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In vitro screening of probiotic properties of Lactobacillus plantarum isolated from fermented milk product

In vitro screening of probiotic properties of Lactobacillus plantarum isolated from fermented... Key words: raw milk; probiotic; Lactobacillus plantarum; gastric juice; auto-aggregation; hydrophobicity. reuteri (L. reuteri), and Lactobacillus rhamnosus (L. rhamnosus) (de Introduction Vos, 2011; Ahmed et al., 2019). Probiotic strains of Lactobacilli par- Probiotics are live microorganisms that, when consumed in suffi- ticularly adhere to the GI tract of the host cells, which is dependent cient amounts by the host, confer health benefits. Elie Metchnikoff, on their aggregation ability (Goh and Klaenhammer, 2010). They while working at the Pasteur Institute in Paris, first pointed out also act as a barrier to prevent the adherence of pathogenic bac- the health benefit of lactic acid bacteria (LAB) present in yogurt teria, by interfering with colonization and proliferation of patho- (Metchnikoff, 1907). Recent studies claimed that probiotics exhibit gens, thereby preventing the manifestation of infections (Patrignani an indispensable role in the treatment of diseases such as diarrhoea et  al., 2019). Lactobacilli species also produces antimicrobial pep- (Lai et  al., 2019), allergy (Huang et  al., 2016), diabetes (Soleimani tides (e.g. bacteriocins), organic acids (e.g. acetic acid and lactic et al., 2017), hypertension (Toral et al., 2018), cancer (Brady et al., acid), and other metabolites (e.g. hydrogen peroxide), which are 2000), and genetic disorders (Kiely et  al., 2017; Toral et  al., 2019) reported to inhibit the growth of pathogenic bacteria (Li and Gu, and also boost immunity (Tsai et  al., 2012). The probiotic micro- 2018). Probiotic strains of L. plantarum also participate in the ab- organism should be acid-resistant, bile-tolerant, non-carcinogenic, sorption of water and sodium in the colon and decrease diarrhoeal non-pathogenic, adhere to host epithelial tissue, enrich the intestinal symptoms (Mortensen and Clausen, 1996). They also exert holistic microflora, reduce pathogenic adherence, and able to produce of sec- health benefits for medical conditions such as psoriasis, allergies, and ondary metabolites antagonistic to pathogen microorganisms (Pan nervous system diseases (Hessle et al., 1999; Das and Goyal, 2015; et al., 2009; Prabhurajeshwar and Chandrakanth, 2017). Beneficial Kim et al., 2015). microorganism interacts with the raw food (substrate) and produces The excessive application of antibiotics, immunosuppressive valuable fermentation product of medical advantages such as en- therapy, and irradiation may alter the gut composition and adversely zymes and vitamins. Moreover, the consumption of probiotic mi- affect the gut microbiota. Consequently, the introduction of pro- crobes produces a protective effect on the gut environment. It is to biotic bacteria in the GI tract has recently become an effective op- be noted that probiotic impacts are strain-dependent, and hence, not tion to ascertain a healthy microbial equilibrium. The US Food and all strains are effective for efficient fermentation and treatment of Drug Administration is researching a way to administer live micro- all diseases. organisms in animal and human feeds, which is termed as ‘direct- The most familiar and recognized LAB belongs to the genus fed microbial’ (Sanders, 2008). A study conducted by Katafiasz and Lactobacillus, which are conventionally used in the fermentation of Bartlett (2012) in Michigan reported the beliefs associated with the vegetables, fruits, and milk (Saito et al., 2019). Lactobacilli are also consumption of raw milk. The consumers choose to drink raw milk present as a normal mucosal microbiota of humans and animals. for the desired flavour and as a means of support to the local sus- They are primarily facultative or anaerobic, non-pathogenic, and tainable farms. Some strains of L. plantarum have a preventive effect prefer an acidic condition of the gastrointestinal (GI) tract. Studies on milder GI symptoms associated with antibiotic treatment, and a have shown that consumption of probiotic Lactobacilli maintains GI continuous intake may exert a protective effect (Lönnermark et al., health and prevents intestinal infections (Yue et al., 2020). The most 2010). commonly used Lactobacillus species in probiotic settings include It has been previously reported that L. plantarum can tolerate pH Lactobacillus acidophilus (L. acidophilus), Lactobacillus plantarum 3.0 and below, after 2 h of exposure (Mathara et al., 2008). They are (L. plantarum), Lactobacillus casei (L. casei), Lactobacillus paracasei able to survive in the presence of bile salts, pancreatin, and pepsin (L. paracasei), Lactobacillus johnsonii (L. johnsonii), Lactobacillus Downloaded from https://academic.oup.com/fqs/article/4/4/213/5918439 by DeepDyve user on 22 December 2020 Probiotic strain of L. Plantarum in fermented milk, 2020, Vol. 4, No. 4 215 (Maragkoudakis et al., 2006; Mahmoudi et al., 2016). In vitro ad- Clustal W. A phylogenetic tree was constructed using PhyML, using hesion of L.  plantarum to the mucus-secreting cell lines has been Geneious R8 software package (Biomatters Ltd, Auckland, New reported by Zoumpopoulou et  al. (2018). Lactobacillus plantarum Zealand) (Nath et al., 2019). exhibits a diverse susceptibility to commonly used antibiotics, pos- sesses antibacterial activity against pathogens, and is non-haemolytic Screening of probiotic properties of isolated (Jiang et  al., 2016; Mahmoudi et  al., 2016; Zoumpopoulou et  al., bacteria 2018). Test for pH tolerance In context to our studies, it has been observed that the most fre- In vitro assay to determine the resistance of isolates to the acidic quently used fermented dairy product in rural areas of Cachar dis- condition of the stomach is an essential attribute in probiotic selec- trict of Assam, India is curd, skimmed milk, and fermented raw milk. tion. As the food remains in the stomach for at least 3 h (Thakkar The traditional belief of consumption of fermented raw milk and the et  al., 2015), this time limit was taken under consideration for pH role of indigenous bacteria to improve digestion, relieving constipa- tolerance test. pH tolerance was determined by inoculating 0.5  ml tion, and to maintain good health remains scientifically unexplored. of 20-h-old broth culture to 5 ml of phosphate buffer saline (PBS), Raw milk usually refers to unpasteurized, non-homogenized, or un- adjusting the pH to 3 and 7.2, with 1N HCl. The sample was then processed milk. Many studies reported that fermented milk acts as a incubated aerobically at 37 °C for 3 h, and their total viable count carrier for L. plantarum strains and shows a higher bacterial load at was measured at every 1 h interval by spreading 100 µl of bacterial the time of consumption. However, there is a possible reduction of suspension on the MRS agar plate. Optical density (OD 600  nm) the probiotic bacteria during passage through the GI tract, so a thera- was recorded at a regular interval to determine their viability and 8 9 peutic daily dose of 10 –10 CFU/g is recommended (Kailasapathy growth pattern (Hassanzadazar et al., 2012). and Rybka, 1997; Shah, 2000). Some studies also report the ques- tionable benefit of fermented raw milk on human health (Wouters Simulated gastric juice tolerance test et al., 2002; Claeys et al., 2013). Therefore, the study aims to isolate Simulated gastric juice was prepared by using 3 g/L pepsin, 7 mM and identify bacteria from fermented raw milk samples that exhibit KCl, 45 mM NaHCO , and 125 mM NaCl, adjusting at pH 3 (assay) health benefits upon consumption. and pH 7 (control) with 1 M HCl and 1 M NaOH, respectively (Archer and Halami, 2015). Overnight grown bacterial broth cul- ture was taken and centrifuged at 5000 rpm at 5 °C for 15 min. The Materials and Methods bacterial pellet was re-suspended in 10 ml PBS buffer, followed by Isolation and characterization of bacteria from incubation in simulated gastric juice (both assay and control). The milk sample viable cell counts at 1, 2, and 3  h were recorded, and the survival Collection of samples and isolation of bacteria percentage of the isolate was determined by the following formula: Fermented milk samples were aseptically collected in a pre-sterilized container and immediately bought to the laboratory. Milk samples CFU assay Bacterial survival rate (%) = × 100 –3 were serially diluted (10 fold); 0.1  ml of aliquot was poured on CFU control freshly prepared Man Rogosa Sharpe (MRS) agar plates and incu- bated at 30 °C for 24 h (Khagwal et al., 2014). Isolated distinct col- onies were further sub-cultured on freshly prepared MRS agar plates Bile tolerance test to obtain the pure culture. The bile tolerance test was conducted following the protocol of Gilliland et al. (1984). One hundred microlitres of overnight grown bacterial culture was inoculated in freshly prepared MRS broth con- Morphological and biochemical characterization of bacteria taining 0.3% bile salts (Himedia Pvt. Ltd). Test isolates were also Preliminary identification of the isolates was performed by colony inoculated in MRS broth without bile, which acts as a control. Both morphology, cultural characteristics, and microscopic observa- the test tubes (with and without bile) containing test isolates were in- tions. The isolates that were gram-negative, non-spore forming, cubated at 37 ºC for 4 h, and their growth at a different time interval and catalase-negative were selected and screened by biochem- and percentage resistance was noted by measuring the absorbance ical tests, which include indole production test, methyl red test, of MRS broth at 600  nm. After the incubation period of 4  h, the Voges–Proskauer test, citrate utilization test, oxidase test, starch hy- viability of bacteria in 0.3% bile was also evaluated by spreading drolysis test, and