Probiotics and Antimicrobial Proteins

https://doi.org/10.1007/s12602-019-09610-0

In Vitro Evaluation of Probiotic Potential and Safety Assessment

of Lactobacillus mucosae Strains Isolated from Donkey’s Lactation
Sonakshi Rastogi 1 & Vineeta Mittal 2 & Aditi Singh 1

# Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract
The study, for the first time, reports the efficacy, safety and probiotic properties of two Lactobacillus mucosae strains, Lact.
mucosae SRV5 and Lact. mucosae SRV10 isolated from donkey milk. All major in vitro screening assays were employed to
evaluate studied strains. Both strains displayed good survivability at gastric pH 2.0, 0.3% bile and simulated oro-gastrointestinal
fluid (above 88%). Also, cultures demonstrated good cell surface hydrophobicity and auto-aggregation ability, clearly indicating
their effective cell adhesion ability. Furthermore, functional attributes for both strains demonstrated their efficient bile salt hydrolase
and cholesterol-reducing ability in spent broth. In addition to this, both strains expressed significant DPPH-radical scavenging
ability of both culture supernatant and intact cells. Another auxiliary health benefit exhibited by both these strains is their antimi-
crobial potential against 18 enteric and 5 multidrug-resistant clinical pathogens with significant inhibition zone size. Extracellular
enzyme production such as lipase, amylase, protease and esterase was also studied. Detailed safety evaluation study showed the
presence of innate antibiotic resistance and absence of haemolysis, DNAse and gelatinase activity in both the strains. Also, none of
the strains possessed toxic mucinolytic activity in mucin degradation assay. To conclude, both donkey milk isolates, Lact. mucosae
SRV5[Accession number: MK990014] and Lact. mucosae SRV10 [Accession number: MN064860], exhibited excellent probiotic
ability with tolerance to simulated oro-gastrointestinal fluids, cellular hydrophobicity, auto-aggregation, bile salt hydrolase, cho-
lesterol reduction, high antioxidant activity and antimicrobial potential especially against multidrug-resistant pathogens.

Keywords Probiotic . Lactobacillus mucosae . Donkey milk . Multidrug resistant pathogens . Antioxidant activity . Cholesterol
reducing ability

Introduction administered in sufficient quantity provide numerous health

Since time immemorial, milk has served as a major ecological
reservoir for isolation of numerous beneficial microorgan-
isms, such as lactic acid bacteria (LAB). Among the LAB,
several species of lactobacilli have been found to be safe for
consumption owing to their long history of food fermentation,
thus are incorporated as probiotics in functional foods.
Probiotics are defined as live microorganisms which when
* Aditi Singh
asingh3@lko.amity.edu
1
Amity Institute of Biotechnology,, Amity University Uttar Pradesh,
Lucknow Campus, Gomti Nagar Extension, Near Malhaur Railway
Station, Lucknow, Uttar Pradesh 226028, India
2
Department of Microbiology, Dr. Ram Manohar Lohia Institute of
Medical Sciences, Vibhuti Khand, Lucknow 226010, India
benefits to the consumer [1]. Recently, several reports docu-
mented their potential benefits such as lowering serum cho-
lesterol level [2], stimulation of immune response, alleviation
of urogenital and intestinal disorders such as severe gastroen-
teritis [3], antibiotic-associated diarrhoea [4], colorectal cancer
[5], lactose intolerance [6], constipation [7], allergic reactions
[8] and anti-diabetes [9]. WHO stipulated guidelines for
probiotics evaluation in food, which incorporates all criteria
for preclinical assessment of probiotics, clinical trials and la-
belling [1]. Among them, few requirements to classify isolates
to be potent probiotic include tolerance to stress such as gas-
tric and intestinal enzymes, gastric acid, bile, adherence to
intestinal epithelial cells, antimicrobial substances production
[10] and safety aspects such as antibiotic sensitivity, toxin
production and mucin degradation [11]. Though in vitro work
only mimics in situ environment of gut ecosystem, these stud-
ies can be important tool for screening new microbes from
different samples as probiotics.

