation, another isolate of B. sonorensis the present report. Further, Bacillus spp. seemed to be the
CM2H3L from the gut of C. mrigala has been reported to most common probiotics as they can be stored easily in
inhibit only A. salmonicida, however, neither A. hydrophi- the spore form for an indefinite period (Hong et al. 2005),
la nor A. veronii was inhibited (Dutta and Ghosh 2015). To and therefore, convenient for easy preparation to add into
the authors’ knowledge, extracellular enzyme-producing the artificial diets (Bisht et al. 2012). With a combined
ability and/or pathogen inhibitory potential of B. aerius approach of different screening strategies, the strains B.
from fish gut depicted in the present study has not been aerius CCH1A and B. sonorensis CCH1Ph were select-
reported previously. ed as putative probiotics in the presently reported study.
Apart from the functional attributes (e.g., enzyme se- A combination of two screening strategies for selecting
cretion, pathogen inhibition etc.) a probiotic bacterium probiotics might be far superior to normally prevalent sin-
should have the capacity to withstand the fish GI tract gle screening strategy as they could mutually complement
conditions. Therefore, growth in fish mucus and toler- each other (Luo et al. 2014). While probiotic attributes
ance to fish bile were considered as additional criteria of B. sonorensis from other fish species (C. mrigala) has
to verify the probiotic features. Both of the selected gut been documented very recently (Dutta and Ghosh 2015),
bacteria grew well in fish mucus, although, minor differ- to the authors’ knowledge, this is the first report pertaining
ences were noted in bacterial growth rate which might likely probiotic potential of B. aerius isolated from the fish
be due to specific nutritional requirements of the bacteria gut. Although, postulations based on in vitro experiments
(Geraylou et al. 2014). In accordance to the observation might not act in accordance with in vivo conditions, in-
made by Mukherjee and Ghosh (2014), the presently digenous strains originating from the fish GI tract might
reported study also revealed that intestinal mucus is a ensure viability and colonization of the selected strains to
better growth medium for the selected putative probiot- confer a health benefit. However, long term in vivo studies
ics than the skin mucus. Further, both B. aerius CCH1A are warranted with these autochthonous strains to deter-
and B. sonorensis CCH1Ph were found to tolerate 9% mine their effects on growth and likely health benefits of
and 10.5% of the diluted bile juice, respectively. The Catla catla.
physiological concentration of bile was reported to be
nearly 0.4%–1.3% within the fish GI tract (Balcázar et ACKNOWLEDGEMENTS
al. 2008). Thus, tolerance to the bile juice represented by Sincere thanks to the Head, Department of Zoology,
the gut isolates in the present study was relatively high. The University of Burdwan, West Bengal, India; The De-
Similar observations were recorded by several authors to partment of Science and Technology (FIST programme),
establish bile tolerance of the putative probiotic bacte- New Delhi, India and The University Grants Commission
ria against fish bile (Nikoskelainen et al. 2001, Burbank (UGC-SAP-DRS programme), New Delhi, India for pro-
et al. 2012, Mukherjee and Ghosh 2014) or commercial viding research facilities. The first author is grateful to
bile salts (Allameh et al. 2014, Geraylou et al. 2014). The Council of Scientific and Industrial Research, New
Finally, bio-safety towards the host is an indispensable Delhi, India for awarding the Junior Research Fellowship.
382 Dutta et al.
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Volume 9, Number 1, March .2016
ISSN 1995-6673
Pages 69 - 78
Jordan Journal of Biological Sciences
Non-Starch Polysaccharide Degrading Gut Bacteria in Indian

Major Carps and Exotic Carps
Sudeshna Banerjee, Anjan Mukherjee, Dipanjan Dutta and Koushik Ghosh *
Aquaculture Laboratory, Department of Zoology, The University of Burdwan, Golapbag, Burdwan 713 104, West Bengal, India
Received: Novermber 7, 2015 Revised: December 26, 2015 Accepted: January 2, 2016
Abstract
Cellulose and xylan are the most common Non-Starch Polysaccharides (NSPs); they are available in plants and they exhibit
anti-nutritional effects. The present study is intended to detect cellulose and xylan degrading autochthonous gut bacteria in
Indian major carps (Labeo rohita, Catla catla, and Cirrhinus mrigala) and exotic carps (Hypophthalmichthys molitrix,
Ctenopharyngodon idella, and Cyprinus carpio); it is also meant to identify the most promising strains by molecular
methods. The promising strains were also tested for likely antagonism against few pathogenic Aeromonas strains.
Altogether, 432 microbial strains were isolated on media containing either cellulose or xylan. Seventy strains were
primarily selected through qualitative enzyme assay. Finally, the quantitative assay led to the selection of 5 promising
NSP-degrading strains (LFR1X, CMF1C, HMF6X, CtIF1C, and CMH8X). Amongst these, LRF1X was the best cellulase-
and xylanase-producer. Analyses of 16S rRNA partial gene sequence revealed that strains LRF1X and CMF1C were
closely related to Bacillus pumilus (Accession numbers; KF640221, KF640223, respectively), whereas HMF6X, CtIF1C,
and CMH8X were similar to B. tequilensis (KF640219), B. megaterium (KF640220) and B. altitudinis (KF640222),
respectively. The culture of the selected microorganisms with autochthonous bacteria and yeasts indicated their co-
existence within the fish gut. An appraisal of antagonism against four pathogenic Aeromonas species by the cross-streaking
method revealed that the selected NSP-degrading strains (except CMH8X) were antagonistic to at least 2 pathogens. In
vivo bio-safety assessment through intra-peritoneal injection of the isolates showed no induction of pathological lesions or
mortality in healthy laboratory acclimatized rohu, L. rohita.
Keywords: : Cellulose, Xylan, Carps, Bacteria, Aeromonas.
cakes. The predominant endogenous polysaccharide