carbohydrate fermentation test (Holt et al., 1994; 100 µl of the bacterial sample onto the MRS agar plate. Cappuccino and Sherman, 1996). Pancreatin tolerance test Molecular identification One hundred microlitres of overnight grown bacterial culture was Molecular characterization of the isolate was done by extracting inoculated in 10 ml of MRS broth containing 0.5% (v/w) pancreatin the genomic DNA, following the protocol of Green and Sambrook and without pancreatin (control). Inoculated test tubes were kept in (2012). Thereafter, polymerase chain reaction (PCR) amplifica- a shaker incubator for 48 h at 37 °C. Pancreatin tolerance was de- tion of 16S rDNA gene was performed by forward primer (704F, termined by measuring the OD (at 600 nm) at an interval of 0, 24, 5′-GTAGCGGTGAAATGCGTAGA-3′) and reverse primer (907R, and 48 h. The viable cell count of test and control cultures in MRS 5′-CCGTCAATTCMTTTRAGTTT-3′) (Madison et  al., 2017). agar plates after 48 h of incubation was also determined (Khagwal Sequencing of the amplified gene was performed at Xcelris Labs et al., 2014). Limited, Gujarat, India using ABI 3730xl 96 capillary system with Big Dye Terminator v3.1 kit. The 1463 bp consensus sequence was used to perform BLAST for finding the closest homologous sequence Assessment of cell surface hydrophobicity present in the non-redundant database. Based on the maximum The ability of the bacteria to stick with hydrocarbons determines identity score, the first 10 sequences were selected and aligned using the extent of adhesion to the epithelial cells in the GI tract known Downloaded from https://academic.oup.com/fqs/article/4/4/213/5918439 by DeepDyve user on 22 December 2020 216 S. Nath et al., 2020, Vol. 4, No. 4 as cell surface hydrophobicity. To determine cell surface hydropho- Antibiotic susceptibility test bicity of the isolate, 20-h-old broth culture was centrifuged at 12 Antibiotic susceptibility test was performed by Kirby–Bauer disk 000 rpm for 5 min, and the cell pellets were collected. Cell pellets diffusion method using Mueller-Hinton agar (MHA) media (Baccer were washed twice with PBS buffer (pH 7.2) and re-suspended in et  al., 1966). Bacterial samples were spread and antibiotic discs 6  ml of PBS buffer, followed by measuring the initial absorbance were placed equidistantly onto the surface of freshly prepared MHA 600 nm. Thereafter, 3 ml of the bacterial suspension was mixed with plates. The plates were incubated at 37 °C for 24–48 h and thereafter, 1 ml of hydrocarbons (n-Hexadecane and Toluene) and vortexed for the diameter of the zone of inhibition was measured. This antibiotic 2 min. The mixture was incubated and left undisturbed for 1 h for resistance property of the isolate was classified according to the phase separation. The aqueous phase was then removed carefully, guidelines of the Clinical and Laboratory Standards Institute (CLSI, and the final absorbance was recorded at 600 nm (Rosenberg et al., 2016). The antibiotics used in the present study include Gentamicin 1980). The rate of cell surface hydrophobicity was calculated by the (120  µg), Vancomycin (30  µg), Tetracycline (30  µg), Polymixin-B formula: (300  µg), Kanamycin (30  µg), Ofloxacin (5  µg), Co-trimoxazole (25  µg), Meropenem (10  µg), Ceftriaxone (30  µg), Clindamycin OD − OD (2 µg), Ampicillin (10 µg), Norfloxacin (10  µg), Rifampicin (5 µg), initial final Rate of hydrophobicity (%) = × 100 OD Amikacin (30 µg), Penicillin-G (10 µg), Cefdinir (5 µg), Ciprofloxacin initial (5 µg), Azithromycin (15 µg), Methicillin (5 µg), and Streptomycin (10 µg), which were procured from ‘HiMedia’. Cellular auto-aggregation assay The specific cell–cell interaction, known as auto-aggregation, was Extraction of antibacterial agents and evaluation of their determined by the method described by Xu et al. (2009). The over- antagonistic activity night grown bacterial broth was centrifuged at 5000 rpm for 10 min Isolation of antibacterial agents was performed by following the to harvest the cell pellets. Pellets were repeatedly washed with PBS protocol of Hussein et  al. (2018). About 5  ml of 48-h-old bac- (pH 7.2), re-suspended in PBS buffer, and the initial absorbance was terial isolates GCC_19M1 was mixed with an equal volume of noted at 600 nm. The bacterial suspension was incubated at 37 °C ethyl acetate and shaken at 20 rpm for 10 min in a rotary shaker. for 2 h, and the final absorbance of the supernatant was measured at The sample was centrifuged, and the supernatant was transferred 600 nm. The percentage of cellular auto-aggregation was measured into a fresh tube to evaporate the ethyl acetate. The remaining con- by the formula: tent was used to study the antagonistic effect on the selected test pathogen. OD − OD initial final The test pathogen includes Bacillus cereus (MH482928), Auto-aggregation rate (%) = × 100 OD initial Acinetobacter johnsonii (MH482927), Pseudomonas aeruginosa (MN066610), Stenotrophomonas maltophilia (MN066611), Cedecea davisae (MN066609), and Achromobacter spanius Glucose fermentation test (MK000623), obtained from the culture collection of Institutional Glucose fermentation test was performed by following the methods Biotech Hub, Gurucharan College, Silchar. of Gupta et  al. (2011). Eighteen-hour-old bacterial cultures were The effectiveness of bacterial metabolites was studied by well centrifuged at 4000  rpm for 15  min, and the bacterial pellets diffusion method (Shakhatreh et  al., 2017). MHA plates of each were recovered. The pellets were washed twice by PBS buffer and test pathogen were prepared, and wells were made using a sterile re-suspended in the PBS buffer. Thereafter, 500 µl of PBS buffer con- cork borer. The wells were loaded with aliquots of 60  μl of the taining the bacterial cells were inoculated into MRS broth supple- supernatant and incubated at 37  °C for 24  h. Dimethyl sulfoxide mented by 1% glucose and 0.5% phenol red (dye) and incubated was used as a control. The diameters of the zones of growth inhib- at 37 °C for 24 h. Change in colour from purple to yellow indicates ition were measured and classified as sensitive (<20 mm), moderate positive glucose fermentation, whereas no change in colour indicates (10–20 mm), and low or resistant (>10 mm) (Prabhurajeshwar and negative glucose fermentation. Chandrakanth, 2017). Statistical analysis NaCl tolerance test All the experiments were performed in triplicates, and the results MRS agar plates were prepared in eight different concentrations of were expressed as the means ± standard deviations of three inde- NaCl (0%, 0.5%, 1%, 5%, 7.5%, 10%, 12.5%, and 15%). Isolated pendent replicates. The gathered data were analysed using Microsoft bacteria were streaked in each plate and incubated at 37 °C for 24 h. Excel 2007 and SPSS version 16. The influence of NaCl concentrations on the degree of inhibition of bacterial growth was recorded. Results Biochemical and molecular identification Safety assessment and antimicrobial activity The present study reports gram-negative rod-shaped bacteria, Haemolytic activity showing white and creamy colonies with a jelly-like surface on MRS Haemolytic activity is a determining factor for probiotic bacteria, agar plates without pigmentation. The growth characteristic was ob- and the absence of haemolytic activity indicates that the particular served at the temperature range of 10–40 °C. The gram-negative and bacteria were none virulent. Bacterial culture was streaked on sheep rod-shaped isolates showed negative results for indole test, methyl blood agar and incubated at 37 ºC for 48 h. The zone formed around red test, Voges–Proskauer test, citrate utilization test, oxidase test, the colonies was observed, which is categorized as β-haemolysis and catalase test (Table 1). BLAST result showed that the 16S rDNA (clear zone), α-haemolysis (green-hued zone), or γ-haemolysis (no sequence of the isolated bacteria has 98–99% identity and 98% zone; Gerhardt et al., 1981). Downloaded from https://academic.oup.com/fqs/article/4/4/213/5918439 by DeepDyve user on 22 December 2020 Probiotic strain of L. Plantarum in fermented milk, 2020, Vol. 4, No. 4 217 query coverage with the strains of Lactobacillus plantarum that are NCBI GenBank, with a given name Lactobacillus plantarum strain recorded in the GenBank. The phylogenetic tree represents the degree GCC_19M1, and archiving accession number MN988925. of relatedness with the retrieved database sequences, which is shown in Figure 1. The 1463 bp 16S rDNA sequence has been submitted to Tolerance to low pH Probiotic microorganisms must survive in the GI tract at pH 3 or below, where food has to pass for a period of 2–3 h. During the incu- bation period of 3 h, L. plantarum strain GCC_19M1 demonstrated Table 1. Morphological and biochemical characteristics of test analogous growth pattern at pH 3 and 7.2 (Figure 2). However, the isolate GCC_19M1. isolate demonstrated a 10.72% decrease in the bacterial count at pH Parameters Observations 3 as compared to pH 7.2. General characteristics Tolerance to simulated gastric juice MRS broth Turbid In the presence of simulated gastric juice (pH 3), L.  plantarum MRS agar Dry, rough, irregular colony Colour White and creamy colonies strain GCC_19M1 showed significant growth with a survival rate Surface Jelly-like appearance of 96.97% after 1  h of incubation. However, the trend gradually Pigmentation No decreased to 93.48% after 2  h, and later a marginal increase was Growth temperature 10–40 °C observed after 3 h of incubation (Figure 3). Gram staining Gram negative, rod Endospore test Negative Biochemical characteristics Bile tolerance of L. plantarum stain GCC_19M1 Indole test − Bile disrupts lipids and fatty acids of the cell membrane, which even- Methyl red test − tually decreases the survival rate of bacteria (Khagwal et al., 2014). Voges–Proskauer test − The present study reports a much higher viability of L.  plantarum Citrate utilization test − strain GCC_19M1 in 0.3% bile, with