LWT - Food Science and Technology 176 (2023) 114553

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

Probiotics from the bovine raw milk of Lahaul valley showed cis-9, trans-11
conjugated linoleic acid isomer and antioxidant activity with food
formulation ability
Neha Baliyan a, b, Antim Kumar Maurya b, c, Anil Kumar a, b, Vijai Kant Agnihotri b, c,
Rakshak Kumar a, b, *
a
Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, 176 061, India
b
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India
c
Chemical Technology Division, CSIR-Institute of Himalayan Bioresource and Technology, Palampur, 176061, India

A R T I C L E I N F O A B S T R A C T

Keywords: Dairy foods, especially of bovine origin, are the most common carriers of probiotics and conjugated linoleic acid
Probiotic attributes (CLA)-producing microorganisms. The present study isolated probiotic strains from raw cow milk of the Lahaul
Conjugated linoleic acid (CLA) valley capable of producing cis-9, trans-11 CLA isomer, besides exhibiting antioxidant properties with food
Linoleic acid
formulation abilities. After preliminary probiotic screening, 34 strains were obtained, with 21 qualifying all the
Safety parameter
probiotic attributes and passing safety analysis. The seven most statistically promising probiotics were further
Curdled milk
explored for CLA production (16.87–28.01 μg/mL), and showed cis-9, trans-11 CLA isomer production as
confirmed by GC-MS. To establish genomic evidence for safety analysis, whole genome sequencing was con­
ducted for Enterococcus faecalis LJM:05, resulting in the deduction of antibiotic-resistance and virulence genes
(lower identity scores). Based on the superior probiotic attributes, antioxidant, and CLA-producing properties,
the strain Latilactobacillus curvatus LGM:16 was used to develop the curdled milk, with a legal, viable count (7.55
log10 CFU/mL) under pH ~4.15 for 28 days of storage periods (4 ◦ C). Scanning electron microscopic exami­
nation revealed microstructural changes after fermentation in the curdled milk compared to the control. The
findings highlighted the role of indigenous probiotic strains with superior probiotic features, antioxidant activity,
and CLA-producing ability in functional food formulation.

1. Introduction
Probiotics are gaining widespread interest regarding gut microflora,
immune systems, metabolic disorders, and cardiovascular diseases
(Ayivi et al., 2020). The specific health effects might vary between
different strains of the same species, which might depend upon their
prebiotic combination and geographical area of origin (Bhat & Bajaj,
2020; Kumari et al., 2020a). The available commercial probiotics are
mostly of Western or European origin, for example, Lacticaseibacillus
rhamnosus (GR1) and Limosilactobacillus reuteri (RC14), commonly re­
ported curing diseases specifically related to women’s health. i.e., uri­
nary tract infection, bacterial vaginosis, Group B Streptococcus
colonization, vulvovaginal candidiasis infection, etc (Sulin, 2017).
Meanwhile, species like Bacillus subtilis, B. licheniformis, Enterococcus
faecium, Lacticaseibacillus casei, etc., from the European region (Zielińska
& Kolożyn-Krajewska, 2018) are claimed to cause a reduction in
cholesterol level. Commercial strains were also used to develop
dairy-fermented foods, such as Yakult, Vifit, Nestlé’s LC1, fruit juices,
drinks, and soup, and animal nutritional products (Zielińska & Kolo­
żyn-Krajewska, 2018).
The probiotics from the Western region showed poor adaptation and
colonization with the Indian subjects (Bhat & Bajaj, 2020) and are
ineffective in such parts. Knowledge of the probiotic diversity of the
Western Himalayas’ traditional foods is scarce. Despite the stressful
environment of the high altitude, people in these areas are healthier and
live longer due to their diet focusing primarily on traditional fermented
foods (Kanwar et al., 2007). Therefore, there is tremendous scope for
isolating indigenous probiotic strains from traditional foods. Few

* Corresponding author. Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Post Box No. 06, Palampur, 176 061, Himachal Pradesh,
India.
E-mail address: rakshak@ihbt.res.in (R. Kumar).

https://doi.org/10.1016/j.lwt.2023.114553
Received 17 July 2022; Received in revised form 31 January 2023; Accepted 1 February 2023
Available online 2 February 2023
0023-6438/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
N. Baliyan et al.
traditional foods like channg, starter phab, sura (Thakur et al., 2015),
cheese (Kumari et al., 2020), chulli (Sharma, 2017), and lugri (Baliyan
et al., 2021) have been reported for probiotics.
Presumably, no documentation of lactic acid bacteria (LAB) from the
raw cow milk of Lahaul valley as a potential probiotic has been reported.
LAB has the efficiency in converting linoleic acid (LA) to conjugated
linoleic acid (CLA) by isomerization, exhibiting few health benefits like
anti-obesity, antidiabetic, anticancer, antioxidant, and enhancing the
immune system (Hennessy et al., 2016). CLA constitutes a group of 18
carbon-conjugated dienoic acids, a collective term used to define the
positional (C18:2, cis-9, and cis 12) and geometric (cis-9: trans11 and
cis-10: trans12) isomers, whereby the cis-9: trans-11 CLA isomer alone
covers 90% of the total isomers in natural lipids (Vieira et al., 2017).
Moreover, researchers reported that several CLA isomers might have a
mixed influence on the antioxidant characteristics of trans-10, cis-12
CLA isomers and the pro-oxidant qualities of cis-9, trans-11 CLA isomers
(Özer et al., 2016). The antioxidant activities against free radicals
complement the beneficial health properties of LAB.
Previous studies have reported CLA-producing probiotics from
various sources like human breast milk (Dahiya & Puniya, 2018b), khadi
(Dubey et al., 2012), and kimchi (Vieira et al., 2017). Nonetheless, milk
and dairy products are the richest sources of fat and n-3 fatty acids like
CLA. Although CLA isomers are naturally present in ruminant dairy and
meat products, their consumption in the human diet is below the sug­
gested levels (Wang et al., 2007). It is recommended that humans
consume 1.0–3.0 g of CLA per day to gain health advantages (Vieira
et al., 2017). Accordingly, preparing food formulations with efficient
CLA-producing probiotic bacteria could impart nutritional and thera­
peutic values to the food products alongside acceptable texture, odor,
and taste impartment (Wang et al., 2022). In this regard, raw milk from
the pristine and under-explored North-Western Himalayan region could
be an authentic and effective source of CLA-producing probiotic strains.
The current study investigates the indigenous LAB from raw cow milk of
the Lahaul valley for their probiotic attributes and safety evaluation to
screen promising CLA-producing and antioxidant probiotics with the
potential for functional food production.