Multiple drug resistance in clinical pathogens is a major and
growing concern nowadays, as they elevate the incidence of
mortality and morbidity in nosocomial and immunocompro-
mised patients. Treatment regimen is still limited to synthetic
antibiotic usage which has several adverse effects. Therefore,
now probiotics are envisaged to provide safe and stable treat-
ment to deal with the progressing multiple drug resistant (MDR)
strains. Up till now, there are limited studies available that have
shown potential of the Lactobacillus species in inhibiting the
growth of oppressive antibiotic-resistant clinical isolates owing
to their ability to produce numerous antimicrobial metabolites
[12–14]. Also, several reports stated the beneficial role of genus
Lactobacillus in lowering cholesterol [15, 16].
As functional properties of probiotics have been reported to
be strain specific, there is always a need for exploring better
performing strains from unconventional sources. Nowadays,
isolation of probiotics from donkey milk has stimulated sci-
entific interest due to its similarities with human milk in terms
of nutrient, microbial and chemical compositions [17].
Donkey milk is now considered as better alternative to feed
infants with acute IgE antibody-mediated allergy from cow’s
milk protein [18]. Other functional benefits include antimicro-
bial activity due to presence of high content of lysozyme [19],
suppression of human lung tumours [20], treating whooping
cough and acts as strong vasodilator, thus alleviating athero-
sclerosis [21]. Microbiological compositional study by Soto
del Rio et al. [22] demonstrated the presence of huge diversity
of lactobacillus species in raw donkey milk.
For the first time, Roos et al. [23] discovered novel species
of Lactobacillus genus, Lact. mucosae from pig small intes-
tine in the study aimed to isolate Lact. reuteri strains carrying
colonisation factor gene, MUB (mucus-binding). Later, Lact.
mucosae strains were also isolated from human ileal and vag-
inal tract [24], bovine intestine and stool [25], and milk of goat
and sheep [26, 27]. Lact. mucosae are gram positive, non-
motile, catalase negative, non-spore forming and
heterofermentative bacilli [23]. In last decades, probiotic po-
tential of this species was explored by several researchers and
has shown prevalence of numerous genes in its genome such
as adhesion-related [28], beta-galactosidases and bile salt hy-
drolases [26]. Likewise, cholesterol-reducing [29], anti-
inflammatory [27], exopolysaccharide production [25] and
antioxidant activity [27] were also studied earlier. These find-
ings suggest that this relatively recently isolated species of
Lactobacillus, Lact. mucosae, can be selected as probiotic as
it possesses wide spectrum of biological functional attributes.
Thus, the purpose of this report was to investigate the probi-
otic ability of two strains of Lact. mucosae SRV5 and SRV10
isolated for the first time from donkey milk, specifically fo-
cusing on its gastrointestinal tolerance, cell surface hydropho-
bicity and aggregation capacity, functional attributes such as
antioxidant potential, antagonistic activity against both MDR
and enteric pathogens and cholesterol-reducing ability. Safety
Probiotics & Antimicro. Prot.
aspects were also studied in detail in terms of their antibiotics
susceptibility and production of exocellular enzymes and mu-
cin degradation.
Materials and Methods
Bacterial Cultures and Culture Media
The two Lact. mucosae strains were screened from the pool of
bile and acid-resistant lactobacilli (n = 21) isolated from
jenny’s milk (n = 6), which were collected from local farms
in Lucknow (India). De Man, Rogosa Sharpe (MRS) medium
(HiMedia, India) was used to culture the Lact. mucosae strains
anaerobically. All pathogenic strains were procured from Dr.
Ram Manohar Lohia Institute of Medical Sciences, Lucknow,
and maintained in Brain heart infusion broth (BHI) (Oxoid,
UK). ATCC indicator strains employed for test were
Escherichia coli ATCC-25922, Proteus vulgaris ATCC-
6380, Klebsiella oxytoca ATCC-700324, Enterococcus
faecalis ATCC-51299, Pseudomonas aeruginosa ATCC-
27853 and Staphylococcus aureus ATCC 25923, while clini-
cal strains used were Listeria monocytogenes, Salmonella
typhi T(H), Salmonella paratyphi T(H), Salmonella
typhimurium, Corynebacterium diphtheriae, Shigella sonnei,
Shigella dysenteriae, Shigella boydii, Shigella flexneri type 2,
Serratia fecaria, Stenotrophomonas maltophilia and
Klebsiella pneumonia. Apart from clinical strains, five
multidrug-resistant pathogenic strains used in present study
were Pseudomonas aeruginosa (MDR), Proteus mirabilis
(MDR), Acinetobacter baumannii (MDR), Escherichia coli
(MDR) and Klebsiella pneumonia (MDR).
Identification of Lactobacillus mucosae Strains
Morphological and biochemical tests were conducted to iden-
tify lactobacilli isolates, while confirmatory strain identifica-
tion was done using 16S ribosomal RNA (rRNA) sequence
analysis, using universal primers 27F and 1492R. Genomic
DNA was extracted using DNA extraction kit (HiMedia,
India) according to manufacturer’s protocol. Amplification
mixture (25 μl) contained 1 μl genomic DNA, 1 μl (0.3
pmol/μl) of each primer, 1 μl (0.5 U/μl) Taq DNA polymer-
ase, 400 μM each of deoxynucleotides triphosphates, 5 μl 10x
