Evaluation of the Probiotic Potential of Confusa Isolated From Traditional Fermented Rice

Soumitra Nath (  [email protected] ) Department of Biotechnology, Gurucharan College, Silchar, Assam, India https://orcid.org/0000-0003- 3678-2297 Monisha Roy Gurucharan College, Silchar Jibalok Sikidar Gurucharan College, Silchar Bibhas Deb Gurucharan College, Silchar

Research

Keywords: Fermented rice, Weissella confusa, Probiotic, Artifcial gastric juice, Hydrophobicity

Posted Date: September 21st, 2020

DOI: https://doi.org/10.21203/rs.3.rs-75426/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Version of Record: A version of this preprint was published on April 21st, 2021. See the published version at https://doi.org/10.1016/j.crbiot.2021.04.001.

Page 1/21 Abstract

Background: Probiotic are microorganism that is good for health, especially for the digestive system and can be consumed through fermented foods or supplements. The study aims to identify potential probiotic from fermented rice sample that are commonly found in Cachar district of Assam, India.

Methods: White rice sample of “Ranjit” variety was collected from the local market, cooked in the laboratory and soaked overnight in sterile water for microbial fermentation. Probiotic properties of isolates were tested, and was identifed by biochemical tests and 16S rRNA sequencing. In-vitro tests were also performed to demonstrate their colonisation properties, haemolytic activity and antagonistic activity against other pathogens.

Results: The predominant fermentative-bacteria was identifed as Weissella confusa strain GCC_19R1 (GenBank: MN394112). The isolate showed signifcant growth in the presence of artifcial gastric-juice, bile and pancreatin. A moderate percentage of hydrophobicity (35.8% for n-hexadecane and 32.56% for toluene) and autoaggregation (38.7%) was also recorded. The strain survived well at acidic pH, 12.5% NaCl, and able to ferment glucose. The strain fulflled the safety criteria concerning haemolytic activity, inhibits the growth of other bacteria, and found to be resistant towards antibiotics that are commonly used for GI-tract infections.

Conclusion: The present study reports the prevalence of W. confusa in fermented rice samples. The fnding of also supports the indegenious knowledge of fermented products, and its nutritional health benefts.

1. Introduction

Weissella was identifed as a unique genus in 1993 on the basis of 16S rRNA gene sequence analysis and named after Norbert Weiss, a German microbiologist, for his many contributions to the of (Collins et al. 1993). Phylogenetically, Weissella constitutes a separate from those of other genera of lactic acid bacteria, including Leuconostoc, , and Streptococcus (Flaherty et al. 2003). W. confusa has a diverse environmental distribution. It has been isolated from a variety of food stuffs such as milk (Goh and Philip 2015), fruit juices (Di Cagno et al. 2013), vegetables (Säde et al. 2016), fermented meat (Tanasupawat et al. 2015) and other food products. Some strains are able to produce novel, non-digestible oligosaccharides and extracellular polysaccharides, mainly dextran. These polymers used as potential probiotics and for a wide range of industrial applications, predominantly for bakeries and for the production of cereal-based fermented functional beverages (Fusco et al. 2015).

However, many studies also report their prevalence in clinical settings and human faeces (Fairfax et al. 2014; Lee et al. 2012; Lee et al. 2011). Due to vast diversity and lack of scientifc knowledge, Weissella sp. is still not recognised as GRAS (Generally Recognized as Safe) by the FDA (Food and Drug Administration) neither as QPS (Qualifed Presumption of Safety) by the EFSA (European Food Safety Authority). They are often classifed as opportunistic pathogens, which seems to be a limit for food

Page 2/21 application (Fessard and Remize 2017). Reports of clinical infections due to the vancomycin-resistant, Gram-positive coccobacillus Weissella confusa are rare as it is usually considered a contaminant (Kumar et al. 2011).

The present study aims to isolate and identify potential probiotic bacteria from fermented rice that exerts benefcial health effect to consumers. The leftover cooked rice is often soaked in water and allowed to ferment overnight and consumed in the next morning. The fermented dish is slightly sour in taste with soft texture and is generally consumed in breakfast along with salt, chilly and lemon. Fermented rice is locally known as ‘Panta Bhat’ in the southern part of Assam, India. However, the probiotic properties, safety assessment, nutritional and therapeutic value of traditional fermented rice remains unexplored. In an effort to fnd potential probiotic bacteria in traditional fermented rice of this locality, a total of 17 bacteria were recovered which belong to the genus Weissella and Lactobacillus. Among them, only one isolate was selected based on its overall survival in low pH, gastric juice, bile, pancreatin and NaCl. In- vitro tests were also performed to demonstrate their colonisation properties, haemolytic activity and antagonistic activity against test pathogens.