a survival rate of 83.70%. The Catalase test − growth curve does not show any significant growth in initial hours; Oxidase test − however, after 3  h of incubation, a marked growth was observed Carbohydrate fermentation (Figure 4). Glucose + Lactose + Sucrose + Pancreatin tolerance of L. plantarum stain Xylose + GCC_19M1 Fructose + Pancreatic enzymes notably amylase, lipase, and protease are essen- Ribose + tial for the normal digestion of carbohydrates, fats, and proteins, Galactose + respectively. Therefore, the capability to endure these enzymes is Dextrose + Maltose + a measure for the selection of probiotic bacteria (Rayavarapu and Tallapragada, 2019). Lactobacillus plantarum strain GCC_19M1 ‘+’ indicates a positive result and ‘−’ indicates a negative result; MRS, Man showed significant growth after 24  h of incubation in 0.5% pan- Rogosa Sharpe. creatin. The survival rate of the test isolate was found to be 68.42% Figure 1. Phylogenetic tree of isolate GCC_19M1, generated by aligning 16S rRNA gene sequence of the isolates with the database sequence. Downloaded from https://academic.oup.com/fqs/article/4/4/213/5918439 by DeepDyve user on 22 December 2020 218 S. Nath et al., 2020, Vol. 4, No. 4 0.4 1.4 0.35 1.2 0.3 0.25 0.8 0.2 0.6 0.15 0.1 0.4 0.05 0.2 0 hr 1 hr 2 hrs3 hrs 0 hr 1 hr 2 hrs3hrs4 hrs L. plantarum strain GCC_19M1 (pH-7.2) L. plantarum strain GCC_19M1 (pH-3.0) L. plantarum strain GCC_19M1 (control) L. plantarum strain GCC_19M1 (0.3% bile) Figure 2. Growth of Lactobacillus plantarum strain GCC_19M1 in pH 7.2 and Figure 4. Growth of Lactobacillus  plantarum strain GCC_19M1 in the pH 3, after 3 h of incubation. Values are mean ± standard deviation of three presence of 0.3% bile, and their growth comparison with control set (without replicates. bile). Values are mean ± standard deviation of three replicates. 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 hr 24 hrs 48 hrs L. plantarum strain GCC_19M1 (control) L. plantarum strain GCC_19M1 (pancrean) Figure 5. Growth of Lactobacillus  plantarum strain GCC_19M1 in 0.5% Figure 3. Survival percentage of Lactobacillus  plantarum strain GCC_19M1 pancreatin, and their growth comparison with control set (without pancreatin). in the presence of simulated gastric juice, determined by comparing with the control set without gastric juice. Values are mean ± standard deviation of three replicates. and hydrophobicity of the strain (Zhou et al., 2013). Lactobacillus plantarum strain GCC_19M1 exhibited 29.60% auto-aggregation, and 55.11% after 24 and 48 h of incubation, respectively. A decrease which signifies a lower ability to colonize and attach with intestinal in the viable bacterial count was also observed with extending incu- epithelium (Montoro et  al., 2016). Outcomes of the present study bation time. Figure 5 illustrates the growth kinetics of test isolates, demonstrated that collectively auto-aggregation and hydrophobicity which was determined by measuring the absorbance after 24 and might expand the adherence capacity of probiotic strains. 48 h of incubation. Glucose fermentation by L. plantarum stain Cell surface hydrophobicity of L. plantarum stain GCC_19M1 GCC_19M1 Lactobacillus plantarum strain GCC_19M1 changes the medium Cell surface hydrophobicity indicates bacterial adhesion to human colour from red to yellow, which indicates fermentation of glucose intestinal cells which is a prerequisite for probiotic activity and con- and production of organic acid by reducing the pH. sidered as an important selection criterion for potential probiotic strains. The rate hydrophobicity of Lactobacillus  plantarum strain NaCl tolerance test by L. plantarum stain GCC_19M1 GCC_19M1 was found to be 31.60% and 25.20% for the hydro- Lactobacillus plantarum strain GCC_19M1 showed tolerance to carbons n-hexadecane and toluene, respectively. This hydrophobic 5% NaCl. However, upon increasing the NaCl concentration to nature of microbes is probably involved in the attachment of bac- 7.5%, viability in bacteria decreases. The strain showed no sign of teria to epithelial tissue that confers bacterial maintenance in human growth at 10% NaCl or above, even after 48 h of incubation. GI tract (García et al., 2017). Haemolytic activity of L. plantarum stain GCC_19M1 Cellular auto-aggregation of L. plantarum stain Lactobacillus plantarum strain GCC_19M1 exhibited γ-haemolytic GCC_19M1 activity (no haemolysis) when grown in sheep blood agar. Absence Auto-aggregation is an imperative probiotic quality that helps in of haemolytic activity ensures the non-virulent nature of the isolate the development of ecological niches particularly in the human gut, which may be regarded as a selecting criterion for potential probiotic and the accumulation of bacteria depends upon auto-aggregation strains (Peres et al., 2014). OD at 600 nm OD 600 nm OD at 600 nm Downloaded from https://academic.oup.com/fqs/article/4/4/213/5918439 by DeepDyve user on 22 December 2020 Probiotic strain of L. Plantarum in fermented milk, 2020, Vol. 4, No. 4 219 Antibiotic susceptibility and resistance pattern of pH of the gastric mucus layer (Sanhueza et  al., 2015; Montoro et al., 2018). A study conducted by Zielińska et al. (2015) reported L. plantarum stain GCC_19M1 30–100% survival rate of probiotic strains of Lactobacillus, when Lactobacillus plantarum strain GCC_19M1 was found to be sus- inoculated in a gastric juice at pH 3 (Zielińska et al., 2015). The pre- ceptible to gentamicin, tetracycline, kanamycin, meropenem, and sent study reports a much higher survival rate (<90%), which not- ceftriaxone, and a moderate susceptibility was observed against ably signifies their efficacy and further use as a potential probiotic vancomycin, polymyxin-B, ofloxacin, ampicillin, norfloxacin, ri- strain. The viability of L. plantarum strain GCC_19M1 at pH 3 and fampicin, amikacin, penicillin-G, streptomycin, ciprofloxacin, and acidic gastric juice was found to be similar with other reported pro- azithromycin. However, out of all the tested antibiotics, the strain biotic strains that are commonly reported in traditional food prod- showed resistance towards co-trimoxazole, clindamycin, methicillin, ucts (Haghshenas et al., 2015; Mortuza, 2016; Sharma et al., 2018; and cefdinir. Singhal et al., 2019). In the duodenum, the physiological concentra- tion of human bile is around 0.3%, and therefore this concentration Antagonistic activity of L. plantarum strain is usually selected as an essential criterion for the assessment and se- GCC_19M1 against pathogens lection of probiotic strains (Dunne et al., 2001; Shehata et al., 2016). Antimicrobial agents of L.  plantarum strain GCC_19M1 inhibited Lactobacillus plantarum strain GCC_19M1 reported in the present the growth of B. cereus strain SN_SA, A. johnsonii strain SB_SK, and study showed considerable growth in 0.3% bile with a survival rate P. aeruginosa strain GCC_19W1, with the zone of inhibition of 8 ± of 83.70%. The growth abilities of probiotic strains of L. plantarum 0.33 mm, 10 ± 0.67 mm, and 11 ± 0.88 mm, respectively (Table 2). in a wide bile concentration (0.05–0.3%) are well assessed by These bacteria are often found as foodborne microorganisms and other researchers (Balasingham et  al., 2017; Asan-Ozusaglam and manifest infections upon human exposure. A lesser degree of inhib- Gunyakti, 2019). A decrease in the viability of probiotic organisms ition (≥6  mm) was observed against other tested bacteria that are has been evidenced by Balasingham et  al. (2017) with the increase often found in a polluted environment and seldom colonize human in bile salt concentration. A high concentration of bile salts dissoci- GI tract and cause clinical infections. ates the lipid bilayer and integral protein of their cell membranes, resulting in the disruption of cellular homeostasis, bacterial content leakage, and sometimes cell death (Mandal et al., 2006). Discussion In the presence of 0.5% pancreatin, the isolate showed good growth upon 24  h incubation; however, the trend decreases upon The present study was designed to identify the bacteria found in fer- further incubation of 48 h. Similar growth characteristics were also mented raw milk and to explore its probiotic potential. Lactobacillus obtained by Pan et  al. (2009) in probiotic L.  acidophilus NIT, iso- plantarum strain GCC_19M1 was found to be gram-negative bacilli, lated from infant faeces. In recent times, many studies have been non-spore forming, and tested negative for catalase, oxidase, and conducted to demonstrate the interaction of bacteria with pancre- IMViC tests. 