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Probiotic characterization of lactic acid bacteria isolated from fermented

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DOI: 10.1016/j.lwt.2015.10.057

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 LWT - Food Science and Technology 66 (2016) 428e435

Contents lists available at ScienceDirect

LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Probiotic characterization of lactic acid bacteria isolated from

fermented foods and beverage of Ladakh
Kunzes Angmo, Anila Kumari, Savitri, Tek Chand Bhalla*
Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla 171005, India

a r t i c l e i n f o a b s t r a c t

Article history: The present research was focused on probiotic characterization of lactic acid bacteria from fermented
Received 12 February 2015 foods and beverage of Ladakh. Twenty five lactic acid bacteria were examined in vitro for potential
Received in revised form probiotic properties based on their low pH tolerance, bile-salt resistance, lysozyme tolerance, cholesterol
7 September 2015
removal, hydrophobicity, autoaggragation, production of antimicrobial substances, exopolysaccharide
Accepted 24 October 2015
production, b-galactosidase activity and haemolytic activity. The outcome of these studied parameters
Available online 30 October 2015
was used as input data for a principal component analysis (PCA) to select the most promising isolate and
ten most potential probiotic isolates were identified through 16S rDNA sequencing. On the basis of PCA,
Keywords:
Fermented food
isolate 84 (Lactobacillus plantarum KJ722784) showed similar trend to Lactobacillus casei Shirota used as
Beverage reference strain in terms of probiotic properties. Fermented milk sample inoculated with L. plantarum
Lactic acid bacteria KJ722784 exhibited decrease in viable count during storage at 4
C on day 28. However, the survival
Probiotic count is greater than 7 log CFU/ml which is higher than the requirement of 6 log CFU/ml to exhibit health
Probiotic properties benefit.
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction known to play an essential role in food preservation and inhibit

Probiotics are non-pathogenic microorganisms, which on
ingested in adequate amount exert a positive health benefit on host
(FAO/WHO, 2006). Consumption of probiotic have been shown to
be helpful in overcome various clinical conditions ranging from
infantile diarrhoea, antibiotic associated diarrhoea, relapsing Clos-
tridium difficile colitis, Helicobacter pylori infections, inflammatory
bowel disease to cancer and female uro-genital infections (Reid,
Jass, Sebulsky, & McCormick, 2003). Other beneficial effects of
probiotics include improving lactose intolerance, lowering serum
cholesterol level, increasing utilization of nutrients and decreasing
use of antibiotics (Guo, Kim, Namb, Park, & Kim, 2010). In general,
log 6- log 7 of probiotic bacteria per ml or g of food has been rec-
ommended for exhibition of health benefit (Lahteinen et al., 2010).
Lactic acid bacteria (LAB) are most commonly studied probiotic
for the past few decades. These are desirable microflora of the
gastro intestinal tract (GIT) and are thus ‘generally regarded as safe’
(Tannock, 1997). Secondly they are involved in the fermentation
and are dominant microflora of fermented products. They are
* Corresponding author.
E-mail address: bhallatc@rediffmail.com (T.C. Bhalla).
spoilage microorganisms or food borne pathogens by production of
lactic acid, acetic acid, H2O2, bacteriocin, diacetyl and CO2 (Nur &
Aslim, 2010).
Worldwide a variety of traditional fermented foods and bever-
ages are prepared and consumed. Fermented products are associ-
ated with desired and edible microbes which are beneficial for
health. Research in the field of exploring an interesting strain with
probiotic potential from fermented food products as a source of
new isolates are blooming. In Asia, an array of fermented foods and
beverages are produced. The people of the Himalayan region of
India particularly Ladakh region consume a variety of indigenous
fermented foods such as tagi khambir/skyurchuk (brown sourdough
bread), zho (curd), tara (buttermilk), labo (cottage cheese), chhurphe
(dried cottage cheese), etc. (Angchok, Dwivedi, & Ahmed, 2009)
and fermented vegetables and alcoholic beverages like chhang and
aarak (Angmo & Bhalla, 2014). However, no work has been carried
out to explore the microflora of fermented foods and beverages of
Ladakh and study their proposed health attributes. Therefore pre-
sent study aimed on isolation of lactic acid bacteria from traditional
fermented foods and beverage of Ladakh and examination of
various phenotypic characteristics, widely used in the screening
and selection of probiotic bacteria.