2. Materials and methods
2.1. Sample collection and proximate analysis
The raw milk samples of the cow were collected from Jundha
(32.64◦ N–76.84◦ E), Ghosal (32.54◦ N–76.96◦ E), and Sissu (32′ 29◦ N-
77′ 7◦ E) villages of Lahaul valley of the North-western Himalaya in
sterile vials under hygienic conditions and stored at 4 ◦ C for further
experimentation. To measure the electric conductivity (E.C.) and pH of
the samples, a digital pH meter (Eutech, India) was used. The moisture
content of the samples was analysed using a moisture analyser (MOC63u
Shimadzu, Japan). Five grams of the sample was weighed and heated to
between 550 and 600 ◦ C in an electric muffle furnace for at least 5 h to
quantify the ash content (Gundidza et al., 2011). The crude protein
content in the raw milk samples was measured using the Kjeldahl ni­
trogen method (Lynch et al., 2002). The crude fat content was measured
using the Soxhlet extraction apparatus (Dutta et al., 2014).
2.2. Isolation and physicochemical characterization of bacterial strains
The probiotic isolation from raw milk samples was carried out using
the spread plate technique described by Yadav et al. (2016). The diluted
sample (100 μl) was spread on the de-Mann Rogosa Sharpe (MRS) agar
plate (Hi-media Lab., India) under sterile conditions and incubated at
37 ◦ C for 24–48 h. The unique morphotype isolates were subcultured on
the MRS media to obtain pure colonies. Each isolate’s glycerol stock was
maintained at − 80 ◦ C for further use.
The selected discrete morphotypes strains were qualitatively
assessed at acidic conditions (2–4 pH) and different bile salt (300–3000
2
LWT 176 (2023) 114553
mg/dL) concentrations. The positively grown bacterial isolates were
further checked for physicochemical parameters on MRS media at
different pH (4–8), temperature (4–55 ◦ C), NaCl concentration
(2000–6000 mg/dL), and phenol concentration (400 and 600 mg/dL),
respectively (Somashekaraiah et al., 2019). The strain Lactiplantibacillus
plantarum (MTCC 1407) was used as a reference control in all the tests.
2.3. Molecular characterization of the bacterial strains and nucleotide
sequence GenBank accession numbers
Using the 16S rRNA gene sequencing, the selected bacterial strains
were identified by PCR amplification with the universal primers 27F (5′ -
AGAGTTTGATCCTGGCTCAG-3′ ) and 1492R (5′ -TACGGTACCTTGT­
TACGACTT-3′ ). The nearest neighbor of the strains was determined
using the Basic Local Alignment Search Tool (BLAST) analysis. The
phylogenetic tree was constructed using the software Molecular Evolu­
tionary Genetics Analysis (MEGA version X), using the inbuilt Clustal W
algorithm. The 16S rRNA gene sequences of the isolated probiotic strains
were submitted to the NCBI GenBank, and accession numbers are given
in Table 1.
2.4. Evaluation of thestrains for probiotic attributes
2.4.1. Acid and bile tolerance
The strains were grown overnight for the acid tolerance assay,
washed thrice with buffer (phosphate buffer saline, PBS, pH 7.2), and
resuspended in the same PBS buffer. The simulated gastric juice was
prepared with 3000 mg/dL pepsin (Sigma-Aldrich) in 500 mg/dL NaCl
with pH 2.0 and 3.0, respectively. The prepared cell suspensions (1 mL)
were mixed with simulated gastric juice (5 mL) and incubated at 37 ◦ C.
The aliquots of 100 μl were taken every 0, 1, and 3h, respectively, and
spread plated on MRS agar to determine the viability of the bacterial
isolates (Yadav et al., 2016).
The bile tolerance of the tested isolates was performed using (Lee
et al., 2016) method. The tolerance was determined by the percentage of
the culture viability after incubation in MRS broth with bile salt and the
control and was calculated using the following equation Eq. (1);
Bile resistance (%) = (A(t)620nm) / (A(0)620nm) × 100 (1)
Where A(t) culture’s grown in MRS broth with bile salt and A(0) control
contained culture’s in MRS broth without bile salt.
2.5. Adhesion properties of the probiotic strains
2.5.1. Cell auto-aggregation and cell-surface hydrophobicity
The cell auto-aggregation assay was performed using a previously
known method (Kumari et al., 2020).
The following calculation expressed the results of auto-aggregation
Eq. (2);
Cell auto-aggregation (%) = [A0 - At/A0] × 100 (2)
Where, A0 denoted for the initial culture optical density (OD) (time 0),
At is the OD (time 1h, 2h, 24h) of the tested samples. The cell-surface
hydrophobicity of the strains was determined using the method by
(Mallappa et al., 2019) The washed cultures were adjusted 0.7 ± 0.1 OD
and mixed with chloroform, and toluene (A0, at time 0) followed by
incubation at 37 ◦ C. The OD of the cultures was measured at 600 nm
after 1h, 2h, and 24h (At).
The results were expressed by using the following Eq. (3);
Cell surface hydrophobicity (%) = [A0 - At/A0] × 100 (3)
N. Baliyan et al. LWT 176 (2023) 114553

Table 1 2.6.2. Biofilm assay, haemolysis activity, and DNase test

Identification of probiotic strains isolated from raw cow milk of Lahaul valley of The biofilm and hemolysis assays were performed for the safety
North Western Himalayan. evaluation of the bacterial isolates using the method described (Yadav
Closest Match (type Sequenced nta % simb % Accession et al., 2016).
strain) Strains Comptc no. The bacterial cultures were spotted on the deoxyribonuclease
Leuconostoc (DNase) agar medium at 37 ◦ C for two days to check the DNase enzyme
Leuconostoc lactis JCM HMM: 02 1418 100.00 96.3 OM949938 production (Somashekaraiah et al., 2019). The appearance of a pinkish
6123(T) HMM: 04 1418 100.00 96.3 OM949940 zone around the colonies was considered positive for DNase activity.
Leuconostoc LJM: 01 1305 99.85 89.3 OM956177
pseudomesenteroides LJM: 02 1315 99.85 89.3 OM956178
NRIC 1777(T) LJM: 08 1315 99.85 89.3 OM956182 2.7. Genome assembly, annotation, and analysis for safety-related genes
Weissella
Weissella cibaria KACC HMM: 03 1444 99.86 96.3 OM949939 The genome of one of the representative strain, Enterococcus faecalis
11862(T) HMM: 12 1424 99.93 95.0 OM949942 (LJM:05), was sequenced by the Illumina Miseq platform to check for
HMM: 15 1443 99.93 96.3 OM949943
Levilactobacillus
antibiotic resistance and virulence factors related genes. The raw reads
Levilactobacillus brevis HMM: 11 1331 100.00 89.4 OM949941 were assembled by Unicycler assembler v0.5.0 (Wick et al., 2017). The
ATCC 14869(T) LGM: 08 1295 100.00 87.0 OM956159 genome was annotated using Rapid Annotations using the Subsystems
LGM: 13 1409 100.00 94.2 OM956164 Technology (RAST) server. The genes predicted in the genome of the
LGM: 21 1421 100.00 95.5 OM956169
strain LJM:05 were searched for antibiotic resistance and virulence
LGM: 22 1421 100.00 95.5 OM956170
Latilactobacillus factors using the comprehensive antibiotic resistance database (CARD)
Latilactobacillus LGM: 04 1420 100.00 95.1 OM956157 and virulence factors database (VFDB) (Liu et al., 2019), respectively,
curvatus JCM LGM: 09 1299 100.00 87.1 OM956160 using default parameters. The genome of the strain Enterococcus faecalis
1096(T) LGM: 11 1435 100.00 96.2 OM956162 (LJM:05) was submitted to the NCBI GenBank with accession number
LGM: 12 1437 100.00 96.3 OM956163
LGM: 16 1429 100.00 95.7 OM956166
JAODJD000000000.
