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Current IssueEditorial BoardOnline First Articles
Asian Journal of
Dairy and Food Research
Print ISSN: 0971-4456
Online ISSN: 0976-0563
NASS Rating
5.44
SJR
0.176
CiteScore
0.357

Chief Editor:
Harjinder Singh
Massey Institute of Food Science and Technology, NEW ZEALAND
Frequency:Bi-Monthly
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Full Research Article

Screening of Probiotic Isolates from Fermented Rice Water and Their Antibacterial Efficacy against Food-borne Pathogens

Balasubramanian Ramasamy1, Arunachalam Kalirajan2, Krishnasanthiya Murugesan1, Gayathri Ramkumar1, Mohamed Jubeer Peer Oli1, Prasannaa Sankar1, Neha Sharma3,*
  • https://orcid.org/0000-0003-4841-4352
1Department of Biotechnology, Sri Paramakalyani College, M.S. University, Tirunelveli-627 412, Tamil Nadu, India.
2Department of Chemistry and Biology, School of Natural and Applied Sciences, Mulungushi University, Kabwe-80415, Zambia.
3Department of Research Innovation Sponsored Projects and Entrepreneurship (RISE), Chandigarh Engineering College, Chandigarh Group of Colleges, Landran, Mohali-140 307, Punjab, India.

ABSTRACT

Background: Probiotics are viable microorganisms being consumed as food additives/supplements. They significantly contribute to health benefits in terms of gut replenishment by improving the intestinal microbial balance of the host. Improper eating habits, consumption of chlorinated drinking water, stress and health adversities, consumption of alcohol and unabated use of antibiotics are known to alter the composition and activities of gut flora. Fermentation is one of the primary approach known to augment palatability, shelf life and nutritional value of food products.

Methods: Rice water is known to exhibit probiotic properties. Rigorous screening strategy was adopted to isolate prospective probiotic isolates. For microbiological studies, differential media MRS agar was used, followed by evaluation of antimicrobial potential of screened isolates by agar well diffusion and agar overlay methods against food borne pathogens.

Result: A total of 11 bacterial isolates exhibiting probiotic properties were assessed for antibacterial activity against food borne pathogens Staphylococcus sp., Escherichia coli, Bacillus sp., Enterococcus sp. and Pseudomonas sp. Probiotic characterization was done using acid pH tolerance test and Bile Salt Hydrolase (BSH) activity.

INTRODUCTION

Probiotics, by virtue of their immense health benefits including regulation of intestinal balance, are consumed as food supplements (Fuller, 1989). They are live microorganisms which when administered in adequate amounts confer health benefits on the host (WHO, 2001). The term probiotic is originated from Greek words “pro” and “bios”, meaning life (Gismondo et al., 1999). Probiotics, when supplemented in food contributes to balanced bowel movements (Bekkali et al., 2007). Rice, commonly referred to as the grain of life is considered a prospective source of probiotics (Srinivasa et al., 2013). Referring to the Indian demographic trends, it is the staple food catering to two thirds of the population (Muthayya et al., 2014). Globally, India occupies the second position both as the producer and consumer of rice (Gross and Zhao, 2014). Rice production (around 4,000 varieties) has significantly crossed 100 million in 2011-2012 accounting for 22.81% of global production.
               
Fermentation has long been known to augment the organoleptic properties of functional foods including probiotics (Salovaara and Simpson, 2004). As an age old practice, agrarian communities in Asian ethnicities relied on fermented rice water as one of the nutritional beverages (Yong-Jin et al., 2010). An array of primary metabolites, lactic acid is known to lower down the pH, thus enhancing shelf life of fermented food (Jagadeeswari et al., 2010). A recent research report has suggested the role of antibacterial and anti-colon cancer potentials of Lb. acidophilus-fermented milk supplemented with water-soluble curcumin (Hamad et al., 2025). Fermentation leads enrichment of rice by supplementing it with different essential amino acids, vitamins, minerals, prebiotics, probiotic organisms and leads to breakdown of phytic acid, tannins and polyphenols. This contributes both to fortification and enhancing therapeutic potential of rice. A recent report has suggested the role of dairy waste water as a prospective source of probiotic strains (Sharma et al., 2018). Synthetic food colors have witnessed a significant usage in bakery products, confectioneries, jellies and beverages, which has been linked to food induced toxicity (Saleem et al., 2013). Long term adverse health hazards ranging from anaemia, allergic reactions, mutations, metastasis, sleep disorders, hepatotoxicity, nephrotoxicity and brocho-pumonary ailments amongst others (Ashfaq and Masud, 2002; Bachalla, 2016; Nath et al., 2015; Shinde and Shinde, 2013; Rowe and Rowe, 1994; Tuormaa, 1994). Indiscriminate use of permitted synthetic colors is not regarded as safe (Das and Mukherjee, 2004). The present study was aimed at isolation the probiotics from fermented rice water and to screen, characterize and to reveal the food colorant degradative capability of the isolates.