2. Materials And Methods

2.1. Collection of samples and isolation of bacteria

A preliminary survey was made to identify the potential food products that are frequently consumed by the people of Cachar district of Assam, India. The study strictly relies on indigenous fermented rice product, commonly reported to have a benefcial effect during indigestion and constipation. White rice sample of “Ranjit” variety was collected, cooked and soaked overnight for 10-12 hrs in sterile water (Goswami et al. 2016). Samples were serially diluted (10-1-10-3 fold), 0.1 ml of aliquot was poured on MRS (De Man, Rogosa and Sharpe) agar plates and incubated anaerobically at 37 °C for 24 hrs. Dense white colonies were formed on the surface of agar plates which were counted using a colony counter.

2.2. Identifcation of bacteria

Individual distinct colonies were sub-cultured and identifed by colony morphology, Gram’s staining and biochemical tests (indole production test, methyl red test, Voges–Proskauer test, citrate utilization test, oxidase test, catalase test, starch hydrolysis test etc.) (Holt et al. 1994). Genomic DNA was extracted from isolated bacterial cultures (Green and Sambrook 2012), and PCR amplifcation of 16S rDNA gene was achieved by 704F (5'-GTAGCGGTGAAATGCGTAGA-3') and 907R (5'-CCGTCAATTCMTTTRAGTTT -3') (Madison et al. 2017). Sequencing of 16S rDNA was carried out at Xcelris Labs Limited, Gujrat, India using ABI 3730xl 96 capillary system using Big Dye Terminator v3.1 kit. The consensus sequence of the 16S rDNA gene was generated from forward and reverse sequence data using aligner software and EMBOSS merger. A BLAST search was performed with the 16s ribosomal RNA sequence database to fnd the closest homologous sequence. Based on the maximum identity score, frst ten sequences were selected, and aligned using Clustal-W. The aligned nucleotide sequence was used to construct a

Page 3/21 phylogenetic tree using PhyML (Guindon et al. 2010; Nath et al. 2018). Geneious R8 software package was used to perform the above analysis (Biomatters Ltd., Auckland, New Zealand).

2.3. Assessment of probiotic properties

2.3.1. Test for resistance to low pH

Tolerance to pH is one of the essential attributes in in-vitro assays to determine the resistance to the acidic condition of the stomach. As the food remains in the stomach for at least 3 hrs (Thakkar et al. 2015); this time limit was taken under consideration for in vitro assay. pH tolerance was determined by inoculating 0.5 ml of 20 hrs old bacterial suspension to 5 ml of sterile phosphate buffer saline (PBS), adjusting the pH to 3 and 7.2, with 1N HCl. The sample was then incubated aerobically at 37 °C for 3 hrs, and their total viable count was measured at every 1 hr interval by spreading 100 µl of bacterial suspension on MRS agar plate. Optical density (OD 600 nm) was recorded at a regular interval to determine their viability and growth pattern (Hassanzadazar et al. 2012).

2.3.2. Simulated Gastric juice tolerance test

Overnight grown bacterial broth culture was taken and centrifuged at 5000 rpm for 15 min at 5 °C. The bacterial pellet was re-suspended in 10 ml PBS buffer and kept for 10 mins. Resistance to gastric juice and survival percentage was determined by incubating the isolates in simulated gastric juice and determining the viable cell counts at 1, 2 and 3 hrs.

Simulated gastric juice was prepared by using 3 g/l pepsin, 7 mM KCl, 45 mM NaHCO3 and 125 mM NaCl, adjusting at pH 3 (assay) and pH 7 (control) with 1M HCl and 1M NaOH respectively (Archer and Halami 2015).

2.3.3. Bile tolerance test

The bile tolerance test was conducted according to the method described by Gilliland et al. (1984). 100 µl of overnight grown bacterial culture was inoculated in MRS broth containing 0.3 % bile salts (Himedia Pvt. Ltd) and incubated at 37 ºC for 4 hrs. After the incubation period, 100 µl of the bacterial sample was spread onto MRS agar plate to determine the viability of bacteria in 0.3 % bile. Samples were also inoculated in MRS broth without bile, which acts as a control. Growth at a different time interval and percentage resistance of bacteria was determined by measuring the absorbance of MRS broth at 600 nm.

