Chemical Physics Impact

In-vitro Evaluation of Probiotic Properties of Lactic Acid Bacteria Isolated from

Indigenous Fermented Food Products
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Manuscript Number:

Full Title: In-vitro Evaluation of Probiotic Properties of Lactic Acid Bacteria Isolated from
Indigenous Fermented Food Products

Article Type: VSI: Energy & Environmental sustainability

Keywords: In vitro, Probiotic Properties, Lactic Acid Bacteria, Indigenous Fermented Foods,
Lactobacillus

Corresponding Author: saurabh jain, M.Tech

Sharda School of Engineering and Technology
INDIA

Corresponding Author Secondary

Information:

Corresponding Author's Institution: Sharda School of Engineering and Technology

Corresponding Author's Secondary

Institution:

First Author: saurabh jain, M.Tech

First Author Secondary Information:

Order of Authors: saurabh jain, M.Tech

Pramod K. Singh

Kalpana Singh

Faisal Islam Chowdhury

M. Z. A Yahya

Markus Diantoro

Neeraj Jha

Order of Authors Secondary Information:

Abstract: Lactic acid bacteria (LAB) are traditionally associated with the fermentation of local
foods, yet their probiotic potential has not been thoroughly investigated. This study
evaluates LAB isolated from fermented Indian meals for their probiotic attributes. Sixty
LAB isolates were selectively isolated through micro-aerophilic enrichment in MRS
Broth, followed by cultivation on MRS agar. Selected colonies showing transparent
halos were assessed for antibacterial activity, phenotypic and biochemical traits, acidic
yield, and tolerance to acid and bile. Of the 60 LAB isolates, 12 exhibited robust growth
at various pH levels, especially at pH 4, and six isolates showed good tolerance to 2%
bile salt concentration. Seventy-nine percent of the isolates demonstrated variable
antibacterial activity against at least one pathogen. Susceptibility testing revealed
sensitivity to tetracycline, amoxicillin, and gentamicin, with variable resistance to
ampicillin and co-trimoxazole. Most isolates were susceptible to most antibiotics, with
occasional resistance to kanamycin. Three isolates showed increased hydrophobicity,
and isolates SCA-5, SMB-3, SML-6, and SCS-1 had low auto-aggregation.
Phylogenetic analysis identified isolates SCS-1, SMB-5, and SMS-1 as closely related
to Lactobacillus acidophilus and Lactobacillus delbrueckii, while SMY-4 was related to
Lactobacillus rhamnosus. Four isolates, SCS-1, SMB-5, SMS-1, and SMY-4,
demonstrated potential probiotic properties. Further research is needed to explore their
suitability in fortifying fruit and vegetable juices to create probiotic beverages.

Suggested Reviewers: Ashwani Kumar, PhD

Associate Professor, Central University of Haryana
ashwanindri@gmail.com

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 Expert in the field of probiotics

Shashank Sharma
Sharda University
shashank.enviro@gmail.com
Expert of Biochemical Analysis

Opposed Reviewers:

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Manuscript Click here to view linked References

1 1 In-vitro Evaluation of Probiotic Properties of Lactic Acid Bacteria Isolated from

