Antibacterial Activity of Indigenous Fermented Rice Beverage of West Garo Hills, Meghalaya, India

Indigenous fermented beverage brewed from rice is an integral part of the rich tribal diet and culture in the region of West Garo Hills, Meghalaya. The beverage is fermented using locally available rice varieties and traditional rice starter cultures containing a mixed population of indigenous microbes (yeast, bacteria and moulds) and topical medicinal plants. The aim of the present study was to evaluate the antibacterial property of the traditionally fermented rice beverage with respect to five different medicinal plants used individually in the starter culture namely Plumbago zeylanica, Thelypteris clarkei C.F. Reed, Clorodendrum D. Don, Leucas lavandulaefolia and Scoparia dulcis. All the beverages exhibited good antibacterial property in agar well diffusion assay with the beverage brewed using S. dulcis and L. lavandulaefolia showing the highest inhibition zone of 39.50 ± 0.70mm against B. cereus Control beverage brewed from the starter without medicinal plants showed the least inhibition zones ranging from 8.75±0.21mm to 15.67±0.45mm. Hence the investigations testified a potential increase antibacterial property of the traditional rice beverage which can be attributed to the addition of medicinal plants thus plausibly contributing to its curative and health-promoting property claimed by the local tribal folks of West Garo Hills, Meghalaya.

Fermented beverages from cereals especially rice are very popular across the Asian countries namely China, Japan, Korea, Philippines, Vietnam and India in particular the central and north-eastern states of India. Across the various cultures around the world, they sustain a significant position spiritually (Dung, 2013) and are widely consumed during social and cultural events. Among the tribal communities, they are a part of offerings for a good harvest, traditional medicine and postnatal recovery (Chiang et al. 2006). The reason they are greatly relished and revered is because they possess high medicinal value and nutritional benefits besides having low alcohol content. The brewing process uses medicinal plants, herbs and spices containing natural antibacterial agents that apparently enhance the antimicrobial potency of the product (Kim et al. 2013). There have been several claims that the health-promoting effects of alcoholic rice beverages may be related to their antioxidant and anti-bacterial activity. Japanese rice wine sake has also been widely also known for its antimicrobial and anticancer properties. Similar to sake, the Korean rice wine has recently been reported to have beneficial health effects, with gastroprotective, antioxidant and antibacterial properties (Que et al. 2006). There are reports of the beverages fermented from rice being used as a drug and have been conceived to be effective against insomnia, headache, body ache, inflammation of body parts, diarrhoea, urinary problems, expelling worms and as a treatment for cholera (Deka and Sarma, 2010 Although beneficial effects associated with medicinal plants and herbs have been widely studied, limited research has been conducted to determine the positive health effects associated with herbal ingredients when added to alcoholic beverages. Thus, the main objective of the present study was to evaluate the beverages with respect to its antibacterial property and understand the rationale that the curative properties of the beverages conceived by the local tribes could be due to the addition of medicinal plants. To establish the above claim, the antibacterial property of the traditional beverages brewed with and without medicinal plants added to the starter culture were assessed and compared.

Sample collection, materials and reagents
Traditionally brewed rice beverages by the different tribes of West Garo Hills were collected in sterile culture bottles (200ml). Also rice beverage brewed with starter culture without any added medicinal plants was collected. All the beverage samples collected were refrigerated at 4 o C in the laboratory for analysis. Nutrient agar and Mueller Hinton agar media were purchased from HiMedia laboratories, India. Ethanol was purchased from Merck Specialities Pvt., Ltd., India. All the materials and chemicals used in this work were of analytically pure grade.

Antibacterial assay
In vitro antibacterial activity of the rice beverages was carried out using the agar well diffusion method of Cheruiyot et al. (2009) with some modifications. Fermented rice beverages by and large contain alcohol in the rage of 7 to 15 %. Thus 15% ethanol was used as a positive control. The bacterial indicator strains used as test organisms included two gram positive bacteria (Bacillus cereus and Staphylococcus aureus) and two gram negative bacteria (Salmonella typhi and Escherichia coli). The antibacterial assay test was performed at the Department of Dairy Technology, Anand Agricultural University (AAU), Anand, Gujarat, India. The test pathogens are maintained by the department on nutrient agar slants (HiMedia, Mumbai) and stored at 4°C and in glycerol stocks. The strains were subcultured and cross checked for purity before use.