1. Introduction -
glycosic linkages of starch to yield glucose. Presence of
Non-Starch Polysaccharides (NSPs) are complex - c linkages is the reason why cellulose is
polysaccharides that are polymers of hexoses and indigestible by monogastric animals due to the lack of the
pentoses (e.g., galactose, glucose, arabinose, xylose, enzyme cellulase in their gastrointestinal (GI) tract.
-glucans (Van Similarly, the other enzymes for NSP digestion, such as
Barneveld, 1999). The NSPs comprise up to 90% of the - -xylanases, are also either scarce or not
plant’s cell wall (Selvendran and DuPont, 1980), wherein present in fish (Kuz’mina, 1996). Consequently, the
cellulose, hemicelluloses, and pectins are the most dietary NSPs remain mostly indigestible and cannot be
abundant (Sinha et al., 2011). Cellulose is the basic used as a nutritional source. In addition, being partially
structural component of plant cell walls and constitutes soluble in water, NSPs increase the viscosity of the
about 33% of all vegetable materials. Xylan is the most digesta, leading to changes in the physiology and the
common hemicellulose and represents the major non- ecosystem of the gut, thus, exerting anti-nutritive effects
cellulosic cell wall polysaccharide in plants. In contrast, (Sinha et al., 2011). Therefore, it appears that unless
pectic polysaccharides (pectins) are only present in hydrolyzed or degraded by exogenous enzymes, NSPs
modest amounts in plants (Sinha et al., 2011). Being an would decrease the nutritive value of the plant feedstuffs.
integral part of plant ingredients, NSPs are represented in The contribution of endosymbionts in the digestive
aquaculture through natural food and supplementary feeds process requires understanding the relative importance of
in the form of phytoplankton, algae, aquatic macrophytes, both, endogenous (produced by self) and exogenous
detritus, husks (rice bran or wheat bran) and different oil (produced by endosymbionts) enzymes (Clements, 1997).
*
Corresponding author. e-mail: kghosh@zoo.buruniv.ac.in.
70 © 2016 Jordan Journal of Biological Sciences. All rights reserved - Volume 9, Number 1
During the last few decades, there has been an improved enzyme-producing strains were characterized through 16S
understanding of the enzymes produced by the resident rRNA partial gene sequence analysis to corroborate their
endosymbiotic microbial community in fish (Ringø et al., identity. Bio-safety in fish was evaluated in vivo by
2010). Several studies have reported the occurrence of injecting healthy laboratory acclimatized rohu, L. rohita,
cellulase-producing microorganisms within the GI tract of with fresh cultures of the isolates. In addition, the
diverse fish species, including carps.1 promising enzyme-producers were evaluated for
In contrast, reports on xylanase production by fish gut antagonism against few well-known fish pathogens
microorganisms are scarce (German and Bittong, 2009; belonging to the genus Aeromonas. Motile aeromonads,
Banerjee and Ghosh, 2014). In the previous studies, being the major bacterial pathogens among tropical
association of cellulase (Saha and Ray, 1998; Bairagi et freshwater fish, are reported to be the main causative
al., 2002; Ghosh et al., 2002, 2010; Saha et al., 2006; factor behind mass mortalities associated with increased
Mondal et al., 2008), phytase (Roy et al., 2009; Khan et stocking in composite carp culture (Karunasagar et al.
al., 2011; Khan and Ghosh, 2012) and tannase (Mandal 1986). Aeromonas outbreaks in aquaculture are common
and Ghosh, 2013) producing microorganisms were in tropical countries and, therefore, the use of pathogen
demonstrated as an ecological adaptation of the carps inhibitory microorganisms has been suggested as
towards herbivory. Likewise, the existence of the gut- probiotic bio-control agents to substitute the use of
associated symbiotic microbial community capable of antimicrobial drugs (Balcázar et al., 2006; Dutta and
producing NSP-degrading enzymes (in addition to Ghosh, 2015). Finally, the selected bacterial isolates were
cellulase) could not be excluded in carps. cultured with the previously isolated autochthonous gut
Carps are the freshwater fish of the family Cyprinidae. bacteria and yeasts to substantiate their co-existence as
Various species of carps have been included in gut microbiota in fish in view of their likely application in
aquaculture as food fish across Europe and Asia for aquaculture.
centuries. Three Indian major carp species (Labeo rohita,
Catla catla, and Cirrhinus mrigala) along with three 2. Materials and Methods
Chinese carps (Hypophthalmichthys molitrix,
Ctenopharyngodon idella, and Cyprinus carpio) 2.1. Experimental fishes
constitute the composite carp culture in India. All these
carp species are mostly herbivorous or omnivorous in Six freshwater carp species consisting of three Indian
feeding aptitude (Jhingran, 1997). Intensive carp major carps (rohu, Labeo rohita; catla, Catla catla;
polyculture demands improvements in feed efficiency, mrigal, Cirrhinus mrigala) and three exotic carps (silver
while the replacement of animal protein sources (e.g., fish carp, Hypophthalmichthys molitrix; (grass carp,
meal) with the non-conventional plant ingredients is Ctenopharyngodon idella; common carp, Cyprinus
already a concern to trim down the production costs. carpio) were selected for the present study. Three
Enumeration of exo-enzyme producing gut specimens of each species were collected by gill net from
microorganisms capable of degrading complex three different composite fish culture farms at and around
polysaccharides and their utilization as either feed Burdwan (23°14'N, 87°39'E), West Bengal, India during
supplement or in vitro processing of plant feedstuffs has June to September, 2012, and kept in 350L Fiber-
been recommended in some of the recent investigations Reinforced Plastic (FRP) aquaria. Specimens were
(Khan and Ghosh, 2013; Das and Ghosh, 2015). brought to the laboratory within oxygen-packed bags.
Since, cellulose and xylans are the major NSPs in Descriptions of the fishes examined along with their
plant feedstuffs, the present study is undertaken to feeding habits are depicted in Table1.
appraise the occurrence of cellulase and xylanase-
producing autochthonous microorganisms within the GI
tracts of six freshwater carps. The most promising
1
Table 1.Food habits, average live weight, average fish length, average gut weight and gut length of the fishes examined.
Average fish Average

Average gut Gut length (L G )
Fish Species Food habits* fish length
live weight (g) weight (g) (cm.)
(cm)
Rohu, L.rohita Omnivorous, mostly plant matter 260±13.44 30.5±2.61 11.32±0.62 271.7±8.51
Catla, C.catla Zooplanktophagous 370±10.97 29.4±2.34 12.18±0.59 224.5±7.76
Mrigal, C. mrigala Detrivorous 330±12.33 30.7±2.70 8.29±0.57 431.3±10.27
Silver carp, H.
Phytoplanktophagous 440±14.42 26.6±3.84 8.38±0.68 218.3±8.68
molitrix
Grass carp,C. idella Herbivorous, mostly macrophytes 450±10.88 28.9±2.21 16.7±0.55 63.2±8.39
Common carp,
Detrivorous 375±13.44 27.4±2.37 7.81±0.58 47.3±9.81
C. carpio
Data are means ± S.D. of three determinations. * adapted from Jhingran (1997)
1
For more information, see Ray et al. (2012).
© 2016 Jordan Journal of Biological Sciences. All rights reserved - Volume 9, Number 1 71
2.2. Processing of Specimens isolates were distinguished after flooding the XY