2
3
4 2 Indigenous Fermented Food Products
5
6
3 Saurabh Jain1,2, Pramod K. Singh3, Kalpana Singh4 , Faisal Islam Chowdhury5, M. Z. A
7
8 4 Yahya6, Markus Diantoro7, Neeraj Jha1
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10 5
11
12 1
6 Department of Biotechnology, Sharda School of Engineering & Technology, Sharda
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14 7 University Greater Noida, India
15
2
16 8 Department of Biotechnology, MGIMT, Lucknow, India
17
18 3
9 Center for Solar-cells and Renewable Energy (CSRE), Department of Physics, SSBSR,
19
20 10 Sharda University, Greater Noida, 201310, India
21
4
22 11 , Lloyd Institute of Management and Technology, Plot No.-11, Knowledge Park-II,
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24 12 Greater Noida, Uttar Pradesh, India
25
26 5
27 13 Department of Chemistry, University of Chittagong, Chattogram, Bangladesh
28
6
29 14 Faculty of Defence Science and Technology, Universiti Pertahanan Nasional Malaysia
30 15 (UPNM), 57000 Kuala Lumpur, Malaysia
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32 7
33 16 Department of Physics, Faculty of Mathematics and Natural Science, Universitas Negeri
34 17 Malang, Semarang 5, Malang 65145, Indonesia
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36 18
37
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39
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19 Abstract
41
42
20 Lactic acid bacteria (LAB) are traditionally associated with the fermentation of local foods, yet their
43
44
45 21 probiotic potential has not been thoroughly investigated. This study evaluates LAB isolated from
46
47 22 fermented Indian meals for their probiotic attributes. Sixty LAB isolates were selectively isolated
48
49
50 23 through micro-aerophilic enrichment in MRS Broth, followed by cultivation on MRS agar. Selected
51
52 24 colonies showing transparent halos were assessed for antibacterial activity, phenotypic and
53
54 25 biochemical traits, acidic yield, and tolerance to acid and bile. Of the 60 LAB isolates, 12 exhibited
55
56
57 26 robust growth at various pH levels, especially at pH 4, and six isolates showed good tolerance to 2%
58
59 27 bile salt concentration. Seventy-nine percent of the isolates demonstrated variable antibacterial
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61
62 1
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 28 activity against at least one pathogen. Susceptibility testing revealed sensitivity to tetracycline,
1
2 29 amoxicillin, and gentamicin, with variable resistance to ampicillin and co-trimoxazole. Most isolates
3
4
5 30 were susceptible to most antibiotics, with occasional resistance to kanamycin. Three isolates showed
6
7 31 increased hydrophobicity, and isolates SCA-5, SMB-3, SML-6, and SCS-1 had low auto-aggregation.
8
9 32 Phylogenetic analysis identified isolates SCS-1, SMB-5, and SMS-1 as closely related to Lactobacillus
10
11
12 33 acidophilus and Lactobacillus delbrueckii, while SMY-4 was related to Lactobacillus rhamnosus. Four
13
14 34 isolates, SCS-1, SMB-5, SMS-1, and SMY-4, demonstrated potential probiotic properties. Further
15
16
17
35 research is needed to explore their suitability in fortifying fruit and vegetable juices to create probiotic
18
19 36 beverages.
20
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22 37 Keywords: In vitro, Probiotic Properties, Lactic Acid Bacteria, Indigenous Fermented Foods,
23
24 38 Lactobacillus
25
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27 39
28
29
30 40 Introduction
31
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33 41 With its diverse cultures, religions, and climates, India boasts various fermented foods crafted through
34
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42 ancestral knowledge from different ethnic groups [1]. Fermented foods have deep cultural roots,
36
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38 43 contributing to meals with delightful flavours, consistency, and colours, utilizing locally available raw
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40 44 ingredients (grains, cereals, milk, plant leaves, roots, etc.). Traditional fermentation methods involve
41
42
43 45 using microbes or starter cultures, representing age-old biotechnological practices to augment the
44
45 46 shelf-life of perishable items [2]. Probiotics, primarily lactic acid bacteria (LAB), are incorporated into
46
47 47 fermented foods to enhance their nutritional content and promote gut health [3]. The safe ingestion
48
49
50 48 of these microorganisms holds medicinal potential for treating various conditions.
51
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53 49 Probiotics, comprising beneficial microorganisms, offer medicinal properties whenever ingested in
54
55 50 adequate amounts. They contribute to the protection of the gut ecosystem and play a crucial part in
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57 51 the management of various ailments, including diarrhoea [4], allergies [5], diabetes [6], hypertension
58
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60 52 [7], inflammatory bowel syndrome, intestinal bowel syndrome [8], genetic disorders [7,9], cancer [10],
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62 2
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 53 and ulcerative colitis [8]. Pan et al. highlight that effective probiotic bacteria should possess the ability
1
2 54 to synthesize secondary metabolites with antagonistic effects against infectious microbes, adhere to
3
4
5 55 intestinal epithelial tissue, exhibit bile and acid resistance, exhibit non-carcinogenic properties, and
6
7 56 reduce pathogenic adherence [11]. Beneficial bacteria interact with raw food substrates, generating
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9 57 fermentation products such as vitamins and enzymes that benefit health [12]. It is important to note
10
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12 58 Probiotics' effectiveness depends on the isolates, and not all strains can uniformly treat disorders or
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14 59 facilitate optimal fermentation [13].
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17 60 Rod-shaped, non-spore-forming, nonmotile, catalase-negative, and Gram-positive LAB [14], such as
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19 61 Lactobacillus are vital for fermentations [15]. Other critical species like Lactobacillus rhamnosus,
20
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22 62 Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus johnsonii, Lactobacillus paracasei,
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24 63 Lactobacillus casei, and Lactobacillus acidophilus, are frequently employed in probiotic settings
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26
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64 [16,17]. These non-pathogenic bacteria thriving in acidic GI environments participate in preserving GI
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29 65 well-being by inhibiting pathogen growth via producing antibacterial peptides and organic acid
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31 66 compounds [13,18,19].
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34 67 Fermented foods, like Dahi (curd) products [20], milk [21], and yogurt [22], are recognized sources of
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37 68 probiotics. The probiotic potential of a Bifidobacterium strain was recently identified in the traditional
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39 69 Indian cuisine Haria (rice beer), suggesting its applicability in fortifying cereal-based foods [23].
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41 70 Probiotics from conventional fermenting dairy products may act as a starting point for massive
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44 71 manufacturing, offering favourable functional characteristics as probiotics. Our investigation aims to
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46 72 sequester and ap-praise the probable probiotic attributes of Lactobacillus acidophilus (LAB) from
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48 73 various traditionally fermented food products, including fermented cereal foods (idli, dhokla,
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51 74 sourdough bread, appam, uttapam) and fermented milk products (curd, buttermilk, lassi, shrikhand,
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53 75 yogurt).
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56 76
57
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59 77 Materials And Method
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 78 Specimen Collection
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3 79 Fermented cereal products like Idli, dhokla, sourdough bread, appam, and uttapam, as well as