LGM: 19 1431 99.93 96.0 OM956168
LGM: 25 1437 100.00 96.2 OM956171 2.8. Functional attributes
Latilactobacillus sakei LGM: 14 1441 99.93 96.3 OM956165
subsp. Sakei JCM LGM: 07 1376 100.00 92.0 OM956158
2.8.1. Antimicrobial activity
1157(T) LGM: 01 1374 100.00 91.8 OM956155
LGM: 17 1428 100.00 95.5 OM956167 The probiotic strains were tested against gram-positive [Micrococcus
Latiplantibacillus luteus (MTCC 2470), Staphylococcus aureus (MTCC 96), Bacillus subtilis
Lactiplantibacillus LGM: 02 1328 99.77 89.3 OM956156 (MTCC 121)] and gram-negative [Klebsiella pneumoniae (MTCC 109),
paraplantarum LGM: 10 1329 100.00 89.3 OM956161 Pseudomonas aeruginosa (MTCC 2453), Escherichia coli (MTCC 43)]
DSM10667(T)
Lactiplantibacillus LJM: 06 1401 100.00 94.1 OM956180
opportunistic pathogen type strains. The results were expressed by
pentosus DSM LJM: 07 1302 99.92 87.4 OM956181 measuring the zone of inhibition’s diameter after incubation for 24 h at
20314(T) 37 ◦ C.
Loigolactobacillus
Loigolactobacillus LGM: 24 1437 99.93 96.3 OM956286
2.8.2. Exopolysaccharide production (EPS)
coryniformis subsp. LGM: 23 1422 100.00 95.3 OM956285
torquens KCTC The overnight grown bacterial isolates were spotted on the modified
3535(T) MRS plates containing sucrose and lactose (5000 and 10000 mg/dL
Enterococcus concentrations, respectively) as the carbon sources and incubated for 3
Enterococcus durans LJM: 09 1427 99.86 96.2 OM956183 day at 37 ◦ C to determine the EPS production (Kumari et al., 2020).
NBRC 100479(T) LJM: 10 1421 99.79 95.9 OM956184
LJM: 12 1424 99.86 96.0 OM956217
Enterococcus faecalis LJM: 05 1402 100.00 94.2 OM956179 2.9. Screening of probiotic strains for conjugated linoleic acid (CLA)
ATCC 19433(T) production
nta: Length of 16S rRNA gene sequence; % simb: Percentage sequence similarity
of isolated strains with type strains of validly published prokaryotic names The CLA production ability of probiotic bacteria was measured ac­
(available online http://eztaxon-e.ezbiocloud.net/); %Comptc: Completeness- cording to a method given by Ribeiro et al. (2018). The bacterial isolates
Percentage of the degree of coverage of a query 16S rRNA gene sequence with were inoculated in MRS broth containing free linoleic acid (L.A.) (0.5
respect to the full-length, complete 16S rRNA gene sequence. mg/mL) with 2000 mg/dL Tween 80 (Himedia) and incubated at 37 ◦ C
for 48 h. The culture was then centrifuged at 18,000×g for 3 min, and
2.6. Safety assessment of bacterial isolates cell-free supernatant was collected. Then, isopropanol (2 mL) was added
to the collected supernatant (1 mL), and the solution was mixed prop­
2.6.1. Antibiotic susceptibility assay erly. This mixture was incubated for 3 min and vigorously vortexed
The growth ability of probiotic strains against selected antibiotics before adding 1.5 mL hexane to the solution for fatty acid extraction.
was measured by disc diffusion assay (Kumari et al., 2020). The absor­ The presence of CLA in the test culture supernatant was measured at
bance of the strains was adjusted at 0.5 McFarland, and 100 μl cultures 233 nm using a UV-transparent spectrophotometer (Synergy LX multi­
were spread plate on the MRS agar. The activity of the strains was tested mode reader, BioTek) by taking 230 μl of fat-soluble hexane layer.
against Penicillin G (10 units), Ciprofloxacin (5 mcg), Ampicillin (10 The cis-9, trans-11 conjugated linoleic acid isomer (Sigma-Aldrich)
mcg), Rifampicin (5 mcg), Erythromycin (15 mcg), Gentamycin (10 was used for the standard curve preparation. The potential isolates were
mcg), Chloramphenicol (30 mcg), Streptomycin (10 mcg), Azithromycin analysed with Gas Chromatography-Mass Spectrometry (GC-MS) to
(15 mcg), Kanamycin (30 mcg), Tetracycline (30 mcg), and Vancomycin identify the produced CLA isomer. For further analysis, the extracted
(30 mcg) antibiotics. Based on the zone of inhibition, the results were lipid layer from the bacterial supernatant was methylated with
expressed as sensitive (S), moderately sensitive (M.S.), and resistant (R) NaOH–BF3 in methanol to convert the fatty acids into fatty acid methyl
(according to the guideline given by the Clinical and Laboratory Stan­ ester (FAME), as given in Ribeiro et al. (2018). The final sample (FAME)
dards Institute (CLSI, 2016) after the incubation at 37 ◦ C for 24 h. was stored at − 20 ◦ C to confirm CLA content (isomers) using GC-MS.
Separations of FAMEs metabolites from samples were carried out
using a model instrument Shimadzu Q.P. 2010 attached with a non-

3
N. Baliyan et al.
polar capillary column ZB-5MS, dimension: 30 m, ID: 0.25 mm, and film
thickness 0.25 μm. The G.C. oven temperature was maintained at 70 ◦ C
for 4 min, then gradually raised to 280 ◦ C at 4 ◦ C/min and held for 5
min. The run time was 45 min, and Helium (He) was applied as carrier
gas. Other parameters were programmed as follows: injector tempera­
ture, interface temperature, acquisition mass range, and ionization en­
ergy (270 ◦ C, 280 ◦ C, 800–50 amu, and 70 eV), respectively.
2.10. Preparation of curdled milk
The potential probiotic strains were further accessed for the pro­
duction of curdled milk. The experiment was performed with the
method described by Baliyan et al. (2021). The bacterial culture (2000
mg/dL) was inoculated in the sterile skim milk powder (4000 mg/dL)
with 0.5 mg/mL linoleic acid and incubated at 37 ◦ C for 24 h. Then, the
sample was stored at 4 ◦ C for four weeks to check the viability of the
strain, pH, and enumeration of pathogenic microorganisms. Addition­
ally, scanning electron microscopy (SEM) was done on the curdled milk
using the method (Cui et al., 2021).
