Professional Documents
Culture Documents
Characterization of Proteases of Bacillus Subtilis Strain 38 Isolated From Traditionally Fermented Soybean in Northern Thailand
Characterization of Proteases of Bacillus Subtilis Strain 38 Isolated From Traditionally Fermented Soybean in Northern Thailand
C , 50
C , an d
6 0
C ) , wi th th e B. subtilis p ro teases b ei n g m o st
affected at 60
C an d 50
C
sh owed sli gh t effect on en zym e acti vi ty as m ore th an
80 an d 50% of p roteolyti c acti vi ty resp ecti vely cou ld
sti ll be d etected after 60 m i n assay F i g 4B ) .
P rotease en zym es of B. subtilis gen erally can be
categori zed i n to two grou p s wi th resp ect to th ei r
o p ti m al p H n eu tral an d alk ali n e p ro teases. T h e
form er exh i bi t op ti m al p H at 7.0, wh ereas th e latter
h ave p H op ti m a between 9 1 1 .
20
T h erefore, th e
cru d e p rotease en zym es p rod u ced from B. subtilis
strai n 38 belon g to th e n eu tral grou p si n ce th ey h ad
o p ti m al p H at 6 . 5 . To fu rth er ch aracteri ze th e
Fig 1. C om p arati ve resu lts of p roteolyti c acti vi ty of B. subtilis
strai n s i solated from thua nao, T h ai ferm en ted soybean s.
T h e p roteolyti c acti vi ty was assayed u si n g sk i m m i lk agar
an d exp ressed as a squ are of th e clear zon e d i am eter.
1 8
Fig 2. R elati on sh i p between cell growth an d p rotease p rod u cti on
of B. subtilisstrai n 38. C ell growth I was m easu red by
ab so rb an ce at 6 6 0 n m an d p ro tease acti vi ty I) was
d eterm i n ed by p resen ce of th e clear zon e on sk i m m i lk
agar.
Fig 3. E ffect of p H on acti vi ty of th e Bacillus subtilis 38 p roteases.
G ood 's bi ologi cal bu ffers M E S, P I P E S, T E S, TAP S, C H E S
Si gm a) ) were u sed to cover a p H ran ge 5.5-1 0 followi n g
th e m an u factu re's recom m en d ati on . T h e cru d e extract
u sed for each bu ffer was 20 l.
244 ScienceAsia 28 (2002)
p ro teases, vari o u s typ es o f i n h i b i to rs were u sed .
O n ly 1 , 1 0-p h en an th roli n e was clearly able to i n h i bi t
th e en zym e acti vi ty T ab le 1 ) i n d i cati n g th at th e
p rotease i s a m etallop rotease s .
F or m an y d ecad es, thua nao h as been p rod u ced
i n a con ven ti on al m an n er. T h e ferm en tati on p rocess
th u s m ai n ly d ep en d s on a m i xtu re of bacteri al sp eci es
alth ou gh , n ot su rp ri si n gly, th e Bacillus sp eci es h ave
p layed a k ey role. T h ere i s also a lack of system ati c
p roced u re i n clu d i n g n o tem p eratu re con trol. H en ce,
th e qu ali ty of th e ferm en ted p rod u cts i s n ot u n i form .
U si n g a su i table B. subtilis sp eci es as a p u re starter
cu ltu re i s a p ro m i si n g ap p ro ach to i m p ro ve th e
ferm en tati on p rocess, wh i ch m ay lead to com m erci al-
scale p rod u cti on . C on sequ en tly, we attem p ted to
u ti li ze B. subtilis strai n 38 i solated i n a laboratory-
scale soybean ferm en tati on . F or th i s, ferm en tati on
of soybean s was carri ed ou t i n both trad i ti on al T h ai
an d natto styles at 3 7
C an d 4 5
C . D u ri n g th e
ferm en tati on p rocess, ch an ges i n p H were m on i tored
every 6 h . G en erally, th e tren d s of p H ch an ge of
each ferm en tati on batch were si m i lar wi th i n i ti al p H
of 6.0 an d grad u ally i n creased to ap p roxi m ately 8.0
after 72 h ferm en tati on F i g 5) . T h e i n creased i n
p H of each batch was p ossi bly d u e to p roteolysi s
an d am m on i a release.