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5 80 fermented milk products like curd, buttermilk, lassi, shrikhand, and yoghurt, were procured locally in
6
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8 81 Lucknow, India, and the neighbouring areas (Table 1). About 200 g of each sample was aseptically
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10 82 collected using sterilized containers and transported to the laboratory under refrigerated conditions
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12 83 for subsequent analysis. To prepare idli batter, polished parboiled rice and decorticated black gram are
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15 84 soaked in water for four hrs at 30±1 °C. Bengal gram dal and rice are soaked, ground separately,
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17 85 combined, and subjected to natural fermentation before being cooked into the popular Indian savoury
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19 86 dish dhokla [24]. Sourdough (SD) fermentation is a conventional biotechnological method to enhance
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22 87 baked items’ qualities [25].
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25 88 Selective Isolation of LAB isolates
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28 89 The MRS medium (HiMedia Laboratories Pvt. Limited, India) isolated LAB. To achieve LAB isolation, 225
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30 90 ml of sterile peptone water (0.1% W/V) was combined with 25 mL or 25 g of samples of conventionally
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32 91 fermented foods. After serial dilution (10–3 fold), 0.1 ml of a sample was placed on freshly made MRS
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35 92 agar plates and placed in an anaerobic container (BBL, Gas Pak Anaerobic Frameworks) at 37 °C for 24-
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37 93 48 hrs [26]. Next, 10-20 colonies were randomly selected from measurable MRS plates for subsequent
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94 refining and isolation. After being separated into roughly 5 ml of MRS broth, isolated colonies of LAB
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42 95 were repeatedly streaked over MRS agar to purify them. After streaking pure LAB cultures onto MRS
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44 96 agar slants, they were kept at -4 °C subjected to further in vitro screening [27].
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47 97 Tests for morphology, biochemistry, and physiological processes
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50 98 Microscopic investigations, colony morphology, and cultural traits were carried out during the
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52 99 preliminary detection of samples. Biochemical analyses, such as the carbohydrate fermentation test,
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55 100 growth at various temperatures, ammonia production, and development at variable NaCl
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57 101 concentrations, were used to identify and screen catalase-negative, non-spore-forming, and gram-
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102 negative strains [28,29].
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 103 Cell Morphology
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3 104 Wet-mounted overnight cultures on tiny slides were observed using oil immersion objectives under a
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5 105 light microscope. Cell shape and configuration were considered cellular morphological parameters
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8 106 throughout the assessment.
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107 Growth at Different NaCl Concentrations
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13 108 MRS broth tubes were prepared at three various concentrations of NaCl (2%, 4%, and 6.5%). Tubes
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15
16 109 were then inoculated with isolated bacteria and cultured at 37 °C for 24 hrs. The effect of the amount
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18 110 of sodium chloride on restricting microbial proliferation was measured using Optical density (OD600
19
20
111 nm) [30].
21
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23 112 Growth at Different Temperatures
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26 113 Overnight LAB culture (50 μL) was placed in 4 distinct vials containing 5 mL MRS broth and bromocresol
27
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29 114 purple. Following inoculation, half of the vials underwent incubation at 15 °C for 7 days, while the
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31 115 remaining two were maintained at 45 °C. Growth progress was monitored by changing the growth
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33 116 media colour from purple to yellow at either temperature.
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36 117 Catalase Test
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39 118 Isolate colonies were cultivated in anaerobic environments for 24 hrs at 37 °C on MRS agar. The
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42 119 catalase test involved applying two droplets of 3% H2O2 on a glass slide to cultures grown for at least
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44 120 24 hrs. The catalase test revealed a positive result, shown by the generation of oxygen bubbles, a sign
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46 121 that the test bacteria are producing catalase. The strains which failed to generate air bubbles were
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49 122 selected for additional tests [31].
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52 123
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124 Characterization of Probiotic Properties in vitro
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57 125 Tolerance to Low pH
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 126 In anaerobic environments, the strains were cultured independently overnight at 37 °C in 5 ml MRS
1
2 127 broth. To achieve a starter inoculum level of log 6 CFU/mL, 1 mL of log 7 CFU/mL of each overnight
3
4
5 128 grown population was added to 10 ml MRS broth. After centrifuging the culture for 10 min at 4 °C at
6
7 129 5000 rpm, the pellets were twice rinsed in phosphate buffer (HiMedia Laboratories Pvt. Limited, India)
8
9 130 with a pH of 7.2. The resulting pellets were resuspended in 5 mL of sterile MRS broth that had been
10
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12 131 pH-corrected, utilizing 1 N HCl to mimic the stomach habitat. At 37 °C, test tubes were allowed to
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14 132 incubate for 3-6 h. Grosu-Tudor and Zamfir employed ordinary MRS broth injected with the culture as
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133 a positive control [32]. The ratio of the number of LAB colonies grown in the MRS medium compared
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19 134 to the original quantity of microbes was utilized to compute the percentage of colonies that survived.
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21 135 After incubating aerobically for 6 hrs at 37 °C, the optical density of MRS broth was measured at 600
22
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24 136 nm. Three duplicates of the test were run [33].
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137 Bile salt intolerance
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138 The Gilliland et al. methodology was followed for performing the bile tolerance test [34]. After
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32 139 inoculating the 18-hrs culture with bile into MRS broth and incubating it anaerobically for 24 hrs at 35
33
34 140 °C, the growth was computed using absorbance at 600 nm [35]. After that, the cultures were spun in
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37 141 a centrifuge for 10 min at 4 °C at 5000 rpm with a starting dosage of 106 CFU/ml. The pellets
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39 142 underwent two rounds of washing in phosphate-buffered saline (PBS) at 7.2 pH. 100 µl of a bacterial
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41 143 culture developed overnight was added to freshly prepared MRS broth that included 2% and 4% (w/v)
42
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44 144 bile salts (HiMedia Laboratories Pvt. Limited, India). As a control, test isolates were also injected into