MATERIALS AND METHODS

Experimental strategy
 
It is represented in Fig 1.

Fig 1: Screening of probiotic isolates from fermented rice water.


 
Source of probiotics
 
Preparation of fermented rice water: Rice samples were procured from a nearby grocery stores. R1: Yellowish White (Grocery Store 1): Control; R2: White (Grocery Store 2): Experimental; H1: Yellow (Home Made): Experimental: H2: White (Home Made): Experimental. Fermentation was carried out by method devised by (Dharsini et al., 2023) (Fig 2).

Fig 2: Rice samples and subsequent fermented rice water as probiotic source.


 
Isolation of probiotic isolates
 
Considering conventional serial dilution approach, 1 ml fermented rice samples were serially diluted up to 10-10 dilutions and were plated onto MRS agar (de Man, Rogosa and Sharpe) media. Incubation was carried out at 37oC for 24-48 hours. Pin point bacterial colonies were inoculated by consecutive streaking on freshly prepared MRS agar. Pure cultures were maintained on agar slants at 4oC.
 
Preparation of test inoculums
 
Probiotic cultures
 
From the stock cultures, pure strains were thawed by sub culturing in to MRS broth under the same incubation conditions as described previously.
 
Test pathogens
 
Five pathogenic isolates (Escherichia coli, Pseudomonas sp. Enterococcus sp., Bacillus sp., Staphylococcus sp.) were obtained from PG and Research Department of Microbiology, Sri Paramakalyani College, Alwarkurichi. After thawing, inoculation (O.D.600= 0.1/ McFarland Standard: 0.5) was carried out in nutrient broth and incubated as described previously.
 
Antimicrobial activity
 
The anti-bacterial activity was carried out by modified agar overlay method and agar well diffusion method.
 
Agar overlay method
 
Briefly, against pathogenic organisms, screened probiotic isolates were inoculated into Mueller Hinton agar medium in gridded plates by tooth pick method and incubated as described previously. 5 ml of molten agar were added with 5 μl of test organisms and poured to gridded plates. Following incubation, the plates were observed for halo formation around the colony in accordance with the protocol devised by (Hossain et al., 2022).
 
Agar well diffusion assay
 
10 mm wells were made in test organisms  and swabbed in Mueller Hinton agar plates followed by addition of 100ìl probiotic soup into each well and incubated accordingly followed by halo formation (Balouiri et al., 2016).
 
Probiotic characterization
 
Assay for BSH activity of probiotics
 
BSH activity was carried out by replica plating technique. Briefly, culture was streaked on MRS agar containing 0.0%, 0.3% and 0.5% conjugated bile salt, 100 g amp ml-1 and 0.5 mm IPTG. Incubation was carried out anaerobic conditions for 24-48 hours. Halo formation associated with precipitation of deoxycholic acid was positive for BSH activity (Hernández-Gómez  et al., 2021).
 
Acid tolerance test for probiotics
 
Fresh cultures (overnight) were inoculated into MRS broth with pH previously adjusted to 2,3,4,5 with HCl or NaOH. At 0 and after 2 hours incubation, the strains were re- inoculated in modified MRS agar and was incubated as described previously. Results were expressed in terms of % viable strains with respect to the plate count at 2 hours (Somashekaraiah  et al., 2019).