reaction buffer and water. The PCR amplification was carried
out by initial incubation at 80 °C for 5 min, denaturation at 94
°
C for 2 min, twenty-five cycles of denaturation at 98 °C for 10
s, annealing at 53 °C for 30 s and extension at 68 °C for 1 min.
PCR products were separated by electrophoresis in 0.8%
(w/v) agarose gels in 0.5x TAE buffer at 100 V for 1 h. Gels
were stained in 0.5x TAE buffer containing 0.5 μg/ml
ethidium bromide (Sigma Diagnostics, St. Louis, MO,USA),
and resulting amplicons obtained by PCR, targeting the full
Probiotics & Antimicro. Prot.
length 1.5 Kb 16S rRNA gene, were purified using standard
method and directly sequenced with primers 785F and 907R
using BigDye® Terminator v3.1 Cycle Sequencing Kit
(Applied Biosystems). The sequences obtained were aligned
and compared to known sequences in GenBank using online
tool BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The
strains were identified as Lactobacillus mucosae.
Low pH and 0.3% Bile Tolerance
Lact. mucosae strains survival at pH 2.0 using 1 M HCl
(Merck, Bangalore, India) and 0.3% ox gall (Sigma-Aldrich)
were determined according to modified protocol by Kaushik
et al. [30]. Briefly, 900 μl of MRS broth adjusted to pH 2.0 or
supplemented with 0.3 % (w/v) ox gall was inoculated with
100 μl of 48 h bacterial culture. After 120 min anaerobic
incubation at 37 °C, 50 μl of diluted cultures were plated onto
MRS agar and kept for 48 h in anaerobic conditions to obtain
viable counts in CFU/ml. MRS broth without bile or at pH 6.7
was served as control. Assay was performed independently in
triplicates and expressed as mean ± SD. After calculating log
cfu/ml, percent survivability was taken out using formula:
% survivability
¼ ðviable log count at time t=viable log count at t ¼ 0Þ  100
Simulated Gastric and Intestinal Stress Tolerance
Lact. mucosae strains’ resistance to simulated gastrointestinal
environment was evaluated according to Moreas et al. [26] with
slight modifications. Simulated gastric fluid was prepared freshly
by suspending 2.0 g/L pepsin (Himedia, India) in sterile saline
(0.9% w/v) adjusted to pH 2.0 with 1 M HCl. Gastric tolerance
assay was performed by mixing 1 ml cellular suspension con-
taining approx. 108 cfu/ml with 4 ml of simulated gastric fluid
and statically incubating at 37 °C for 120 min in anaerobic con-
dition. Similarly, simulated intestinal fluid was prepared by
adding 250 mg/L pancreatin (HiMedia) and (0.8 % w/v) ox gall
(Sigma-Aldrich) to sterile saline solution (0.9% w/v), adjusted to
pH 8.0 with 1 M NaOH. Evaluation of strain’s enteric tolerance
was done by mixing 1 ml bacterial suspension and 4 ml simu-
lated intestinal fluid, followed by anaerobic incubation at 37 °C
for 120 min. Bacterial viability was determined by surface plat-
ing on MRS agar before and after exposure to test conditions.
The independent assays were performed in triplicates for both
strains and expressed as mean ± SD. Percent viability was calcu-
lated using formula:
% survivability
¼ ðviable log count at time t=viable log count at t ¼ 0Þ  100
Simulated Salivary Tolerance
Lact. mucosae strains’ ability to tolerate salivary enzymes was
evaluated by Sirichokchatchawan et al. [31]. Briefly, 100 μl
aliquot of overnight cultures of both Lact. mucosae strains
(~108CFU/ml) were inoculated in 900 μl simulated saliva-
containing sterile electrolyte solution [SES: 0.02%(w/v)
CaCl2, 0.2% (w/v) KCl, 0.12%(w/v) NaHCO3, 0.6%(w/v)
NaCl] supplemented with 100 mg/L Lysozyme (HiMedia,
India). After 90 min anaerobic incubation at 37 °C, 50 μl of
diluted cultures were plated onto MRS agar, and survival of
viable cell was calculated as log cfu/ml. Assays were per-
formed as three independent replicates, and results are
expressed as mean ± SD. Percent viability was calculated
using formula:
% survivability
¼ ðviable log count at time t=viable log count at t ¼ 0Þ  100
Cell Surface Hydrophobicity
To check cell surface hydrophobicity, Lact . mucosae
strains and two pathogens E. coli and Salm. typhimurium
were cultured overnight, and stationary-phase cells were
collected by centrifugation (10,000× g for 15 min at 4
°C), washed twice with PBS (50 mM KH2PO4/K2HPO4,
pH 6.5) and resuspended in PBS and absorbance (A0) at
600 nm recorded. 3 ml microbial suspension was mixed
with 1 ml of hydrocarbon xylene, followed by thorough
vortexing and 1 h incubation at 37 °C. Thereafter, A600
value (A) of aqueous layer was determined using
NanoDrop Spectrophotometer (DS-11, Denovix, USA)
and results expressed as percentage of hydrophobicity
(%H) = (A0-A)/ A0 x 100, where A0 and A were OD value
before and after extraction with organic solvent, respec-
tively. Test was carried out thrice for both strains.
Auto-Aggregation Ability
For auto-aggregation assay, 24 h grown Lact. mucosae strains
were harvested by centrifugation (10,000× g for 15 min,4
°C), washed twice with PBS (50 mM KH2PO4/K2HPO4,
pH 6.8) and resuspended in PBS to obtain absorbance (A0)
around 0.8 at 600 nm. 3 ml bacterial suspension was vortexed
for 1 min and incubated at room temperature for 4 h. Every
hour, 0.1 ml of upper suspension was transferred to another
3.9 ml PBS and OD measured at 600 nm. PBS was used as
blank. Auto-aggregation percentage was calculated as
[A0−At]/A0 × 100, where At is the OD at time t (t = 1, 2, 3,
4) and A0 is OD at t = 0.