16S rRNA sequence of the isolate showed 98–99% atic enzymes, and their survival in in vitro screening becomes an similarity to L. plantarum, as recorded in the GenBank. The isolated important criterion for probiotic selection (Junnarkar et  al., 2018; strain was able to ferment all the tested sugars (Table  1), which is Oh et al., 2018; Mantzourani et al., 2019). found to be similar to the studies of Yadav et  al. (2016). The cul- The specific attachment of bacteria with host epithelial mucosa tural, morphological, and biochemical characteristics of isolated is greatly affected by their hydrophobicity and auto-aggregation L. plantarum strain GCC_19M1 showed resemblance with other re- abilities (García-Cayuela et  al., 2014). However, in most cases, ported probiotic strains of L. plantarum, which were isolated from a bacterial adhesiveness is species- or strain-dependent (Jacobs and wide variety of fermented foods (Palachum et al., 2018; Nwachukwu Chenia, 2011). It is also dependent upon the growth stage of bac- et al., 2019). teria, its cell surface composition, and the surrounding environment The preliminary investigation involves the screening of the test (Sorongon et al., 1991; Wu and Shah, 2014; Nivoliez et al., 2015). isolate, mimicking the stress conditions found in the GI tract of hu- The percentage hydrophobicity of L. plantarum strain GCC_19M1 mans. The common barrier that may hinder the growth of probiotic was found to be 31.60% for n-hexadecane and 25.20% for toluene, bacteria includes low pH, gastric juice, bile salts, and pancreatin. which indicates the moderate tendency of the strain to adhere to Probiotic microorganisms should be tolerant to pH 3, which is the non-polar materials in preference to water. Similar results were also reported on probiotic strains L.  plantarum, L.  acidophilus, Table 2. Antagonistic activity of L.  plantarum strain GCC_19M1 against indicated bacterial strains. and L.  johnsonii using n-hexadecane and xylene (Kaushik et  al., 2009; García-Cayuela et  al., 2014). Upon adherence, the probiotic L. plantarum strain aggregates and colonizes in the gut and confers health bene- Test pathogens strain fits. Lactobacillus plantarum strain GCC_19M1 exhibited moderate GCC_19M1 auto-aggregation of 29.6%; however, a higher degree of cellular Bacillus cereus strain SN_SA 8.07 ± 0.13 auto-aggregation by some probiotic strains has also been reported, Acinetobacter johnsonii strain SB_SK 10.07 ± 0.18 which includes L.  acidophilus LA7 (46.5%), L.  johnsonii LA1 Pseudomonas aeruginosa strain GCC_19W1 11.10 ± 0.23 (40.4%), and L.  plantarum Lp9 (31.0%) (Kaushik et  al., 2009). Stenotrophomonas maltophilia strain GCC_19W2 4.07 ± 0.18 Bacterial auto-aggregation plays an important role in their adhesion, Cedecea davisae strain GCC_19S1 5.23 ± 0.15 colonization, and persistence to the oral cavity, GI, and urogenital Achromobacter spanius strain GCCSB1 3.20 ± 0.12 tract (Nikolic et al., 2010). The presence of proteins, glycoproteins, Dimethyl sulfoxide (control) 0 teichoic, and lipoteichoic acids on the cell wall is mainly involved in aggregation ability of bacteria (Li et al., 2015). Zone of inhibition is measured in mm, and the results are expressed as the Auto-aggregation is mediated by reducing the pH, followed by means ± standard deviations of three independent replicates. L. plantarum, glucose fermentation and lactic acid production (Saito et al., 2019), Lactobacillus plantarum. Downloaded from https://academic.oup.com/fqs/article/4/4/213/5918439 by DeepDyve user on 22 December 2020 220 S. Nath et al., 2020, Vol. 4, No. 4 which is also evident in our studies. The efficient fermentation of ability of L.  plantarum to grow and reproduce at this temperature glucose and other carbohydrates is linked to the synthesis of ATP range has been evidenced by other researchers (Longhi et al., 2013; by substrate-level phosphorylation which is subsequently used in Matejčeková et al., 2016). Inactivation of L. plantarum has been ob- the biosynthetic pathway (Plumed-Ferrer et al., 2008). Lactobacillus served when the temperature has increased from 40 to 55 °C, which plantarum strain GCC_19M1 was also able to utilize different types is also evidenced by Sampedro et al. (2006). The result of our studies of carbohydrates such as glucose, lactose, sucrose, xylose, fructose, also indicates the non-pathogenic nature of the L. plantarum strain ribose, galactose, dextrose, and maltose, indicating their ability to GCC_19M1, as evidenced by γ-haemolytic in sheep blood agar grow in varied habitats. Lactobacillus plantarum is able to fer- plates. Negative results in haemolysis assay have been previously ment hexoses via glycolysis and pentoses via the phosphoketolase reported in many strains of Lactobacillus sp. (García et  al., 2017; pathway, leading to homo- or heterolactic fermentation, respect- Nwachukwu et al., 2019). ively (Axelsson, 2004). Glucose fermentation by probiotic strains A traditional belief of consumption of fermented raw milk of L.  plantarum isolated from various fermented food products during GI tract infections, urogenital tract infections, and traveller’s such as milk, corn–soyabean, and cereal-based feed has been re- diarrhoea has been noticed in the study area. People also choose ported by many researchers (Plumed-Ferrer et  al., 2008; Kalui to drink fermented milk during antibiotic therapy without knowing et  al., 2009; Kumar and Murugalatha, 2012; Yadav et  al., 2016; their interaction and possible therapeutic effect. Our finding Nwachukwu et al., 2019). NaCl is an inhibitory substance that in- was in accordance with other studies that documented the resist- hibits the growth of certain types of bacteria, and probiotic strains ance of L.  plantarum against vancomycin (Palachum et  al., 2018), must be able to withstand high salt concentration in the human gut penicillin-G (Belletti et al., 2009; Lapsiri et al., 2011), streptomycin (Prabhurajeshwar and Chandrakanth, 2017). Our results revealed (Chang et al., 2009; Lapsiri et al., 2011), ciprofloxacin (Yadav et al., that L. plantarum strain GCC_19M1 isolated from milk sample was 2016), and co-trimoxazole (Cebeci and Gürakan, 2003). The con- able to tolerate 5% of NaCl, whereas a moderate growth was per- current therapy of Lactobacillus strains along with the antibiotic ceived up to 7.5% NaCl. Similar NaCl sensitivity of probiotic strains to which they are intrinsically resistant may give better therapeutic of Lactobacillus sp. and Bifidobacterium sp. has been reported by effect, by decreasing the growth of the infectious organism and pro- other researchers (De Castro-Cislaghi et  al., 2012; Ahmed et  al., moting the growth of beneficial microflora (Cebeci and Gürakan, 2019). A  high concentration of NaCl may cause lethal injuries in 2003; Chavez-Tapia et al., 2015; Yadav et al., 2016). Probiotic bac- cells, which probably occurs due to osmotic stress, and causing tem- teria also produce antimicrobial or inhibitory substances as sec- porary permeabilization of the cell membranes of these cells (Sunny- ondary metabolites, which inhibits the growth of harmful microbes Roberts and Knorr, 2009). As per the reports gathered from the local during fermentation of various food products (Charlier et al., 2008; community, temperature variation greatly influences bacterial via- Prabhurajeshwar and Chandrakanth, 2017; Li and Gu, 2018). The bility, fermentation process, and the overall product quality. In vitro results of the present study also demonstrate the antagonistic ac- test results demonstrated the growth of L. plantarum at a tempera- tivity of L. plantarum strain GCC_19M1 against tested pathogens. ture range of 10–40 °C, which is conducive for the study site. The Probiotic bacteria may also indirectly inhibit or reduce the growth of Figure 6. Probable interaction of L. plantarum strain GCC_19M1 in the gastrointestinal tract of human, evaluated by in vitro assays. Downloaded from https://academic.oup.com/fqs/article/4/4/213/5918439 by DeepDyve user on 22 December 2020 Probiotic strain of L. Plantarum in fermented milk, 2020, Vol. 4, No. 4 221 Archer,  A.  C., Halami,  P.  M. 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Antibiotic resist- ditions of the GI tract, absence of haemolytic activity, antibiotic re- ance of probiotic strains of lactic acid bacteria isolated from marketed sistance, and antagonistic activity against test pathogens. The study foods and drugs. Biomedical and Environmental Sciences, 22(5): 401–412. Charlier, C., Even, S., Gautier, M., et al. (2008). Acidification is not involved in affirms the general belief of traditional people of Cachar district the early inhibition of Staphylococcus aureus growth by Lactococcus lactis of Assam, India, that the consumption of fermented raw milk im- in milk. International Dairy Journal, 18(2): 197–203. proves digestion, relieves constipation, and maintains good health. Chavez-Tapia, N. C., González-Rodríguez, L., Jeong, M., et al. (2015). Current However, further in vivo studies are needed to explicate its potential evidence on the use of probiotics in liver diseases. Journal of Functional in real-life situations for human health benefits. Foods, 17: 137–151. Claeys, W. L., Cardoen, S., Daube, G., et al. (2013). Raw or heated cow milk consumption: review of risks and benefits. Food Control, 31(1): 251–262. Acknowledgements Clinical Lab Standards Institute. (2016). Performance Standards for Anti- The authors extend their thanks to the Department of Biotechnology, microbial Susceptibility Testing. Clinical Lab Standards Institute, Wayne, Institutional Biotech Hub, and Bioinformatics Centre of Gurucharan College, PA. Silchar for providing laboratory facilities to conduct the study. The study Das,  D.¸ Goyal,  A. (2015). Antioxidant activity and γ-aminobutyric acid was supported by the Department of Biotechnology (DBT), New Delhi. The (GABA) producing ability of probiotic Lactobacillus plantarum DM5 iso- authors are also grateful to Mehdi Hasan Chowdhury, Assistant Professor, lated from Marcha of Sikkim. LWT—Food Science and Technology, 61(1): Gurucharan College, Silchar for providing valuable suggestions in writing the 263–268. manuscript. De Castro-Cislaghi, F. P., Carina Dos Reis, E. S., Fritzen-Freire, C. B., et  al. (2012). Bifidobacterium Bb-12 microencapsulated by spray drying with whey: survival under simulated gastrointestinal conditions, tolerance to Conflict of Interest NaCl, and viability during storage. Journal of Food Engineering, 113(2): 186–193. The authors declare no conflicts of interest. Dunne, C., O’Mahony, L., Murphy, L., et al. (2001). In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings. The American Journal of Clinical Nutrition, 73(2): 386s–392s. Ethics Statement García, A., Navarro, K., Sanhueza, E., et  al. (2017). Characterization of The study did not include any human subjects and animal experiments. Lactobacillus fermentum UCO-979C, a probiotic strain with a potent anti-Helicobacter pylori activity. 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In vitro screening of probiotic properties of Lactobacillus plantarum isolated from fermented milk product

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Oxford University Press
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© The Author(s) 2020. Published by Oxford University Press on behalf of Zhejiang University Press.