plates with Congo red solution [0.5% Congo red (w/v)
Prior to sacrifice, experimental fishes were starved for and 5% ethanol (v/v)] 5 min., followed by repeated
48 h to clear their gastrointestinal (GI) tracts and to decolorizaion with 1 M NaCl (Ninawe et al., 2006). The
remove traces of any undigested food or fecal matter appearance of halo, owing to the hydrolyzed XY
therein (Mondal et al., 2010). Specimens were surrounding the bacterial colony, indicated xylanase
anesthetized using 0.03% tricainemethanesulfonate (MS- production in the medium. Isolates producing a halo of
222). Ventral surfaces were sterilized using 70% ethanol
and fishes were dissected aseptically to remove the GI selected for quantitative enzyme activity.
tracts (Ghosh et al., 2010). GI tracts were divided into Proficient NSP-degrading strains were identified
proximal (PI) and distal (DI) regions, cut into pieces, and through the quantitative assay of the extracellular
flushed carefully three times with Sterile Saline Solution cellulase and xylanase production. Respective selective
(SSS) using an injection syringe in order to remove non- broth media were used to obtain the enzymes. The culture
adherent (allochthonous) microbiota (Ghosh et al., 2010; flasks were incubated (37°C, 72 h) with vigorous shaking
Khan and Ghosh, 2012). Gut segments from 3 specimens (150–170 rpm), centrifuged at 10,000g (4°C, 10 min), and
of each species, collected from the same pond, were the cell-free supernatant containing the enzymes was
pooled together region-wise for each replicate, therefore collected (Bairagi et al., 2002). The quantitative assay for
providing 3 replicates for each fish species. Pooled cellulase production was performed following the method
samples were utilized to avoid erroneous conclusions due described by Denison and Koehn (1977) using 1% CMC
to individual variation in gut microorganisms as described in sodium citrate buffer (0.1 M, pH 5.0) as substrate.
elsewhere (Ringø et al., 1995; Spanggaard et al., 2000; Xylanase activity was assayed using 1% birch-wood
Ringø et al., 2006). xylan as the substrate as described by Bailey et al. (1992)
2.3. Microbial Culture and using D-xylose as the standard. Production of
Pooled segments for each replicate were homogenized reducing sugar (glucose) from the substrate due to
separately with pre-chilled SSS (1:10; weight: volume), cellulolytic or xylanolytic activity was measured at 540
serially diluted (1:10) up to 10 , and used as inoculums nm by the dinitrosalicylic acid method (Miller, 1959)
for isolation of gut microorganisms (Beveridge et al., using glucose or D-Xylose as the standard. Unit activity
1991). Diluted samples (100 L) were spread aseptically (U) of cellulase was defined as the glucose liberated
onto sterilized tryptone soya agar (TSA; HiMedia mL-1 of enzyme extract min-1. Xylanase activity (U) was
Laboratories, Mumbai, India) plates to obtain the defined as the mg of D-xylose liberated mL-1 of enzyme
culturable heterotrophic aerobic/facultative anaerobic extract min-1.
autochthonous microbial population. In order to isolate 2.5. Identification of Isolates by 16S rRNAGene Sequence
cellulase and xylanase-producing microorganisms, diluted Analysis
samples were plated onto carboxy-methyl-cellulose The most promising NSP-degrading strains (bacteria)
(CMC, gL : carboxy-methyl-cellulose 5, Yeast extract 5, were identified through 16S rRNA partial gene sequence
peptone 5, NaCl 5, agar 20) and xylan (XY, gL : peptone analysis after isolation and PCR (polymerase chain
5, yeast extract 2, MgSO 4 .7H 2 O 0.5, NaCl 0.5, CaCl 2 reaction) amplification following the methods described
0.15, Birchwood xylan 20, agar 20) supplemented in Das et al. (2014). To prepare template DNA, pure
selective media plates as described in Dutta and Ghosh colonies were suspended in sterilized saline, centrifuged
(2015). The culture plates were incubated at 30°C for 24 (12,000g, 10 min), supernatants removed and the pellets
h. Colony counts were determined utilizing the dilution suspended in InstaGene Matrix (Bio-Rad, USA). DNA
plate count technique. The average values of the replicates isolation was carried out following the manufacturer
were expressed as log viable count g-1 GI tract (LVC). recommendations. PCR amplification of the 16S rRNA
The well-separated colonies were randomly collected with gene was performed using universal primers, 27f (5´-
inoculation loop and streaked individually onto respective AGAGTTTGATCCTGGCTCAG-3´) and 1492r (5´-
media plates repeatedly to acquire pure cultures. Pure GGTTACCTTGTTACGACTT-3´). PCR was executed
cultures were maintained on slants in a refrigerator (4°C) using a PCR mix containing 200 µM of deoxynucleotides
for further study. (dNTPs), 0.2 µM of each primer, 2.5 mM MgCl 2 , 1X
2.4. Screening of Potent Cellulase and Xylanase- PCR buffer, 0.2 U of Taq DNA polymerase (Invitrogen)
Producing Isolates by Qualitative and Quantitative and 1 µL of template DNA. The cycle used for PCR was:
Enzyme Assay initial denaturation at 95°C for 3 minutes, followed by 35
Gut isolates were primarily evaluated for a qualitative cycles of denaturation at 94°C for 45 sec, annealing at
determination of extracellular cellulase and xylanase- 55°C for 1 min, extension at 72°C for 1 min, and a final
producing capacities following growth (30°C, 48 h) on extension at 72°C for 3 minutes (Lane, 1991). E. coli
the selective media plates containing respective genomic DNA was included as positive control. PCR
substrates. The cellulase-producing capacity was products were purified using Montage PCR Clean up kit
determined on CMC plates flooded with Congo red (Millipore, USA). The sequencing of the purified PCR
prepared with 0.7% agarose (Teather and Wood, 1982). products was performed using the Big Dye terminator
Congo red selectively binds with unhydrolyzed cycle sequencing kit (Applied BioSystems, USA).
carbohydrate polymers. The appearance of a clear zone Sequencing products were resolved on an automated
(halo), due to the presence of hydrolyzed CMC, indicated DNA sequencing system (Applied BioSystems 3730XL,
a cellulase production in the medium. Positive xylanolytic USA). Sequence data were edited using BioEdit Sequence
Alignment Editor (Version 7.2.0); then they were aligned 1 fishes were kept as control, and each fish received Intra-
and analyzed to find the closest homolog using Basic Peritoneal (IP) injection of 1.0 mL SSS (Mesalhy et al.,
Local Alignment Search Tool (BLAST) in National 2008). The five selected NSP-degrading bacterial isolates
Centre for Biotechnology Information (NCBI) GenBank were grown in Tryptone Soya Broth (TSB) at 30°C for
and Ribosomal Database Project (RDP) databases. 24 h, centrifuged (2800g for 15 min, 4°C), and cell pellets
Sequences were deposited in the NCBI GenBank and were suspended in SSS. Each fish in the experimental
accession numbers were obtained. A phylogenetic tree groups (groups 2, 3, 4, 5, and 6) was given IP injection of
was constructed incorporating 16S rRNA partial gene 1.0 mL saline containing 109 cells ml-1 of each test
sequences of the closest type strains using the MEGA bacterium. All groups were kept under observation for 21
5.1Beta4 software following the Minimum Evolution days for likely development of any disease symptom.
Method. 2.9. Statistical Analysis
2.6. Fish Pathogens and Culture Maintenance Statistical analysis of quantitative enzyme activity data
Four fish pathogenic strains: Aeromonas salmonicida was performed using the analysis of variance (ANOVA)
MTCC-1945 (AS), Aeromonas sobria MTCC-3613(AB), followed by Tukey’s test according to Zar (1999) using
Aeromonas hydrophila MTCC-1739 (AH), and Statistical Package for the Social Sciences (SPSS)
Aeromonas veronii (KT737240) (AV) were used to Version 10 (International Business Machines Corporation,
evaluate pathogen inhibitory activity of the promising Armonk, New York) (Kinnear and Gray, 2000).
cellulase and xylanase-producing strains. The pathogenic
strains were maintained in the laboratory on TSA 3. Results
(HiMedia, Mumbai, India) slants at 4°C. Stock cultures in
Tryptone Soya Broth (TSB) were stored at -20°C in Enumeration of gut microbial communities in the GI
0.85% NaCl with 20% glycerol to provide stable tracts revealed that considerable amounts of
inoculums throughout the study (Sugita et al., 1998). autochthonous aerobic or facultative anaerobic culturable
2.7. In Vitro Antagonistic Activity and Test of heterotrophic, as well as cellulose and xylan-degrading
Compatibility microorganisms, were present in both the PI and DI
regions of all the fish species studied (Table 2). The