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46 145 MRS broth without bile. The test isolates were cultured at 37 °C for 3 hrs and 6 hrs in test tubes that
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48 146 contained both bile and no bile. By evaluating the absorbance of MRS broth at 600 nm, the growth at
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51 147 2% and 4% (w/v) bile salts after 3 and 6 hrs was observed, together with the percentage resistance
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53 148 [32].
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56 149 Antimicrobial Activity
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 150 Employing an earlier reported triplicate agar well-diffusion experiment, the antimicrobial efficacy of
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2 151 the isolates was assessed [36]. As indicator bacteria, we employed Klebsiella sp., E. faecalis, S. aureus,
3
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5 152 P. alcaligenes, P. aeruginosa, E. coli, B. subtilus, and B. cereus. A layer of 6 mL MHA agar, which had
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7 153 been inoculated with 60 µL of indicator bacteria using 0.5 McFarland, was placed over a layer of pre-
8
9 154 prepared solidified Muller-Hinton agar (HiMedia Laboratories Pvt. Limited, India) 0.8% agar. Following
10
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12 155 that, the agar was punctured with a 6 mm hole. Overnight, isolated LAB was grown in MRS broth. Cell-
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14 156 free culture supernatants were obtained by centrifugating overnight bacterial culture at 12,000 × g for
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157 20 min at 4 °C [37]. After seeding the hole with 100 µL of the supernatant, it was incubated for 24 hrs
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19 158 at 37 °C. A measurement and record of the growth inhibition zone were made. Each test was
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21 159 conducted in duplicate, and the results were shown as mean standard deviation (SD).
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24 160 Tests for antibiotic susceptibility
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27 161 The Kirby-Bauer disc diffusion technique used a Mueller-Hinton agar (MHA) medium to screen for
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162 antibiotic resistance [38]. Freshly made MHA plates were covered with bacterial populations, and
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32 163 antibiotic discs were spaced equally throughout their surface. After the dishes were incubated for 24
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34 164 to 48 hrs at 37 °C, the zone of inhibition (ZOI) width was determined. The Clinical and Laboratory
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36
37 165 Standards Institute's (CLSI, 2016) classification rules were used to classify the isolate's antibiotic
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39 166 resistance attribute. The antibiotics utilized in this investigation were Gentamicin (120 µg), Tetracycline
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41 167 (30 µg), Kanamycin (30 µg), Ampicillin (10 µg), Amoxycillin (30 µg), Co-trimoxazole (5 µg), and
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44 168 Streptomycin (10 µg). The resulting ZOI values were determined following CLSI 2016 and categorized
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46 169 as follows [39,40]: resistant (ZOI: 15 mm), sensitive (ZOI: 21 mm), or intermediately susceptible (ZOI:
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48 170 16–20 mm). Three replications of the observations were made, and the results were recorded as mean
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51 171 standard deviation (SD).
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54 172 Evaluation of Hydrophobicity and Auto-Aggregation
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57 173 The microbial Adhesion to Hydrocarbons (MATH) technique was utilized to ascertain the hydrophobic
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59 174 nature of the microbes that were obtained [41]. Using this technique, overnight-grown microbial
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 175 suspensions in MRS medium were spun down by centrifugation at 6000 rpm for 8 min at the ambient
1
2 176 temperature. The cells were set to an absorbance of 0.55–0.60 at 600 nm (H0) after being twice rinsed
3
4
5 177 in phosphate buffer (pH 6.6). The hydrophobic properties of the cell surface were assessed using n-
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7 178 hexadecane, xylene, and chloroform as solvents. The bacterial suspension was mixed with solvents,
8
9 179 and the mixture was vortexed for one minute. Following ten minutes at laboratory temperature for
10
11
12 180 phase stabilization and separation, the optical density of the aqueous phase was calculated at 600 nm
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14 181 (Ht). The following formula was used to compute the hydrophobicity:
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16
17 H = ((H0 – Ht)/H0) × 100 (1)
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20 182 Auto-aggregation
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23 183 To determine the auto-aggregation, the method of Zuo et al. was used [42]. In brief, bacterial
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184 suspensions grown overnight in an MRS broth were harvested by centrifugation at 6000 rpm for 8 min
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28 185 at room temperature. The cells were washed twice using phosphate buffer (pH 6.6) and adjusted to
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30 186 the optical density of 0.55–0.60 at 600 nm (A0). For the auto-aggregation assay, each bacterial
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33 187 suspension (8 mL) was incubated at 37 °C, and the auto-aggregation values were measured at 6 hrs
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35 188 and 24 hrs (At) [43]. The values of auto-aggregation were calculated according to the following formula:
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37 A = (1 - At/A0) × 100 (2)
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39
40
41 189 Identification of Probiotic LAB Isolates Using 16S rRNA Gene Sequencing
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44 190 The isolates were molecularly characterized by obtaining the bacterial genomes [44] and performing,
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46 191 PCR-based transcription of 16S rDNA gene using forward primer (704F, 5’-GTAGCGGTGAAATGCGTAGA-
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48
49 192 3’) and reverse primer (907R, 5’-CCGTCAATTCMTTTRAGTTT-3’) [45].
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51
193 Analyses Phylogenetic
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54 194 The closest comparable sequence found in the non-redundant database was located utilizing BLAST
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57 195 using the 1463 bp consensus sequence. Selecting and aligning the top ten sequences was based on
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59 196 the maximum identity score. The neighbor-joining approach was used to estimate the evolutionary
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62 8
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 197 history [46]. Following the branches, the bootstrap values indicate the per cent replicated trees in
1
2 198 which the associated taxa formed a cluster, as determined by the bootstrap test (conducted with 1000
3
4
5 199 repetitions). The branch lengths are presented in identical components as the distances across
6
7 200 evolution used to generate the tree of phylogeny, and the tree is displayed to scale [47]. The
8
9 201 evolutionary distances are expressed in base substitutions per site and were calculated using the
10
11
12 202 greatest composite likelihood approach. Every position with incomplete data and gaps was removed.
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14 203 The evolutionary analyses were carried out employing MEGA11 software.
15
16
17 204 Statistical Analysis
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19
20 205 Experiments were performed in three batches, and findings were presented as mean standard
21
22 206 deviation (SD). The statistical software SAS (version 9.2, Raleigh, NC) was used to analyze the data
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25 207 using one-way ANOVA and post-hoc Duncan's test. The hierarchy of trees was generated with MEGA11
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27 208 (version 11.0). The level of significance was set at p < 0.05.