ISSN
2399-1399
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2399-1402
DOI
10.1093/fqsafe/fyaa026
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Abstract

Key words: raw milk; probiotic; Lactobacillus plantarum; gastric juice; auto-aggregation; hydrophobicity. reuteri (L. reuteri), and Lactobacillus rhamnosus (L. rhamnosus) (de Introduction Vos, 2011; Ahmed et al., 2019). Probiotic strains of Lactobacilli par- Probiotics are live microorganisms that, when consumed in suffi- ticularly adhere to the GI tract of the host cells, which is dependent cient amounts by the host, confer health benefits. Elie Metchnikoff, on their aggregation ability (Goh and Klaenhammer, 2010). They while working at the Pasteur Institute in Paris, first pointed out also act as a barrier to prevent the adherence of pathogenic bac- the health benefit of lactic acid bacteria (LAB) present in yogurt teria, by interfering with colonization and proliferation of patho- (Metchnikoff, 1907). Recent studies claimed that probiotics exhibit gens, thereby preventing the manifestation of infections (Patrignani an indispensable role in the treatment of diseases such as diarrhoea et  al., 2019). Lactobacilli species also produces antimicrobial pep- (Lai et  al., 2019), allergy (Huang et  al., 2016), diabetes (Soleimani tides (e.g. bacteriocins), organic acids (e.g. acetic acid and lactic et al., 2017), hypertension (Toral et al., 2018), cancer (Brady et al., acid), and other metabolites (e.g. hydrogen peroxide), which are 2000), and genetic disorders (Kiely et  al., 2017; Toral et  al., 2019) reported to inhibit the growth of pathogenic bacteria (Li and Gu, and also boost immunity (Tsai et  al., 2012). The probiotic micro- 2018). Probiotic strains of L. plantarum also participate in the ab- organism should be acid-resistant, bile-tolerant, non-carcinogenic, sorption of water and sodium in the colon and decrease diarrhoeal non-pathogenic, adhere to host epithelial tissue, enrich the intestinal symptoms (Mortensen and Clausen, 1996). They also exert holistic microflora, reduce pathogenic adherence, and able to produce of sec- health benefits for medical conditions such as psoriasis, allergies, and ondary metabolites antagonistic to pathogen microorganisms (Pan nervous system diseases (Hessle et al., 1999; Das and Goyal, 2015; et al., 2009; Prabhurajeshwar and Chandrakanth, 2017). Beneficial Kim et al., 2015). microorganism interacts with the raw food (substrate) and produces The excessive application of antibiotics, immunosuppressive valuable fermentation product of medical advantages such as en- therapy, and irradiation may alter the gut composition and adversely zymes and vitamins. Moreover, the consumption of probiotic mi- affect the gut microbiota. Consequently, the introduction of pro- crobes produces a protective effect on the gut environment. It is to biotic bacteria in the GI tract has recently become an effective op- be noted that probiotic impacts are strain-dependent, and hence, not tion to ascertain a healthy microbial equilibrium. The US Food and all strains are effective for efficient fermentation and treatment of Drug Administration is researching a way to administer live micro- all diseases. organisms in animal and human feeds, which is termed as ‘direct- The most familiar and recognized LAB belongs to the genus fed microbial’ (Sanders, 2008). A study conducted by Katafiasz and Lactobacillus, which are conventionally used in the fermentation of Bartlett (2012) in Michigan reported the beliefs associated with the vegetables, fruits, and milk (Saito et al., 2019). Lactobacilli are also consumption of raw milk. The consumers choose to drink raw milk present as a normal mucosal microbiota of humans and animals. for the desired flavour and as a means of support to the local sus- They are primarily facultative or anaerobic, non-pathogenic, and tainable farms. Some strains of L. plantarum have a preventive effect prefer an acidic condition of the gastrointestinal (GI) tract. Studies on milder GI symptoms associated with antibiotic treatment, and a have shown that consumption of probiotic Lactobacilli maintains GI continuous intake may exert a protective effect (Lönnermark et al., health and prevents intestinal infections (Yue et al., 2020). The most 2010). commonly used Lactobacillus species in probiotic settings include It has been previously reported that L. plantarum can tolerate pH Lactobacillus acidophilus (L. acidophilus), Lactobacillus plantarum 3.0 and below, after 2 h of exposure (Mathara et al., 2008). They are (L. plantarum), Lactobacillus casei (L. casei), Lactobacillus paracasei able to survive in the presence of bile salts, pancreatin, and pepsin (L. paracasei), Lactobacillus johnsonii (L. johnsonii), Lactobacillus Downloaded from https://academic.oup.com/fqs/article/4/4/213/5918439 by DeepDyve user on 22 December 2020 Probiotic strain of L. Plantarum in fermented milk, 2020, Vol. 4, No. 4 215 (Maragkoudakis et al., 2006; Mahmoudi et al., 2016). In vitro ad- Clustal W. A phylogenetic tree was constructed using PhyML, using hesion of L.  plantarum to the mucus-secreting cell lines has been Geneious R8 software package (Biomatters Ltd, Auckland, New reported by Zoumpopoulou et  al. (2018). Lactobacillus plantarum Zealand) (Nath et al., 2019). exhibits a diverse susceptibility to commonly used antibiotics, pos- sesses antibacterial activity against pathogens, and is non-haemolytic Screening of probiotic properties of isolated (Jiang et  al., 2016; Mahmoudi et  al., 2016; Zoumpopoulou et  al., bacteria 2018). Test for pH tolerance In context to our studies, it has been observed that the most fre- In vitro assay to determine the resistance of isolates to the acidic quently used fermented dairy product in rural areas of Cachar dis- condition of the stomach is an essential attribute in probiotic selec- trict of Assam, India is curd, skimmed milk, and fermented raw milk. tion. As the food remains in the stomach for at least 3 h (Thakkar The traditional belief of consumption of fermented raw milk and the et  al., 2015), this time limit was taken under consideration for pH role of indigenous bacteria to improve digestion, relieving constipa- tolerance test. pH tolerance was determined by inoculating 0.5  ml tion, and to maintain good health remains scientifically unexplored. of 20-h-old broth culture to 5 ml of phosphate buffer saline (PBS), Raw milk usually refers to unpasteurized, non-homogenized, or un- adjusting the pH to 3 and 7.2, with 1N HCl. The sample was then processed milk. Many studies reported that fermented milk acts as a incubated aerobically at 37 °C for 3 h, and their total viable count carrier for L. plantarum strains and shows a higher bacterial load at was measured at every 1 h interval by spreading 100 µl of bacterial the time of consumption. However, there is a possible reduction of suspension on the MRS agar plate. Optical density (OD 600  nm) the probiotic bacteria during passage through the GI tract, so a thera- was recorded at a regular interval to determine their viability and 8 9 peutic daily dose of 10 –10 CFU/g is recommended (Kailasapathy growth pattern (Hassanzadazar et al., 2012). and Rybka, 1997; Shah, 2000). Some studies also report the ques- tionable benefit of fermented raw milk on human health (Wouters Simulated gastric juice tolerance test et al., 2002; Claeys et al., 2013). Therefore, the study aims to isolate Simulated gastric juice was prepared by using 3 g/L pepsin, 7 mM and identify bacteria from fermented raw milk samples that exhibit KCl, 45 mM NaHCO , and 125 mM NaCl, adjusting at pH 3 (assay) health benefits upon consumption. and pH 7 (control) with 1 M HCl and 1 M NaOH, respectively (Archer and Halami, 2015). Overnight grown bacterial broth cul- ture was taken and centrifuged at 5000 rpm at 5 °C for 15 min. The Materials and Methods bacterial pellet was re-suspended in 10 ml PBS buffer, followed by Isolation and characterization of bacteria from incubation in simulated gastric juice (both assay and control). The milk sample viable cell counts at 1, 2, and 3  h were recorded, and the survival Collection of samples and isolation of bacteria percentage of the isolate was determined by the following formula: Fermented milk samples were aseptically collected in a pre-sterilized container and immediately bought to the laboratory. Milk samples CFU assay Bacterial survival rate (%) = × 100 –3 were serially diluted (10 fold); 0.1  ml of aliquot was poured on CFU control freshly prepared Man Rogosa Sharpe (MRS) agar plates and incu- bated at 30 °C for 24 h (Khagwal et al., 2014). Isolated distinct col- onies were further sub-cultured on freshly prepared MRS agar plates Bile tolerance test to obtain the pure culture. The bile tolerance test was conducted following the protocol of Gilliland et al. (1984). One hundred microlitres of overnight grown bacterial culture was inoculated in freshly prepared MRS broth con- Morphological and biochemical characterization of bacteria taining 0.3% bile salts (Himedia Pvt. Ltd). Test isolates were also Preliminary identification of the isolates was performed by colony inoculated in MRS broth without bile, which acts as a control. Both morphology, cultural characteristics, and microscopic observa- the test tubes (with and without bile) containing test isolates were in- tions. The isolates that were gram-negative, non-spore forming, cubated at 37 ºC for 4 h, and their growth at a different time interval and catalase-negative were selected and screened by biochem- and percentage resistance was noted by measuring the absorbance ical tests, which include indole production test, methyl red test, of MRS broth at 600  nm. After the incubation period of 4  h, the Voges–Proskauer test, citrate utilization test, oxidase test, starch hy- viability of bacteria in 0.3% bile was also evaluated by spreading