http://dx.doi.org/10.1016/j.lwt.2015.10.057
0023-6438/© 2015 Elsevier Ltd. All rights reserved.
 K. Angmo et al. / LWT - Food Science and Technology 66 (2016) 428e435
2. Materials and methods
2.1. Sample collection
Samples of chhurphe (dried cottage cheese), vegetable pickle
and chhang (barley based beer) were collected in sterile vials from
different villages (Nyemo, Saspol, Sankar, Thiksay, Zanskar) and Leh
town of Ladakh, India and were used for isolation of lactic acid
bacteria (LAB).
2.2. Isolation of lactic acid bacteria
LAB were isolated by spread plating on MRS (de Mann Rogosa
Sharpe) agar (Himedia), and the plates were incubated at 30
C for
48 h. Colonies with different morphologies on the MRS agar plate
were selected and further subcultured in order to obtain a pure
colony. Glycerol stock of LAB isolates were prepared and stored
at 80
C.
Provisional or tentative identification of genera was made by
Gram staining, cell morphology and catalase reaction. Physiological
properties such as growth at different temperature and different
NaCl concentration, gas production from glucose, hydrolysis of
arginine and carbohydrate fermentation were also studied.
2.3. Strains
Reference strain: Lactobacillus casei Shirota (P1) was from Uni-
versity of Helsinki, Finland. Test strains: Bacillus cereus, Escherichia
coli, Staphylococcus aureus, Shigella dysenteriae and Pseudomonas sp.
were obtained from Indira Gandhi Medical College (IGMC) Shimla,
India and Listeria monocytogenes was procured from Microbial Type
Culture Collection (MTCC), Institute of Microbial Technology,
Chandigarh, India.
2.4. Evaluation of probiotic properties
2.4.1. Acid and bile tolerance
The experiment for tolerance of isolate to pH 2.0, 3.0 and 7.0
(control) was performed following the method described by Yu
et al. (2013).
The ability of the isolates to grow in the presence of 0.5% and 1%
of bile (w/v) was determined according to the method of Vinderola
and Reinheimer (2003). Resistance was evaluated by plate count on
MRS agar.
2.4.2. Lysozyme tolerance
Tolerance to lysozyme was assessed following the method re-
ported by Vizoso-Pinto, Franz, Schillinger, and Holzapfel (2006).
2.4.3. Cholesterol removal
Cholesterol removal was determined using o-phthalaldehyde
method according to Liong and Shah (2005). Three different bile
salts such as deconjugated bile (cholic acid), conjugated bile (so-
dium taurocholate) and mixture of conjugated (97%) and decon-
jugated (3%) bile (oxbile) were used in this study.
2.4.4. Cell surface hydrophobicity
This assay was done by the method given by Mishra and Prasad
(2005).
2.4.5. Autoaggregation
Autoaggregation was carried out according to Collado,
Meriluoto, and Salminen (2008) with little modifications. Over-
night grown cells were suspended in PBS buffer to obtain an optical
density (O.D.) of 0.25 ± 0.05 at 600 nm. The bacterial suspension
429
(4 ml) was vortexed for 10 s and incubated at 30
C. Samples were
monitored during different time intervals (0 h, 3 h and 24 h) and
autoaggregation percentage was expressed as [1  At/A0]  100,
where At represents the absorbance at time t and A0 as the absor-
bance at t ¼ 0.
2.4.6. Antibacterial activity
Agar well diffusion method was used to test the antimicrobial
activity as described by Mishra and Prasad (2005). The superna-
tants of 18e20 h grown LAB cells were tested against B. cereus, E.
coli, S. aureus, S. dysenteriae and Pseudomonas sp. and L.
monocytogenes.
2.4.7. Antibiotic resistance
MRS agar plate was overlaid with 100 ml of LAB culture con-
taining 108 CFU/ml and antibiotic discs containing penicillin G (10
units), clindamycin (2 mcg), co-trimoxazole (25 mcg), erythro-
mycin (15 mcg), vancomycin (30 mcg) and ampicillin (10/10 mcg)
were placed on inoculated plates under sterile conditions. After
incubation for 24 h at 30
C, the diameter (mm) of inhibition zone
was measured.
2.4.8. b-Galactosidase activity
For b-galactosidase activity, bacterial cultures were streaked on
MRS agar plates containing 60 ml X-gal (5-bromo-4-chloro-3-
indolyl-b-D-galactopyranoside) and 10 ml of IPTG (iso-propyl-thio-
b-D galactopyranoside) solution as inducer.
2.4.9. Exopolysaccharide production