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30 209
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33 210 Results and Discussion
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35
36 211 Isolation Lactic Acid Bacteria Morphological, Biochemical, and Physiological Characterization
37
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39 212 An aggregate of 60 LABs was obtained from 10 traditionally fermenting Indian food items (Table 1). All
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41 213 strains were examined for morphology, while biochemical tests were performed with all isolates to
42
43 214 identify Lactobacillus (Table 2). Among them, 12 (20%) bacteria were found to be Gram-positive,
44
45
46 215 endospore-negative, and catalase-negative. Microscopic observations of these 12 isolates revealed
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48 216 their non-motile nature, exhibiting growth in the confined stab line, and were non-endospore forming.
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50
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217 The catalase test revealed 12 positive isolates, while other isolates were negative. Each species
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53 218 exhibited homofermentative behaviour, fermenting glucose into acid. Further, the identification of
54
55 219 probiotic bacteria by morphological and biochemical features was also reported by Menconi et al. [48]
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57
58 220 and Fontana et al. [49], where gram-positive, catalase-negative, oxidase-negative features were used
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60 221 as the first selective criteria in the detection of LAB as a probiotic.
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62 9
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 222 After incubating for 48 hrs, the strains displayed modest to robust multiplication on MRS agar. Multiple
1
2 223 isolates of LAB were identified from camel milk [50] and Garris (Sudanese traditional fermented camel
3
4
5 224 milk) [51]. Further, Hassanzadazar and Ehsani isolated 720 bacterial isolates from traditional Koopeh
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7 225 cheese [52]. Similarly, different strains of LAB were obtained from curd [53,54]. Most of these strains
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9 226 were non-spore-forming Gram-positive bacteria, while few were catalase-negative [54].
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11
12 227 Ability to Sustain % NaCl and Variable Temperature
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15 228 The data (Table 3) clearly show the ability of 12 isolates tested qualitatively to grow in MRS medium
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18 229 incubated at 15 °C and 45 oC. At 15 oC, 70% of isolates showed the ability to grow, whereas 83.3% of
19
20 230 strains thrived at 45 °C as evidenced by turbidity formed after 24 hrs. Every strain proliferated
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22 231 profoundly in another set of experiments at 4% NaCl levels. In comparison, the change in concentration
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25 232 to 2% and 6.5% reduced the capability to grow by 66.6% and 58.3%, respectively (Table 3).
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27 233 Our results conform with the earlier studies and indicate that the bacteria preferred high temperatures
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234 to grow and metabolize. Earlier studies also showed a similar trend, where 50% of the LAB obtained
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32 235 from the humped camel milk thrived at 40 °C, while only 0.5% of strains grew at 10 °C [55]. In another
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34 236 investigation, Menconi et al. reported that LAB strains ‘18 and 48d’ could grow at 15 °C and 45 °C at 2
35
36
37 237 and 4 hrs of incubation [48]. This demonstrates that few strains can withstand a broad spectrum of
38
39 238 temperatures yet persist and grow. Furthermore, the capacity of the isolates to reproduce at elevated
40
41 239 temperatures is a desirable trait since this is sometimes linked to elevated lactic acid generation.
42
43
44 240 Additionally, growing at increased temperatures reduces the infiltration from various microbes [56].
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46 241 Further, in another study, it has been reported that isolated lactic acid bacteria strain ‘18 and 48d’
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48 242 were able to grow in the presence of 3.5% and 6.5% NaCl up to 4 hrs, and that indicates the
49
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51 243 osmotolerance ability of LAB strains [48]. Previous authors have reported that LAB isolates can survive
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53 244 within the 25-40 °C range but cannot withstand extreme temperatures. The study isolates were used
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55
56
245 as poultry probiotics [57].
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58 246 In vitro Characterization of Probiotic Properties
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62 10
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 247 Probiotic assessments involve antibiotic resistance, antimicrobial compounds, coaggregation, auto-
1
2 248 aggregation, low bile, NaCl tolerance, and pH [58]. For a microbe to be considered a probiotic, it must
3
4
5 249 withstand acidic pH [59,60] and high bile salt concentration [61] and should show antimicrobial
6
7 250 resistance [62]. Hence, the 12 strains were evaluated through various probiotic tests.
8
9
10 251 Tolerance to Low pH
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12
13 252 The study assessed the viability of LAB isolates under low pH conditions resembling the stomach
14
15 253 environment, which is crucial for their endurance in acid trauma. The impact of acidity on the survival
16
17
18 254 of all 12 isolates was evaluated by their growth in various incubation pH levels in MRS broth. Figure 1
19
20 255 shows the acid tolerance of different isolates subjected to distinctive pH conditions (2, 4, and 8) for
21
22 256 varying durations (3 and 6 hrs) at 37 ºC; pH 7.0 is taken as a control. After 3 hrs of incubation at various
23
24
25 257 pH, it was detected that isolate SCD-2 showed maximum growth at different pH conditions; SCA-5
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27 258 showed poor growth at pH 2 and pH 8, while SMY-4 exhibited poor growth at pH 4. The remaining
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259 isolates reported average growth at pH 2 and good growth at pH 4 and 8. After 6 hrs of incubation,
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32 260 SCD-2 shows maximum growth at all pH. However, nine isolated displayed average growth; three
33
34 261 exhibited poor proliferation at pH 2. At pH 4, at least 10 strains showed excellent growth, and two
35
36
37 262 isolates showed above-average growth. Lastly, at pH 8, three isolates show decent growth, and nine
38
39 263 show mediocre expansion.
40
41
42 264 The current results suggest that the LAB isolates demonstrate robust endurance in the highly acidic
43
44 265 environments of the GI tract, taking into account the maximum passage interval of meals through the
45
46
47 266 GI tract, which is approximately 3 hrs [63]. Unlike LAB, Bifidobacterium spp. is susceptible to pH 2.0-
48
49 267 3.0 [49]. It can be inferred that LABs are inherently acidophilic. However, it is essential to distinguish
50
51 268 this from elevated concentrations of free acids (H+), as excessive free acids may lead to growth
52
53
54 269 inhibition [64]. Furthermore, the capability of the isolates to thrive at pH 1.5 indicates their ability to
55
56 270 endure the passage throughout the digestive tract, wherein the pH could fall as low as 1.5-2.0 [65].
57
58 271 This resilience ensures their 3 hrs or more viability before entering the GI tract [66]. Aswathy et al.
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60
61
62 11
63
64
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 272 examined the effect of salt concentration on LAB strains, revealing their optimal growth at pH 5, with
1
2 273 five isolates thriving well even at pH 2.5 [67]. In another study, LAB isolated from curd and cucumber
3
4
5 274 demonstrated excellent growth at all pH ranges tested [27]. The investigation into the pH tolerance of
6
7 275 LAB revealed that all evaluated isolates could endure incubation periods of 2-6 hrs at both pH 2.0 and
8
9 276 pH 3.0. However, there was a decrease in survival percentage with prolonged exposure time [68].
10
11