2.3.4. Pancreatin tolerance test

Page 4/21 100 µl of 24 hrs old bacterial culture was inoculated in 10 ml of MRS broth containing 0.5 % (v/w) pancreatin and without pancreatin (control). Inoculated test tubes were kept in a shaker incubator for 48 hrs at 37 °C. Pancreatin tolerance was determined by comparing the viable cell count of test and control cultures in MRS agar plates (Khagwal et al. 2014). Pancreatin tolerance was also determined by measuring the OD (at 600 nm) at an interval of 0, 24 and 48 hrs.

2.3.5. Cell surface hydrophobicity

Adhesion is one of the important characteristics of probiotic bacteria. The ability of the bacteria to stick with hydrocarbons determines the extent of adhesion to the epithelial cells in the gastrointestinal tract known as cell surface hydrophobicity (Pan et al. 2006). 20 hrs old bacterial cultures were centrifuged at 12000 rpm for 5 mins at 4 °C. Pellets were washed twice by PBS buffer (pH 7.2) and re-suspended in 6 ml of PBS buffer. The initial absorbance was noted at 600 nm. After that, 3 ml of the bacterial suspension was mixed with 1 ml of hydrocarbons (n- Hexadecane and Toluene) and vortexed for 2 mins. The mixture was incubated and left undisturbed for 1 hr for phase separation. The aqueous phase was then removed carefully with a micropipette, and the fnal absorbance was measured at 600 nm (Rosenberg et al. 1980). The percentage of cell surface hydrophobicity was measured by the formula:

2.3.6. Cellular autoaggregation

Many bacteria have the ability to self-recognise surface structures and bind to themselves; this self- binding is known as autoaggregation or autoagglutination. The specifc cell-cell interaction or autoaggregation was determined by the method described by Xu et al. (2009). Bacterial cultures were grown overnight at 37 °C and cells were harvested by centrifugation at 5000 rpm for 10 mins. Pellets were washed with PBS (pH 7.2), re-suspended in PBS buffer and the initial absorbance was noted at 600 nm. The suspension was incubated for 2 hrs at 37 °C. The fnal absorbance of the supernatant was measured at 600 nm. The percentage of cellular autoaggregation was measured by the formula:

2.3.7. Glucose fermentation test

Glucose fermentation test was conducted according to the method described by Gupta et al. (2011). 18 hrs old bacterial cultures were centrifuged at 4000 rpm for 15 min. Pellets were washed by PBS buffer and re-suspended in the PBS buffer. 500 µl of bacterial suspension was inoculated into MRS broth supplemented by 1 % glucose and 0.5 % phenol red as a dye and incubated for 24 hrs at 37 °C. Change in

Page 5/21 colour from purple to yellow indicates positive glucose fermentation, whereas no change in colour indicates negative glucose fermentation.

2.3.8. NaCl tolerance test

The growth of any bacteria can be affected by the amount of water entering or leaving the cell. If the medium has a low amount of solute, it is hypotonic and has high osmotic pressure on the cell. On the other hand, if the solute is high, the medium is hypertonic, and growth is considerably inhibited. In such cases, cytoplasm dehydrates and shrinks away from the cell wall. MRS agar plates were prepared in fve different concentrations of NaCl. The concentration of NaCl was maintained at 0.5 %, 1 %, 5 %, 10 % and 15 % and one kept as a control (0 % NaCl). The MRS media was poured in Petri plates, bacterial strains were streaked on each plate and incubated at 37 °C for 24 hrs. The infuence of NaCl concentrations on the degree of inhibition of bacterial growth was recorded.

2.4. Safety Assessment

2.4.1. Haemolytic Activity

Bacterial culture was streaked on sheep blood agar and incubated for 48 hrs at 37 ºC. The blood agar plate was observed and examined for signs of β-haemolysis (clear zone around the colonies), α- haemolysis (green-hued zones around colonies) or γ-haemolysis (no zones around the colonies) (Gerhardt et al. 1981).