2.11. Antioxidant activity
The antioxidant activity of selected seven probiotic strains was
assessed using 2,2-diphenyl-2- picrylhydrazyl hydrate (DPPH) and 2,2
-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. More­
over, ferric thiocyanate and reducing power assays were also performed
using the previously known method (Mukhia et al., 2021).
2.12. Statistical analysis
The results were analysed using the IBM SPSS Statistics (version 26)
software. The principal component analysis (PCA) plot was built with
the quantitative results of probiotic attributes viz., acid, bile, cell auto-
aggregation, and cell surface hydrophobicity of bacterial isolates using
XLSTAT (v2020.3.1) software. The significant level was set at p < 0.05.
All the experiments were recorded in triplicate. The differences between
means were evaluated using 2-sided Tukey’s HSD.
3. Results
3.1. Proximate analysis of milk samples
The proximate analysis of the raw milk samples collected from
different villages of Lahaul valley was evaluated for pH, electrical con­
ductivity (EC), ash, moisture, crude fat, and protein content. The pH and
EC values of the samples slightly varied from 6.12 ± 0.21 to 6.23 ± 0.28
and 11.4 ± 0.10 to 13.2 ± 0.2. Meanwhile, the moisture content ranged
from 86.97g ± 0.10/100g (wet basis) to 87.73g ± 0.01/100g (wet
basis). The results of ash, crude fat, and protein content are given in
Supplementary Table S1. The microbial load of the samples yielded from
7.4 ± 0.04 × 107 to 6.54 ± 0.06 × 108 CFU/mL (Supplementary
Table S1).
After isolation, 321 probiotic strains were obtained from the three
raw milk samples. A total of 72 discrete morphotypes showing a unique
appearance on the MRS medium were sub-cultured. Subsequently, we
screened for the qualitative test with acid and bile tolerance. Among the
72 tested bacterial isolates, 34 strains showed better growth under
different pH values (2.0, 2.5, and 3.0) and bile salt concentrations (300
mg/dL). The Gram-positive and catalase-negative strains with optimum
growth at 37 ◦ C, pH 6, 4000 mg/dL NaCl, and 400 mg/dL phenol con­
centrations were selected (Supplementary Table S2). After the qualita­
tive screening, 21 out of 34 identified strains were chosen for further
probiotic attributes, functional properties, and safety assessment (Sup­
plementary Table S2).
4
LWT 176 (2023) 114553
3.2. Molecular identification and phylogenetic analysis
The 34 bacterial strains (based on their probiotic attributes, func­
tional, and safety evaluation) were characterized by molecular identi­
fication. The sequences of the selected strains presented >99%
similarity to the GenBank sequences. Based on the available 16S rRNA
gene sequencing, the 34 probiotic isolates were characterized and
belonged to 7 distinct genera and 11 different species (Table 1). A
phylogenetic tree was constructed to classify the isolates to the species
level based on the neighbor-joining method (Supplementary Fig. S1).
3.3. Evaluation of probiotic attributes
3.3.1. Gastro-intestinal transit tolerance assay
The probiotic isolates were grown under simulated in vitro gastric
juice at pH 2.0 and 3.0 at different intervals, i.e., 0, 1, and 3 h. The
isolates retained about one log viability level, and the strains LGM:02,
LGM:04, LGM:13, LGM:16, and LGM:25 showed maximum survivability
(7 log values) when exposed to pH 2.0 for up to 3 h (Supplementary
Table S3). Likewise, the same isolates showed optimum survivability
when grown at pH 3.0 for 3 h, with LGM:02 and LGM:16 exhibiting the
highest viability level (7.30 ± 0.08 and 7.34 ± 0.16 log10 CFU/mL;
Supplementary Table S3). For bile tolerance, strains LGM:02 and
LGM:16 survived with 88.28 ± 0.14% and 88.47 ± 0.21% viability, as
presented in Supplementary Table S3, different subscripts upper case
letters indicate significantly different at p < 0.05.
3.3.2. Cell auto-aggregation and cell surface hydrophobicity activity
The results of cell auto-aggregation range from 14.38 ± 0.44% to
96.30 ± 0.38% at different periods (1, 2, and 3 h). The highest auto
aggregation ability was found in the strain LJM:05, while the other
strains exhibited auto aggregation activity of ≥90% (Supplementary
Table S4).
The hydrophobic ability of the tested strains ranges from 13.36 ±
0.28% to 97.46 ± 0.34% for chloroform and 5.45 ± 0.40% to 95.39 ±
0.40% for polar solvent toluene. The strains showed high hydropho­
bicity in chloroform compared to toluene at different intervals (1, 2, and
24 h; Supplementary Table S5 different subscripts upper case letters
indicate significantly different at p < 0.05).
3.4. Safety assessment
3.4.1. Antibiotic susceptibility, biofilm assay, hemolysis, and DNase assay
The selected strains were checked for their susceptibility against
different antibiotic discs. Most isolates were resistant to vancomycin,
azithromycin, kanamycin, and ciprofloxacin antibiotics. Meanwhile, the
remaining isolates vary in susceptibility, resistance, and sensitivity
(Supplementary Table S6).
The strains’ biofilm formation was determined based on the optical
density (OD) values. The isolates LJM:05, LGM:02, LGM:10, and LGM:16
showed the highest value of ≥0.82 ± 0.40, while the strains HMM:01,
HMM:04, HMM:15, LGM:13, and HMM:21 exhibit moderate biofilm
formation. The remaining isolates possessed weak biofilm formation
(Supplementary Table S7). The hemolysis and DNase activity were also
assessed to determine the safety of the bacterial isolates, with no zone
formation observed on their representative media plate, revealing the
strains’ non-pathogenic nature.
3.4.2. Antibiotic resistance and virulence factors related genes
The genome of the LJM:05 strain contains genes associated with
resistance to diaminopyrimidine, tetracycline, and glycopeptide antibi­
otics. The assessments of antibiotic resistance include antibiotic target
replacement, target protection, and target alteration (Supplementary
Table S8). Moreover, the genome contains various putative virulence
factors such as adherence, capsule formation, biofilm, cytolysin trans­
port, and enterococcal surface protein (Supplementary Table S9). The
N. Baliyan et al.
similarity percentage of most virulence factors detected in the genome is
less than 60%, with the virulence factors database indicating low simi­
larity to the genes in the database.
3.5. Functional attributes
3.5.1. Antimicrobial activity and EPS production
The inhibition ability of 21 probiotic strains was determined against
6 opportunistic pathogen-type strains. The maximum inhibition (>21
mm) was shown against Micrococcus luteus and Klebsiella pneumoniae,
whereas the least inhibition (11–15 mm) was detected for B. subtilis and
Pseudomonas aeruginosa. The isolates exhibit a different degree of inhi­
bition (Supplementary Table S10). All the tested bacterial strains
showed mucoid colony formation on the sugar-containing MRS agar
plate, indicating their Exopolysaccharide (EPS)-producing ability.