12 277 Tolerance to Bile Salts
13
14
15 278 Bile interferes with lipids and fats in the microbial membrane, reducing bacterial mortality rates [26].
16
17
18 279 The isolate's capacity to adapt to varying levels of bile acids aids the LAB in maintaining a healthy gut
19
20 280 microbiota [35]. To thrive, the bacteria must endure a gut bile concentration of 0.3% [30]. Figure 2
21
22 281 illustrates the isolated LAB's survival capabilities, demonstrating varying tolerance to salt
23
24
25 282 concentration, ranging from poor to high. In this study, at 3 hrs SCI-1 shows the highest tolerance at
26
27 283 2% bile salt concentration, and SMB-5 shows the least tolerance. At 4% salt concentration, SMC-2
28
29
284 shows the highest tolerance, while the least tolerance was observed in SML-6 and SMY-4. However,
30
31
32 285 after 6 hrs at 2% concentration, the growth of the isolate was approximately the same, while at 4%
33
34 286 bile concentration, the growth of the isolates was suppressed.
35
36
37 287 Bacteria that can endure bile salts may employ them for metabolism, proliferation, and establishment
38
39
288 in the gut, resulting in advantageous outcomes for the host [35]. Menconi et al. documented the bile
40
41
42 289 salt tolerance of ‘LAB18’ and ‘LAB48’ strains when cultivated at 0.4%, 0.5%, and 0.6% bile salt
43
44 290 concentrations during 2, 4, and 24 hrs of incubation [48]. Bile salt concentrations in the small intestine
45
46
47 291 range from 0.2% to 0.3%, reaching up to 2% (w/v) depending on diet and individual factors [66]. Bile
48
49 292 resistance in isolates has been associated with the enzyme bile salt hydrolase (BSH), which breaks
50
51 293 down bile acids and reduces their harmful properties [69]. This BSH function is frequently observed in
52
53
54 294 bacteria isolated from animal intestines or excrement [70]. Garriga et al. discovered that certain LAB
55
56 295 isolates exhibited resilience to 4% bile acids [71].
57
58
59 296 NaCl concentration
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61
62 12
63
64
65
 297 The GI tract contains both bile salts as well as significantly elevated levels of salt [72]. After 3 hrs of
1
2 298 incubation, bacterial isolates showed varied growth patterns at 4% NaCl concentration. The growth
3
4
5 299 rates of isolates SMB-3, SMB-5, SMS-1, and SCS-1 were comparatively higher than others, suggesting
6
7 300 that these isolates are more salt-tolerant Figure 3. In contrast, the growth rates of isolates SCA-5, SMC-
8
9 301 2, SMY-4, and SML-6 were comparatively lower, indicating a decreased capability to flourish at high
10
11
12 302 NaCl concentrations. Compared to other isolates, isolates SMB-3 and SMB-5 displayed comparatively
13
14 303 better growth at 8% NaCl, indicating their greater tolerance to increasing salt concentrations. At the
15
16
17
304 same salt concentration, SCA-5, SMC-2, SMY-4, and SMY-6 showed decreased growth, suggesting they
18
19 305 were susceptible to higher NaCl concentrations.
20
21
22 306 The capability of LAB strains to thrive at varying salt concentrations has been explored in different
23
24 307 studies. Jayakumar et al. investigated LAB strains from milk-based products and found that cell
25
26
27
308 proliferation ceased at a saline concentration of 8% [73];. In comparison, Aswathy et al. identified LAB
28
29 309 strains capable of growing at 12% NaCl concentration [67]. Ibourahema et al., studied LAB from the
30
31 310 poultry isolates of Senegal [74]. They found that the isolates Spo5 grew up to 7.5% NaCl concentration,
32
33
34 311 with others unable to grow beyond 5% salt concentration. Our findings align with a previous study
35
36 312 reporting bacterial isolates thriving at a 4–6% salt concentration. However, it is essential to note that
37
38 313 some LAB strains are highly susceptible to excessive salt quantities and cannot thrive in saline
39
40
41 314 concentrations exceeding 2% [75].
42
43
44 315 Salt tolerance is a critical attribute for LAB in various food applications. A study by Coulibaly et al.
45
46 316 emphasizes the significance of high salt tolerance in LAB for commercial purposes [37]. Elevated salt
47
48 317 concentrations in cultures can cause a reduction in turgor pressure, which affects enzymatic activities,
49
50
51 318 water metabolism, and general physiological functions of cells [37]. In fermentation processes, where
52
53 319 LAB produces lactic acid, an alkaline solution is frequently added to the culture media to counteract
54
55
56
320 acidic conditions. This process converts free acids to their salt forms, elevating the cells' osmotic
57
58 321 pressure [76].
59
60
61
62 13
63
64
65
 322 Antimicrobial Activities
1
2
3 323 An additional goal of the present investigation sought to discover LAB strains with antibacterial
4
5 324 properties. For instance, isolates SCS-1, SCU-3, SMB-3, SML-6, and SMS-1 possess limited inhibitory
6
7
8 325 activity against some bacterial strains (Table 4). Conversely, isolates with potent antimicrobial
9
10 326 potential, such as SCD-2, SMY-4, and SMY-6, display greater inhibitory zones (between 5.8 and 9.3
11
12 327 mm). These isolates demonstrate robust and consistent antibacterial effects against diverse test
13
14
15 328 strains. Remarkably, isolates such as SCI-1, SMC-2, and SMB-5 demonstrate varying degrees of
16
17 329 inhibition depending on specific bacterial strains, indicating their strain-specific antagonistic activities,
18
19 330 with inhibitory zone diameters ranging from mild to high against particular test strains.
20
21
22 331 It has been documented that LAB can prevent the proliferation of S. enteritidis, E. coli (O157:H7), and
23
24
25 332 C. jejun [48]. Further, potential probiotics such as L. reuteri, L. plantarum, L. mucosae, L. rossiae
26
27 333 extracted from fecal matter were tested for antimicrobial properties against many pathogens. This
28
29
334 study demonstrated that the cell-free extracts suppressed all pathogenic microbes except B.
30
31
32 335 hyodysenteriae [77].
33
34
35 336 Antibiotic Susceptibility Test
36
37
38 337 The antibiotic resistance profiles of different LAB isolates make it evident that most of them show
39
40 338 resistance (R) to streptomycin and some degree of resistance to kanamycin. The isolates generally
41
42 339 exhibit susceptibility towards tetracycline, amoxicillin, and gentamicin. Resistance to ampicillin and co-
43
44
45 340 trimoxazole among the isolates. Notably, there is variability in resistance to ampicillin and co-
46
47 341 trimoxazole. Except for sporadic variable resistance to kanamycin, most of the isolates, including SCI-
48
49
342 1, SCD-2, SCA-5, SCU-3, SMC-2, SMB-3, SMB-5, SML-6, and SMS-1, demonstrate susceptibility to the
50
51
52 343 majority of the antibiotics examined (Table 5). These isolates might be helpful in situations when
53
54 344 antibiotic susceptibility is essential. Conversely, isolates SCS-1, SMY-4, and SMY-6 exhibit more diverse
55
56
57 345 resistance patterns. They exhibit varying resistance levels to co-trimoxazole, amoxicillin, kanamycin,
58
59 346 and streptomycin.
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61
62 14
63
64
65
 347 Resistance to antibiotics in probiotics improves GI tract survivability following therapy with antibiotics.
1
2 348 In resistant bacteria, several resistance-related genes and their pathways remain debatable [78,79].
3
4
5 349 When probiotic microbes and antibiotic medications are administered together, probiotics must be
6
7 350 resilient to specific drugs to thrive in the GI tract [80,81]. It has been revealed that Lactobacillus species
8
9 351 have genes resistant to antibiotics and that plasmids and conjugative transposons are transferred to
10
11
12 352 and from LAB [82]. Most of the time, resistance to glycopeptides, aminoglycosides, and
13
14 353 sulfamethoxazole in LAB is linked to their inherent immunity resulting from membrane impenetrability,
15
16
17
354 supplemented by an efflux system [39,83,84]. Tetracycline, erythromycin, and ampicillin were effective
18
19 355 against LAB isolates [85,86]. Conversely, some LAB isolates were reportedly resistant to erythromycin
20
21 356 [87]. In a similar study, five Lactobacillus strains were found to be susceptible to kanamycin. At the
22
23
24 357 same time, seven Lactobacillus species exhibited resistance to erythromycin, nine strains were resilient
25
26 358 towards ampicillin, and eight specimens showed resistance to tetracycline [88].
27
28
29 359 Hydrophobicity percentage of the LAB isolates
30
31
32 360 Cell surface hydrophobicity and bacterial attachment to the surfaces are closely associated. The MATH
33
34 361 method is commonly utilized to measure the hydrophobic nature of the cell surface of LAB [89]. This
35
36
37 362 method assesses hydrophobicity by determining the microorganisms’ affinity for organic solvents.
38