RESULTS AND DISCUSSION

Isolation of probiotic organisms
 
A total of 11 bacterial isolates were isolated from 5 rice water samples (Fig 3). Morphological, staining (Table 1) and biochemical characterization (Table 2) was carried out. Sakthika et al., 2021 reported Lactobacillus acidophilus and Lactobacillus fermentum capable of degrading food based colorants. (Soumitra et al., 2021) reported a novel Weissella  confusa strain GCC_19R1 isolated from fermented sour rice.

Fig 3: Probiotic isolates screened from fermented rice water samples.



Table 1: Morphological (colony) characteristics of probiotic isolates.



​

Table 2: Biochemical characterization of probiotic isolates.


 
Antimicrobial activity
 
Primary screening
 
Agar overlay method was carried out to test the antimicrobial property of the eleven isolates from fermented rice water against 5 pathogenic organisms (Fig 4) and findings were in accordance with (Acharjee et al., 2022) (Table 3).

Secondary screening of  screened probiotic isolates against test pathogens is highlighted in Table 4.

Fig 4: Primary screening of screened probiotic isolates against test pathogens.



​

Table 3: Primary screening of probiotic isolates against test pathogens.



Table 4: Secondary screening of probiotic isolates against test pathogens.


       
The isolate B3 show positive for Pseudomonas sp. and Enterococcus sp. and negative for other three pathogens in harmony with (Patra and Das, 2011). Reports pertaining to antibacterial efficacy of probiotic isolates against food pathogens is available in literature (Prabhurajeshwar and Chandrakanth, 2017; Manzoor et al., 2016).
       
The isolates exhibited effective zone of inhibition against the test pathogens in accordance with (Goudarzvand et al., 2016). Agar well diffusion is employed to evaluate the lowest concentration of probiotic samples (rice water), capable of lowering the viability of test pathogens (Carson et al., 1995).
 
Probiotic characterization
 
Acid pH tolerance test
 
According to general phenomena of application and establishment, probiotic bacteria challenge to successive high acidic environments in stomach and intestine respectively (Khushboo et al., 2023). The potent probiotic grew well expressed as CFU/ml at pH 2, 3, 4 and 5 (Table 5), in accordance with findings of (M’hamed  et al., 2022).

Table 5: Cell density of probiotic isolates at different pH (CFU/ml).


 
BSH activity
 
Bile Salt Hydrolase is an important indicator which is related to lowering of serum cholesterol levels in hyper-cholesterolemic patients (Hernández-Gómez  et al., 2021). The white precipitates seen around the colonies with halo formation is indicative of BSH activity (Frappier et al., 2022).  Findings are recorded in a tabulated form (Table 6). the isolate S1 from R1 rice sample show best survivability of 8.21 CFU/ml at a concentration of 0.50% and the isolate B1 from H2 rice sample show second higher survivability of 7.2 CFU/ml at a concentration of 0.50% Lactobacillus plantarum strains are known to harbor BSH activity (O’Flaherty  et al., 2018).

Table 6: Bile Salt Concentration and survivability of probiotic isolates.

CONCLUSION

With an unabated use of antibiotics and their adversities, probiotics have gained momentum in recent times. They have been employed both as nutraceuticals and dietary supplements for gut replenishment. In this study, we have reported rice water as the prospective source of probiotics, corroborating its importance as an age old practice. 11 probiotic isolates were screened from fermented rice samples and biochemically characterized.  Five commonly encountered food borne pathogens (Staphylococcus sp., Escherichia coli, Bacillus sp., Enterococcus sp. and Pseudomonas sp.)  were evaluated for establishing anti-bacterial efficacy of probiotic isolates. Isolates B1 and S1 exemplified antibacterial activity, capable of eliminating pathogen. This pilot study remarkably aims to develop a bio-fortified probiotic supplement, offering an array of therapeutic properties considering fermented rice as the benchmark candidate.

ACKNOWLEDGEMENT

The present study was carried out at Sri Paramakalyani College, Alwarkurichi, Tirunelveli, Tamil Nadu, for providing necessary infrastructure to carry out the bench work and Department of Research Innovation Sponsored Projects and Entrepreneurship (RISE), Chandigarh Group of Colleges, Landran (Mohali) for providing necessary information resources
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
Not applicable to this study.
 

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish or preparation of the manuscript.

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