2.4.2. Antibiotic susceptibility test

Antibiotic susceptibility test was performed by the Kirby-Bauer disk diffusion test (Baccer et al. 1966). Antibiotic discs were placed on freshly prepared lawns of each isolate on Mueller-Hinton agar (MHA) plates and incubated for 24–48 hrs at 37 °C. The diameter of the inhibition zones was measured, and the strains were classifed following the standard antibiotic disc chart. Standard antibiotic discs were procured from ‘HiMedia’ which includes Gentamicin (120 µg), Vancomycin (30 µg), Tetracycline (30 µg), Polymixin-B (300 µg), Kanamycin (30 µg), Ofoxacin (5 µg), Co-Trimoxazole (25 µg), Meropenem (10 µg), Ceftriaxone (30 µg), Clindamycin (2 µg), Ampicillin (10 µg), Norfoxacin (10 µg), Rifampicin (5 µg), Amikacin (30 µg), Penicillin-G 10 µg), Cefdinir (5 µg), Ciprofoxacin (5 µg), Azithromycin (15 µg), Methicilin (5 µg), Streptomycin (10 µg).

2.4.3. Extraction of antibacterial agents and evaluation of their antagonistic activity

Isolation of antibacterial agents was performed by following the protocol of Hussein et al. (2018). 5 ml of 48 hrs old broth culture of test isolate GCC_19R1 was mixed with an equal volume of ethyl acetate and shaken well in a rotary shaker at 20 rpm for 10 mins. The isolates were centrifuged, and the supernatant was transferred into a centrifuge tube. Ethyl acetate was allowed to evaporate, and the remaining content was used to study the antagonistic effect on other bacterial isolates.

Page 6/21 The effectiveness of bacterial metabolites was studied by well diffusion method (Shakhatreh et al. 2017). MHA plates were prepared, and wells were made using a sterile syringe puncture. The bacterial sample was spread on to the Petri plates using a sterile spreader, which includes Bacillus cereus strain SN_SA, Acinetobacter johnsonii strain SB_SK, Pseudomonas aeruginosa strain GCC_19W1, Stenotrophomonas maltophilia strain GCC_19W2, Cedecea davisae strain GCC_19S1 and Achromobacter spanius strain GCCSB1. Thereafter, wells were flled with test bacterial extracts, and plates were incubated for 24-48 hrs at 37 °C. The antibacterial activity was evaluated by measuring the zone of inhibition in mm.

2.5. Statistical analysis

All the above experiments were performed in triplicates, and the results were expressed as the means ± standard deviations of three independent replicates. The gathered data were analyzed using Microsoft Excel 2007 and SPSS version 16.

3. Results And Discussion

3.1. Identifcation of bacteria

The present study reports the prevalence of gram-positive coccobacilli in traditional fermented rice (panta bhaat). The isolate GCC­_19R1 shows white colonies on MRS agar plates without pigmentation. Biochemical test results demonstrated positive results for indole production test and methyl red test. Negative results were observed for Voges-Proskauer test, nitrate reduction test, citrate utilization test, catalase test and starch hydrolysis test. Taxonomically, W. confusa is most closely related to W. cibaria. Both are able to produce acid and gas from glucose in MRS broth and NH3 from arginine (Kumar et al. 2011). 16S rDNA sequence of the isolated bacteria was aligned using BLAST-N algorithm, which showed that the query sequence has 99.93 % identity and 100 % query coverage with the 16S rDNA of the bacterium recorded in the GenBank. Phylogenetic tree inferred the degree of relatedness between 16S rDNA sequence of the isolates with other closely related 16S rDNA sequences retrieved from the database (Fig.1). Based on these data, the isolate was identifed as Weissella confusa strain GCC­_19R1. 16S rDNA sequence was submitted to NCBI, archiving GenBank accession numbers MN394112.

3.2. Tolerance to low pH

Weissella confusa reported in the present study was stable at pH 3 without any signifcant loss in viability. The survival percentage was found to be 94.98 % after 3 hrs of incubation. Spectroscopic analysis revealed a signifcant increase in growth upto 2 hrs of incubation; however, the trend marginally decreases with further incubation time (Fig. 2). As compared with previously reported W. confusa KR78067 isolated from traditional Indian fermented food (Idli batter) (Sharma et al. 2018), W. confusa strain GCC­_19R1 isolated from fermented rice showed improved growth and low pH tolerance. Another study conducted by Lee et al. (2013) reported that W. cibaria JW15 was not able to survive at pH 2, but survived at pH 3; thereby illustrating the fact that lower pH negatively affects viable growth of bacteria.