3
Accord i n g to F i g 5, th e p H
ch an ges were m ost d ram ati c at 1 2 h of ferm en tati on .
T h e d i fferen ce d u e to tem p eratu re con d i ti on s 37 C
an d 45
C ) was also clearly observed at th i s p eri od ,
both ferm en ted soybean sam p les obtai n ed by th e
thua nao an d nattom eth od s sh owed h i gh er p H 7.59
an d 7.85) at 45
-thua nao, 45 C ;
O -natto, 37 C ; -natto, 45 C .
ScienceAsia 28 (2002) 245
wou ld be exp ected . T h e n eu tral p roteases p rod u ced
d u ri n g th e ou tgrowth or log p h ase p resu m ably p lay
an i m p ortan t role i n soybean ferm en tati on . T h i s
strai n h o ld s great p ro m i se fo r th e p ro d u ct, an d
provides attractive possibilities for fu rth er optim ization
as an i n d u stri al strai n to i m p rove th e soci o-econ om i c
ben efi ts of trad i ti on al food s.
ACKNOWLEDGEMENTS
We wo u ld li k e to ack n o wled ge th e P ro f D r
N atth B h am arap ravati research fu n d , C h i an g M ai
U n i versi ty, C h i an g M ai , T h ai lan d , fo r fi n an ci al
su p p o rt. T h an k s also to A sso ci ate P ro fesso r D r
Arayar Jati sati en r for h er ad vi ce i n stati sti cal an alysi s.
I n ad d i ti on , we wou ld li k e to exp ress ou r th an k s to
D ep artm en t of B i ology, F acu lty of Sci en ce, C h i an g
M ai U n i versi ty, C h i an g M ai , T h ai lan d for p rovi d i n g
laboratory faci li ti es.
REFERENCES
1 . Su n d h agu l M , Sm an m ath u roj P an d B h od ach aroen W 1 972)
T h u a-n ao: a ferm en ted soybean food of N orth ern T h ai lan d .
Thai J Agric Sci 5, 43-56.
2. C h an tawan n ak u l P 1 9 9 5 ) Microorganisms related in Thai
fermented soybeansB Sc D i ssertati on , D ep artm en t of B i ology,
F acu lty of Sci en ce, C h i an g M ai U n i versi ty i n T h ai ) .
3. Sark ar P K , C o o k P E an d O wen s JD 1 9 9 3 ) Bacillus fer-
m en tati on of soybean s. World J Microbiol Biotechnol 9, 295-9.
4. Sark ar P K , T am an g JP, C o o k P E an d O wen s JD 1 9 9 4 )
K i n em aa trad i ti o n al so yb ean ferm en ted fo o d : p ro x i m ate
com p osi ti on an d m i croflora. Food Microbiol 11, 47-55.
5. N ou t M JR , B ak sh i D an d Sark ar P K 1 998) M i crobi ologi cal
safety of k i n em a, a ferm en ted soya been food . Food Control 9,
357-62.
6. Yo k o tsu k a T 1 9 9 1 ) P ro tei n aceo u s ferm en ted fo o d s an d
co n d i m en ts p rep ared wi th k o ji m o u ld s. I n : Handbook of
Applied Mycology Vol 3, Foods and Feeds E d i ted by Arora D K ,
M u k erji K G an d M arth E H ) , p p 329-73. M arcel D ek k er, N ew
York , N Y.
7. O h ta T 1 9 8 6 ) N atto . I n : Legume-based Fermented Foods
E d i ted by R ed d y N R , P i erson M D an d Salu n k h e D K ) , p p 85-
93. C R C P ress.
8. O d u n fa SA 1 985) Afri can ferm en ted food s. I n : Microbiology
of Fermented Foods Vol 2 E d i ted by Wood B JB ) , p p 1 55-91 .
E lsevi er, L on d on .
9. N jok u H O , O gbu li e JN an d N n u bi a C 1 990) M i crobi ologi cal
stu d y o f th e trad i ti o n al p ro cessi n g o f A fri can o i l b ean
Penackethra macrophyllaB en th am ) for u gba p rod u cti on . Food
Microbiol 7, 1 3-26.
1 0. O gbad u L J, O k agbu e R N an d Ah m ad AA 1 990) G lu tam i c
aci d p rod u cti on by Bacillusi solates from N i geri an ferm en ted
vegetable p rotei n s. World J Microbiol Biotechnol 6, 377-82.