3.6. Principal component analysis (PCA)
The PCA analysis was conducted to select the most potential bacte­
rial isolates based on their positive probiotic attributes. The PCA
revealed 39.52% of the total variation in two principal components, and
the variable homogenous distribution on the principal plane component
showed F1 and F2, with 21.85% and 17.66% variation (Fig. 1). The
highest number of bacterial isolates correlates with the F1 and F2
components, implying that these variables have a role in strain selection
(Supplementary Table S11). The seven bacterial strains were found in
quadrant I, which displayed the maximum correlation to the variables.
From the seven selected isolates, LGM:02, LGM:16, LGM:19, LGM:23,
and LGM:25 strains were from the Ghosal milk sample, LJM:05 from
Jhundha, and only one strain, HMM:03, from the Sissu milk sample.
3.7. CLA spectrometric assay and GC-MS chromatography analysis
The CLA-converting ability of the probiotics from Linoleic acid (L.A.)
was determined using the spectrophotometric assay, and the results (μg/
mL) are presented in Fig. 2. The CLA isomer was confirmed using GC-MS
by identifying the high abundance of the 9,11-octadecadienoic acid
methyl ester peaks, with the percentage shown in Supplementary
Table S12. The cis-9, trans-11 octadecanoic acid level is significantly
high in the strains LGM:16, LGM:02, and LJM:05. The strain LGM:16
showed a 6.03% area (Supplementary Fig. S3) compared to LGM:02
(4.97%) and LJM:05 (4.10%).
3.8. Antioxidant activity
The antioxidant activity was performed to determine the free-radical
scavenging ability of the seven potential bacterial strains. The strains
showed ABTS and DPPH radical scavenging activities ranging from
15.15 ± 0.03% to 30.23 ± 0.11% and 30.32 ± 0.21% to 57.22 ± 0.01%
(Fig. 3). Meanwhile, the ferric thiocyanate showed 19.23%–31.15%
5
LWT 176 (2023) 114553
(Supplementary Fig. S3) and reducing power assay revealed
0.109–0.140 (A700nm) (Supplementary Table S13). Out of seven bacte­
rial strains, LGM:02 and LGM:16 showed the highest antioxidant
activity.
3.9. Curdled milk and scanning electron microscopy (SEM)
Based on the isolates’ antioxidant activity and CLA production, the
strain LGM:16 was selected and used to prepare the fermented curdled
milk (Supplementary Fig. S2). The strain could ferment sterile skim milk
(in addition to 0.5 mg/mL LA) with viability ranging from 8.54 to 7.55
log10 CFU/mL in 24 h at 37 ◦ C. The changes in pH and viability of the
strain used to prepare the fermented milk were recorded until four
weeks of storage at 4 ◦ C. The viability and pH changes during the storage
period were found at 8.54 to 7.55 log10 CFU/mL and 5.12 to 4.15,
respectively (Fig. 4). The pathogenicity test indicates the absence of
coliform, fungus, and enterobacteria (Supplementary Table S14) in the
curdled milk. The scanning electron microscopy (SEM) images depict
the microstructural changes in the test and control samples. The
micrograph of the control sample reveals a lumpy shape and smooth
surface with a compact texture (Fig. 5). In comparison, the test sample
depicts a looser network structure with larger holes and cracks, possibly
attributable to fermentation. The fermented milk with the strain LGM:16
shows more structure and larger pore dimensions than the control
sample.
4. Discussion
Several studies reported the biological aspects of probiotics as being
strain-specific, and their functional characteristics cannot be concluded
at the species level. Hence, researchers constantly look for new probiotic
strains with more promising health benefits (Devi et al., 2018). The
present study focused on isolating potential CLA-converting probiotic
strains from raw cow milk of the Lahaul valley with superior probiotic
attributes for potential applications in food formulations. To the best of
our knowledge, the probiotic diversity of raw milk of the Lahaul valley
has never been documented hence, it offered good opportunity to
explore indigenous probiotics. Raw milk contains LAB, which has a
significant role in dairy industries due to its nutritional and therapeutic
properties (Reuben et al., 2020). The proximate analysis of the raw milk
revealed that the ash, moisture, crude fat, pH, and protein contents of
the milk samples fell in the accepted range of 3.08–3.25%,
86.97–87.73%, 3.08–3.25%, 6.12–6.23, and 3.06–4.01% respectively;
indicating its potential nutritive properties, similar to the previous study
(Tallini, 2015). Meanwhile, the microbial count of the samples was in
the range of 7.4 × 107 to 6.54 × 108 CFU/mL, revealing the high bac­
terial load in the milk sample. In the current study, the isolated strains
were examined for their probiotic properties according to the
FAO/WHO guidelines (2002). Firstly, the basic and foremost probiotic
attributes were determined by observing their growth under different
Fig. 1. Principal component analysis (PCA) of the
different probiotic attributes (Acid and bile tolerance
at different pH and concentrations, cell auto-
aggregation, cell surface hydrophobicity) of 34 bac­
terial isolates. (A) Scree biplot (eigenvalue) of prin­
cipal components (F1–F11) for the probiotic potential
of different isolates from raw milk. The plot showed
that only two factors out of sixteen were retained in
the exploratory factor analysis in a principal compo­
nent (B) Principal component analysis (PCA) biplot
projection based on probiotic attributes for selecting
the most promising probiotic strain isolated from raw
milk. Based on PCA analysis seven most promising
bacterial strains were selected for further Conjugated
Linoleic Acid (CLA) production.
N. Baliyan et al.
Fig. 2. CLA production analysis from different bacterial strains on the basis of UV-based Spectrophotometric method in the MRS broth after 48 h, compared to the
control (reference type strain L. plantarum MTCC 1407). Different letters indicate significant differences (p < 0.05) between strains.
acidic conditions and bile salts (Reale et al., 2015). Out of 321 unique
morphotypes, 72 strains could tolerate the low pH and bile salt condi­
tions (qualitative assay), attributing to their survival ability in the
human gastrointestinal (GI) tract.
From the quantitative screening (acid and bile tolerance), 34 strains
were selected and identified using 16S rRNA gene sequencing and
phylogeny construction. The raw milk samples were dominated by LAB,
representing diverse taxonomic affiliations with 7 distinct genera and 11
different species. Out of which the L. lactis has been reported from
Izmir’s (Turkey) raw cow milk (Yerlikaya, 2019). The selected 34 strains
passed all the required probiotic attributes i.e., GI conditions (acid and
bile tolerance), cell-adhesion (auto-aggregation and cell-surface hydro­
phobicity assays), and also possess functional properties (antimicrobial
activity and EPS production). The tolerance ability for acid and bile
6
LWT 176 (2023) 114553
Fig. 3. The antioxidant activity of the seven most
potential probiotics isolated from raw milk of Lahaul
valley using DPPH, ABTS, and Ferric thiocyanate (%)
compared with the control (reference type strain
L. plantarum MTCC 1407). The antioxidant activity
was performed to determine the free-radical scav­
enging ability of the seven potential bacterial strains.