Pathogen inhibitory activity of the selected strains was highest counts of cellulase-producing bacteria were noted
studied against the four Aeromonas spp. utilizing the in the DI region of mrigal, C. mrigala (LVC=6.14 g-1
‘cross-streaking’ method (Madigan et al., 1997). The intestinal tissue), followed by the DI region of catla, C.
NSP-degrading bacterial isolates were cultured with catla (LVC=5.97 g-1 intestinal tissue). Similarly, the
previously isolated eight autochthonous fish gut bacteria highest counts of xylanase-producing bacteria were noted
and two yeasts: Bacillus subtilis subtilis (JX292128), in the DI region of silver carp, H. molitrix (LVC=5.34 g-1
Bacillus atrophaeus (HM246635), Bacillus subtilis intestinal tissue), followed by the DI region of rohu, L.
(HM352551), Bacillus pumilus (KF454036), Bacillus rohita (LVC=5.04 g-1 intestinal tissue).
flexus (KF454035), Bacillus methylotrophicus Table 2. Log viable counts (LVC) of cellulase and xylanase
(KF559344), Bacillus subtilis subsp. spizizenii producing autochthonous (adherent) bacteria isolated from
(KF559346), Enterobacter hormaechei (KF559347), theproximal (PI) and distal (DI) parts of intestine of the fish
Pichia kudriavzevii (KF479403), and Candida rugosa species examined.
(KF479404). Source and description of these
autochthonous gut microorganisms were presented in LVC g -1 intestinal tissue
previous studies: (Das and Ghosh, 2013; Khan and Fish species and
Bacterial Bacterial
intestinal site of Heterotrophic
Ghosh, 2012; Banerjee et al. 2015; Mukherjee and Ghosh count on count on
isolation count on TSA
2014; Banerjee and Ghosh, 2014). Selected strains were cellulase xylanase
plate
inoculated as a line onto TSA media plates and incubated plate plate
at 30°C for 24 h. Subsequently, pure cultures of the PI 6.98 5.12 4.94
L. rohita
autochthonous fish gut microorganisms (8 bacteria, 2 DI 7.11 5.45 5.04
yeasts) were inoculated as a perpendicular line to the PI 6.32 5.45 4.19
NSP-degrading bacterial strains keeping a gap of about 1 C. catla
DI 6.65 5.97 4.54
mm. Following incubation (30°C, 24 h), the growth of PI 6.95 5.07 4.26
C.
microorganisms was examined and the disappearance of mrigala DI 7.54 6.14 4.82
the gap indicated compatibility (co-existence) of the
H. PI 7.25 4.95 4.93
autochthonous yeast and bacteria strains with the NSP-
molitrix DI 7.85 5.26 5.34
degrading bacterial strains.
PI 5.54 4.26 4.14
2.8. Safety of the Selected Isolate C. idella
DI 5.96 4.69 4.28
The safety of the selected isolates (LRF1X, CMF1C, PI 6.48 4.65 3.81
HMF6X, CtIF1C, and CMH8X) was evaluated through in C. carpio
DI 6.89 4.86 4.56
vivo studies conducted in 350 L FRP tanks using 90
Altogether, 432 microbial strains were isolated on the
healthy L. rohita (average body weight: 15±1.7 g) divided
media containing either cellulose or xylan. Seventy strains
into six groups (five experimental and one control), each
were primarily selected through qualitative enzyme assay,
with three replicates. The fish were acclimatized for 2
from which 30 microbial strains were further studied by
weeks in FRP aquaria under laboratory conditions. Group
quantitative assay to select the most promising isolates.
Data pertaining to quantitative assay of cellulase and (24.25±1.29 U), followed by the strain HMF6X
xylanase activities for the 30 promising strains (12 from (23.28±1.37 U). Strains CMF1C (15.41±1.17 U),
PI and 18 from DI), 5 strains from each of the 6 fish CMH8X (16.34±1.21 U), and CtIF1C (13.41±1.29 U)
species, are presented in Table 3. This led to the selection also exhibited a substantial xylanase activity. Considering
of 5 promising NSP-degrading strains (LFR1X, CMF1C, the cumulative activities of the two NSP-degrading
HMF6X, CtIF1C, and CMH8X) enzymes, isolates LRF1X, CMF1C, HMF6X, CtIF1C,
Table 3. Quantitative assay of the enzyme activity (unit activity, and CMH8X were finally selected for identification and
U) by the selected bacterial isolates. Numbers within the for possible future evaluation of use.
parenthesis denote total numbers of isolates from the respective Based on nucleotide homology and phylogenetic
fish species examined. analysis of the 16S rRNA partial gene sequences and
Source and Strains# Activity of NSP-degrading using the nucleotide blast in the NCBI GenBank and RDP
number of enzymes databases, the strains LRF1X and CMF1C were identified
isolates* Cellulase† Xylanase as Bacillus pumilus (Accession no. KF640221 and
L. rohita LRH4X 55.48±2.48f 12.38±1.21d KF640223, respectively), which were closest to the type
(PI-38, LRF1X 64.61±2.39g 24.25±1.29g strain Bacillus pumilus (Accession no. AY876289).
e
DI-47) LRH3C 44.55±1.12 7.07±0.26b Another isolate, HMF6X, was identified as Bacillus
e
LRH5C 48.61±1.04 9.26±0.29c tequilensis (Accession no. KF640219) as it showed a
e
LRH5X 49.14±1.19 8.81±0.31c maximum similarity with the type strain Bacillus
C. catla CCF1C 17.34±1.62ab 11.38±0.44d tequilensis (Accession no. HQ223107). Likewise, NCBI
(PI-25, CCF1X 20.41±1.71b 7.59±0.65b GenBank and RDP databases revealed that CtIF1C and
DI-39) CCF2X 21.51±1.64 b
7.67±0.54b CMH8X were similar to Bacillus megaterium (Accession
CCF3X 23.58±1.81 b
8.48±0.54bc no. KF640220) and Bacillus altitudinis (Accession no.
CCH4C 19.36±1.64 ab
7.73±0.59b KF640222), respectively. The isolate CtIF1C showed a
C. mrigala CMF1C 36.51±1.26 d
15.41±1.17f similarity with Bacillus megaterium (Accession no.
(PI-30, CMF3C 13.35±1.08a 15.18±1.06f D16273), while the isolate CMH8X was closest to
DI-35) CMH2C 16.73±0.75 a
10.58±0.72cd Bacillus altitudinis (Accession no.AJ831842). The
CMH3X 20.68±1.01 b
11.43±1.06f identities of the promising NSP-degrading fish gut
CMH8X 27.62±1.25 c
16.34±1.21f bacteria and their homology with the closest type strains
H. molitrix HMF1C 24.34±1.11bc 10.24±0.27c are presented in Table 4. Phylogenetic relation of the five
(PI- 34, HMF1X 23.44±1.25 b
8.13±0.45bc identified cellulase and xylanase-producing bacteria with
DI-50) HMF6X 48.46±2.15 d
23.28±1.37g closely related type strains, retrieved from the RDP
HMH1C 23.53±1.72 b
13.25±1.27e database, are presented in the dendogram (Figure 1).
HMH5X 22.41±1.29b 8.23±0.31bc To verify the pathogen inhibitory activity, five
C. idella CtIF1C 26.35±1.21c 13.41±1.29e selected NSP-degrading bacterial isolates were screened
(PI-26, CtIF2C 24.46±1.20 bc
11.33±1.01d against four different strains of fish pathogenic
DI-37) CtIH1X 14.41±1.23 a
8.95±0.31c Aeromonas spp. Results of the pathogen inhibitory
CtIH2X 21.48±0.94 b
8.14±0.29bc activity, as revealed by the cross streaking method, are
CtIH3X 29.14±1.18c 4.65±0.28a depicted in Table 5. Four strains (LRF1X, CMF1C,
C. carpio CyCH3C 22.26±1.72 b
7.43±0.31b HMF6X, and CtIF1C) were antagonistic towards two of
(PI-26, CyCH4C 17.89±1.25 ab
6.84±0.29a the tested Aeromonas spp. The strain CMH8X showed
DI-45) CyCH5C 17.39±1.19 ab
7.24±0.27b antagonism only towards Aeromonas veronii. Three
CyCH6C 21.76±1.14b 8.35±0.26bc strains (LRF1X, CMF1C, and HMF6X) were antagonistic
CyFH5X 16.65±1.14 a
6.66±0.27b towards Aeromonas salmonicida.
Values with the same superscripts in the same vertical column
An in vitro co-culture test against eight autochthonous
are not significantly different (P < 0.05). fish gut bacteria and two yeast isolates revealed that the
*PI: Proximal intestine; DI: Distal intestine selected bacterial strains did not affect the growth of the
#
The alphabets ‘F’ and ‘H’ before the numeric value indicate autochthonous gut microbiota. Therefore, the selected
isolates from PI and DI, respectively NSP-degrading bacterial isolates were considered
† -1
of enzyme extract min-1 compatible with commonly occurring autochthonous fish
mg of D-xylose liberated mL-1 of enzyme extract min-1 gut microbiota.
The strain LRF1X, isolated from the PI of L. rohita, After 21 days of a small-scale in vivo experiment
exhibited the highest cellulolytic activity (64.61±2.39 U). involving an intra-peritoneal injection of the selected
A considerable cellulase activity was also noted with bacterial isolates, no pathological signs, disease
other strains isolated from L. rohita. On the other hand, symptoms, or mortalities were observed in either the
the highest xylanase activity was recorded in LRF1X experimental sets or the control set.
Table 4. Identities of the promising NSP-degrading fish gut bacteria and their homology with the closest type strains in RDP.
Strain(s) Closest type strains in RDP S_ab Homology Bacterial species NCBI GenBank accession
score levels number(s)
(Max. indent)
LRF1X Bacillus pumilus 0.967 99% Bacillus pumilus KF640221
AY876289
CMF1C Bacillus pumilusAY876289 0.971 99% Bacillus pumilus KF640223
HMF6X Bacillus 0.972 99% Bacillus tequilensis KF640219