Bile Salt Deconjugation Ability
Lact. mucosae strains’ ability to deconjugate bile salt was
evaluated by following the protocol described by Shehata
et al. [32] with few modifications. 24 h-grown cultures of both
the strains were spotted on MRS agar surface containing 0.5%
(w/v) sodium taurodeoxycholate (Himedia, India) and
0.037% calcium chloride (HiMedia, India). After 72 h of an-
aerobic incubation at 37 °C, the presence of precipitation zone
around colonies was considered as positive result. Test was
performed in triplicates for each strain.
Cholesterol Reduction Ability
Lact. mucosae strains’ ability to reduce cholesterol in spent
broth was determined by modifying method of Archer &
Halami [33]. 1% overnight cultures were inoculated in MRS
broth suspended with 0.8% (w/v) ox gall (Sigma-Aldrich) or
0.4% (w/v) sodium taurodeoxycholate (Himedia, India) and
0.1 g/L water soluble cholesterol (Sigma-Aldrich) and incu-
bated for 72 h under anaerobic condition at 37 °C. Sterile
MRS broth without test condition was served as control.
Culture was then centrifuged (12,000× g for 15 min, 4 °C)
to obtain supernatant, which was then used to quantify cho-
lesterol reduction. 3 ml 95% ethanol and 2 ml 45% (w/v)
KOH was added to 1 ml supernatant with continuous mixing,
followed by heating at 60 °C for 12 min and then cooling
under cold water. To each tube, 5 ml hexane was added and
thoroughly mixed, followed by addition of 1 ml double-
distilled water. Mixture was kept for phase separation for 15
min. 5 ml freshly prepared O-phthalaldehyde (0.5 mg/ml gla-
cial acetic acid) was added to tube containing hexane residue
and kept for 20 mins. Finally, absorbance was read at 552 nm
after addition of 2 ml conc. sulphuric acid and allowing it to
stand for 30 min. Cholesterol reduction was calculated using
formula:
%cholesterol reduction
¼ ½1−A=Ao   100; where A is spent broth and Ao is control broth:
Free Radical Scavenging Ability
By measuring the scavenging ability of 2,2 dipheny-1-
picrylhydrazyl (HiMedia, India) of Lact. mucosae strains, an-
tioxidant potential was determined as per the modified proto-
col of Nithya et al. [34]. 24 h-grown culture of both strains
was centrifuged (12,000× g for 10 min, 4 °C) to obtain both
supernatant and culture filtrate (CF). The test sample was pre-
pared by adding 2 ml DPPH solution (6 mg/100 ml methanol)
to the tube containing 500 μl of supernatant or CF. Blank
solution was prepared by adding 2 ml of methanol to 500 μl
of supernatant/CF. 2 ml each of DPPH in MRS broth/distilled
Probiotics & Antimicro. Prot.
water was served as negative control. L-ascorbic acid
(HiMedia, India) was used as positive control. All tubes were
kept in dark for 30 min after thorough vortexing. OD at
517 nm was taken and scavenging ability was calculated in
percentage using formula:
% scavenging was calculated
¼ f1−½A517 sample−A517 blank=A517 controlg  100:
Antagonistic Activity Against Enteric
and Multidrug-Resistant Pathogen
Lact. mucosae strains’ ability to antagonise potent enteric
pathogens and multidrug-resistant pathogens was carried out
as described by Jabbari et al. [35]. The 48-hour-grown cul-
tures of Lact. mucosae strains were centrifuged (12,000× g for
15 min, 4 °C), and supernatant collected was sterilised to
remove residual bacterial cells using 0.2μ cellulose acetate
filter. These cell-free supernatants (CFS) of both the strains
were then used to assess inhibitory activity. Furthermore,
pathogenic strains were cultured in BHI broth and aerobically
incubated at 37 °C for 24 h to reach the cellular density about
108 CFU/ml. Mueller Hinton agar (MHA) (Oxoid) containing
test pathogens were then punctured using 6 mm hole driller to
make wells, where 80 μl of CFS was put inside each well.
After overnight incubation at 37 °C, plates were observed for
inhibitory zone. Zone sizes were expressed as weak (7–9
mm), intermediate (10–13 mm), strong (14–24 mm) and very
strong (> 17 mm) according to Sirichokchatchawan et al. [31]
Each test was performed in triplicates, and results are given as
mean of inhibition zones±SD.
Exoenzyme Production
The extracellular enzymes protease, lipase, esterase and amy-
lase production was also studied. Modified protocols of
Romero-Luna et al. [36] and Norouzi et al. [37] were
employed, and both the strains were tested for the same. The
modified agar media were prepared and inoculated with 50 μl
aliquot of overnight cultures of both strains, while uninoculat-
ed plate was served as control, followed by anaerobic incuba-
tion for 48 h at 37 °C for observation.
Protease activity: Freshly prepared skimmed milk medium
[1.2%(w/v) skimmed milk, 0.5%(w/v) peptone, 0.1%(w/v)
glucose, 0.3% (w/v) yeast extract, 2% (w/v) agar] was used
to determine protease production. The positive result is pres-
ence of clear zone of digested milk around colonies.
Lipase activity: Tributyrin medium containing 1% (w/v)
tributyrin, 0.5% (w/v) peptone, 0.3% (w/v) yeast extract and
2%(w/v) agar was used to evaluate lipase production. Positive
results indicated clear zone around colonies.
Probiotics & Antimicro. Prot.
Esterase activity: Modified medium (1% (w/v) tween 80,
0.5%(w/v) NaCl, 0.01% (w/v) CaCl2.2H2O, 1%(w/v) pep-
tone, 2%(w/v) agar) was prepared to ensure esterase activity
of strains. Positive result indicated the formation of opaque
halo around growth.
Amylase activity: Starch medium (1.5% (w/v) starch, 0.3%
(w/v) yeast extract, 0.5% (w/v) peptone, 2% (w/v) agar) was
prepared to evaluate amylase activity. After incubation, plates
were flooded with 1% (w/v) iodine solution. Decolourized
halo around cellular growth confirms amylase activity.
Antibiotic Susceptibility
Susceptibility of Lact. mucosae strains to selected antibi-
otics was evaluated by Kirby-Bauer disc diffusion method
with slight modifications. The following antibiotic discs
(Oxoid, Basingstoke, UK) were used: vancomycin (30
μg), cefoxitin (48 μg), ciprofloxacin (5 μg), gentamicin
(120 μg), tetracycline (30 μg), ampicillin (10 μg), penicil-
lin (10 μg), erythromycin (10 μg), cefotaxime (30 μg),
chloramphenicol (30 μg), fosfomycin (200 μg ),
tobramycin (10 μg), linezolid (30 μg) and doxycycline
(30 μg). Each antibiotic disc was dispensed on the MRS
agar previously homogeneously swabbed with 0.5
McFarland turbid cultures of both the strains. After 48 h
anaerobic incubation at 37 °C, inhibitory zones were re-
corded and results interpreted as sensitive or resistant based
on CSLI 2018 guidelines from three independent
experiments.
Hemolytic Activity
Hemolytic activity of Lact. mucosae strains was carried out as
described by Cui et al. [38]. 48 h cultures of both strains were
spotted onto blood agar containing 5% (v/v) sheep blood (BD,
New Jersey, USA) and kept for anaerobic incubation at 37 °C
for 72 h. Plates were then observed for complete or β-
haemolysis (clear halo around each colonies or), α-
haemolysis (greenish halos around each colonies) and γ-
haemolysis (no halo around each colonies). Staph. aureus
ATCC 25923 showing β-haemolysis was served as positive
control.
Gelatinase Activity
Lact. mucosae strains were verified for gelatinase activity as
described by Perin et al. [39].10 μl freshly grown cultures
were spotted on MRS (Himedia, India) agar containing 5%
(w/v) gelatin (HiMedia, India) and incubated anaerobically at
37 °C for 72 h. Thereafter, plates were kept at 4 °C for 4 h and
observed for opaque halo around the colonies.
DNAse Activity:
DNAse-producing ability of Lact. mucosae strains was
assessed by inoculating 10 μl of each cultures on DNAse agar
(Oxoid, UK) and anaerobically incubated for 72 h at 37 °C.
After incubation, plates were flooded with 3% HCl for 8 min
and observed for clear zone around colonies. Positive control
used was Staph. aureus ATCC 25923.
Mucin Degradation Assay
Both the strains of Lact. mucosae were assessed for their tox-
icity in degrading gastric mucin in vitro using plate method as
described by Martin et al.[40]. 0.5% (w/v) partially purified
hog gastric mucin (HGM) (Sigma-Aldrich) and 1.5% (w/v)
agarose (HiMedia, India) were added to anaerobic culture me-
dium without glucose. Plates were then anaerobically incubat-
ed for 72 h at 37 °C. Thereafter, plates were stained with 0.1%
amido black in acetic acid for 30 min, followed by washing
with 1.2 M acetic acid. Mucin lysis zone around colonies was
served as positive control. Salm. typhimurium and Sh. flexneri
were served as positive control. Experiment was performed in
triplicates.
Accession Numbers
The nucleotide sequences of 16S rRNA of both strains were
deposited at the GenBank database under the following acces-
sion numbers: Lact. mucosae SRV5 (MK990014) and Lact.
mucosae SRV10 ( MN064860)
Statistical Analysis
All the experiments were carried out in triplicates, and mean ±
standard deviation (SD) of experimental data was calculated
using Microsoft Excel 2010, Microsoft Corporation (USA).
Results
Acid and Bile Tolerance
Both Lact. mucosae strains exhibited marginal reduction in
initial population in terms of log cfu/ml after 120 min expo-
sure to pH 2.0 and 0.3% ox gall as compared to control (MRS
broth without test condition; Table 1). At pH 2.0, strain SRV5
showed 91.805% and SRV10 showed 95.827% mean survival
rates. Whereas at 0.3% ox gall concentration, strains SRV5
and SRV10 displayed 95.913 and 89.304% mean survivabil-
ity. Only slight interference in growth of Lact. mucosae strains
under harsh test conditions clearly suggests that these strains
can appreciably tolerate low pH and high bile concentration
 Probiotics & Antimicro. Prot.