1 1 . Sn eath P H A 1 986) Secti on 1 3. E n d osp ore-form i n g G ram -
p o si ti ve ro d s an d co cci . I n : Bergeys Manual of Systematic
Bacteriology Vol 2, 9th ed E d i ted by Sn eath P H A, M ai r N S,
Sh arp e M E an d H olt JG ) , p p 1 1 04-39. Wi lli am s & Wi lk i n s,
B alti m ore, M D .
1 2. F u ji ta M , K ei i ch i N , H on g K , I to Y, Asad a A an d N i sh i m u ro S
1 9 9 3 ) P u ri fi cati o n an d ch aracteri zati o n o f a fi b ri n o lyti c
en zym e n attok i n ase) i n th e vegetable ch eese n atto, a p op u lar
soybean ferm en ted food i n Jap an . BiochemBiophys Res Comm
197, 1 340-7.
1 3. F u k u tak e M , T ak ah ash i M , I sh i d a K , K awam u ra H , Su gi m u ra
T an d Wak abayash i K 1 996) Q u an ti fi cati on of gen i stei n an d
gen i sti n i n soybean s an d soybean p rod u cts. Food ChemToxicol
34, 457-61 .
1 4. O k am ato A, H an agata H , K awam u ra Y an d Yan agi d a F 1 995)
An ti -h yp ersen si ti ve su bstan ces i n ferm en ted soybean , n atto.
Plant Foods Human Nutr 47, 39-47.
1 5. O sawa R an d M atsu m oto K 1 997) D i gesti on of stap h ylococcal
en terotoxi n by Bacillus natto. AntonieVan Leeuwenhoek 71,
307-1 1 .
1 6. Su m i H , Yatagai C , Wad a H , Yo sh i d a E an d M aru yam a M
1 995) E ffect of Bacillus nattoferm en ted p rod u ct bi ozym e)
on blood alcoh ol, ald eh yd e con cen trati on after wh i sk y d ri n k -
i n g i n h u m an volu n teers, acu te toxi ci ty of acetald eh yd e i n
m i ce. Arukoru Kenkyuto Yakubutsu Izon 30, 6 9 -7 9 . i n
Jap an ese)
1 7. N orri s JR , B erk eley R C W, L ogan N A an d O D on n ell AG 1 981 )
T h e gen era Bacillusan d Sporolactobacillus. I n : TheProkaryotes
Vol 2 E d i ted by Starr M P, Stolp A, Tru p er AG , B alows A an d
Sch legel H G ) , p p 1 71 1 -42. Sp ri n ger-Vertag, B erli n , G erm an y.
1 8. C oop er K E 1 963) T h e th eory of an ti bi oti c i n h i bi ti on zon es.
I n : Analytical Microbiology E d i ted by K avan agh F ) , p p 1 -86.
Acad em i c P ress, N ew York , N Y.
1 9. B ro ck F M , F o rsb erg C W an d B u ch an an -Sm i th JG 1 9 8 2 )
P roteolyti c acti vi ty of ru m en m i croorgan i sm s an d effects of
p rotei n ase i n h i bi tors. Appl Environ Microbiol 44, 561 -9.
20. K eay L 1 972) P rotease of th e gen u s Bacillus. I n : Fermented
Technology Today E d i ted by Teru i G ) , p p 289-98.
21 . D u n n B M 1 989) D eterm i n ati on of p rotease m ech an i sm . I n :
Proteolytic Proteins; A Practical Approach E d i ted by B eyn on
R J an d B on d JS) , p p 57-81 . I R L P ress.
22. K ram er JM an d G i lbert R J 1 989) Bacillus cereus an d oth er
Bacillus sp eci es. I n : Food-borneBacterial Pathogens E d i ted
by D oyle M P ) , p p 21 -70. M arcel D ek k er, N ew York , N Y.
23. D en n er WH B an d G i llan d ers T G E 1 9 9 6 ) T h e legi slati ve
asp ects of th e u se of i n d u stri al en zym es i n th e m an u factu re of
food an d food i n gred i en ts. I n : Industrial Enzymology, 2nd ed
E d i ted by G od frey T an d West S) , p p 397-41 1 . T h e M acm i llan
P ress L td , B asi n gstok e, U K .