Overnight cultures were streaked on the surface of plates con-
taining ruthenium red milk (10% w/v, skim milk powder, 1% w/v,
sucrose and 0.08 g/l ruthenium red, 1.5% w/v agar).
2.4.10. Haemolytic activity
LAB isolates were streaked on the surface of Columbia blood
agar plates (Oxoid) supplemented with 5% sheep blood. After 48 h
of incubation at 30
C the plates were examined for haemolytic
reaction.
2.4.11. Statistical analysis
Statistical analysis was performed on the data by SPSS 19.0
Bivariate Correlation Analysis (SPSS Inc., Chicago, Ill., U.S.A.). Prin-
cipal component analysis (PCA) was conducted with XLSTAT TM
software (Addinsoft, Paris, France). The results of both qualitative
(antibiotic resistance, b-galactosidase activity and haemolytic) and
quantitative assays (acid, bile and lysozyme tolerance, antimicro-
bial activity, cholesterol removal, hydrophobicity and autoag-
gregation) were converted into three coded values (0, 1 and 2) and
used as input data.
2.5. Identification of the finally selected isolates by 16S rDNA
sequencing
16S rDNA of the selected isolates were amplified by PCR using
primers 27F (50 - AGAGTTTGATCCTGGCTCAG-30 ) and 1492R (50 -
TACGGYTACCTTGTTACGACTT-30 ).
DNA sequencing of amplified fragments was carried out by
sequencing service of Xcelris Labs Limited, Ahmedabad, India. The
fragments of sequences were assembled and edited with software
BioEdit 7.2.5 and consensus sequences were compared with those
deposited in the GenBank DNA database using the Basic Local
Alignment Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov/Blast.
cgi). A phylogenetic tree based on 16S rRNA genes was also con-
structed to determine the closest bacterial species by the neighbor-
joining method (Saitou & Nei, 1987), using MEGA 6.06 (Tamura
430 K. Angmo et al. / LWT - Food Science and Technology 66 (2016) 428e435
et al., 2011).
2.6. Production of fermented milk
The LAB isolate was assessed for the production of fermented
milk product. The preparation and fermentation of milk was per-
formed according to the method of Chiu, Lu, Tseng, and Pan (2006).
Skimmed milk powder was weighed and dissolved in water to
obtain 4% skimmed milk (w/v), which was sterilized in an autoclave
at 121
C for 15 min and cooled to room temperature. The milk was
inoculated with 2% bacterial culture and inoculated milk was
incubated for fermentation (24 h) at 37
C in sealed container. After
fermentation, the fermented milk was stored at 4
C for 28 days,
and the viability of bacteria and changes in pH of fermented milk
was determined at every week during the storage period.
3. Results
3.1. Isolation and physiological properties of isolates
One hundred and four isolates from fermented foods and
beverage of different regions of Ladakh were isolated. Out of these
isolates, twenty five isolates showing typical appearance of lactic
acid bacteria on MRS medium (small pin pointed colonies) were
randomly selected and assayed for physiological properties.
All the selected isolates were Gram positive, catalase negative
and rod shaped bacteria. They were mesophilic and showed good
growth at 1.5% and 2.5% of NaCl concentration. Seven isolates
produced CO2 from glucose. All the isolates hydrolyzed arginine
and showed diversity in their ability to ferment different sugars
(data not shown).
3.2. Evaluation of probiotic properties
3.2.1. Acid and bile tolerance
When exposed to stimulated in vitro gastric juice of pH 2, the
survival of LAB isolates showed a significant (p<0.05) variability
ranging from 3.21 log CFU/ml to 6.75 log CFU/ml (Table 1). At pH 2,
each isolates demonstrated progressive reduction in survival rate
and isolate 45 showed maximum 4.8 log CFU/ml decline in viability.
Whereas, compared to pH 2, pH 3 had little or no impact on the
viability of most isolates. LAB isolates 11, 20, 40, 52, 63, 68, 72 and
75 retained same level of viability after exposure to pH 3 for 3 h.
Among all LAB, isolates 40, 52, 63 and 68 had higher tolerance than
reference strain, L. casei Shirota (P1) at pH 2.
As shown in Table 1, bile salt has a significant influence on the
viability of LAB isolate. With the exception of isolate 40, all the