7.345 ± 0.266
7.937 ± 0.955
After 90 min
with minimum cell count loss which is one of the prerequisite
conditions for probiotics.

Simulated Gastric, Intestinal and Salivary Tolerance

Salivary tolerance*

Initial population

8.079 ± 0.053.
8.140 ± 0.256
Further, both Lact. mucosae strains were subjected to survival
(log cfu/ml) in simulated fluids containing digestive enzymes in order to
explore the resistance of strains to harsh conditions of the
stomach and upper intestine. The studied strains exhibited

8.131 ± 0.0825
only slight reduction in initial cell count in presence of gastric
After 120 min

8.078 ± 0.13
enzyme pepsin (SGF) at pH 2.0 and intestinal enzyme pancre-
atin (SIF) containing 0.8% ox gall at pH 8.0 after 4 h exposure
(Table 1). The present study discerned the good viability of
strain SRV5 in both the gastric and intestinal conditions, with
Intestinal tolerance*

Initial population

mean % survivability of 90.379 and 96.304%, respectively.

9.1875 ± 0.206
8.388 ± 0.093

Another strain SRV10 displayed 92.681% mean viability in

(log cfu/ml)

gastric fluid, while its endurance in intestinal fluid was com-

paratively moderate (88.506%). Also, both Lact. mucosae
In vitro resistance to acid, bile, simulated salivary and gastrointestinal stress of Lact. mucosae strains from donkey milk

strains when exposed to simulated salivary fluid containing

After 120 min

lysozyme (100 mg/l) for 90 min exhibited lesser cease in

8.541 ± 0.462
8.215 ± 0.088

growth (Table 1). The mean survival rates of SRV5 and

SRV10 were 97.5001 and 90.92%, respectively, after 90 min.

Cellular Hydrophobicity and Auto-Aggregation

Gastric tolerance*

Initial population

Capacity
9.216 ± 0.149
9.089 ± 0.205
(log cfu/ml)

The ability of Lact. mucosae strains to adhere to the intestinal

epithelium is evaluated based on their cell surface hydrophobicity
towards of a polar solvent, xylene. Figure 1 shows variable hy-
After 120 min

6.724 ± 0.168
6.126 ± 0.135

drophobicity by both strains towards xylene. The hydrophobicity

of strain SRV5 determined with xylene (54.280%) was higher
than that with strain SRV10 which showed 46.829%.
Auto-aggregation ability of Lact. mucosae strains was
Initial population

measured at four consecutive times intervals 1, 2, 3 and 4 h,

Bile tolerance*

6.832 ± 0.018

and results are expressed in Fig. 2 which show a steady in-

7.010 ± 0.20
(log cfu/ml)

*Values are mean of triplicate experiments ± standard deviation

crease in auto-aggregation by both test strains. After 4 h, strain

SRV5 and SRV10 displayed 64.79 and 43.14% cellular auto-
60
After 120 min

6.738 ± 0.040
6.706 ± 0.150

50
cell surface hydrophobicity %

40

30
Initial population
Acid tolerance*

6.831 ± 0.212
7.031 ± 0.116

20
(log cfu/ml)