Page 7/21 3.3. Tolerance to simulated gastric juice

Weissella confusa strain GCC_19R1 showed stable bacterial count and tolerance when inoculated at gastric juice (pH 3). Survival percentage of W. confusa was found to be 97.81 %, 98.72 % and 98.16 % after 1, 2 and 3 hrs of incubation (Fig. 3). Besides their ability to tolerate acidic pH and gastric juices, Weissella sp. is still not recognized as safe by the FDA (Food and Drug Administration) and EFSA (European Food Safety Authority). They are often classifed as opportunistic pathogens, which seems to be a limit for food application (Fessard and Remize 2017). Many studies report the prevalence of Lactobacillus species in traditional fermented rice that pose probiotic properties (Ghosh et al. 2015; Jeygowri et al. 2015; Mishra et al. 2018; Nocerino et al. 2017). The reported probiotic stain of W. confusa may produce some benefcial effects when they pass through the stomach and duodenum to reach the intestines.

3.4. Tolerance to bile

Bile salt resistance is a primary requirement for survival and colonization in the intestinal tract and hence it is one of the crucial characteristics of probiotic microorganisms (Patel et al. 2014). The present study evidenced a greater extent of bile tolerance by Weissella confusa strain GCC_19R1, exhibiting survival percentage of 94.98 % after 4 hrs. This may be due to the presence of the bile resistant protein in the bacterial cells (Hamon et al. 2011). Growth of test isolates at different time intervals demonstrates steady growth, however, maximum growth was observed in the control set without bile amendments (Fig. 4). Sharma et al. (2018) reported that probiotic strains of W. confusa can resist 1% ox-bile salts for 3 h, thereby demonstrating strong tolerance to bile salts and potential application as a probiotic. Many studies reported the survival of Weissella and other LAB, isolated from human faeces and fermented food in bile salt conditions (Lee et al. 2012; Lee et al. 2013; Patel et al. 2014).

3.5. Tolerance to pancreatin

Weissella confusa strain GCC_19R1 showed a slow but steady growth in MRS culture plates, which were pre-inoculated in MRS broth containing 0.5 % (v/w) pancreatin. As compared to control plate, test isolate showed survival rate of 53.85 % at 24 hrs of incubation. A signifcant increase in colony count was observed after 48 hrs, where the survival rate rises to 86.75 %. W. confusa strain GCC_19R1 showed an exponential growth during 48 hrs of incubation (Fig. 5), illustrating their pancreatin tolerance and ability to remain viable in the gastrointestinal tract. Tolerance of other probiotic strains of Weissella sp. to pancreatin has been reported by other researchers (Ayeni et al. 2011; Hu et al. 2017).

3.6. Cell surface hydrophobicity of bacterial isolates

The interaction of the cell membrane with the host cell surface is essential for the bacterial cell to adhere on the gastrointestinal tract. Therefore, the analysis of cell surface hydrophobicity was an important physicochemical variable required for the colonization of microorganisms on the host cells (Dey et al. 2019). The cell surface hydrophobicity for W. confusa strain GCC_19R1 for the hydrocarbons n-

Page 8/21 hexadecane and toluene was 35.8 % and 32.56 % respectively. A percent hydrophobic index greater than 70% was classifed as hydrophobic (Nostro et al. 2004). A similar study conducted by Shangpliang et al. (2017) on LAB strains of Enterococcus faecalis, E. faecium and Lactococcus lactis subsp. lactis. isolated from dahi and datshi, demonstrated probiotic activities when tested for tolerance against bile, acidic pH, gastric juice and lysozyme. However, none of the isolates showed >70% hydrophobicity. Therefore, these limited technological properties are not enough to validate the potential probiotic uses of these isolates.

3.7. Cellular autoaggregation of bacterial isolates

Autoaggregation aids in the colonization of probiotic bacteria in the intestine and attaches signifcantly with intestinal epithelium, hence preventing pathogen adhesion. Bacteria can exhibit different autoaggregation abilities ranging from high (50% and above), medium (35–50%), and low (16–35%) (Montoro et al. 2016). Autoaggregation of W. confusa strain GCC_19R1 was found to be 38.7 %, signifying their capabilities as potential probiotics and able to colonize intestinal epithelium to some extent. Low to high autoaggregation abilities (1.63–80.5 %) were observed with W. confusa KR780676 isolated from an Indian traditional fermented food (Idli batter), and some strains of Lactobacillus brevis, L. plantarum, L. acidophilus, L. curvatus, L. sake, L. fermentum and P. pentosaceus from fermented food and milk products (Abushelaibi et al. 2017; Angmo et al. 2016; Han et al. 2017).