isolates exhibited reduction in viability at 1% of bile salt. Though,
survival rate of isolates 20, 27, 40, 52, 63 and 84 remained unaf-
fected at 0.5% bile salt hence no significant (p<0.05) difference was
found. However, isolates 11, 20, 40 and 45 showed comparatively
better tolerance than strain P1 at 1% bile salt.
3.2.2. Lysozyme tolerance
In lysozyme test, isolates 11, 20, 22, 40, 68 and 72 exhibited an
excellent resistance to 50 mcg/ml concentration of lysozyme since
no significant (p<0.05) differences in viability was noted after 3 h
(Table 1). While all isolates consistently showed reduce tolerance
towards 100 mcg/ml of lysozyme. In particular, isolates 55 and 72 in
100 mcg/ml concentration of lysozyme demonstrated similar
tolerance to 50 mcg/ml of lysozyme, without any loss in viability.
Strain P1 revealed 2.41 log CFU/ml reduction in viability after 3 h of
incubation in 100 mcg/ml of lysozyme.
3.2.3. Cholesterol removal
Uptake of cholesterol in the medium containing cholic acid,
oxbile and sodium taurocholate was highest, intermediate and
least, respectively with majority of isolates (Fig. 1). Medium having
cholic acid, the overall cholesterol removal was observed to be
highest for isolates 55 and 72 (upto 38%) whereas isolates 70 and 76
showed least cholesterol removal (8.52% and 4.24% respectively).
Cholesterol removal for medium containing oxbile varied from
1.14% to 19% although isolate 11 demonstrated higher cholesterol
assimilation of 19.75%. Strain P1 had significantly highest choles-
terol removal in cholic acid than compared to oxbile and sodium
taurocholate.
3.2.4. Cell surface hydrophobicity and autoaggregation
Cell surface hydrophobicity of the LAB isolates tested was highly
variable (<5%e47%) depending upon bacterial cell (Table 2). In
general, isolates 20, 63 and 72 possessed a high percent hydro-
bhobicity (47%, 45% and 45%, respectively) compared to other iso-
lates investigated. Reference strain P1 had hydrophobicity of 40%.
However, isolates 38, 45 and 66 had lowest percent hydrophobicity.
All isolates investigated showed higher percentages of aggre-
gation after 24 h than at 3 h of incubation (Table 2). The most
autoaggregative isolate was isolate 52 (73%) while isolates 20, 29,
40, 63 and 72 exhibited higher autoaggregation than strain P1
(60%). On the other hand, least autoaggregation ability was shown
by isolates 11 and 12.
3.2.5. Antimicrobial activity
Isolate 40 showed an evident zone of inhibition (>5 mm) against
B. cereus, S. aureus and S. dysenteriae whereas, isolate 11 exhibited
inhibitory activity towards S. aureus and S. dysenteriae (5 mm zone
of inhibition). Isolates 12, 20, 29, 38 and 55 were found to have
antimicrobial activity against B. cereus and L. monocytogenes,
although their inhibitory extents were variable. In addition, a few
LAB isolates were observed to have weak to medium antimicrobial
against E. coli (isolates 30, 45, 52 and 75) and S. aureus (isolates 11,
40 and 68). However, none of the isolates showed any antimicrobial
activity against Pseudomonas sp.
3.2.6. Antibiotic resistance
Most of the LAB isolates were found to be susceptible to all
tested antibiotics such as penicillin G, clindamycin, co-trimoxazole,
erythromycin and ampicillin except vancomycin. Isolates 11, 20 and
40 were moderately resistant to penicillin G and isolates 20, 52, 55
and 63 toward ampicillin.
3.2.7. b-Galactosidase activity
In qualitative b-galactosidase screening, isolates 11, 20, 40, 52,
55, 63, 68, 72 and 84 possessed the presence of b-galactosidase
activity after 42 h of incubation at 30
C.
3.2.8. Exopolysaccharide production
The isolates 20, 27, 30, 32, 40, 68, 72 and 84 gave ropy colonies
and were positive for production of exopolysaccharide.
3.2.9. Haemolytic activity
All the LAB isolates studied for haemolytic activity gave negative
result.
3.3. Principal component analysis (PCA)
Analysis of Principal component analysis (PCA) revealed that
first four principal components (PCs) explaining 73.647% of total
variation, while PC1 and PC2 accounting for 37.7% and 17.4%
respectively. As shown in Table 4, pH2, lysozyme (100 mcg/ml),
 K. Angmo et al. / LWT - Food Science and Technology 66 (2016) 428e435 431