tequilensisHQ223107
CtIF1C Bacillus megaterium D16273 0.915 98% Bacillus KF640220
megaterium
CMH8X Bacillus altitudinis 0.962 99% Bacillus altitudinis KF640222
AJ831842
Table 5. Inhibition area (mm) produced by the selected gut bacteria (in excess of colony growth) incross streaking method against the
tested fish pathogens.
Strains AH AS AB AV
LRF1X - 2 2 -
CMF1C 1 1 - -
CMH8X - - - 2
HMF6X 2 3 - -
CtIF1C - - 2 1
AH: Aeromonashydrophila; AS:Aeromonassalmonicida; AB:Aeromonassobria;
AV: Aeromonasveronii;
Figure 1.Dendogram showing phylogenetic relations of the five most promising bacterial strains, LFR1X, CMF1C, HMF6X, CtIF1C, and
CMH8X, with other closely related type strains retrieved from NCBI GenBank. The GenBank accession numbers of the reference strains
are shown in parentheses. Horizontal bars in the dendogram represent the branch length. Similarity and homology of the neighboring
sequences have been indicated by bootstrap values. Distance matrix was calculated using Kimura 3-parameter model. The scale bar
indicates 0.005 substitutions per nucleotide position. Lactobacillus casei AB626050.1 served as an out group control.
4. Discussion culturable heterotrophic cellulase and xylanase-producing