10

0
Lact. mucosae

SRV5 SRV10 S. typhimurium E.coli

Table 1

SRV10
strains

SRV5

Fig. 1 Cell surface hydrophobicity of studied Lact. mucosae strains and

two enteric pathogens
Probiotics & Antimicro. Prot.
Fig. 2 Cellular auto-aggregation ability of Lact. mucosae strains isolated
from donkey milk
aggregation, respectively. The results of both the strains dem-
onstrated good cell surface hydrophobicity and auto-
aggregation ability, clearly indicating their effective cell adhe-
sion capacity.
Bile Salt Hydrolase and Cholesterol Reduction
Lact. mucosae strains’ ability to produce BSH enzyme was
evaluated in vitro by deconjugation of sodium
taurodeoxycholate. Both the stains SRV5 and SRV10 formed
white opaque colonies and caused precipitation, which is at-
tributed due to BSH enzyme-mediated formation of free bile
salts in MRS medium supplemented with sodium
taurodeoxycholate. Also, strains were studied in vitro for their
cholesterol-lowering capability in spent broth in presence of
both ox gall and sodium taurodeoxycholate. The results are
given in Table 2, along with bile salt deconjugation, and show
that strain SRV5 exhibited highest (66.874%) cholesterol-
reducing ability in presence of ox gall, while strain SRV10
had highest (64.676%) value with sodium taurodeoxycholate
after 72 h anaerobic growth.
Antioxidant Activity
The capacity to scavenge 2,2 diphenyl-1-picrylhydrazyl(DPPH)
free radical is a functional attribute of probiotics indicative of its
antioxidative property. In this assay, both Lact. mucosae strains
culture supernatant and intact cells showed varying degree of
Table 2 Functional attributes of Lact. mucosae strains isolated from donkey milk
Lact. mucosae strains Bile salt deconjugation Cholesterol reduction (%)*
Ox gall
SRV10 g+ 45.686 ± 0.91
SRV5 g+ 66.874 ± 0.57
Bile salt deconjugation: g indicates growth; (+) bile salt deconjugation
*Values are expressed as mean ± SD (n = 3)
scavenging ability, as presented in Table 2. Culture supernatant
of strain SRV5 exhibited stronger DPPH scavenging (89.513%)
than strain SRV10 (73.77%) as compared to control (MRS broth)
which displayed significantly lower radical scavenging activity.
Intact cells of strains SRV5 and SRV10 exhibited lower antiox-
idant activity as compared to culture supernatant with 61.698 and
47.169%, respectively.
Antimicrobial Activity Against Enteric and MDR
Pathogens
The inhibitory activity of Lact. mucosae strains against potent
enteric pathogens is another cogent trait of probiotics in
preventing infection of pathogenic microbes in gastrointesti-
nal tract. In our study, CFS of both the strains showed variable
inhibition zones pertaining to 18 different enteric pathogens
employed (Table 3). Strain SRV5 showed strong inhibition
against E. coli ATCC 25922 and Salm. typhimurium with zone
size of 18 mm and 15 mm, respectively, while rest all patho-
gens were inhibited moderately with zone size in range of 10–
12 mm. As compared to strain SRV5, strain SRV10 displayed
superior antagonistic activity against most of the pathogens
with larger zone size in range of 13–24 mm, with largest
inhibition halo of 20 mm for E. coli ATCC 25922.
Antagonistic activity of Lact. mucosae strains against
multidrug-resistant gram-negative bacteria provides one
of the novel preventive methods in fighting these infec-
tions. In our study, cell-free culture supernatant of both
the strains exhibited significant inhibitory activity towards
five MDR strains which include E.coli, Kl. pneumoniae,
Ac. baumanii, Ps. aeruginosa and Pr. mirabilis as given
in Table 4. Strain SRV5 showed moderate inhibition against
Kl. pneumonia with zone size of 12 mm, while the rest of
the MDR bacteria exhibited smaller zone size in range of 8–
10 mm. Strain SRV10 also possessed similar inhibitory
profile, with maximum zone size of 14 mm against Pr.
mirabilis, while others showed mild zone of inhibition in
range of 9–12 mm. The results obtained by employing well
diffusion assay elucidate the fact that inhibitory metabolites
and/or peptides produced by these test strains were diffus-
ible and extracellular.
DPPH-scavenging activity (%)
TDCA Culture supernatant Intact cells
64.676 ± 0.90 73.770 47.570
34.166 ± 0.30 89.513 61.698
 Probiotics & Antimicro. Prot.

Table 3 Antagonistic activity of Lact. mucosae strains against enteric Antibiotic Susceptibility
pathogens

Indicator strain SRV10 SRV5 For safe use of Lact. mucosae strains as probiotics, it is rec-
ommended to characterise their antibiotic susceptibility pro-
Salm. typhimurium +++ +++ file. In present study, 14 antibiotics were used and results were
Salm. typhi T(H) ++ ++ given in Table 5. Both the tested strains were sensitive to
Sh. dysenteriae +++ ++ ampicillin, chloramphenicol, cefoxitin, ciprofloxacin, linezo-
Sh. flexneri type 2 +++ ++ lid, tetracycline, penicillin, erythromycin, cefotaxime and
Sh. paratyphi T(H) +++ ++ doxycycline with variable and significant zone of inhibition.
Sh. sonei +++ ++ Both the strains SRV5 and SRV10 were resistant to vancomy-
Coryne. diphtheriae ++ - cin and gentamycin. In addition, strain SRV5 was also resis-
Pr. vulgaris ATCC 6380 +++ ++ tant to fosfomycin, while strain SRV10 was found to be resis-
Kl. oxytoca ATCC 700324 +++ ++ tant to tobramycin.
E. coli ATCC 25922 ++++ ++++
Ent. faecalis ATCC 51299 +++ ++
Hemolytic, Gelatinase and DNAse Activity
Staph. aureus ATCC 25923 +++ ++
Sh. boydii +++ +
The Lact. mucosae strains were evaluated for safety property,
Kl. pneumoniae +++ ++
in order to ensure their plausibility as probiotics. In our study,
Ps. aeruginosa ATCC 27853 ++ - both the strains SRV5 and SRV10 showed no haemolysis when
Ser. ficaria + - grown on 5% sheep blood agar and no DNAse-producing abil-
St. maltrophia - + ity when grown on DNAse agar after 72 h anaerobic incubation
L. monocytogenes ++ + as compared to control Staph. aureus ATCC25923. Also, none
of the strains hydrolysed gelatin in vitro.
The antagonistic ability of non-neutralised cell-free culture supernatant
was expressed as (-) no inhibition, (+) weak inhibition (7–9 mm), (++)
intermediate inhibition (10–13 mm), (+++) strong inhibition (14–24 mm) Mucin Degradation Assay
and (++++) very strong inhibition (> 17 mm)