drolysis test, and carbohydrate fermentation test (Holt et al., 1994; 100 µl of the bacterial sample onto the MRS agar plate. Cappuccino and Sherman, 1996). Pancreatin tolerance test Molecular identification One hundred microlitres of overnight grown bacterial culture was Molecular characterization of the isolate was done by extracting inoculated in 10 ml of MRS broth containing 0.5% (v/w) pancreatin the genomic DNA, following the protocol of Green and Sambrook and without pancreatin (control). Inoculated test tubes were kept in (2012). Thereafter, polymerase chain reaction (PCR) amplifica- a shaker incubator for 48 h at 37 °C. Pancreatin tolerance was de- tion of 16S rDNA gene was performed by forward primer (704F, termined by measuring the OD (at 600 nm) at an interval of 0, 24, 5′-GTAGCGGTGAAATGCGTAGA-3′) and reverse primer (907R, and 48 h. The viable cell count of test and control cultures in MRS 5′-CCGTCAATTCMTTTRAGTTT-3′) (Madison et  al., 2017). agar plates after 48 h of incubation was also determined (Khagwal Sequencing of the amplified gene was performed at Xcelris Labs et al., 2014). Limited, Gujarat, India using ABI 3730xl 96 capillary system with Big Dye Terminator v3.1 kit. The 1463 bp consensus sequence was used to perform BLAST for finding the closest homologous sequence Assessment of cell surface hydrophobicity present in the non-redundant database. Based on the maximum The ability of the bacteria to stick with hydrocarbons determines identity score, the first 10 sequences were selected and aligned using the extent of adhesion to the epithelial cells in the GI tract known Downloaded from https://academic.oup.com/fqs/article/4/4/213/5918439 by DeepDyve user on 22 December 2020 216 S. Nath et al., 2020, Vol. 4, No. 4 as cell surface hydrophobicity. To determine cell surface hydropho- Antibiotic susceptibility test bicity of the isolate, 20-h-old broth culture was centrifuged at 12 Antibiotic susceptibility test was performed by Kirby–Bauer disk 000 rpm for 5 min, and the cell pellets were collected. Cell pellets diffusion method using Mueller-Hinton agar (MHA) media (Baccer were washed twice with PBS buffer (pH 7.2) and re-suspended in et  al., 1966). Bacterial samples were spread and antibiotic discs 6  ml of PBS buffer, followed by measuring the initial absorbance were placed equidistantly onto the surface of freshly prepared MHA 600 nm. Thereafter, 3 ml of the bacterial suspension was mixed with plates. The plates were incubated at 37 °C for 24–48 h and thereafter, 1 ml of hydrocarbons (n-Hexadecane and Toluene) and vortexed for the diameter of the zone of inhibition was measured. This antibiotic 2 min. The mixture was incubated and left undisturbed for 1 h for resistance property of the isolate was classified according to the phase separation. The aqueous phase was then removed carefully, guidelines of the Clinical and Laboratory Standards Institute (CLSI, and the final absorbance was recorded at 600 nm (Rosenberg et al., 2016). The antibiotics used in the present study include Gentamicin 1980). The rate of cell surface hydrophobicity was calculated by the (120  µg), Vancomycin (30  µg), Tetracycline (30  µg), Polymixin-B formula: (300  µg), Kanamycin (30  µg), Ofloxacin (5  µg), Co-trimoxazole (25  µg), Meropenem (10  µg), Ceftriaxone (30  µg), Clindamycin OD − OD (2 µg), Ampicillin (10 µg), Norfloxacin (10  µg), Rifampicin (5 µg), initial final Rate of hydrophobicity (%) = × 100 OD Amikacin (30 µg), Penicillin-G (10 µg), Cefdinir (5 µg), Ciprofloxacin initial (5 µg), Azithromycin (15 µg), Methicillin (5 µg), and Streptomycin (10 µg), which were procured from ‘HiMedia’. Cellular auto-aggregation assay The specific cell–cell interaction, known as auto-aggregation, was Extraction of antibacterial agents and evaluation of their determined by the method described by Xu et al. (2009). The over- antagonistic activity night grown bacterial broth was centrifuged at 5000 rpm for 10 min Isolation of antibacterial agents was performed by following the to harvest the cell pellets. Pellets were repeatedly washed with PBS protocol of Hussein et  al. (2018). About 5  ml of 48-h-old bac- (pH 7.2), re-suspended in PBS buffer, and the initial absorbance was terial isolates GCC_19M1 was mixed with an equal volume of noted at 600 nm. The bacterial suspension was incubated at 37 °C ethyl acetate and shaken at 20 rpm for 10 min in a rotary shaker. for 2 h, and the final absorbance of the supernatant was measured at The sample was centrifuged, and the supernatant was transferred 600 nm. The percentage of cellular auto-aggregation was measured into a fresh tube to evaporate the ethyl acetate. The remaining con- by the formula: tent was used to study the antagonistic effect on the selected test pathogen. OD − OD initial final The test pathogen includes Bacillus cereus (MH482928), Auto-aggregation rate (%) = × 100 OD initial Acinetobacter johnsonii (MH482927), Pseudomonas aeruginosa (MN066610), Stenotrophomonas maltophilia (MN066611), Cedecea davisae (MN066609), and Achromobacter spanius Glucose fermentation test (MK000623), obtained from the culture collection of Institutional Glucose fermentation test was performed by following the methods Biotech Hub, Gurucharan College, Silchar. of Gupta et  al. (2011). Eighteen-hour-old bacterial cultures were The effectiveness of bacterial metabolites was studied by well centrifuged at 4000  rpm for 15  min, and the bacterial pellets diffusion method (Shakhatreh et  al., 2017). MHA plates of each were recovered. The pellets were washed twice by PBS buffer and test pathogen were prepared, and wells were made using a sterile re-suspended in the PBS buffer. Thereafter, 500 µl of PBS buffer con- cork borer. The wells were loaded with aliquots of 60  μl of the taining the bacterial cells were inoculated into MRS broth supple- supernatant and incubated at 37  °C for 24  h. Dimethyl sulfoxide mented by 1% glucose and 0.5% phenol red (dye) and incubated was used as a control. The diameters of the zones of growth inhib- at 37 °C for 24 h. Change in colour from purple to yellow indicates ition were measured and classified as sensitive (<20 mm), moderate positive glucose fermentation, whereas no change in colour indicates (10–20 mm), and low or resistant (>10 mm) (Prabhurajeshwar and negative glucose fermentation. Chandrakanth, 2017). Statistical analysis NaCl tolerance test All the experiments were performed in triplicates, and the results MRS agar plates were prepared in eight different concentrations of were expressed as the means ± standard deviations of three inde- NaCl (0%, 0.5%, 1%, 5%, 7.5%, 10%, 12.5%, and 15%). Isolated pendent replicates. The gathered data were analysed using Microsoft bacteria were streaked in each plate and incubated at 37 °C for 24 h. Excel 2007 and SPSS version 16. The influence of NaCl concentrations on the degree of inhibition of bacterial growth was recorded. Results Biochemical and molecular identification Safety assessment and antimicrobial activity The present study reports gram-negative rod-shaped bacteria, Haemolytic activity showing white and creamy colonies with a jelly-like surface on MRS Haemolytic activity is a determining factor for probiotic bacteria, agar plates without pigmentation. The growth characteristic was ob- and the absence of haemolytic activity indicates that the particular served at the temperature range of 10–40 °C. The gram-negative and bacteria were none virulent. Bacterial culture was streaked on sheep rod-shaped isolates showed negative results for indole test, methyl blood agar and incubated at 37 ºC for 48 h. The zone formed around red test, Voges–Proskauer test, citrate utilization test, oxidase test, the colonies was observed, which is categorized as β-haemolysis and catalase test (Table 1). BLAST result showed that the 16S rDNA (clear zone), α-haemolysis (green-hued zone), or γ-haemolysis (no sequence of the isolated bacteria has 98–99% identity and 98% zone; Gerhardt et al., 1981). Downloaded from https://academic.oup.com/fqs/article/4/4/213/5918439 by DeepDyve user on 22 December 2020 Probiotic strain of L. Plantarum in fermented milk, 2020, Vol. 4, No. 4 217 query coverage with the strains of Lactobacillus plantarum that are NCBI GenBank, with a given name Lactobacillus plantarum strain recorded in the GenBank. The phylogenetic tree represents the degree GCC_19M1, and archiving accession number MN988925. of relatedness with the retrieved database sequences, which is shown in Figure 1. The 1463 bp 16S rDNA sequence has been submitted to Tolerance to low pH Probiotic microorganisms must survive in the GI tract at pH 3 or below, where food has to pass for a period of 2–3 h. During the incu- bation period of 3 h, L. plantarum strain GCC_19M1 demonstrated Table 1. Morphological and biochemical characteristics of test analogous growth pattern at pH 3 and 7.2 (Figure 2). However, the isolate GCC_19M1. isolate demonstrated a 10.72% decrease in the bacterial count at pH Parameters Observations 3 as compared to pH 7.2. General characteristics Tolerance to simulated gastric juice MRS broth Turbid In the presence of simulated gastric juice (pH 3), L.  plantarum MRS agar Dry, rough, irregular colony Colour White and creamy colonies strain GCC_19M1 showed significant growth with a survival rate Surface Jelly-like appearance of 96.97% after 1  h of incubation. However, the trend gradually Pigmentation No decreased to 93.48% after 2  h, and later a marginal increase was Growth temperature 10–40 °C observed after 3 h of incubation (Figure 3). Gram staining Gram negative, rod Endospore test Negative Biochemical characteristics Bile tolerance of L. plantarum stain GCC_19M1 Indole test − Bile disrupts lipids and fatty acids of the cell membrane, which even- Methyl red test − tually decreases the survival rate of bacteria (Khagwal et al., 2014). Voges–Proskauer test − The