microbiota in both PI and DI regions of the GI tracts of
Several plant-eating animals require the support of the fish species studied might indicate their probable role
symbiotic microorganisms within their GI tracts to in the degradation of NSPs in the plant feedstuffs.
degrade complex polysaccharides or secondary Cereal by-products, oil cakes, and natural feeds like
metabolites to make the energy in these compounds phytoplankton and aquatic weeds, contain considerable
available to the host (Karasov and Martinez del Rio, amounts of NSPs (cellulose and xylan) in their cell wall
2007). Likewise, the previous studies on fish advocated a material. For instance, rice bran, generally used as an
possible degradation of starch (Ghosh et al., 2002, 2010), essential component in fish feed, contains approximately
phytate (Roy et al., 2009; Khan et al., 2011; Khan and 20-25% NSP, consisting of approximately equal amounts
Ghosh, 2012), tannin (Mandal and Ghosh, 2013), and of arabinoxylans and cellulose (Saunders, 1986). Anti-
cellulose (Saha and Ray, 1998; Bairagi et al., 2002; nutritional effects of NSPs in monogastric animals were
Ghosh et al., 2002, 2010; Saha et al., 2006; Mondal et al., generally associated with the viscous nature of the
2008) by the gut-associated microorganisms in diverse polysaccharides. High gut viscosity decreases the rate of
carp species. Although the existence of a cellulase activity diffusion of substrates and digestive enzymes and hinders
in the digestive system of fish was in conflict with studies their effective interaction at the mucosal surface (Edwards
providing contradictory results, later on it became et al., 1988; Ikegami et al., 1990). The higher viscosity
commonly accepted that fish lack the cellulase enzyme, as can reduce the rate of gastric emptying leading to satiety
is the case with other monogastric and ruminant animals, which then decreases the feed intake (Roberfroid, 1993).
with cellulose degradation being mediated through the NSP degrading enzymes (e.g., cellulase and xylanase)
action of cellulase produced by the fish gut microbiota cleave the large molecules of NSP into smaller polymers,
(Ray et al., 2012). On the other hand, reports on xylanase thereby reducing the thickness of the gut content and
production by fish gut endosymbionts are scanty. Many increasing the nutritive value of the feed (Bedford et al.,
microorganisms, including bacteria (Balakrishnan et al., 1991; Choct and Annison, 1992).Therefore, the strategy
2002; German and Bittong, 2009; Azeri et al., 2010), to detoxify/degrade plant-derived anti-nutrients through
actinomycetes (Techapun et al., 2001; Tuncer et al., the enzymes, produced by gut microbiota, might be
2004), and filamentous fungi (Taneja et al., 2002; regarded as an evolutionary adaptation as is the case for
Angayarkanni et al., 2006; Sudan and Bajaj, 2007), have ruminant and non-ruminant herbivores (McBee, 1971).
been reported to produce xylanase. Whether the fish gut As the major aim of the present investigation was to
sustains any autochthonous xylanase-producing detect the efficient cellulase and xylanase-producing
microorganisms has not been authenticated yet, except for strains within the GI tracts of the freshwater carps (if
one report documenting the occurrence of xylanase- any), the preliminary screening for extracellular cellulase
producing yeasts in some freshwater carps (Banerjee and and xylanase production has resulted in the elimination of
Ghosh, 2014). Nonetheless, both cellulase and xylanase 83.8% (362 out of 432) of the total isolates from current
were assumed to be produced by microbes ingested by the study. Furthermore, 5 efficient NSP-degrading strains
fish with detritus rather than produced by a resident were established through quantitative cellulase and
endosymbiotic community (German and Bittong, 2009). xylanase assay and identified on the basis of 16S rRNA
In the present study, heterotrophic as well as cellulase and partial gene sequence analysis. The strains LRF1X and
xylanase-producing microbial symbionts were detected in CMF1C were identified as strains of Bacillus pumilus
the PI and DI regions of the GI tracts in 6 freshwater carp (KF640221 and KF640223), while the strains HMF6X,
species studied. As the fish were starved for 48 h and their CtIF1C, and CMH8X, were identified as B. tequilensis
GI tracts were carefully cleansed with sterilized and (KF640219), B. megaterium (KF640220), and B.
cooled 0.9% saline prior to the isolation of altitudinis (KF640222), respectively. All promising NSP-
microorganisms, it may be corroborated that the degrading isolates belonged to Bacillus spp., which might
microorganisms isolated in the present study belonged to support the hypothesis that gut bacteria in freshwater
the autochthonous (adherent) microbiota as suggested carps were dominated by diverse strains of Bacillus spp.
elsewhere (Ghosh et al., 2010). The occurrence of a (Ghosh et al., 2010; Ray et al., 2012). The occurrence of
higher proportion of heterotrophic microorganisms in DI B. pumilus (Ghosh et al., 2002) and B. megaterium (Saha
regions, compared to the PI regions, was in agreement et al., 2006) within the gut of freshwater carps was
with the previous reports (Mondal et al., 2008; Das et al., reported previously. However, to the best of the authors’
2014). This suggests that the degradation of the feedstuffs knowledge, B. tequilensis and B. altitudinis have not been
within these parts of the GI tract occurs in assistance with documented from carp gut so far.
the well settled enzyme-producing microbiota (Ghosh et The abundance of cellulase-producing bacteria has
al., 2010). The highest population counts of cellulase and been documented in the GI tracts of grass carp, C. idella
xylanase-producing producing microbial symbionts were (Bairagi et al., 2002; Saha et al., 2006; Li et al., 2009),
noted in the DI regions of mrigal, C. mrigala and silver common carp, C. carpio and silver carp, H. molitrix
carp, H. molitrix, respectively. Both species were either (Bairagi et al., 2002), rohu, L. rohita (Saha and Ray,
herbivores or feeding on detritus arising out from the 1998; Ghosh et al., 2002; Kar and Ghosh, 2008; Ray et
plant feedstuffs (Jhingran, 1997). In addition, other carp al., 2010), catla, C. catla and mrigal, C. mrigala (Ray et
species also harbored substantial amounts of NSP- al., 2010), and bata, L. bata (Mondal et al.,
degrading microorganisms within their GI tracts. 2008,2010).Furthermore, the xylanase-producing ability
Therefore, the presence of an appreciable quantity of by gut inhabiting bacterial symbionts from freshwater
carp species has not been documented to date. Results of NSP-degrading microbiota within the fish GI tract.
the present study might suggest considerable Furthermore, NSP-degrading and pathogen inhibitory
opportunities for using cellulase as well as xylanase- bacteria, noticed in the present study, appear to provide
producing bacterial symbionts from the gut of freshwater the host with some ecological benefits by enabling them
carps as aquaculture probiotics that might aid in the to conquer the adverse effects of NSPs and aeromonads.
degradation of complex polysaccharides in feedstuffs Whether the isolated gut bacteria can contribute to the
within the gut microenvironment. In addition, in vitro host’s nutrition and health has not been dealt with in the
degradation of NSPs in plant ingredients by autochthonus present study; a consideration of their function in vivo
NSP-degrading bacteria could be an alternative approach should be given high precedence in upcoming studies
of processing as the bacteria itself or their metabolites before advocating their utilization in commercial
would not impair the normal function in fish because of aquaculture.
the mutual relationship therein, as suggested previously
for phytate-degrading gut bacteria (Khan and Ghosh, Acknowledgment
2013).
Apart from nutritional benefits, the enzyme-producing The authors are grateful to the Head, Department of
gut bacteria in fish have been assumed to compete Zoology, The University of Burdwan, West Bengal, India;
continuously with pathogens through the competitive The Department of Science and Technology (FIST
exclusion or the production of antimicrobial compounds program), New Delhi, India; and The University Grants
(Ray et al., 2012). Although several strains of Bacilli Commission (UGC-SAP-DRS program), New Delhi,
were demonstrated as probiotics for fish, antagonism of India for providing the research facilities. The first author
pathogens by the bacilli has been seldom indicated. is grateful to UGC for awarding the research fellowship.
B. subtilis SG4 (Ghosh et al., 2007) and
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      
    