39 363 Analysis of the data reveals varying levels of hydrophobicity among the isolates. Higher readings
40
41 364 indicate greater hydrophobicity, implying a stronger affinity for the tested hydrophobic solvents.
42
43
44 365 Greater hydrophobicity is indicated by higher readings, which also mean a larger affinity for the
45
46 366 hydrophobic solvents under test. Among the solvents investigated, isolates SCI-1, SCU-3, and SCD-2
47
48 367 exhibit comparatively higher hydrophobicity. SCU-3 has a comparable high affinity for n-hexadecane
49
50
51 368 (90.2±0.79) and chloroform (35.32±0.04), while SCI-1 shows notable hydrophobic in n-hexadecane
52
53 369 (91.44±0.43) and chloroform (71.14±0.47) Figure 4. The isolate SCD-2 displays moderate to high
54
55
56
370 hydrophobicity in all solvents examined. In contrast, isolates SML-6 and SMS-1 exhibit reduced
57
58 371 hydrophobicity in all solvents, indicating a lower affinity for these hydrophobic compounds. SML-6
59
60 372 consistently shows low hydrophobicity in n-hexadecane (11.72±0.09), xylene (20.56±0.11), and
61
62 15
63
64
65
 373 chloroform (18.88±0.11) among all solvents tested. SMY-4 demonstrates varying levels of
1
2 374 hydrophobicity in different solvents.
3
4
5 375 Probiotics’ propensity to bind to the epithelium has been examined in vitro by measuring their surface
6
7
8 376 hydrophobic nature towards organic solvents [90]. Jamaly et al., determined that the hydrophobicity
9
10 377 for Lactobacillus paracasei, Lactobacillus plantarum, and Lactobacillus brevis ranged from 37.80%-
11
12 378 85.67%, 21.06%-88.00%, and 76.33%, respectively [91]. Similarly, Heat treatment increased the
13
14
15 379 hydrophilic nature of LAB [92]. Furthermore, using various organic solvents, LAB isolated using dairy
16
17 380 items has shown varying degrees of hydrophilic properties [73].
18
19
20 381 Auto Aggregation studies
21
22
23 382 A possible method for adherence assessment is auto-aggregation screening, which has proved to be
24
25 383 significantly more accurate and economical than cell lines [93]. Auto-aggregation is an important
26
27
28 384 probiotic trait which helps in the development of biological niches, particularly in the gastrointestinal
29
30 385 tract of humans. Bacterial aggregation is based on auto-aggregation and the strain's hydrophobic
31
32 386 properties [94]. The isolates’ propensities for auto-aggregation at the two time points differed
33
34
35 387 noticeably upon data analysis. Isolates SMC-2 and SCD-2 show comparatively higher auto-aggregation
36
37 388 after the 5 hrs, indicating noticeably stronger inclinations to form aggregates than other isolates. On
38
39
389 the other hand, at this early stage, isolates SCA-5, SMB-3, SML-6, and SCS-1 show the least amount of
40
41
42 390 auto-aggregation Figure 5. For most isolates, auto-aggregation has significantly increased by the 24 hrs
43
44 391 mark. Notably, isolates SCD-2 and SMY-4 show increased auto-aggregation after 24 hrs incubation,
45
46
47 392 although showing only mild levels at 5 hrs. At later periods, isolates SCI-1 and SMY-2 show significant
48
49 393 increases in auto-aggregation, indicating a stronger tendency to form aggregates. Even after 24 hrs,
50
51 394 isolate SMC-2, which showed a comparatively strong auto-aggregation after 5 hrs, can still exhibit
52
53
54 395 remarkable auto-aggregation skills. On the other hand, isolates SCA-5, SMB-3, and SML-6 exhibit a
55
56 396 limited rise in auto-aggregation throughout 24 hs, maintaining comparatively lower levels. The
57
58 397 isolates' varying auto-aggregation inclinations indicate variations in their surface characteristics and
59
60
61
62 16
63
64
65
 398 aggregate-forming capacities. Collado et al. (2007) [92] indicated that auto-aggregation of LAB is
1
2 399 associated favourably with their adherence potential. The higher the rate of auto-aggregation, the
3
4
5 400 more effectively probiotics can attach to cells in the epithelium and promote therapeutic advantages
6
7 401 [95].
8
9
10 402 Identification of Probiotic LAB Isolates by 16S rRNA Gene Sequencing
11
12
13 403 We performed molecular identification of the four isolates (SCS-1, SMB-5, SMY-4, SMS-1) that
14
15 404 performed comparatively better than other isolates. For this, we amplified 16S rDNA amplification
16
17
18 405 obtained by using universal primers supplementary Figure A1. The BLAST and phylogenetic tree
19
20 406 analysis revealed that strain SCS-1 closely resembles the Lactobacillus acidophilus genus Figure 6A.
21
22 407 The isolates SMB-5 and SMS-1 are strongly linked to L. delbruckii species Figure 6B & Figure 6C,
23
24
25 408 whereas strain SMY-4 is closest associated with L. rhamnosus species. Figure 6D shows all the
26
27 409 recognized strains collectively referred to as LAB. Praet et al. cultured Lactobacillus sp. and Weisella
28
29
410 sp. from honeybee guts [96]. In a prior investigation, enterococcus sp. was identified from H. itama
30
31
32 411 [97]. In addition, Lactobacillus bacteria with putative probiotic characteristics were found in the feces
33
34 412 of nursing piglets employing 16S rRNA gene profiling [98]. Similarly, another investigation utilized
35
36
37 413 phylogenetic analysis of the 16S rDNA sequences to identify strains of different LAB with robust
38
39 414 probiotic characteristics [99]. In our study, the isolate could metabolize each of the sugars evaluated
40
41 415 (Table 2), following an earlier study [100]. The colony morphology, culture conditions, and biochemical
42
43
44 416 parameters of L. plantarum strain GCC_19M1 were similar to previously described beneficial strains of
45
46 417 L. plantarum obtained from a broad range of processed foods [101,102].
47
48
49 418
50
51
52 419 Conclusion
53
54
55 420 In our pursuit of identifying unique probiotics suitable for human and animal use, we focused on
56
57 421 isolating LAB strains, particularly Lactobacilli and Bifidobacteria, with potential probiotic
58
59
422 characteristics. In this investigation, we effectively isolated, identified, and characterized seven LAB
60
61
62 17
63
64
65
 423 strains from fermented foods. These LAB isolates exhibit promising attributes that position them as
1
2 424 viable probiotic sources for humans and animals. Furthermore, their potential application extends to
3
4
5 425 serving as starting cultures in food industries. Our investigation's findings underscore these strains'
6
7 426 significant potential in enhancing human health and well-being, suggesting their suitability for future
8
9 427 incorporation into probiotic formulations. Nevertheless, additional research is imperative to assess the
10
11
12 428 in-vivo probiotic efficacy of LAB strains comprehensively. Through continued exploration and
13
14 429 validation, we anticipate these probiotic cultures will emerge as valuable assets for the food industry,
15
16
17
430 offering additional health benefits beyond conventional probiotic strains. Thus, this study lays a solid
18
19 431 foundation for future endeavours aimed at harnessing the probiotic potential of LAB to improve
20
21 432 human and animal health.
22
23
24 433
25
26
27 434 Author Contributions Conceptualization, S.J. and N.K.J.; methodology, S.J. and N.K.J.; software,
28
29
435 N.K.J.; validation, D.W.; formal analysis, N.K.J.; investigation, S.J.; resources, N.K.J.; data curation, S.J.
30
31
32 436 and N.K.J.; writing—original draft preparation, S.J.; writing—review and editing, M.S.A. and S.P.;
33
34 437 visualization, S.J.; supervision, N.K.J.; project administration, N.K.J. All authors have read and agreed
35
36
37 438 to the published version of the manuscript.
38
39
439
40
41
42 440 Data Availability Statement All data generated or analyzed during this study are included in this
43
44
45 441 article.
46
47
48 442
49
50
51
443 Acknowledgments The authors thank the Department of Biotechnology, Sharda University,
52
53 444 Greater Noida, for supporting vital infrastructure. This support encompasses facilities, equipment, and
54
55 445 resources for their research and academic activities. The authors acknowledge the support through
56
57
58 446 the Researchers Supporting Project number (RSPD2024R724), King Saud University, Riyadh, Saudi
59
60 447 Arabia.
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62 18
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65
 448 This work is partially funded by Universitas Negeri Malang under Ministry of Education, Culture,
1
2 449 Research and Technology of Indonesia.
3
4
5 450
6
7
8 451 Declarations
9
10
11 452 Conflict of interest The authors declare no conflict of interest.
12
13
14 453 Ethical Approval Not applicable as the study does not imply the use of any human or animal samples.
15
16 454 This article does not include any studies on human participants or animals conducted by any of the
17
18
19 455 authors.
20
21
22
456
23
24