In present study, none of strains produced mucinolytic zone

Extracellular Enzyme Production around their colonies, while control strains displayed large
mucin lysis zones, thus clearly demonstrating their nontoxic
The ability of Lact. mucosae strains to produce extracellular behaviour.
enzymes provides auxiliary health benefit to host by improv-
Table 5 Antibiotic susceptibility profile of Lact. mucosae strains from
ing nutrient utilisation in the intestine. Considering this, both donkey milk
the strains were evaluated for lipase, esterase, protease and
amylase-producing ability. Strain SRV5 showed mild amylase Antibiotics Disc potency(μg) SRV10 SRV5
activity, while strain SRV10 exhibited mild protease activity.
Ampicillin 10 S S
None of the strains produced lipase and esterase activity on
Cefoxitin 48 S S
modified agar after 48 h anaerobic growth.
Chloramphenicol 30 S S
Ciprofloxacin 5 I I
Gentamycin 120 R R
Table 4 Antagonistic activity of Lact. mucosae strains against
multidrug resistant pathogens Tetracycline 30 S S
Penicillin 10 S S
Indicator MDR strains SRV10 SRV5
Erythromycin 10 S S
E. coli (MDR) ++ ++ Vancomycin 30 R R
Kl. pneumoniae (MDR) + ++ Fosfomycin 200 S R
Ps. aeruginosa (MDR) ++ ++ Tobramycin 10 R S
Pr. mirabilis (MDR) +++ + Linezolid 30 S S
Ac. baumannii (MDR) + + Cefotaxime 30 S S
Doxycycline 30 S S
The antagonistic ability of non-neutralised cell-free culture supernatant
was expressed as (-) no inhibition, (+) weak inhibition (7–9 mm), (++) R indicates resistant to antibiotics; I is intermediary susceptible to antibi-
intermediate inhibition (10–13 mm), (+++) strong inhibition (14–24 mm) otics; S indicates susceptible. Inhibition halo interpreted according to
and (++++) very strong inhibition (> 17 mm) CLSI 2018 guidelines
Probiotics & Antimicro. Prot.
Discussion
In the present study, we have identified and assessed the pro-
biotic potential of Lact. mucosae strains isolated from donkey
milk. In vitro study demonstrated that both the strains can
appreciably tolerate the adversities of the gastrointestinal tract
which is the prerequisite condition for efficacy and viability of
probiotics. Both the strains at pH 2.0 displayed greater than
90% survivability. In acidic microenvironment, transmem-
brane pH gradient is affected owing to accumulation of pro-
tons inside the cell; strains that can tolerate acidic conditions
may probably have the ability to modulate the homeostasis of
intracellular pH by actively removing protons from the cell by
producing basic compounds that can pair with excess protons
or by proton-translocating ATPase [41]. Gastric stress not only
is caused by low pH but is also due to gastric enzyme pepsin
which aids in digestion. Thus, in our study, both the strains
were subjected to simulated gastric tolerance assay and were
found to have high viable population, around 8.215 log cfu/ml
for SRV5 and 8.541 log cfu/ml for SRV10 after 4 h. The
concentration of human bile in the duodenum varies depend-
ing upon the age, gender, race and physiological conditions of
the host and generally lies in range of 0.3–0.5% [10]. Bile
acids synthesised in the liver then undergo chemical alter-
ations in the duodenum as a result of gut microbial enzymes.
These altered bile acids possess inhibitory activity against
wide range of bacteria. Lysozyme in saliva also kills gram-
positive bacteria. Thus, tolerance of microorganisms to bile
and lysozyme holds prime importance in stating them as
probiotics. In our study, both the strains displayed greater than
89% tolerance to 0.3% bile and greater than 90% survivability
at 0.1% lysozyme. Also, strains showed high viable popula-
tion even in simulated intestinal tolerance assay containing
enzyme pancreatin, with 8.078 log cfu/ml for SRV10 and
8.131 log cfu/ml for SRV5 after 4 h. Lact. mucosae LM1
survived best in 0.1–0.3 % bile and pH 3.0 [42]. de Moraes
et al. [26] also showed tolerance of Lact. mucosae strains at
pH 2.5 and 0.45% bile. Another strain, Lact. mucosae AN1,
exhibited slight reduction in log viable counts at pH 2.0, 0.1–
0.7% bile and 0.2% lysozyme [27]. Bao et al. [43] reported
resistance of Lact. fermentum strains at 0.3% bile and gastric
juice at pH 3.0.
In order to populate the gastrointestinal tract in adequate
number, probiotic microorganisms must adhere firmly to the
intestinal epithelial cells or else will be flushed out by peri-
staltic movement. Cell surface hydrophobicity using polar
solvents, such as n-hexadecane or xylene, therefore, is one
of the crucial criteria used in screening probiotic microorgan-
isms [44]. Earlier studies on Lact. mucosae strains from goat
sample [26] stated surface hydrophobicity to be strain specific
as it showed variability among three strains (24–70%), while
Valeriano et al. [42] reported hydrophobic surface character
greater than 50% by Lact. mucosae strain LM1. Strain-
specific variability in surface hydrophobicity is due to differ-
ences in different proportions of surface components [45]. As
such, strains rich in lipoteichoic acid and surface proteins will
be more hydrophobic as compared to strains which possess
higher content of hydrophilic polysaccharides [46]. Also, sev-
eral authors [47, 48] have reported the intrinsic surface hy-
drophobicity of pathogenic bacteria which favour their adhe-
sion to host epithelial tissue prior to multiplying and causing
infection. The higher the surface hydrophobicity, the greater
is the adhesion of cells to the host tissue. Therefore, compar-
ison of cell surface hydrophobicity of both Lact. mucosae
strains on two enteric pathogens E.coli and Salm.
typhimurium was assayed in present study in order to explore
the efficiency of strains in combating the pathogens’ adhesion
to intestinal mucosa. Our results for both Lact. mucosae
strains demonstrated more than 45% hydrophobic surface
character, while both the pathogens exhibited relatively hy-
drophilic character (< 10%) in xylene. Earlier studies by
Valeriano et al. [42] and Bargeron et al. [49] supported our
results by reporting hydrophilic nature of E.coli and Salm.
typhimurium. Also, auto-aggregation is another property of
probiotics to form cell aggregates, which in turn increases
the adhesion of cells to epithelial lining of the intestine, there-
by allowing colonisation of probiotic microorganisms to gut
lining [50]. The percentage of auto-aggregation of both Lact.
mucosae strains obtained in the present study after 4 h incu-
bation is greater than 48%, indicating their good adhesion
ability.
One of the mechanisms by which intestinal colonisation of
probiotic microorganisms curtails high blood cholesterol
in vivo is through action of deconjugating enzyme, bile salt
hydrolase. Thus, bile salt hydrolase-producing ability has
been postulated to be an essential characteristic of probiotics
in exerting hypocholesterolemic effect. Enzyme exerts its ef-
fect by cleaving amino acids with cholesterol-derived steroi-
dal moiety of bile salt [51]. These deconjugated bile salts
possess greater hydrophobicity than their conjugated counter-
parts, thus less readily reabsorbed in the ilium and augmenting
its excretion in faeces. Also, this action tends to upregulate the
de novo synthesis of bile acids in hepatocytes using cholester-
ol as precursor to compensate for the loss. In present study,
both the strains SRV5 and SRV10 displayed good BSH activ-
ity on sodium taurodeoxycholate supplemented with MRS
agar, leading to precipitation due to deconjugation of
taurodeoxycholate. Apart from its role in cholesterol homeo-
stasis, Choi et al. [52] postulated another very significant func-
tion of microbial BSH enzyme as natural means of attenuating
t h e r i s k o f c o l o n c a n c e r, a s d e c o n j u g a t i o n o f
taurodeoxycholate produces sulfonic moiety and consequent-
ly reduces hydrogen sulphide that is highly toxic to colon
epithelium. Being positive for BSH enzyme production, both
strains were further tested for their cholesterol-reducing ability
in spent broth containing both ox gall and sodium