   
        
       

      
    
                  
     
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             

                    
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                     
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             
                  
                
                    
                     
   
                  
              

  
             
                 
                  
                 
                  
              
              
         
     
      
            
          
                   
                 
                    
                 
                   
                 
                   
                     
                      
               
                     
                     
           
                     
           
        
            
                  
                 

      
            
                  
                
                      
                     
                    
                      
                      
                     
                      
                  
                      
                      
                         
                        
                          
                     
                      
                      
                
                     
                   
                 
          
               
         
                 
                
                   
                   
                    
                   
                    
                   
                  
                          
                      
                    
          
            
                
            
                   
                   
                  
                   
                 
                         
          
            
            
                   
         
                   
                    
           
          
                   
        
                 
                
                    
                         
                      
                      
                        
                    
            
      
                        
                   
                       
                        
                      
                         
                 
                        
                        
                          
             
    
     
  
        
                   
                      
                        
                
                     
                   
                      
                          
                    
          
                 
                        
                           
                       
                            
                     
                       
            
             
             
                     
         
           
                     
                         
                    
                     
                        
                        
                  
                     
                    
     
     

      
             
                   
                       
                    
                      
                     
                          
                     
                  
             
                
                     
                     
                    
                   
             
         
                 
           
                  
          
 
           
           
                      
               
       
        
         
            
       
       
       
  
       
       
                  
               
       
        
       
                  
                  
                  
                    
       
                
                  
                  
                  
       
         
       
                   
           
                
        
          
                
                    
            
 
                
        
    
  
                 
             
             
             
                 
             
             
             
               
           
           
           
               
           
           
           
                 
                
                        
                  
                     
                 
                      
                     
                   
                      
                     
                       
                    
   
      
                   
      
                    
                     
             
          
          
 
            
                    
                         
               
            

        
      
   
                 
                  
                 
       
             
    
          
    
     
     
      
  
   
            

     
           
 
              
                 
                 
                    
                
            
 
      
           
                
                  
                
                
                            

       

       

         

      

        

      
             
                  
                     
                    
                     
                   
                    
                
                      
                    
           
         
 
      
          
                      
                   
                   
            
                               
                              
            
                                
        
                     
           
                      
                  
                   
                      
                 
                
                 
                     
                   
                 
                      
       
         
       
 
           
         
           
         
    
           
            
            
                
                 
    
         
            
                      
                       
         
             
            
            
          
                        
                   
                     
            
          

              
                  
                       
                          
      
           
            
 
        
             
       

       
       
       
                    
          
           
        
        
                        
                     
                    
               
                    
               
                          
            
       
          
       
              
         
          
                   
                      
          
          
  
                        
          
                      
       
                          
                   
                        
          
                 
                      
                     
          
             
                           
                      
            
            
            
               
                       
            
      
  
                     
              
                     
           
           
                      
           
               
                  
                       
               
              
                       
                   
                       
         
   
             
         
   
           
 
          
    
                   
           
             
                     
                   
            
                        
                        
                
                     
                    
            
                           
                       
             
                          
                       
              
                         
          
                   
                         
           
                    
               
                          
                       
             
                           
                    
     
                             
                     
                     
                   
           
                              
                 
                       
              
                      
            
                        
          
                            
                         
         
                            
            
           
                        
                         
        
                          
                  
                       
           
                         
                      
                
                         
               
                         
                     
              
                  
                           
             
                
                     
                     
     
                         
                  
               
                     
                      
               
                
                 
                    
    
                           
                        
       
                        
                        
               
           
          
       


 
       

       
        
 
               
               
        

     
             
             
           
           
              
               
                
         
                
                
        
        
     
        
       
            
                   
                  
               
         
       
        
              
 
        

        
       
      
       

          
       
        
        
          
          
        
                
             

             
             
   
                
                    
              
                
                   
              
                 
                 
             
               
            
                 
                      
                   
             
             
                   
                  
             
                  
                  
           
       
             
      
               
         
         
                
                     
                   
                   
                 
               
                
                   
                 
                  
               
                
                   
                   
                   
            
                  
                
             
      
            
        
                 
                
                 
       
             
                   
                  