457 Appendix A Supplementary Fig. A1 16S rDNA amplification was obtained using universal primers
25
26
27 458 (M-Axygen 1Kb Ladder DNA marker, 1-4 are the selected LAB isolates and C- Lactobacillus DNA as
28
29 459 control
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32 460
33
34
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35 800
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 805 Fig. 2 Bile salt tolerance patterns of probiotic LAB at different Bile salt (%) values after 3 and 6 h
1
2 806 exposure
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 819 Fig. 3 NaCl concentration tolerance patterns of probiotic LAB at different NaCl (%) values after 3 and
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2 820 6 h exposure
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 834 Fig. 4 Hydrophobicity exhibits the cell adhesion ability of the isolates in n-hexadecane, xylene and
1
2 835 chloroform
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 848 Fig. 5 The isolates auto-aggregation percentage after 5h and 24 h of incubation
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55 862 Fig. 6 Phylogenetic tree of isolates, generated by aligning 16S rRNA gene sequence of the isolates
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57 863 with the database sequence. Using the Maximum-likelihood method (MEGA 11) the phylogenetic trees
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60 864 were constructed of isolates, (a) SCS-1, (b) SMB-5, (c) SMY-4, (d) SMS-1
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 865
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13 871 TABLES
14
15 872 Table 1 Isolation of different probiotic micro-organisms from various fermented products
16
17
18 FERMENTED CEREAL-BASED PRODUCTS FERMENTED MILK PRODUCTS
19
20 Sources of Sample Isolate Number Growth on MRS Agar Sources of Sample Isolate Number Growth on MRS Agar
21
22 SCI-1 +++ SMC-1 +++
23
24 SCI-2 +++ SMC-2 +++
25
26 SCI-3 ++ SMC-3 +++
27 Idli Curd
28 SCI-4 +++ SMC-4 ++
29
30 SCI-5 ++ SMC-5 ++
31
32 SCI-6 +++ SMC-6 +++
33
34 SCD-1 +++ SMB-1 ++
35
SCD-2 +++ SMB-2 +++
36
37
SCD-3 +++ SMB-3 +++
38
Dhokla Butter milk
39 SCD-4 ++ SMB-4 +++
40
41 SCD-5 +++ SMB-5 ++
42
43 SCD-6 ++ SMB-6 +++
44
45 SCA-1 ++ SML-1 ++
46
47 SCA-2 +++ SML-2 +++
48
49 SCA-3 +++ SML-3 +++
50 Appam Lassi
51 SCA-4 ++ SML-4 ++
52
53 SCA-5 +++ SML-5 +++
54
55 SCA-6 +++ SML-6 ++
56
57 SCS-1 +++ SMS-1 ++
58 Sourdough Bread Shri Khand
59 SCS-2 +++ SMS-2 ++
60
61
62 40
63
64
65
 SCS-3 ++ SMS-3 +++
1
2 SCS-4 ++ SMS-4 +++
3
4 SCS-5 +++ SMS-5 ++
5
SCS-6 +++ SMS-6 ++
6
7
SCU-1 ++ SMY-1 ++
8
9
SCU-2 +++ SMY-2 +++
10
11
SCU-3 ++ SMY-3 ++
12
Uthappam Yoghurt
13
SCU-4 +++ SMY-4 +++
14
15
SCU-5 ++ SMY-5 +++
16
17
SCU-6 +++ SMY-6 ++
18
19
20 873
21
22 874
23
24
25 875
26
27
876
28
29
30 877
31
32
33 878
34
35 879
36
37
38 880
39
40 881
41
42
43 882
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45
46 883
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48 884
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51 885
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53 886
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56 887
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888
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62 41
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 889
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5 891
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7
8 892
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10 893
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13 894
14
15
895 Table 2 Morphological, Biochemical, and Physiological Characterization of isolates
16
17
18 Isolate No. Gram Reaction Shape Motility Endospore test Catalase Test Sugar Fermentation
19
20 SCI-1 +ve Rods - - - H
21
22 SCD-2 +ve Rods - - - H
23
24 SCA-5 +ve Rods - - - H
25
26 SCS-1 +ve Rods - - - H
27
28 SCU-3 +ve Rods - - - H
29
30 SMC-2 +ve Rods - - - H
31
32 SMB-3 +ve Rods - - - H
33
34 SMB-5 +ve Rods - - - H
35
36 SML-6 +ve Rods - - - H
37
38 SMS-1 +ve Rods - - - H
39
40 SMY-4 +ve Rods - - + H
41
SMY-6 +ve Rods - - - H
42
43
44 896
45
46 897
47
48
49 898
50
51
52 899
53
54 900
55
56
57 901
58
59 902
60
61
62 42
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 903
1
2
3 904
4
5 905
6
7
8 906
9
10 907
11
12
13 908
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15
909 Table 3 Ability to Sustain % NaCl and Variable Temperature
16
17
18 Gas Ammonia
19 2% 4% 6.5%
20 Isolate No. from from Growth at 15 °C Growth at 45 °C
21 NaCl NaCl NaCl
22 Glucose Arginine
23
24 SCI-1 + - + + - + +
25
26 SCD-2 - + + + + - +
27
28 SCA-5 - + - + + + -
29
30 SCS-1 + + + + - + +
31
32 SCU-3 - + + + - + +
33
34 SMC-2 + + - + + + +
35
36 SMB-3 + - + - + - +
37
38 SMB-5 - + - + + + +
39
40 SML-6 - + + + - + +
41
SMS-1 + + + - + + -
42
43
SMY-4 - + + + - - +
44
45
SMY-6 + + - + + + +
46
47
48 910
49
50 911
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52
53 912
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55 913
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58 914
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60
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62 43
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 915
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3 916
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5 917
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7
8 918
9
10 919
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12
13 920
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15
16
17
921 Table 4 Antimicrobial activities of LAB against some food-borne pathogens using zones of inhibition
18
19 922 (mm ± S.D.). The mean ± SD values were significant at p < 0.05
20
21
22 S. No LAB Isolates E. coli S. aureus E. faecalis B. cereus B. subtilus P. aeruginosa P. alcaligenes Klebsiella sp.
23
24 1 SCI-1 7.6±0.7 e,f,g 5.6±0.52 d,e 6.7±0.61 f 5.9±0.55 d 4.7±0.44 b,c 6.5±0.6 c,d 7.2±0.66 c,d 4.9±0.45 b,c,d
25
26 2 SCD-2 7.2±0.66 e,f 5.4±0.49 c,d 5.6±0.52 f 4.8±0.44 c 6.7±0.62 e 9±0.83 f 8.8±0.81 e,f 7.9±0.73 c,d
27
28 3 SCA-5 5.9±0.55 c,d 4.1±0.38 b 1.9±0.18 b 3.7±0.34 b 5.6±0.51 c,d 6.3±0.58 b,c,d 7.1±0.65 b,c,d 5.2±0.48 f
29
30 4 SCS-1 0±0 a 2.9±0.27 a 3±0.28 c 4.8±0.44 c 5.4±0.49 b,c,d 5.6±0.51 b,c 7.7±0.71 c,d,e 9.3±0.86 g
31
5 SCU-3 6.8±0.63 d,e,f 7.7±0.7 f 0±0 a 2.3±0.21 a 2.9±0.27 a 4±0.37 a 5.4±0.49 a 4±0.37 b
32
33
6 SMC-2 7.7±0.71 f,g 6.5±0.6 e 6.6±0.6 f 8.5±0.78 e 7.4±0.68 e 8.4±0.77 f 7.9±0.73 c,d,e 6.9±0.64 e
34
35
7 SMB-3 6.6±0.6 c,d,e 5.9±0.55 d,e 3.9±0.36 d 6.3±0.58 d 4.9±0.45 b,c,d 6.5±0.6 c,d 8.3±0.76 d,e,f 0±0 a
36
37 8 SMB-5 7.4±0.68 e,f,g 6.6±0.6 e 3.4±0.31 c,d 4.9±0.45 c 5.6±0.52 d 7.2±0.66 d,e 5.9±0.55 a,b 5.4±0.49 c,d
38
39 9 SML-6 4.1±0.38 b 4.7±0.44 c,d 0±0 a 2.1±0.19 a 4.5±0.41 b 6.3±0.58 b,c,d 7±0.65 b,c 4.5±0.41 b,c
40
41 10 SMS-1 0±0 a 2.9±0.27 a 2.3±0.21 b 4.2±0.39 b,c 3.6±0.33 a 5.2±0.48 b 5.9±0.55 a,b 5.3±0.49 c,d
42
43 11 SMY-4 8.3±0.76 g 7.5±0.69 f 7.4±0.68 g 8.7±0.8 e 6.6±0.6 e 8.1±0.75 e,f 9.3±0.86 f 5.8±0.54 d
44
45 12 SMY-6 5.7±0.53 c 4.5±0.41 b 4.7±0.44 e 7.8±0.72 e 6.7±0.61 e 8.4±0.77 f 7.6±0.7 c,d 5.8±0.54 d
46
47
48 923
49
50
51 924
52
53
54 925
55
56
57
926
58
59 927
60
61
62 44
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65
 928
1
2
3 929
4
5 930
6
7
8 931
9
10 932
11
12
13 933
14
15
934 Table 5 Antibiotic Resistance pattern of isolates against different antibiotics
16
17
18 S. No LAB Isolates Tetracycline Amoxycillin Gentamycin Kanamycin Co-trimoxazole Ampicillin Streptomycin
19
20 1 SCI-1 S S S R S S R
21
22 2 SCD-2 S S R R R S R
23
24 3 SCA-5 S S S R S S R
25
26 4 SCS-1 S R R R R S R
27
28 5 SCU-3 S S S R S S R
29
30 6 SMC-2 S S R R R S R
31
32 7 SMB-3 S S S R S S R
33
34 8 SMB-5 S S S R S S R
35
36 9 SML-6 S S S R S S R
37
38 10 SMS-1 S S S R S S R
39
40 11 SMY-4 S R R R R S R
41
42 12 SMY-6 S R R R R S R
43
44 935
45
46
47 936
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50 937
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52 938
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55 939
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940
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60 941
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949 Supplementary Fig. A1 16S rDNA amplification obtained by using universal primers (M-Axygen 1Kb
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55 950 Ladder DNA marker, 1-4 are the selected LAB isolates and C- Lactobacillus DNA as control
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Declaration of Interest Statement

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☒ The author is an Editorial Board Member/Editor-in-Chief/Associate Editor/Guest Editor Chemical

Physics Impact and was not involved in the editorial review or the decision to publish this article.

☒ The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:

There is no conflict of interest

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