present study reports a much higher viability of L.  plantarum Citrate utilization test − strain GCC_19M1 in 0.3% bile, with a survival rate of 83.70%. The Catalase test − growth curve does not show any significant growth in initial hours; Oxidase test − however, after 3  h of incubation, a marked growth was observed Carbohydrate fermentation (Figure 4). Glucose + Lactose + Sucrose + Pancreatin tolerance of L. plantarum stain Xylose + GCC_19M1 Fructose + Pancreatic enzymes notably amylase, lipase, and protease are essen- Ribose + tial for the normal digestion of carbohydrates, fats, and proteins, Galactose + respectively. Therefore, the capability to endure these enzymes is Dextrose + Maltose + a measure for the selection of probiotic bacteria (Rayavarapu and Tallapragada, 2019). Lactobacillus plantarum strain GCC_19M1 ‘+’ indicates a positive result and ‘−’ indicates a negative result; MRS, Man showed significant growth after 24  h of incubation in 0.5% pan- Rogosa Sharpe. creatin. The survival rate of the test isolate was found to be 68.42% Figure 1. Phylogenetic tree of isolate GCC_19M1, generated by aligning 16S rRNA gene sequence of the isolates with the database sequence. Downloaded from https://academic.oup.com/fqs/article/4/4/213/5918439 by DeepDyve user on 22 December 2020 218 S. Nath et al., 2020, Vol. 4, No. 4 0.4 1.4 0.35 1.2 0.3 0.25 0.8 0.2 0.6 0.15 0.1 0.4 0.05 0.2 0 hr 1 hr 2 hrs3 hrs 0 hr 1 hr 2 hrs3hrs4 hrs L. plantarum strain GCC_19M1 (pH-7.2) L. plantarum strain GCC_19M1 (pH-3.0) L. plantarum strain GCC_19M1 (control) L. plantarum strain GCC_19M1 (0.3% bile) Figure 2. Growth of Lactobacillus plantarum strain GCC_19M1 in pH 7.2 and Figure 4. Growth of Lactobacillus  plantarum strain GCC_19M1 in the pH 3, after 3 h of incubation. Values are mean ± standard deviation of three presence of 0.3% bile, and their growth comparison with control set (without replicates. bile). Values are mean ± standard deviation of three replicates. 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 hr 24 hrs 48 hrs L. plantarum strain GCC_19M1 (control) L. plantarum strain GCC_19M1 (pancrean) Figure 5. Growth of Lactobacillus  plantarum strain GCC_19M1 in 0.5% Figure 3. Survival percentage of Lactobacillus  plantarum strain GCC_19M1 pancreatin, and their growth comparison with control set (without pancreatin). in the presence of simulated gastric juice, determined by comparing with the control set without gastric juice. Values are mean ± standard deviation of three replicates. and hydrophobicity of the strain (Zhou et al., 2013). Lactobacillus plantarum strain GCC_19M1 exhibited 29.60% auto-aggregation, and 55.11% after 24 and 48 h of incubation, respectively. A decrease which signifies a lower ability to colonize and attach with intestinal in the viable bacterial count was also observed with extending incu- epithelium (Montoro et  al., 2016). Outcomes of the present study bation time. Figure 5 illustrates the growth kinetics of test isolates, demonstrated that collectively auto-aggregation and hydrophobicity which was determined by measuring the absorbance after 24 and might expand the adherence capacity of probiotic strains. 48 h of incubation. Glucose fermentation by L. plantarum stain Cell surface hydrophobicity of L. plantarum stain GCC_19M1 GCC_19M1 Lactobacillus plantarum strain GCC_19M1 changes the medium Cell surface hydrophobicity indicates bacterial adhesion to human colour from red to yellow, which indicates fermentation of glucose intestinal cells which is a prerequisite for probiotic activity and con- and production of organic acid by reducing the pH. sidered as an important selection criterion for potential probiotic strains. The rate hydrophobicity of Lactobacillus  plantarum strain NaCl tolerance test by L. plantarum stain GCC_19M1 GCC_19M1 was found to be 31.60% and 25.20% for the hydro- Lactobacillus plantarum strain GCC_19M1 showed tolerance to carbons n-hexadecane and toluene, respectively. This hydrophobic 5% NaCl. However, upon increasing the NaCl concentration to nature of microbes is probably involved in the attachment of bac- 7.5%, viability in bacteria decreases. The strain showed no sign of teria to epithelial tissue that confers bacterial maintenance in human growth at 10% NaCl or above, even after 48 h of incubation. GI tract (García et al., 2017). Haemolytic activity of L. plantarum stain GCC_19M1 Cellular auto-aggregation of L. plantarum stain Lactobacillus plantarum strain GCC_19M1 exhibited γ-haemolytic GCC_19M1 activity (no haemolysis) when grown in sheep blood agar. Absence Auto-aggregation is an imperative probiotic quality that helps in of haemolytic activity ensures the non-virulent nature of the isolate the development of ecological niches particularly in the human gut, which may be regarded as a selecting criterion for potential probiotic and the accumulation of bacteria depends upon auto-aggregation strains (Peres et al., 2014). OD at 600 nm OD 600 nm OD at 600 nm Downloaded from https://academic.oup.com/fqs/article/4/4/213/5918439 by DeepDyve user on 22 December 2020 Probiotic strain of L. Plantarum in fermented milk, 2020, Vol. 4, No. 4 219 Antibiotic susceptibility and resistance pattern of pH of the gastric mucus layer (Sanhueza et  al., 2015; Montoro et al., 2018). A study conducted by Zielińska et al. (2015) reported L. plantarum stain GCC_19M1 30–100% survival rate of probiotic strains of Lactobacillus, when Lactobacillus plantarum strain GCC_19M1 was found to be sus- inoculated in a gastric juice at pH 3 (Zielińska et al., 2015). The pre- ceptible to gentamicin, tetracycline, kanamycin, meropenem, and sent study reports a much higher survival rate (<90%), which not- ceftriaxone, and a moderate susceptibility was observed against ably signifies their efficacy and further use as a potential probiotic vancomycin, polymyxin-B, ofloxacin, ampicillin, norfloxacin, ri- strain. The viability of L. plantarum strain GCC_19M1 at pH 3 and fampicin, amikacin, penicillin-G, streptomycin, ciprofloxacin, and acidic gastric juice was found to be similar with other reported pro- azithromycin. However, out of all the tested antibiotics, the strain biotic strains that are commonly reported in traditional food prod- showed resistance towards co-trimoxazole, clindamycin, methicillin, ucts (Haghshenas et al., 2015; Mortuza, 2016; Sharma et al., 2018; and cefdinir. Singhal et al., 2019). In the duodenum, the physiological concentra- tion of human bile is around 0.3%, and therefore this concentration Antagonistic activity of L. plantarum strain is usually selected as an essential criterion for the assessment and se- GCC_19M1 against pathogens lection of probiotic strains (Dunne et al., 2001; Shehata et al., 2016). Antimicrobial agents of L.  plantarum strain GCC_19M1 inhibited Lactobacillus plantarum strain GCC_19M1 reported in the present the growth of B. cereus strain SN_SA, A. johnsonii strain SB_SK, and study showed considerable growth in 0.3% bile with a survival rate P. aeruginosa strain GCC_19W1, with the zone of inhibition of 8 ± of 83.70%. The growth abilities of probiotic strains of L. plantarum 0.33 mm, 10 ± 0.67 mm, and 11 ± 0.88 mm, respectively (Table 2). in a wide bile concentration (0.05–0.3%) are well assessed by These bacteria are often found as foodborne microorganisms and other researchers (Balasingham et  al., 2017; Asan-Ozusaglam and manifest infections upon human exposure. A lesser degree of inhib- Gunyakti, 2019). A decrease in the viability of probiotic organisms ition (≥6  mm) was observed against other tested bacteria that are has been evidenced by Balasingham et  al. (2017) with the increase often found in a polluted environment and seldom colonize human in bile salt concentration. A high concentration of bile salts dissoci- GI tract and cause clinical infections. ates the lipid bilayer and integral protein of their cell membranes, resulting in the disruption of cellular homeostasis, bacterial content leakage, and sometimes cell death (Mandal et al., 2006). Discussion In the presence of 0.5% pancreatin, the isolate showed good growth upon 24  h incubation; however, the trend decreases upon The present study was designed to identify the bacteria found in fer- further incubation of 48 h. Similar growth characteristics were also mented raw milk and to explore its probiotic potential. Lactobacillus obtained by Pan et  al. (2009) in probiotic L.  acidophilus NIT, iso- plantarum strain GCC_19M1 was found to be gram-negative bacilli, lated from infant faeces. In recent times, many studies have been non-spore forming, and tested negative for catalase, oxidase, and conducted to demonstrate the interaction of bacteria with pancre- IMViC tests. 