                
                 
              
                   
                   
                 
                
                     
                   
                  
                   
                 
             
          
            
        
              
       
               
                    
                 
           
          
           
               
       
   
             
        
             
        
           
      
                
               
               
            
           
          
           
                
      
    
    
        
                
       
                   
                      
                
   
               
                    
                      
               
                   
                    
                
                
                   
             
        
              
        
                    
                
                  
                  
               
                
                     
                 
                  
               
       
            
          
                 
                  
                 
                  
                 
                 
             
                 
                
           
            
              
                   
                  
                  
                   
             
         
      
             
        
            
       
                 
                 
            
                     
       
    
      

    
    
   
    

      

   
     
       
     
  

     
     























           
         
            
                 
                    
   

 
     
 
   
 
 
 
 
 
 
 


 
 
 
 
 
 
 
 
 
 
 
 
                       
   
   

 
     
  
  
 
 
 
 
 
 
 


 
 
 
 
 
 
 
 
 
 
 
 
                       
   
                        
   
   

 
  

     

    
  

  
       
    
 
    
  
     
 
     
             
      

 
   
      

    
  

  
       
    
  
    
  
     
 
     
             
      

 

  
      

    
 


 
       
    
  
    
  
     
 
     
             
      

 
   
      

    



  
       
    
  
    
  
     
 
     
             
    
 
       
  

           
           

            
       
        
  
            
            
          
              
           

        
           

   
      

       
                  
            
           
                
             
     
          

         
        
           
               
        
              
        
              
       
                  
                
                     
                   
           
          
       
                
    
                
                
    
                  
                            

       

       

         

      

         

      
   
         

  
        
            
       
   
        
   
       
  
           
  
        
   
         
             
             
            
            
          
          
            
                
           
        
         
                
 
        
      
             
           
             
                
               
        
             
        
            
                   
                    
                
           
       
                 
         
              
             
             
              
         
                   
               
                 
                
                 
                 
                 
            
       
   

    
   
  
    
    
   
    
   
   
    
   
    
    
   
 
                
              
            
                
               
               
                   
                   
                
                  
                 
                
                   
                
                 
                  
                
                 
               
                
              
                 
                
                 
                
          
               
                
                  
   
               
       
                   
                        
                    
               
                
                
                  
                
                
                  
                   
                  
               
                    
                  
                
                 
                  
                  
                  
                     
                   
                
                  
                  
                  
              
                
                
        
            
               
               
                   
                
                 
                   
                
                 
                  
                   
                  
                
                     
                  
                   
                
                    
                  
                  
                    
       
         

         
                         
                 
        
       
     
                     
                 
                  
                
      
                   
                  
                  
             
         
       
        
                
                
           
                  
         
                  
        
                     
            
         
        
 
                  
       
                   
              
  
         
          
                 
                  
               

       
          
         
              
             
                  
       
         
 
                   
                
       
                
 
                  
                 
             
                
         
        
         
           
              
                  
              
                
                   
              
                       
   
                      
                
                
         
                     
                      
                 
           
                 
                   
             
                 
                    
         
                      
           
                  
            
                  
                 
              
               
                    
                 
                   
         
                 
               
                
   
                  
                   
         
              
                 
                 
                 
     
                     
       
                    
                
         
                    
                
                  
              
   
                    
                   
                  
                  
              
                    
               
                    
                    
                     
                 
       
                        
                    
          
                   
                
      
       
                       
                
                    
                
                   
         
           
                    
          
                     
              
              
         
                   
                  
          
               
    
                     
            
            
                  
                 


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Appendix

List of Publications and GenBank Submissions

1. Dutta D and Ghosh K (2015) Screening of extracellular enzyme-producing and

NCBI GenBank Submission

Strains Amylase(U)1 Protease(U)2 Lipase(U)3 Cellulase(U)4 Phytase (U)5 Xylanase(U)6

ND= Not detected

4. Discussion observations recorded from the GI tracts of the Indian major

Sudeshna Banerjee, Anjan Mukherjee, Dipanjan Dutta and Koushik Ghosh*

*Corresponding author: kghoshbu@gmail.com ; kghosh@zoo.buruniv.ac.in doi: 10.15414/jmbfs.2015.5.1.12-19

ARTICLE INFO ABSTRACT

Keywords: Bacillus pumilus, Bacillus flexus, chitinase, fish gut bacteria

*adapted from Jhingran, 1997

SELECTION AND PROBIOTIC CHARACTERIZATION OF EXOENZYME-PRODUCING

Dipanjan DUTTA, Sudeshna BANERJEE, Anjan MUKHERJEE, and Koushik GHOSH*

Morphological, physiological, and biochemical char- RESULTS

sequence analysis after isolation and PCR ampliﬁcation 4

following the methods described in Das et al. (2014). The 3

gene encoding 16S rRNA was ampliﬁed from the iso- 2

lates by polymerase chain reaction (PCR) using univer- 1

sal primers (27f, 5´-AGAGTTTGATCCTGGCTCAG-3´ 0

and 1492r, 5´-GGTTACCTTGTTACGACTT-3´). The Heterotrophic

PCR reactions were performed using 20 μL of PCR mix PI DI

and 1 μL of template DNA. To extract genomic DNA for

Enzyme activity [U]

against A. hydrophila, A. salmonicida, and A. veronii Table 3

93 Bacillus aerius AJ831843.1

Bacillus sonorensis AF302118.1

Bacillus amyloliquefaciens AB255669.1

100 Bacillus subtilis subsp. spizizenii AF074970.1

92 Bacillus tequilensis HQ223107.1

Bacillus humi AJ627210.1

50 Bacillus firmus D16268.1

86 Bacillus niacini AB021194.1

93 Bacillus selenatarsenatis AB262082.1

Bacillus algicola FR775437.1

REFERENCES Bier M. 1955. [106] Lipases: RCOOR′ + H2O → RCOOH

Non-Starch Polysaccharide Degrading Gut Bacteria in Indian

Sudeshna Banerjee, Anjan Mukherjee, Dipanjan Dutta and Koushik Ghosh *

Keywords: : Cellulose, Xylan, Carps, Bacteria, Aeromonas.

cakes. The predominant endogenous polysaccharide

Average fish Average

2.2. Processing of Specimens isolates were distinguished after flooding the XY

HMF6X Bacillus 0.972 99% Bacillus tequilensis KF640219

4. Discussion culturable heterotrophic cellulase and xylanase-producing

    

   

        

       

      

          

     

        

       

       

        

   

   

  

          

          

       

         

   

         