Table 1
Effect of low pH, bile salt and lysozyme on the viability of LAB isolates during 3 h incubation.

Isolate pH tolerance Bile tolerance Lysozyme tolerance

pH 7 pH 3 pH 2 Control 0.5% 1% Control 50 mcg/ml 100 mcg/ml

b b a c b a b b
11 8.52 ± 0.17 8.11 ± 0.30 5.70 ± 0.17 8.50 ± 0.15 7.49 ± 0.08 6.99 ± 0.09 8.25 ± 0.05 8.10 ± 0.03 6.47 ± 0.23a
12 8.42 ± 0.08c 6.66 ± 0.12b 4.63 ± 0.90a 8.58 ± 0.06c 7.26 ± 0.11b 5.33 ± 0.07a 8.64 ± 0.29c 7.44 ± 0.11b 5.78 ± 0.14a
17 9.12 ± 0.05c 7.95 ± 0.08b 3.73 ± 0.12a 8.96 ± 0.10c 8.60 ± 0.04b 6.10 ± 0.09a 8.86 ± 0.11c 7.46 ± 0.23b 6.12 ± 0.09a
19 9.04 ± 0.06c 7.88 ± 0.10b 5.08 ± 0.06a 8.89 ± 0.03c 7.58 ± 0.12b 6.78 ± 0.04a 8.93 ± 0.21c 6.96 ± 0.17b 5.09 ± 0.07a
20 8.65 ± 0.10b 8.60 ± 0.11b 6.03 ± 0.09a 8.40 ± 0.06b 8.11 ± 0.05b 7.38 ± 0.04a 8.41 ± 0.05b 8.15 ± 0.0.03b 6.54 ± 0.21a
22 8.50 ± 0.14c 7.94 ± 0.08b 4.02 ± 0.16a 8.47 ± 0.06c 5.29 ± 0.06b 3.81 ± 0.07a 8.04 ± 0.23b 7.60 ± 0.15b 5.01 ± 0.15a
24 8.93 ± 0.10c 8.44 ± 0.09b 3.73 ± 0.12a 8.83 ± 0.08c 7.96 ± 0.09b 6.59 ± 0.13a 8.83 ± 0.08c 7.96 ± 0.09b 6.59 ± 0.13a
27 8.88 ± 0.07c 7.77 ± 0.11b 4.97 ± 0.15a 8.09 ± 0.04b 7.86 ± 0.04b 5.68 ± 0.07a 8.69 ± 0.04c 7.16 ± 0.04b 5.68 ± 0.07a
29 8.95 ± 0.05c 7.91 ± 0.09b 4.19 ± 0.07a 8.84 ± 0.09c 6.33 ± 0.06b 4.95 ± 0.10a 8.63 ± 0.17c 7.35 ± 0.03b 6.81 ± 0.17a
30 8.70 ± 0.07c 6.27 ± 0.07b 3.93 ± 0.07a 8.69 ± 0.06c 7.99 ± 0.13b 4.66 ± 0.05a 8.96 ± 0.19c 7.78 ± 0.18b 5.85 ± 0.40a
32 8.48 ± 0.08c 7.73 ± 0.10b 4.14 ± 0.07a 8.35 ± 0.07c 6.66 ± 0.04b 5.36 ± 0.04a 8.08 ± 0.07c 6.73 ± 0.28b 4.13 ± 0.14a
38 8.03 ± 0.06c 6.86 ± 0.04b 3.04 ± 0.12a 8.11 ± 0.11c 7.65 ± 0.09b 5.79 ± 0.09a 8.23 ± 0.23c 7.57 ± 0.12b 6.88 ± 0.12a
40 8.87 ± 0.08b 8.79 ± 0.06b 6.69 ± 0.05a 7.19 ± 0.06a 6.97 ± 0.15a 6.79 ± 0.03a 8.64 ± 0.21b 8.30 ± 0.03b 7.84 ± 0.12a
44 8.88 ± 0.05c 7.91 ± 0.04b 5.43 ± 0.17a 8.74 ± 0.14c 7.06 ± 0.05b 5.73 ± 0.12a 8.80 ± 0.16c 6.80 ± 0.17b 5.42 ± 0.07a
45 8.04 ± 0.10c 6.87 ± 0.05b 3.21 ± 0.04a 8.15 ± 0.08b 6.51 ± 0.04a 6.48 ± 0.06a 8.42 ± 0.21c 7.37 ± 0.16b 4.56 ± 0.12a
52 8.62 ± 0.08b 8.45 ± 0.06b 6.54 ± 0.11a 8.20 ± 0.06b 7.97 ± 0.06b 5.84 ± 0.05a 8.35 ± 0.13b 8.21 ± 0.22b 6.86 ± 0.11a
55 8.84 ± 0.05c 7.89 ± 0.02b 6.40 ± 0.09a 8.63 ± 0.07c 7.32 ± 0.09b 5.91 ± 0.08a 8.68 ± 0.09c 7.01 ± 0.11b 6.65 ± 0.24b
63 8.52 ± 0.07b 8.29 ± 0.17b 6.63 ± 0.10a 8.12 ± 0.08b 7.80 ± 0.10b 5.61 ± 0.07a 8.13 ± 0.13c 7.45 ± 0.22b 6.23 ± 0.13a
66 9.12 ± 0.04c 8.76 ± 0.09b 4.43 ± 0.11a 8.85 ± 0.08c 7.03 ± 0.05b 6.68 ± 0.07a 8.78 ± 0.16c 7.13 ± 0.13b 5.48 ± 0.17a
68 8.75 ± 0.56b 8.65 ± 0.14b 6.75 ± 0.12a 8.77 ± 0.09c 7.37 ± 0.07b 6.05 ± 0.12a 8.47 ± 0.03b 8.24 ± 0.04b 7.50 ± 0.13a
70 7.97 ± 0.04c 6.88 ± 0.09b 4.35 ± 0.05a 8.10 ± 0.06c 6.03 ± 0.07b 4.69 ± 0.06a 8.44 ± 0.21c 7.83 ± 0.15b 6.12 ± 0.13a
72 8.95 ± 0.05b 8.82 ± 0.10b 6.53 ± 0.06a 8.71 ± 0.03c 7.69 ± 0.04b 6.06 ± 0.05a 8.40 ± 0.05b 8.27 ± 0.04ba 8.16 ± 0.04a
75 8.36 ± 1.07b 8.10 ± 0.10b 4.46 ± 0.07a 8.45 ± 0.05c 7.37 ± 0.06b 4.90 ± 0.07a 8.81 ± 0.04c 8.24 ± 0.07b 5.12 ± 0.10a
76 8.89 ± 0.04c 8.29 ± 0.06b 4.88 ± 0.06a 8.96 ± 0.09c 8.08 ± 0.12b 4.98 ± 0.13a 8.77 ± 0.11c 6.85 ± 0.16b 5.26 ± 0.16a
84 8.75 ± 0.09c 7.81 ± 0.15b 4.94 ± 0.08a 8.08 ± 0.08b 7.70 ± 0.10b 5.77 ± 0.05a 8.33 ± 0.12c 7.49 ± 0.17b 5.33 ± 0.10a
P1 8.12 ± 0.24c 7.94 ± 0.21b 5.69 ± 0.45a 8.32 ± 0.09c 7.81 ± 0.16b 6.12 ± 0.16a 8.75 ± 0.09c 7.65 ± 0.13b 6.34 ± 0.19a

Values represented as mean ± SD; for each column, different subscripts lowercase letters indicate significantly different at p<0.05, as measured by 2-sided Tukey's HSD;
Reference strain P1 is Lactobacillus casei Shirota.