Agar well diffusion method
This method is based on the principle that the antibacterial constituents present in the rice beverages are allowed to diffuse out into the medium and interact in a plate freshly seeded with the test organisms. If the test organisms are susceptible, it would result in formation of zones of inhibition that would be uniformly circular as there will be an affluent lawn of bacterial growth. The diameter of inhibition zones can be measured in millimetre (mm). Bacterial strains preserved in nutrient agar at 4°C were revived in nutrient broth and incubated at 37±1°C overnight, and the suspensions were checked to provide approximately 10 5 colony forming units (CFU)/ml. Active culture (100 µl) of each indicator strains were added to 100ml of nutrient agar (1.6 percent) and pour plated. The agar was allowed to solidify. The plates were refrigerated at 4°C for 10-15 minutes before several wells (6 mm diameter, 4 mm deep and 2 cm apart) were punched out of the agar with sterile 6mm cork borer (HiMedia). 100 µl of filter sterilised rice beverages were filled into the wells and the plates were once again refrigerated at 5°C for 1-2 hours to facilitate the diffusion of supernatant after which they were incubated at 37°C for 24-48 hours. The inhibition activities of rice beverages on the test bacterial strains would be indicated by the presence of a clear zone surrounding the agar wells.
In addition, minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC) of rice beverage samples were determined using the macro broth dilution method (Barchiesi et al. 1994

Statistical analysis
The experimental determinations were executed in triplicate. The data was recorded as Mean ± Standard deviation using Microsoft Excel Windows10.
Comparison of means was performed by oneway ANOVA with post-hoc Scheffe's test and the differences were considered significant at p ≤0.05.

Antibacterial activity of fermented rice beverages
The present study revealed that all rice beverages brewed with the medicinal plants showed good antibacterial activity against all the test organisms ( Very few literatures are available on antimicrobial assessments of fermented rice beverages. A study by Chang et al., (2015) on the antimicrobial activities of commercial rice wine extracts of Taiwanese Allium fistulosum by using agar disc diffusion and tube dilution tests showed that all extracts exhibited antibacterial activities against Bacillus subtilis and Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The antimicrobial property of ethanol in beer was studied by the prominent brewing microbiologist Shimwell (1935) who had noted that as low as 5% ethanol inhibits cell membrane functions, and inactivates bacteria by inducing cell membrane leakage leaving bacterial cells unable to maintain pH homeostasis. As a result the morphology and a range of cell functions may be affected. Jana et al. (2004) studied the antibacterial activity of Handia against five pathogenic bacterial  Mulay and Khale (2011) observed that fermentation ruptures the cells of herbs actively and that the bacterial enzymes breakdown the cell walls of herbal materials to further enhance the leaching process. Thus fermentation is known to enhance antibacterial activities of some plant product by releasing functional ingredients and enhancing various bioactive properties of the herbal and medicinal plants used (Wen et al. 2013).
Some scientific publications have reported on the antibacterial and other therapeutic properties of the medicinal plants used the starter culture wanti. A review by Makhija et al. (2011) gives accounts of various in vivo studies carried on L. lavandulaefolia that report the plant to possess hepatoprotectve, hypoglycemic, antipyretic, antidiarrheal, antitussive, wound-healing, psychopharmacological and antimicrobial properties. Likewise Pamunuwa et al. (2016) discussed the anti-diabetic, antioxidant, anti-inflammatory, analgesic, antimalarial, hepatoprotective, sedative, hypnotic, antiulcer, anti-sickling, and antimicrobial properties of S. dulcis and concluded that the plant could be used as an alternative and complementary to therapy for diabetes. Abera et al. (2015) investigated antimicrobial activities of crude extracts of Plumbago zeylanica and showed that the plant possesses significant antimicrobial activity against S. aureus, S. pneumoniae, K. pneumoniae, S. boydii, E. coli and antifungal activity against C. albicans and suggested that P. zeylanica plant may be used effectively as preventive agent in the pathogenesis of some diseases. Wang et al. (2017) in their review highlighted the phytoconstituents isolated and reported from the genus Clerodendrum anddiscussed the pharmacological activities (anti-inflammatory and anti-nociceptive, antioxidant, antihypertensive, anticancer, antimicrobial, antidiarrheal, hepatoprotective, hypoglycemic and hypolipidemic, memory enhancing and neuroprotective) of these compounds. Studies on antimicrobial activity of Thelypteris genus are still naive, hitherto in the present study, the beverage brewed with T. clarkei C.F. Reed showed good inhibition against S. aureus (In. z -34.00 mm, MIC/MBC -50/80) suggesting potential antimicrobial property possessed by the fern. Thus, we can adjudicate that the fermented rice beverages assessed in our study possess antimicrobial properties primarily due to addition of medicinal plants in the starter culture and also probably due to other components such as secondary metabolites produced by the fermenting yeast , fungi and bacteriocins produced by lactic acid bacteria present in the beverage samples.

CONCLUSION
The present study on the indigenous fermented rice beverages brewed in the region of West Garo Hills endorses the conception that the medicinal plants added in the starter rice cake (wanti) aid to increase the antimicrobial potential of the rice beverage. It has been noted that that the beverage brewed with S. dulcis & L. lavandulaefolia showed excellent antibacterial activity against all the test pathogens used in the study with the highest antibacterial activity against B. cereus (diameter of inhibition zone -39.50±0.70mm). Hence it can be considered as a good source of natural potent antibacterial constituents. Nevertheless, all the medicinal plants used in the starter culture wanti exhibited good inhibition towards the test organisms used. Hence, it and could be ascertained that these plants emphatically contributed to increase the antibacterial property of the rice beverage thus plausibly conferring to its curative and health-promoting value claimed by the humble tribes of West Garo Hills, Meghalaya.