3.8. Glucose Fermentation test of bacterial isolates

W. confusa strain GCC_19R1 changes the medium colour from red to yellow, which indicates that glucose is fermented by the isolates and organic acid is produced by reducing the pH. Genus Weissella do not possess cytochromes and ferment glucose heterofermentatively via the hexose-monophosphate and phosphoketolase pathways. The end products of glucose heterofermentation include lactic acid, gas

(CO2) and ethanol and/or acetate (Fusco et al. 2015). The ability to ferment glucose by probiotic strains of Lactobacillus like L. plantarum, L. delbruekii, L. paracasei, L. acidophilus, L. oris and L. fermentum have been reported by Mousavi et al. (2011) and Hossen (2015).

3.9. Tolerance to NaCl

Weissella confusa strain GCC_19R1showed tolerance to 7.5% NaCl. However, increasing the NaCl concentration to 10 % and 12.5%, viability in culture plates was lowered. On further increase in NaCl concentration in MRS agar plates, no traces of growth and viability was observed, even after 48 hrs of incubation. Growth kinetics of W. confusa at varying concentration of NaCl has been reported by Lee et al. (2012), where most of the strains were able to withstand 6.5% of NaCl concentration, but failed to grow at 8 % NaCl or above. In contrast to previous studies, Weissella strains reported in the present study could grow in many fermented preparations where the salt concentration is around 10 % (w/v).

3.10. Haemolytic activity of bacterial isolates

Page 9/21 W. confusa does not exhibit any sign of haemolysis when grown on blood agar for 24 hrs under anaerobic conditions. Absence of haemolytic activity is a selecting criterion for probiotic strains, ensuring that these bacteria are non-virulent in nature. Negative results in haemolysis assay has been previously reported in many strains of W. confusa (Dubey and Jeevaratnam 2015; Sharma et al. 2018) and some strains of Lactobacillus sp. (Wang et al. 2016).

3.11. Antibiotic susceptibility test of bacterial isolates

Weissella confusa strain GCC_19R1 was sensitive to gentamicin, tetracycline, polymyxin-B, kanamycin, co-trimoxazole, ceftriaxone, ampicillin, amikacin, clindamycin, penicillin-G, ciprofoxacin, azithromycin and polymyxin-B. However, the strain showed resistance towards vancomycin, ofoxacin, meropenem, norfoxacin, rifampcin, streptomycin, methicillin and cefdinir. Many studies report clinical concern on vancomycin resintance by W. confusa (Kamboj et al. 2015; Kumar et al. 2011). However, sepsis caused by W. confusa is rarely life-threatening and usually susceptible to all classes of antibiotics with the exception of vancomycin (Kumar et al. 2011). Some antibiotic resistant Lactobacilli strains are being classifed as potential probiotic and they do not usually create a safety concern. In these cases, the antibiotic resistance is not of the transmissible type and possess health beneft to consumers (Rojo- Bezares et al. 2006). Despite of all the fact, W. confusa strain GCC_19R1 isolated from fermented rice exhibits health benefts to consumers, a more detailed study on transferable antibiotic resistance genes is necessary to establish their safety criteria.

3.12. Antagonistic activity of W. confusa strain GCC_19R1

Antimicrobial agents of Weissella confusa strain GCC_19R1 showed considerable zone of inhibition against all the tested bacteria (Table 1), which are often found as food- and water-borne microorganisms and manifest infections upon human exposure. Previously reported data shows a lesser degree of inhibition (≥ 6 mm) against other pathogenic bacteria that are often found in polluted environment and seldom colonize human GI tract and cause clinical infections. Probiotics strains produce substances like organic acids, hydrogen peroxide, bacteriocins or other inhibitory substances, which inhibits the pathogenic and harmful microbes during fermentation of various food products (Ahmadova et al. 2013). 6 bacteriocins have so far been reported for Weissella strains, which includes Weissellicin 110 (Srionnual et al. 2007), Weissellin A (Papagianni and Papamichael 2011), Weissellicin L (Leong et al. 2013), Weissellicin D (Chen et al. 2014), Weissellicin M and Weissellicin Y (Masuda et al. 2012). Among these, bacteriocin weissellin A is widely used in fermented sausages (Papagianni and Sergelidis 2013). It has been reported that, adherence of W. confusa to the intestinal epithelial cells and gastric mucous cells, may inhibit the adherence of H. pylori (Nam et al. 2002). W. confusa also restricts the growth of Bacillus cereus, Pediococcus acidilactici and Staphylococcus aureus (Chavasirikunton et al. 2006; Shah et al. 2016). Result of the present study was found to be similar to L. acidophilus strain LA1, which demonstrates antibacterial activity against Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, and Enterobacter cloacae (Kaboosi 2011).