Fig. 1. Cholesterol removal (%) by LAB isolates.

cholic acid, n-hexadecane, S. aures, S. dysenteriae, penicillin G and b- respect to reference strain. Therefore isolate 84 was selected for the
galactosidase were correlated to PC1 and PC2, suggesting that these preparation of milk based fermented probiotic drink.
variables are contributing for the selection of the most relevant
technological isolates.
3.4. Identification by 16S rDNA sequencing
Fig. 2(a) represents the distribution plots of variables on the
plane of first two principal components. Projection of LAB isolates
All ten potential probiotic isolates (based on PCA) were identi-
in two-dimensional space of the PC1 and PC2 loading factors could
fied by 16S rRNA gene sequence and PCR amplification of the 16S
be differentiated three main clusters (Fig. 2(b). It can be inferred
rRNA gene resulted in amplicon of 1300e1500 bp. Alignments were
that LAB isolates present in quadrant I and IV were significant as
performed using the BLAST and lactobacilli identified were desig-
they showed correlation with respect to variables. Isolates 11 and
nated as PLA (Probiotic lactic acid bacteria) followed by the
40 of quadrant I was related with antimicrobial activity against S.
numbers 22 to 37.
aures and S. dysenteriae and resistant toward penicillin G. On the
In an effort to identify LAB isolates at the species level, molec-
other hand, isolates 29, 84, 72, 20, 63, 55 and 52 were the most
ular phylogeny analysis was conducted and phylogenetic tree was
potential candidate as they showed highest probiotic character.
constructed based on the 16S rDNA sequences from evolutionary
While isolate 84 demonstrated higher resemblances with strain P1
distances by the neighbor-joining method (Fig. 3). Analysis of the
explaining the similar trend in probiotic activities of isolate 84 with
sequences depicted that the 50% of isolates clustered with 16S rRNA
432 K. Angmo et al. / LWT - Food Science and Technology 66 (2016) 428e435

Table 2 sequences of Lactobacillus brevis while 40% isolates clustered with

Hydrophobicity and autoaggregation ability of LAB isolates. sequences of Lactobacillus plantarum, and only one isolate clustered
Isolate Hydrophobicity % Autoaggregation % with Lactobacillus fermentum. The GenBank accession numbers for
n-Hexadecane 3h 24 h
the 16S rRNA gene sequence of isolates are given in Table 3.
bcde a
11 12.02 ± 1.70 1.84 ± 0.10 5.04 ± 0.10a
12 7.44 ± 0.67ab 1.76 ± 0.11a 8.24 ± 0.22b 3.5. Production of fermented milk
17 16.52 ± 1.21efg 11.54 ± 0.10def 25.33 ± 0.10e
19 35.93 ± 2.30i 17.52 ± 0.10jkl 33.96 ± 0.10gh Isolate 84 (Lactobacillus plantarum KJ722784) was used for the
20 46.59 ± 2.12kl 15.24 ± 0.11ghij 63.25 ± 0.11no
preparation of milk based fermented probiotic drink. The isolate 84
22 11.66 ± 0.67bcde 5.55 ± 0.10b 28.01 ± 0.10f
24 8.47 ± 0.56ab 11.80 ± 0.44def 29.94 ± 0.11f and reference strain P1 grew well on sterilized milk and their count
27 9.70 ± 0.56abc 13.75 ± 0.10fghi 35.16 ± 0.10hi reached 8.68 log CFU/ml and 8.83 log CFU/ml, respectively after
29 20.28 ± 0.45g 16.15 ± 0.13ijk 62.18 ± 0.13n 24 h of fermentation at 30
C (Fig. 4). During the fermentation
30 15.74 ± 0.87efg 13.35 ± 0.09efghi 19.88 ± 0.09d process gradual decrease in pH was observed and after fermenta-
32 7.59 ± 0.56ab 6.40 ± 0.09bc 28.98 ± 0.09f
tion pH of sample inoculated with isolate 84 and strain P1 was 4.62
38 5.81 ± 0.67a 8.89 ± 0.09cd 33.06 ± 0.09g
40 26.96 ± 0.88h 18.42 ± 0.23kl 64.71 ± 0.23


and 4.46, respectively. However, a change in the viable count of
44 11.26 ± 0.56abcde 10.55 ± 0.63de 36.18 ± 0.31i isolate during 28 day of storage at 4
C was found. Isolate 84 and
45 5.69 ± 0.45a 26.78 ± 0.79m 40.23 ± 0.79j strain P1 revealed 0.98 log CFU/ml and 0.93 log CFU/ml reduction in
52 38.90 ± 0.89i 24.56 ± 0.09m 73.09 ± 0.18p
viability during the 28 d of storage. pH of fermented milk continued
55 30.47 ± 4.33i 5.27 ± 0.25b 24.78 ± 0.37e
63 44.56 ± 2.33jk 18.42 ± 0.23kl 64.78 ± 0.11

to decrease and reached 4.3 (isolate 84) and 4.24 (P1) on 28 d of
66 5.96 ± 0.56a 1.63 ± 0.11a 16.73 ± 0.21c
68 11.63 ± 1.10bcde 5.68 ± 0.14b 15.71 ± 0.29c
70 15.34 ± 0.86defg 12.82 ± 2.22efgh 40.06 ± 0.11j
72 44.74 ± 2.93l 12.46 ± 0.13efg 61.91 ± 1.27mn
75 10.43 ± 1.10abcd 19.88 ± 1.01l 42.69 ± 1.01k
76 13.66 ± 0.65cde 24.01 ± 0.10m 41.36 ± 1.07l


84 35.74 ± 1.43i 15.45 ± 0.18hij 58.37 ± 0.09
P1 40.16 ± 1.56ij 15.04 ± 1.58ghij 60.00 ± 2.00lm

Values represented as mean ± SD of triplicate analyses; for each column, different

subscripts lowercase letters indicate significantly different at p<0.001 as measured
by 2-sided Tukey's HSD between different strains.