Out of seven bacterial strains, LGM:16 showed the
highest antioxidant activity. Different letters denote
statistically significant (p < 0.05) differences.
assay mimics bacterial strains’ viability under a gastrointestinal pH
environment. The tested bacterial strains displayed high
auto-aggregation ability, rendering their vital role in adhering to the
intestinal cells and exerting beneficial effects for inhibiting pathogenic
microbes. The strains showed higher adhesion towards chloroform than
toluene (monopolar aliphatic and aromatic solvents) in the hydropho­
bicity assay, depicting the presence of low electron acceptor/acidic
surface components on the cell surface of most strains, congruent to a
previous study (Rokana et al., 2018). The bacterial adhesion to the in­
testinal epithelium depends on various factors, such as surface electro­
static charges, gravitational forces, and van der Waals attraction
(Vinderola & Reinheimer, 2003).
Several probiotic species have been designated as ‘generally recog­
nized as safe’ (GRAS notices can be obtained at https://www.fda.gov),
N. Baliyan et al. LWT 176 (2023) 114553

Fig. 4. The change in the viability and pH of the curdled milk (containing linoleic acid (0.5 mg/mL) prepared using strain LGM:16 compared to the reference strain,
i.e., L. plantarum MTCC 1407. The pH and viability of the curdled milk were checked for 4 weeks and decreased in both the control and test samples. Different letters
indicated the significant difference (p < 0.05) in the viability and pH of the strains.

Fig. 5. Scanning electron micrographs of the (A) control (without any probiotic strain) and (B) curdled milk fermented with LGM:16, at ×50.0 μm magnification.
The fermented milk with the strain LGM:16 showed structural breakage and porous structure compared to the control.

while many species carry the qualified presumption of safety (QPS)
status. The main safety concern with putative probiotic strains is their
antibiotic resistance (Kumari et al., 2020). Additionally, none of the
strains exerts hemolytic and DNase activity, and the isolates showed
varied biofilm formation ability, indicating their safe use for future food
formulation. Among all the species, E. faecalis is controversial, as it is
known as a probiotic that enhances health benefits while at the same
time behaving as a nosocomial pathogen (Cirrincione et al., 2019).
Therefore, in the present study, we analysed the safety-related genes,
including antibiotic resistance and virulence genes, of the representative
strain E. faecalis LJM:05. A few virulence genes were detected in the
LJM:05 genome, which were responsible for adherence, capsule for­
mation, and other enzymatic activities. The adhesion and capsule
forming genes, including asa1 and ace, have been reported previously in
other probiotic E. faecalis genomes (Panthee et al., 2021). Such genes are
common in probiotic bacterial strains where they are crucial for
persistence, colonization and resisting its removal from the host (Pillar
& Gilmore, 2004). A very few antibiotic resistance genes were detected
in the LJM:05 genome, among which dfrE and tet(M) genes were found
in other probiotic E. faecalis strains as well (Panthee et al., 2021). The
absence of other antibiotic resistance and virulence genes in the
E. faecalis thus supported its safety.
Previous studies have revealed that the production of bioactive fatty
acids is an additional effect of a probiotic trait (Ribeiro et al., 2018). The
seven promising probiotic strains were further evaluated for
CLA-producing ability using a systematic three-step screening procedure
for CLA-producing probiotics, i.e., LA tolerance, UV based spec­
trophotometeric assay, Fatty acid methyl esters (FAME), and GC-MS
chromatogram. The study positively found three higher
CLA-converting probiotic strains, i.e., Lactiplantibacillus paraplantarum
(LGM:02), Latilactobacillus curvatus (LGM:16), and Enterococcus faecalis
(LJM:05). It has been proposed that the conversion of LA to CLA by
bacterial strains is a detoxifying mechanism positively connected to CLA
synthesis (Ribeiro et al., 2018). In the present study, the strains LGM:02,
LGM:16, LGM:25, and LJM:05 showed similar significant differences in
CLA production (μg/mL); whereby the strains LGM:19, LGM:23,
HMM:03 produced lower quantities of CLA. The characteristics of
CLA-producing strains also depend upon various factors, such as LA
concentration, media components, incubation period, time duration, etc
(Kuhl & De Dea Lindner, 2016). In the current study, a higher CLA
content was found in the supernatant of the tested strains, similar to a
previous report (Dahiya & Puniya, 2018a). After GC-MS analysis, a

7
N. Baliyan et al.
higher cis-9, trans-11 CLA isomer was detected (6.03%) in the LGM:16
strain, followed by LGM:02 and LJM:05 at 4.10% and 4.97%. Probiotic
strains, e.g., L. plantarum and E. faecalis have been reported to produce
the cis-9, trans-11 CLA isomer (Mishra & Ghosh, 2020; Ribeiro et al.,
2018). Probiotics mainly produce cis-9, trans-11 of all the CLA isomers
because it alone comprises of 90% of the total CLA isomers (Vieira et al.,
2017), besides having health benefits in humans, such as anti-obesity,
antidiabetic, anticancer, antioxidant, and improved immune system
(Hwang et al., 2021).
The additional properties of the probiotic strains were further
confirmed by the exertion of strong antioxidant effects, protecting the GI
tract against free radicals and alleviating oxidative stress. The obtained
antioxidant results are comparable to a previous study (Kim et al.,
2022). Furthermore, the most promising CLA-producing strain,
L. curvatus LGM:16, with the highest antioxidant activity, could ferment
skim milk in the presence of LA (0.5 mg/mL) to produce the curdled milk
drink. The viability and pH range of the tested strain (LGM:16), and no
pathogenic growth during the storage period (28 days) at 4 ◦ C were
noted. The significant difference between the control and the test sample
in Fig. 4 shows a higher microbial count at lesser pH than the tested
bacterial sample. The obtained microbial count and pH range were
similar to the study by (Kumari et al., 2016). The SEM analysis of the
curdled milk revealed that fermentation changed the sugar-ordered
structures (i.e., lactose) and broke the uniform layer compared to the
control sample (Fig. 5). Therefore, the most potent CLA-producing strain
LGM:16 with superior antioxidant activity could be a future potential
probiotic strain that can be used for development of functional food.
Besides, seven strains (LJM:05, HMM:03, LGM:04, LGM:16, LGM:19,
LGM:23, and LGM:25) revealed superior probiotics properties and
functional attributes with safe use for future study. They could also be
promising candidates for functional food formulation with therapeutic
properties. Present study is presumably the first to explore the combi­
nation of nutritional profiling of probiotic attributes and CLA-producing
property of probiotic strains from raw cow milk of the Lahaul region.
5. Conclusion
The current study was conducted to obtain possible probiotic strains,
with comprehensive analysis for probiotic qualities, functional charac­
teristics, and safety. After statistical analysis, seven most promising
probiotic strains were investigated for CLA production. The whole
genome analysis of a representative potential probiotic strain revealed
antibiotic resistance and virulence genes, which complemented their
probiotic traits. Among seven most potential strains, Latilactobacillus
curvatus LGM:16 showed the maximum ability to convert linoleic acid
into CLA (cis 9, trans 11 octadecanoic acid) isomer and antioxidant
properties. Therefore, LGM:16 was used to develop the curdled milk,
which showed that viability remained above the required legal viable
count (6 log10 CFU/mL) at lower pH ~4.1, for four weeks of storage at
4 ◦ C, also confirmed by the microstructure changes through SEM anal­
ysis. Therefore, the strain LGM:16 can be incorporated into food for­
mulations to boost CLA intake and confer health benefits. The study
highlighted the importance of documenting indigenous probiotics for
health applications. The use of the obtained strains as a starter culture
for future fermented food formulation complies with the application-
based research approach.
