Evaluation of the Probiotic Potential of Weissella 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, Articial 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 bacteria 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 identied 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 identied as Weissella confusa strain GCC_19R1 (GenBank: MN394112). The isolate showed signicant growth in the presence of articial 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 fullled 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 nding of also supports the indegenious knowledge of fermented products, and its nutritional health benets. 1. Introduction Weissella was identied 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 taxonomy of lactic acid bacteria (Collins et al. 1993). Phylogenetically, Weissella constitutes a separate from those of other genera of lactic acid bacteria, including Leuconostoc, Lactobacillus, 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 scientic knowledge, Weissella sp. is still not recognised as GRAS (Generally Recognized as Safe) by the FDA (Food and Drug Administration) neither as QPS (Qualied Presumption of Safety) by the EFSA (European Food Safety Authority). They are often classied 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 benecial 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 nd 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 benecial 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. Identication of bacteria Individual distinct colonies were sub-cultured and identied 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 amplication 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 nd the closest homologous sequence. Based on the maximum identity score, rst 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.