Page 10/21 Overall study demonstrated that, W. confusa strain GCC_19R1 isolated from fermented rice (panta bhat) are able to grow in artifcial gastric juice, bile and pancreatin. The strain survived well at low pH, 12.5 % NaCl, and able to ferment glucose. A moderate percentage of hydrophobicity (35.8 % for n-hexadecane and 32.56 % for toluene) and autoaggregation (38.7 %) was also recorded. The strain fulflled the safety criteria concerning haemolytic activity, inhibits the growth of other bacteria, and found to be resistant towards antibiotics that are commonly used for GI tract infections.

4. Conclusion

It is evident from the present study that consumption of fermented rice by the some communities of Cachar district of Assam, India exerts health benefts to the consumers. The fnding of the present study supports the indegenious knowledge of fermented products, and its nutritional and health benefts. The present study demonstrated that strain of W. confusa, predominantly found in fermented rice samples are able to to survive in the gastrointestinal tract and provide defence against other pathogens. However, further studies are needed to better understand their safety aspects, nutritional parameters and health beneft properties, before their application and formulation of functional probiotics for human use.

5. Declarations

Acknowledgements: The authors extend their thanks to the Department of Biotechnology, Institutional Biotech Hub and Bioinformatics Centre of Gurucharan College, Silchar for providing laboratory facilities to conduct the study. The study was supported by Department of Biotechnology (DBT), New Delhi.

Authors’contributions: SN was responsible for study design and guided the experiments. Both MR and JS were responsible for data collection and carrying out experiments. MR and JS both wrote the manuscript, and fnally edited by SN and BD. All authors read and approved the fnal manuscript.

Funding: Not applicable.

Availability of data and materials: All data generated or analysed during this study are included in thispublished article

Ethics approval and consent to participate: The study did not include any human subjects and animal experiments

Consent to publication: Not applicable

Confict of interest: None

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7. Tables

Table 1: Antagonistic activity of Weissella confusa strain GCC_19R1 against laboratory isolates.

Page 16/21 Laboratory isolates Weissella confusa strain GCC_19R1

Bacillus cereus strain SN_SA 8 ± 0.42 mm (GenBank Acc. No. MH482928)

Acinetobacter johnsonii strain SB_SK 9 ± 1.10 mm (GenBank Acc. No. MH482927)

Pseudomonas aeruginosa strain GCC_19W1 8 ± 0.54 mm (GenBank Acc. No. MN066610)

Stenotrophomonas maltophilia strain GCC_19W2 5 ± 0.65 mm

(GenBank Acc. No. MN066611)

Cedecea davisae strain GCC_19S1 6 ± 0.50 mm

(GenBank Acc. No. MN066609)

Achromobacter spanius strain GCCSB1 4 0.34 mm

(GenBank Acc. No. MK000623)

DMSO (control) 0 mm

Zone of inhibition are measured in mm, and

The results are expressed as the means ± standard deviations of three independent replicates

Figures

Page 17/21 Figure 1

Phylogenetic tree of the bacterial isolate GCC_19R1, isolated from fermented rice

Page 18/21 Figure 2

Growth of Weissella confusa strain GCC_19R1 in pH 7.2 and pH 3, after 3 hrs of incubation

Figure 3

Page 19/21 Growth of Weissella confusa strain GCC_19R1 in the presence of simulated gastric juice, determined by comparing with control set without gastric juice

Figure 4

Growth of Weissella confusa strain GCC_19R1 in the presence of 0.3 % bile, determined by comparing with control set without bile.

Page 20/21 Figure 5

Growth of Weissella confusa strain GCC_19R1 in 0.5% pancreatin

Supplementary Files

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GraphicalAbstract.docx

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