CRediT authorship contribution statement
Neha Baliyan: Methodology, Validation, Data curation, and,
Writing – original draft. Antim Kumar Maurya: Data curation, of FAME
and GC-MS. Anil Kumar: Genome, Formal analysis, and, writing. Vijai
Kant Agnihotri: GC-MS, Data curation. Rakshak Kumar: Conceptual­
ization, Writing – review & editing.
8
LWT 176 (2023) 114553
Declaration of competing interest
Authors have no conflict of interest.
Data availability
No data was used for the research described in the article.
Acknowledgement
N.B. is thankful to Indian Council of Medical Research (ICMR),
Government of India, for ‘Senior Research Fellowship’ award (letter no.
3/1/2 (8) Obs./2021-NCD-II). A.K. acknowledges Department of
Biotechnology (DBT), Government of India for the PhD studentship
award (letter no. DBT/JRF/BET-17/I/2017/AL/367). RK acknowledges
the financial support by DBT, Government of India through grant no.BT/
NER/95/SP39801/2020. The authors gratefully acknowledge Dr Sanjay
Kumar, Director, CSIR-IHBT Palampur (H.P.), India, for continuous
encouragement and for providing the necessary facilities during the
course of the study. The authors also duly acknowledge the technical
support provided by Mr Anil Chaudhary, 16S rRNA gene sequencing and
Mrs Vijaylata Pathania for performing the GC-MS run. This manuscript
represents CSIR-IHBT communication no. 5191.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.lwt.2023.114553.
References
Ayivi, R. D., Gyawali, R., Krastanov, A., Aljaloud, S. O., Worku, M., Tahergorabi, R.,
Silva, R. C. D., & Ibrahim, S. A. (2020). Lactic acid bacteria: Food safety and human
health applications. Dairy, 1(3), 202–232.
Baliyan, N., Dindhoria, K., Kumar, A., Thakur, A., & Kumar, R. (2021). Comprehensive
substrate-based exploration of probiotics from undistilled traditional fermented
alcoholic beverage ‘lugri. Frontiers in Microbiology, 12, Article 626964.
Bhat, B., & Bajaj, B. K. (2020). Multifarious cholesterol lowering potential of lactic acid
bacteria equipped with desired probiotic functional attributes. 3 Biotech, 10(5),
1–16.
Cirrincione, S., Neumann, B., Zühlke, D., Riedel, K., & Pessione, E. (2019). Detailed
soluble proteome analyses of a dairy-isolated Enterococcus faecalis: A possible
approach to assess food safety and potential probiotic value. Frontiers in Nutrition, 6,
71.
CLSI. Clinical and Laboratory Standards Institute. (2016). Performance standards for
antimicrobial susceptibility testing; twenty-fifth informational supplement M100-S26.
Wayne, PA.
Cui, L., Chang, S. K. C., & Nannapaneni, R. (2021). Comparative studies on the effect of
probiotic additions on the physicochemical and microbiological properties of
yoghurt made from soymilk and cow’s milk during refrigeration storage (R2). Food
Control, 119, Article 107474.
Dahiya, D. K., & Puniya, A. K. (2018a). Conjugated linoleic acid enriched skim milk
prepared with Lactobacillus fermentum DDHI27 endorsed antiobesity in mice, 09
Future Microbiology, 13, 1007–1020.
Dahiya, D. K., & Puniya, A. K. (2018b). Optimisation of fermentation variables for
conjugated linoleic acid bioconversion by Lactobacillus fermentum DDHI 27 in
modified skim milk. International Journal of Dairy Technology, 71(1), 46–55.
Devi, S. M., Kurrey, N. K., & Halami, P. M. (2018). In vitro anti-inflammatory activity
among probiotic Lactobacillus species isolated from fermented foods. Journal of
Functional Foods, 47, 19–27.
Dubey, V., Ghosh, A. R., & Mandal, B. K. (2012). Appraisal of conjugated linoleic acid
production by probiotic potential of Pediococcus spp. GS4. Applied Biochemistry And
Biotechnology, 168(5), 1265–1276.
Dutta, R., Sarkar, U., & Mukherjee, A. (2014). Extraction of oil from Crotalaria Juncea
seeds in a modified Soxhlet apparatus: Physical and chemical characterization of a
prospective bio-fuel. Fuel, 116, 794–802.
FAO, WHO. (2002). Guidelines for the evaluation of probiotics in food. London, ON, Canada:
WHO Technical Report Series.
Gundidza, M., Mmbengwa, V., Sibambo, S. R., Magwa, M. L., Mushisha, O.,
Benhura, M. A., Gundidza, E., & Samie, A. (2011). Rheological, moisture and ash
content analyses of a gum resin from Commiphora Africana. African Journal of Food
Science, 5, 188–193.
Hennessy, A. A., Ross, P. R., Fitzgerald, G. F., & Stanton, C. (2016). Sources and bioactive
properties of conjugated dietary fatty acids. Lipids, 51(4), 377–397.
Hwang, C. E., Kim, S. C., Lee, H. Y., Suh, H. K., Cho, K. M., & Lee, J. H. (2021).
Enhancement of isoflavone aglycone, amino acid, and CLA contents in fermented
N. Baliyan et al.
soybean yogurts using different strains: Screening of antioxidant and digestive
enzyme inhibition properties. Food Chemistry, 340, Article 128199.
Kanwar, S. S., Gupta, M. K., Katoch, C., Kumar, R., & Kanwar, P. (2007). Traditional
fermented foods of Lahaul and Spiti area of Himachal Pradesh.
Kim, S., Lee, J. Y., Jeong, Y., & Kang, C.-H. (2022). Antioxidant activity and probiotic
properties of lactic acid bacteria. Fermentation, 8(1), 29.
Kuhl, G. C., & De Dea Lindner, J. (2016). Biohydrogenation of linoleic acid by lactic acid
bacteria for the production of functional cultured dairy products: A review. Foods, 5
(1), 13.
Kumari, A., Angmo, K., & Bhalla, T. C. (2016). Probiotic attributes of indigenous
Lactobacillus spp. isolated from traditional fermented foods and beverages of north-
western Himalayas using in vitro screening and principal component analysis.
Journal of Food Science & Technology, 53(5), 2463–2475.
Kumari, M., Kumar, R., Singh, D., Bhatt, S., & Gupta, M. (2020). Physiological and
genomic characterization of an exopolysaccharide-producing Weissella cibaria CH2
from cheese of the western Himalayas. Food Bioscience, 35, Article 100570.
Lee, K. W., Shim, J. M., Park, S.-K., Heo, H.-J., Kim, H.-J., Ham, K.-S., & Kim, J. H.