taurodeoxycholate in vitro. Results showed that both the
strains reduced cholesterol significantly but to varying extent
for both these substrates. Similar studies confirmed the pres-
ence of BSH gene in Lact. mucosae strains [26]. Lact.
mucosae DPC 6426 is highly BSH-active and showed good
cholesterol reduction in vivo [25, 29].
Our study also suggests therapeutic role of Lact. mucosae
strains SRV5 and SRV10 as natural antioxidants, as they sig-
nificantly scavenged DPPH free radical (45–90%). Thus,
hypothesising the role of these strains in alleviating the prog-
ress of many chronic diseases which may result from oxida-
tive stress due to elevated level of reactive oxygen species
(ROS) activity through oxidation process. Also, results ob-
tained indicate stronger antioxidant potential of culture super-
natant as compared to intact cells, clearly documenting the
fact that these strains may produce metabolites with free rad-
ical scavenging ability such as glutathione (GSH), folate and
butyrate as evidenced by previous reports [53]. Ahire et al.
[54] explored antioxidant potential of folate producing Lact.
helveticus CD6, while Kullisaar et al. [55] studied about two
antioxidative Lact. fermentum strains, E-3 and E-18, that pos-
sess elevated level of GSH. Lact. mucosae AN1 exhibited
66.53% DPPH scavenging ability [27]. Presently, very few
published studies on antioxidant ability by Lact. mucosae
are currently available, thus emphasising their relatively novel
beneficial role.
Another cogent health claim for probiotics is their puta-
tive prevention and reduction of infectious diseases in the
gastrointestinal tract. The antagonistic property of
probiotics is due to production of exocellular substances
such fatty acids, organic acids (acetic acid, lactic acid),
acetaldehyde, diacetyl, hydrogen peroxide and the protein
(bacteriocins) or lipophilic factors [56, 57] that not only
affect the pathogen viability but also affect its toxin produc-
tion or metabolism. Our study represented good antimicro-
bial activity of both Lact. mucosae strains SRV5 and
SRV10 against broad range of enteric as well as
multidrug-resistant clinical pathogens. The studied strains
inhibited the growth of almost all eighteen enteric patho-
gens and five multidrug-resistant pathogens to varying de-
gree; this is in agreement with earlier reports [58–60] that
confirmed their strain-specific nature. The pH of culture
supernatant of both the strains was measured prior to agar
diffusion assay and was found to lie in range of 3.1–4.3.
This acidic pH of the 24 h broth may be the probable reason
for strong antibacterial activity in our study. Martin et al.
[61] earlier observed the production of high amount of lac-
tic acids by Lact. gasseri CECT5715, Lact. gasseri
CECT5714 and Lact. fermentum CECT5716 when grown
in MRS broth which imparted to their inhibitory activity.
Lact. mucosae strain LM1 have previously proven to inhibit
Salm. typhimurium KCCM 40253 and E. coli K88 [42]. So
far, this is the first report that demonstrated the prudent role
Probiotics & Antimicro. Prot.
of Lact. mucosae strains in inhibiting multidrug-resistant
bacteria.
Safety evaluation of probiotic strains is of prime concern
among scientific community and food regulators [1]. Within
Lactobacillus genus, resistance towards different antibiotics is
an inherent or acquired trait. In case of acquired resistance, there
is high risk of horizontal transfer of antibiotic resistant gene
through plasmids from probiotics to pathogens in host gut.
Hence, in order to restrain this, probiotic Lactobacillus strains
must be susceptible to most of the antibiotics currently available.
Current study showed that the two Lact. mucosae strains SRV5
and SRV10 were susceptible to almost all antibiotics tested ex-
cept vancomycin and gentamycin. These results are in accor-
dance with previous reports by other authors [34, 62, 63].
Resistance to vancomycin is considered to be innate to
Lactobacillus genus as genes are present in chromosomal DNA
and thus are not horizontally transmissible type [64]. Also, ac-
cording to Franz et al. [64], resistance to aminoglycoside such as
gentamycin and streptomycin is also common among
Lactobacillus genus and is mainly due to the absence of
cytochrome-mediated electron transport, which mediates drug
uptake. Both Lact. mucosae isolates were found negative for
red blood cell lysis which makes them safe for human consump-
tion. These results were similar with previous findings by de
Moraes et al. [26]. Also, both strains did not degrade gastric
mucin in vitro, clearly signifying their safe use.
Conclusion
This is the first study reporting isolation of Lact. mucosae
from donkey milk. Taken together, the techno-functional dif-
ferentiation of studied Lact. mucosae strains SRV5 and
SRV10 has successfully demonstrated their potential as
probiotics. It is also one of the few reports showing antioxi-
dant ability of Lact. mucosae strains, which clearly indicate
the fact that as long as it is consumed, it will benefit the host,
particularly in fighting against damage caused by reactive ox-
ygen species. Also, present report is novel in terms of high
antagonistic activity of Lact. mucosae strains against not only
wide range of enteric pathogens but also deadly MDR clinical
strains isolated from hospitalised patients. Considering the
role of high cholesterol in cardiovascular diseases, our study
significantly proved the cholesterol-reducing ability of both
the strains. Although our work substantially proved excellent
probiotic ability of both strains, but for possible health claim
in humans, in vivo animal model experimentations need to be
done in future.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of
interest.
Probiotics & Antimicro. Prot.
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