Table 3
Identified LAB isolates by 16S rRNA gene sequencing and their GenBank accession
number.

Isolate Species NCBI accession No

11 Lactobacillus brevis KJ722770

20 Lactobacillus brevis KJ722771
29 Lactobacillus brevis KJ722772
68 Lactobacillus brevis KJ722773
40 Lactobacillus plantarum KJ722780
63 Lactobacillus brevis KJ722781
52 Lactobacillus plantarum KJ722782
55 Lactobacillus plantarum KJ722783
84 Lactobacillus plantarum KJ722784
72 Lactobacillus fermentum KJ722785

Table 4
Correlation of variables to the factors of the PCA analysis based on factor loadings.

Variables PC 1 PC 2 PC 3 PC 4

pH2 0.625 0.155 0.644 0.137

Bile 1% 0.386 0.212 0.736 0.259
Lysozyme 100 mcg/ml 0.698 0.132 0.043 0.081
Oxbile 0.586 0.336 0.082 0.522
Cholic Acid 0.712 0.091 0.063 0.165
Hexadecane 0.715 0.530 0.215 0.067
Autoaggregation 24 h 0.554 0.513 0.337 0.079
B. cereus 0.595 0.134 0.161 0.586
S. aures 0.560 0.738 0.186 0.072
S. dysenteriae 0.483 0.739 0.007 0.029
Pseudomonas sp. 0.000 0.000 0.000 0.000
Penicillin G 0.634 0.527 0.202 0.023
Co-Trimoxazole 0.000 0.000 0.000 0.000
Vancomycin 0.485 0.046 0.193 0.611
Ampicillin 0.540 0.531 0.178 0.374
b-Galactosidase 0.868 0.141 0.142 0.056
Fig. 2. (a). Projection of the variables on the plane formed by PC1 and PC2 analyzed by
Values in bold within the same factor indicate the variable with the largest principal component analysis. (b). Projection of the LAB isolates and reference strain L.
correlation. casei Shirota (P1) in the space of PC1 and PC2.
 K. Angmo et al. / LWT - Food Science and Technology 66 (2016) 428e435
Fig. 3. Neighbor joining phylogenetic tree based on 16S rDNA sequences. Numbers in parentheses are accession numbers of published sequences. Filled circles are the reference
strains from NCBI and empty circle is the out group used for tree construction.
Fig. 4. pH and viable count of Lactobacillus plantarum (KJ722784) and reference strain
Lactobacillus casei Shirota during milk fermentation (24 h at 37
C) and cold storage (28
days at 4
C). Error bars are standard deviations with respect to the mean values of
triplicate analyses.
storage.
4. Discussion
In this paper, significant effort has been made to select lactic
acid bacteria originating from the indigenous Ladakhi fermented
products on the basis of the most important technological criteria
in order to obtained probiotic lactic acid bacteria. Indeed, it is
worthwhile to isolate and identify probiotic strain from fermented
products as they are safe and cause various health benefits.
Microorganisms to be applied as probiotic must overcome the
inhospitable condition of human gastrointestinal tract (GIT) and
subsequently colonize the intestinal tract. In order to reach active
and viable enough through GIT, they should be resistant to acid,
lysozyme and bile. Acid tolerance of bacteria is important not only
for withstanding gastric stresses, but it also enables the strain to
survive for longer periods in high acid carrier foods, such as yogurt,
433
without reduction in their number (Wang, Lin, Ng, & Shyu, 2010).
The in vitro low pH tolerance study revealed that several isolates at
pH 3 showed equal percent of viability as compared to pH 7 (con-
trol). However, incubation at pH 2 resulted in significant decrease
in the survival rate of all LAB isolates. These results are in agree-
ment with the finding of Guo et al. (2010). They reported that the
viable counts of all lactic acid bacteria were significantly affected by
the low acidity, especially at pH 2. Corcoran, Stanton, Fitzgerald,
and Ross (2005) reported that resistance to low pH by Lactoba-
cillus is due to the presence of F0F1-ATPase activity. Bile salts are
toxic for living cells and bile salt tolerance is considered one of the
essential properties required for lactic acid bacteria to survive in
the small intestine (Succi et al., 2005). In this study, most of the
isolates showed resistance to 0.5% bile concentration. But at 1% bile
salt only isolate 40 demonstrated full tolerance. This decrease in
viability caused by bile salts is implicated with its effect on cell
membrane of the microorganisms (Papamanoli, Tzanetakis,
Litopoulou-Tzanetaki, & Kotzekidou, 2003). The variation in bile
sensitivity observed in this study is consistent with many previous
reports (Jacobsen et al. 1999; Maldonado, de-Ruiz, Otero, Sesma, &
Nader-Macias, 2012; Mishra & Prasad, 2005).
Another important property of probiotic is its hypocholester-
olemic effect on host, however it is not essential but one of the
desired quality/character of the probiotic strain. We found that
overall cholesterol removal was highest in cholic acid and least in
sodium taurocholate. The decrease in cholesterol suggests that a
significant amount of cholesterol in the medium precipitated after
addition of deconjugated bile acids (cholic acid). No or very little
precipitation of cholesterol was observed in the presence of con-
jugated bile acids sodium taurocholate. This coprecipitation effect
of cholesterol and deconjugated bile acids vis-a-vis reduction in
cholesterol level has been reported in Lactobacilli and Bifidobacte-
rium bifidum (Klaver & van der Meer, 1993) and L. fermentum KC5b
(Pereira, McCartney, & Gibson, 2003).
LAB exerts their health promoting effects by several mecha-
nisms. The property of adherence to intestinal epithelial cell is one