(2016). Isolation of lactic acid bacteria with probiotic potentials from kimchi,
traditional Korean fermented vegetable. LWT–Food Science and Technology, 71,
130–137.
Liu, B., Zheng, D., Jin, Q., Chen, L., & Yang, J. (2019). Vfdb 2019: A comparative
pathogenomic platform with an interactive web interface. Nucleic Acids Research, 47
(D1), D687–D692.
Lynch, J. M., Barbano, D. M., Fleming, J. R., Collaborators, Laboratory, B.,
Agriculture, C. D. of F., One, D., Dairy Quality Control Institute, I., O’Lakes, L., &
Agriculture, S. (2002). Determination of the total nitrogen content of hard,
semihard, and processed cheese by the Kjeldahl method: Collaborative study. Journal
of AOAC International, 85(2), 445–455.
Mallappa, R. H., Singh, D. K., Rokana, N., Pradhan, D., Batish, V. K., & Grover, S. (2019).
Screening and selection of probiotic Lactobacillus strains of Indian gut origin based
on assessment of desired probiotic attributes combined with principal component
and heatmap analysis. Lebensmittel-Wissenschaft und -Technologie, 105, 272–281.
Mishra, A. K., & Ghosh, A. R. (2020). Probiotic Enterococcus faecalis AG5 mitigated high
fat diet induced obesity and produced propionic acid stimulated apoptosis in 3T3-L1
pre-adipocyte. Life Sciences, 261, Article 118292.
Mukhia, S., Kumar, A., & Kumar, R. (2021). Generation of antioxidant peptides from soy
protein isolate through psychrotrophic Chryseobacterium sp. derived alkaline broad
temperature active protease. Lebensmittel-Wissenschaft und -Technologie, 143. https://
doi.org/10.1016/j.lwt.2021.111152
Özer, C. O., Kılıç, B., & Kılıç, G. B. (2016). In-vitro microbial production of conjugated
linoleic acid by probiotic L. plantarum strains: Utilization as a functional starter
culture in sucuk fermentation. Meat Science, 114, 24–31.
Panthee, S., Paudel, A., Hamamoto, H., Ogasawara, A. A., Iwasa, T., Blom, J., &
Sekimizu, K. (2021). Complete genome sequence and comparative genomic analysis
of Enterococcus faecalis EF-2001, a probiotic bacterium. Genomics, 113(3),
1534–1542.
Pillar, C. M., & Gilmore, M. S. (2004). Enterococcal virulence–pathogenicity island of E.
Faecalis. Frontiers in Bioscience-Landmark, 9(3), 2335–2346.
Reale, A., Di Renzo, T., Rossi, F., Zotta, T., Iacumin, L., Preziuso, M., Parente, E.,
Sorrentino, E., & Coppola, R. (2015). Tolerance of Lactobacillus casei, Lactobacillus
paracasei and Lactobacillus rhamnosus strains to stress factors encountered in food
LWT 176 (2023) 114553
processing and in the gastro-intestinal tract. LWT–Food Science and Technology, 60
(2), 721–728.
Reuben, R. C., Roy, P. C., Sarkar, S. L., Alam, A. S. M. R. U., & Jahid, I. K. (2020).
Characterization and evaluation of lactic acid bacteria from indigenous raw milk for
potential probiotic properties. Journal of Dairy Science, 103(2), 1223–1237.
Ribeiro, S. C., Stanton, C., Yang, B., Ross, R. P., & Silva, C. C. G. (2018). Conjugated
linoleic acid production and probiotic assessment of Lactobacillus plantarum
isolated from Pico cheese. Lebensmittel-Wissenschaft und -Technologie, 90, 403–411.
Rokana, N., Singh, B. P., Thakur, N., Sharma, C., Gulhane, R. D., & Panwar, H. (2018).
Screening of cell surface properties of potential probiotic lactobacilli isolated from
human milk. Journal of Dairy Research, 85(3), 347–354.
Sharma, N. (2017). Evaluation of Enterococcus Faecium CH-1 isolated from Chuli-A
traditional fermented apricot product of Trans Himalayan region. Journal of Food
Science & Technology, –2, 165–176.
Somashekaraiah, R., Shruthi, B., Deepthi, B. V., & Sreenivasa, M. Y. (2019). Probiotic
properties of lactic acid bacteria isolated from neera: A naturally fermenting coconut
palm nectar. Frontiers in Microbiology, 10, 1382.
Sulin, C. (2017). Isolation and characterisation of lactobacilli from the female anogenital tract
in healthy malayasian women and its probiotic potentail.
Tallini, R. A. (2015). Effects of pasteurization and ultra-high temperature processes on
proximate composition and fatty acid profile in bovine milk. American Journal of
Food Technology, 10(6), 265–272.
Thakur, N., Savitri Saris, P. E. J., & Bhalla, T. C. (2015). Microorganisms associated with
amylolytic starters and traditional fermented alcoholic beverages of North Western
Himalayas in India. Food Bioscience, 11, 92–96. https://doi.org/10.1016/j.
fbio.2015.05.002
Vieira, C. P., Cabral, C. C., da Costa Lima, B. R. C., Paschoalin, V. M. F., Leandro, K. C., &
Conte-Junior, C. A. (2017). Lactococcus lactis ssp. cremoris MRS47, a potential
probiotic strain isolated from kefir grains, increases cis-9, trans-11-CLA and PUFA
contents in fermented milk. Journal of Functional Foods, 31, 172–178.
Vinderola, C. G., & Reinheimer, J. A. (2003). Lactic acid starter and probiotic bacteria: A
comparative “in vitro” study of probiotic characteristics and biological barrier
resistance. Food Research International, 36(9–10), 895–904.
Wang, J., Li, H., Meng, X., Tong, P., & Liu, X. (2022). Biosynthesis of c9, t11-conjugated
linoleic acid and the effect on characteristics in fermented soy milk. Food Chemistry,
368, Article 130866.
Wang, L.-M., Lv, J.-P., Chu, Z.-Q., Cui, Y.-Y., & Ren, X.-H. (2007). Production of
conjugated linoleic acid by Propionibacterium freudenreichii. Food Chemistry, 103
(2), 313–318.
Wick, R. R., Judd, L. M., Gorrie, C. L., & Holt, K. E. (2017). Unicycler: Resolving bacterial
genome assemblies from short and long sequencing reads. PLoS Computational
Biology, 13(6), Article e1005595.
Yadav, R., Puniya, A. K., & Shukla, P. (2016). Probiotic properties of Lactobacillus
plantarum RYPR1 from an indigenous fermented beverage Raabadi. Frontiers in
Microbiology, 7, 1683.
Yerlikaya, O. (2019). Probiotic potential and biochemical and technological properties of
Lactococcus lactis ssp. lactis strains isolated from raw milk and kefir grains. Journal
of Dairy Science, 102(1), 124–134.
Zielińska, D., & Kolożyn-Krajewska, D. (2018). Food-origin lactic acid bacteria may
exhibit probiotic properties. BioMed Research International, 15, 5063185.