16S rRNA sequence of the isolate showed 98–99% atic enzymes, and their survival in in vitro screening becomes an similarity to L. plantarum, as recorded in the GenBank. The isolated important criterion for probiotic selection (Junnarkar et  al., 2018; strain was able to ferment all the tested sugars (Table  1), which is Oh et al., 2018; Mantzourani et al., 2019). found to be similar to the studies of Yadav et  al. (2016). The cul- The specific attachment of bacteria with host epithelial mucosa tural, morphological, and biochemical characteristics of isolated is greatly affected by their hydrophobicity and auto-aggregation L. plantarum strain GCC_19M1 showed resemblance with other re- abilities (García-Cayuela et  al., 2014). However, in most cases, ported probiotic strains of L. plantarum, which were isolated from a bacterial adhesiveness is species- or strain-dependent (Jacobs and wide variety of fermented foods (Palachum et al., 2018; Nwachukwu Chenia, 2011). It is also dependent upon the growth stage of bac- et al., 2019). teria, its cell surface composition, and the surrounding environment The preliminary investigation involves the screening of the test (Sorongon et al., 1991; Wu and Shah, 2014; Nivoliez et al., 2015). isolate, mimicking the stress conditions found in the GI tract of hu- The percentage hydrophobicity of L. plantarum strain GCC_19M1 mans. The common barrier that may hinder the growth of probiotic was found to be 31.60% for n-hexadecane and 25.20% for toluene, bacteria includes low pH, gastric juice, bile salts, and pancreatin. which indicates the moderate tendency of the strain to adhere to Probiotic microorganisms should be tolerant to pH 3, which is the non-polar materials in preference to water. Similar results were also reported on probiotic strains L.  plantarum, L.  acidophilus, Table 2. Antagonistic activity of L.  plantarum strain GCC_19M1 against indicated bacterial strains. and L.  johnsonii using n-hexadecane and xylene (Kaushik et  al., 2009; García-Cayuela et  al., 2014). Upon adherence, the probiotic L. plantarum strain aggregates and colonizes in the gut and confers health bene- Test pathogens strain fits. Lactobacillus plantarum strain GCC_19M1 exhibited moderate GCC_19M1 auto-aggregation of 29.6%; however, a higher degree of cellular Bacillus cereus strain SN_SA 8.07 ± 0.13 auto-aggregation by some probiotic strains has also been reported, Acinetobacter johnsonii strain SB_SK 10.07 ± 0.18 which includes L.  acidophilus LA7 (46.5%), L.  johnsonii LA1 Pseudomonas aeruginosa strain GCC_19W1 11.10 ± 0.23 (40.4%), and L.  plantarum Lp9 (31.0%) (Kaushik et  al., 2009). Stenotrophomonas maltophilia strain GCC_19W2 4.07 ± 0.18 Bacterial auto-aggregation plays an important role in their adhesion, Cedecea davisae strain GCC_19S1 5.23 ± 0.15 colonization, and persistence to the oral cavity, GI, and urogenital Achromobacter spanius strain GCCSB1 3.20 ± 0.12 tract (Nikolic et al., 2010). The presence of proteins, glycoproteins, Dimethyl sulfoxide (control) 0 teichoic, and lipoteichoic acids on the cell wall is mainly involved in aggregation ability of bacteria (Li et al., 2015). Zone of inhibition is measured in mm, and the results are expressed as the Auto-aggregation is mediated by reducing the pH, followed by means ± standard deviations of three independent replicates. L. plantarum, glucose fermentation and lactic acid production (Saito et al., 2019), Lactobacillus plantarum. Downloaded from https://academic.oup.com/fqs/article/4/4/213/5918439 by DeepDyve user on 22 December 2020 220 S. Nath et al., 2020, Vol. 4, No. 4 which is also evident in our studies. The efficient fermentation of ability of L.  plantarum to grow and reproduce at this temperature glucose and other carbohydrates is linked to the synthesis of ATP range has been evidenced by other researchers (Longhi et al., 2013; by substrate-level phosphorylation which is subsequently used in Matejčeková et al., 2016). Inactivation of L. plantarum has been ob- the biosynthetic pathway (Plumed-Ferrer et al., 2008). Lactobacillus served when the temperature has increased from 40 to 55 °C, which plantarum strain GCC_19M1 was also able to utilize different types is also evidenced by Sampedro et al. (2006). The result of our studies of carbohydrates such as glucose, lactose, sucrose, xylose, fructose, also indicates the non-pathogenic nature of the L. plantarum strain ribose, galactose, dextrose, and maltose, indicating their ability to GCC_19M1, as evidenced by γ-haemolytic in sheep blood agar grow in varied habitats. Lactobacillus plantarum is able to fer- plates. Negative results in haemolysis assay have been previously ment hexoses via glycolysis and pentoses via the phosphoketolase reported in many strains of Lactobacillus sp. (García et  al., 2017; pathway, leading to homo- or heterolactic fermentation, respect- Nwachukwu et al., 2019). ively (Axelsson, 2004). Glucose fermentation by probiotic strains A traditional belief of consumption of fermented raw milk of L.  plantarum isolated from various fermented food products during GI tract infections, urogenital tract infections, and traveller’s such as milk, corn–soyabean, and cereal-based feed has been re- diarrhoea has been noticed in the study area. People also choose ported by many researchers (Plumed-Ferrer et  al., 2008; Kalui to drink fermented milk during antibiotic therapy without knowing et  al., 2009; Kumar and Murugalatha, 2012; Yadav et  al., 2016; their interaction and possible therapeutic effect. Our finding Nwachukwu et al., 2019). NaCl is an inhibitory substance that in- was in accordance with other studies that documented the resist- hibits the growth of certain types of bacteria, and probiotic strains ance of L.  plantarum against vancomycin (Palachum et  al., 2018), must be able to withstand high salt concentration in the human gut penicillin-G (Belletti et al., 2009; Lapsiri et al., 2011), streptomycin (Prabhurajeshwar and Chandrakanth, 2017). Our results revealed (Chang et al., 2009; Lapsiri et al., 2011), ciprofloxacin (Yadav et al., that L. plantarum strain GCC_19M1 isolated from milk sample was 2016), and co-trimoxazole (Cebeci and Gürakan, 2003). The con- able to tolerate 5% of NaCl, whereas a moderate growth was per- current therapy of Lactobacillus strains along with the antibiotic ceived up to 7.5% NaCl. Similar NaCl sensitivity of probiotic strains to which they are intrinsically resistant may give better therapeutic of Lactobacillus sp. and Bifidobacterium sp. has been reported by effect, by decreasing the growth of the infectious organism and pro- other researchers (De Castro-Cislaghi et  al., 2012; Ahmed et  al., moting the growth of beneficial microflora (Cebeci and Gürakan, 2019). A  high concentration of NaCl may cause lethal injuries in 2003; Chavez-Tapia et al., 2015; Yadav et al., 2016). Probiotic bac- cells, which probably occurs due to osmotic stress, and causing tem- teria also produce antimicrobial or inhibitory substances as sec- porary permeabilization of the cell membranes of these cells (Sunny- ondary metabolites, which inhibits the growth of harmful microbes Roberts and Knorr, 2009). As per the reports gathered from the local during fermentation of various food products (Charlier et al., 2008; community, temperature variation greatly influences bacterial via- Prabhurajeshwar and Chandrakanth, 2017; Li and Gu, 2018). The bility, fermentation process, and the overall product quality. In vitro results of the present study also demonstrate the antagonistic ac- test results demonstrated the growth of L. plantarum at a tempera- tivity of L. plantarum strain GCC_19M1 against tested pathogens. ture range of 10–40 °C, which is conducive for the study site. The Probiotic bacteria may also indirectly inhibit or reduce the growth of Figure 6. Probable interaction of L. plantarum strain GCC_19M1 in the gastrointestinal tract of human, evaluated by in vitro assays. Downloaded from https://academic.oup.com/fqs/article/4/4/213/5918439 by DeepDyve user on 22 December 2020 Probiotic strain of L. Plantarum in fermented milk, 2020, Vol. 4, No. 4 221 Archer,  A.  C., Halami,  P.  M. 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Antibiotic resist- ditions of the GI tract, absence of haemolytic activity, antibiotic re- ance of probiotic strains of lactic acid bacteria isolated from marketed sistance, and antagonistic activity against test pathogens. The study foods and drugs. Biomedical and Environmental Sciences, 22(5): 401–412. Charlier, C., Even, S., Gautier, M., et al. (2008). Acidification is not involved in affirms the general belief of traditional people of Cachar district the early inhibition of Staphylococcus aureus growth by Lactococcus lactis of Assam, India, that the consumption of fermented raw milk im- in milk. International Dairy Journal, 18(2): 197–203. proves digestion, relieves constipation, and maintains good health. Chavez-Tapia, N. C., González-Rodríguez, L., Jeong, M., et al. (2015). Current However, further in vivo studies are needed to explicate its potential evidence on the use of probiotics in liver diseases. Journal of Functional in real-life situations for human health benefits. Foods, 17: 137–151. Claeys, W. L., Cardoen, S., Daube, G., et al. (2013). Raw or heated cow milk consumption: review of risks and benefits. Food Control, 31(1): 251–262. Acknowledgements Clinical Lab Standards Institute. (2016). Performance Standards for Anti- The authors extend their thanks to the Department of Biotechnology, microbial Susceptibility Testing. Clinical Lab Standards Institute, Wayne, Institutional Biotech Hub, and Bioinformatics Centre of Gurucharan College, PA. Silchar for providing laboratory facilities to conduct the study. The study Das,  D.¸ Goyal,  A. (2015). Antioxidant activity and γ-aminobutyric acid was supported by the Department of Biotechnology (DBT), New Delhi. The (GABA) producing ability of probiotic Lactobacillus plantarum DM5 iso- authors are also grateful to Mehdi Hasan Chowdhury, Assistant Professor, lated from Marcha of Sikkim. LWT—Food Science and Technology, 61(1): Gurucharan College, Silchar for providing valuable suggestions in writing the 263–268. manuscript. De Castro-Cislaghi, F. P., Carina Dos Reis, E. S., Fritzen-Freire, C. B., et  al. (2012). Bifidobacterium Bb-12 microencapsulated by spray drying with whey: survival under simulated gastrointestinal conditions, tolerance to Conflict of Interest NaCl, and viability during storage. Journal of Food Engineering, 113(2): 186–193. The authors declare no conflicts of interest. Dunne, C., O’Mahony, L., Murphy, L., et al. (2001). In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings. The American Journal of Clinical Nutrition, 73(2): 386s–392s. Ethics Statement García, A., Navarro, K., Sanhueza, E., et  al. (2017). Characterization of The study did not include any human subjects and animal experiments. Lactobacillus fermentum UCO-979C, a probiotic strain with a potent anti-Helicobacter pylori activity. 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