of the mechanisms which involved different type of interaction.
Several workers have reported that hydrophobicity (Botes, Loos,
van Reenen, & Dicks, 2008; Duary, Rajput, Batish, & Grover, 2011)
and aggregation ability (Collado et al., 2008; Jankovic, Frece, Abram,
& Gobin, 2012) are correlated to cell adherence properties. With
this regard, LAB isolates were examined for degree of hydropho-
bicity and autoaggregation ability. In our study, LAB isolates
434 K. Angmo et al. / LWT - Food Science and Technology 66 (2016) 428e435
showed wide differences in their hydrophobicity and isolates viz.
20, 63 and 72 demonstrated higher hydrophobicity toward n-
hexadecane. This suggest that the complexity of the cell surface
mosaic resulting from hydrophobic and hydrophilic appendages
and other macromolecule components might give rise to differ-
ential hydrophobicity toward hydrocarbons. Mishra and Prasad
(2005) reported that the strain NCDC17 had maximum hydropho-
bicity towards hexadecane while NCDC19 possesses maximum
hydrophobicity for octane. In another study of assessing the
adhesion of faecal isolates, L. plantarum Lp91 showed maximum
percentage hydrophobicity for n-hexadecane and toluene which
was followed by L. plantarum Lp9 (Duary et al., 2011). As far as
aggregartion is concerned, all the isolates in the present study
exhibited some degree of autoaggregation. Although, autoag-
gregation ability was enhanced with time and was higher at 24 h of
incubation than at 3 h. It can be explained that aggregation pro-
moting factors increase self-aggregation with incubation (Goh &
Klaenhammer, 2010). Similar finding was also reported by Dias,
Duarte, and Schwan (2013), that the autoaggregation ability of L.
plantarum strains improved with the increase in time of incubation.
Other important characteristics require to be screened pro-
gressively for selection of probiotic is the absence of undesirable
properties (virulence factors and transmissible antibiotic re-
sistances) and safety aspect of probiotic isolates. In this regard, the
antibiotic resistance, antimicrobial acitivity and haemolytic activity
of the isolates were assessed. Furthermore, b-galactosidase activity
and exopolysaccharide production were also studied.
Multivariate data analysis was used for the selection of most
promising probiotic potential isolates with respect to technological
activities assayed. The most potential probiotic isolate was isolates
20, 52, 55 and 72 as they exhibited survival at pH 2, lysozyme
(100 mcg/ml), cholesterol removal (cholic acid), antagonistic aci-
tivity against B. cereus and b-galactosidase activity while isolate 63
showed autoaggregation ability (24 h), hydrophobicity and resis-
tant toward ampicillin. However, isolate 84 provide an interesting
perspective as it demonstrated same trend as reference strain and
selected for the production of milk based probiotic product.
The last parameter of this study was development of milk based
probiotic product presupposes the application of probiotic lactic
acid bacteria which not only grows in milk but also show survival in
acidic fermented milk. Meanwhile, the extent of health benefit on
consumption of fermented milk containing probiotic depends upon
the high viability of the probiotic microorganisms. In our study, L.
plantarum KJ722784 was selected for the production of milk based
probiotic product after its extensive characterization. The viable
cell counts of L. plantarum KJ722784 showed decrease over the
storage period, but its viable cell numbers in fermented milk
remained above the legal requirement of 6 log CFU/ml to exhibit
health benefit. Similar result of fermented milk containing pro-
biotic was reported by Yerlikaya, Ender, Torunoglu, and Akbulut
(2013).
5. Conclusions
The emerging demand for the products having health benefits
beyond nutrition has provided ample opportunity to explore rela-
tively unexplored foods and beverages for isolation of lactic acid
bacteria for their potential role in probiotic research. Traditional
fermented foods and beverages of Ladakh region of India have been
explored for isolation of lactic acid bacteria to be used as probiotic.
A promising isolate, L. plantarum KJ722784 having good probiotic
traits has been identified as favorable candidate for the production
of probiotic products. These isolates were originated from fer-
mented products and considered safe for consumption, thus
traditional foods can serve as potential source of lactic acid bacteria
to be used as probiotic. Additionally, these foods and beverages can
be proposed as delivery vehicle for probiotics, leading to increased
demand of these traditional foods which indirectly lead to
improvement of rural economy.
Acknowledgment
The present study was supported by Department of Biotech-
nology (DBT), Ministry of Science and Technology, Government of
India (Grant No. DBT/JRF/14/FIN/503) and University Grants Com-
mission, New Delhi (Grant No. F. 15-13/12 (SA-II). The authors are
thankful to Dr. R. B. Srivastava, Director, DIHAR, DRDO, Leh-Ladakh,
and Dr. Tsering Stobdan, Horticulture Division, DIHAR, DRDO, Leh-
Ladakh for giving permission and providing facilities to do part of
research work in their Laboratory.
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