Evaluation of Health Benefits of Lassi (Buttermilk): A Traditional Non Alcoholic Beverage of Northern India
Shweta Handa* Nivedita Sharma
Microbiology Research Laboratory, Department of Basic Sciences,Dr Y S Parmar University of Horticulture and Forestry, Nauni, Solan-173230, HP, India
J Innov Biol (2016) Volume 3, Issue 3: Pages: 297-301
Abstract: Lassi is a traditional fermented buttermilk product indigenous to India having its history dating back to ancient times. In this study organoleptic qualities of lassi viz., colour and appearance, body and texture, flavour, acidity and in chemical qualities included fat, protein, lactose, sucrose, total solids, total sugar, acidity and pH were determined and studied. A new bacterial strain, Lactobacillus acidophilus F14 was isolated from this buttermilk. The isolate was identified by conventional and molecular techniques and tested for different probiotic properties. The 16S rRNA sequence of the isolate was registered in National Centre for Biotechnology Information (NCBI) under accession number KT865225. Further, L. acidophilus F14 was evaluated for its probiotic potential viz., autoaggregation capacity, hydrophobicity, acidity tolerance, antibiotic susceptibility and cumulative probiotic potential and was found to posses’ good probiotic potential with cumulative probiotic score of 100%. L. acidophilus F14 have been proved to be highly effective, therefore this lassi can contributes in its nutritional as well as probiotic effects upon consumption.
Received: 22 May 2016
Accepted: 22 August 2016
Published Online: 01 September 2016
Dr Y S Parmar University of Horticulture and Forestry, Nauni, Solan-173230, HP, India
Keywords: Lassi, Health benefits, Lactobacillus acidophilus, Probiotics
Probiotics are the major discussion topic of health today. Due to its increasing biomedical benefits, these were widely used by various industries for the formation of nutraceutical products. Probiotics are described as ‘live microorganisms which, when ad¬ministered in adequate numbers, confer a health benefit on the host (Fuller, 1989). Among these, lactic acid bacteria are regarded as the most beneficial major group of probiotic microorganisms (Collins et al. 1998). They are non-pathogenic, technologically suitable for industrial processes, acid fast; bile tolerant, adhere to the gut epithelial tissue and produce antimicrobial substances, including, organic acids, hydrogen peroxide and bacteriocins (biologically active proteins) (Dunne et al. 2001). Various fermented beverages are exploited today for the isolation of potential probiotic bacteria.
By considering the nutritional significance and economical importance of lassi, it becomes essential to find out and check the health benefits. Keeping these points in view, it was proposed to carry out research work on evaluation of the physicochemical properties of Lassi and probiotic effect of lactic acid bacteria isolated from Lassi.
Material and methods
Collection of Samples
Lassi (Buttermilk) was collected from nearby village of Nauni, Solan (Himachal Pradesh, India) and evaluated for its nutritional and bio functional properties. The samples were taken and stored at 4 ºC for further study.
Nutritional and Physicochemical analysis
Moisture content, ash analysis, protein content, fat content, total carbohydrates, dietary fibre and caloric value were measured as per different standard methods (AOAC, 2007; Ranganna, 1997; Sadasivam and Manickam, 1992; Hofrieter and Hedge, 1962; Folch et al. 1957). The aluminium chloride method was used for the determination of the total flavonoid content of the sample (Madaan et al. 2012). The concentrations of flavonoid in the test samples was calculated from the calibration plot and expressed as mg quercetin equivalent /g of sample. DPPH (2, 2-diphenyl-1-picrylhydrazyl) was used as a source of free radical (Brand et al. 1995). The amount of total phenols in the sample was determined with the Folin-Ciocalteau reagent according to method of Bray and Thorpe (1954).
Isolation Screening and identification of Lactic acid bacteria
Lactic acid bacterial strain was isolated from lassi using the serial dilution and spread plate method on sterilized petriplates containing solidified media Man, Rogosa, Sharpe (MRS) at 37oC for 48 h under anaerobic conditions (Aneja, 2003). Anaerobic conditions were maintained under anaerobic gas jars by using gas pack system (Hi-media). The identification of the isolates was performed according to the criteria of Bergey’sMannual of Determinative Bacteriology (7thEdn.). The studies included Gram’s staining, catalase test, carbohydrate utilization, casein hydrolysis, MRVP test and Growth conditions. Food borne/spoilage causing bacteria viz., Staphylococcus aureus from Indra Gandhi Medical College, Enterococcus faecalis MTCC 2729, Listeria monocytogens MTCC 839, Clostridium perfringens MTCC 1739, Leucononstoc mesenteroids MTCC 107 and Bacillus cereus from Central Research Institute were used to study antagonistic potential . Antagonistic activity of isolates was studied by the Bit/Disc method (Barefoot and Klanhammer, 1983). In this method the culture bits are cut down from the fully confluent plate of F14 isolated and inversely placed over the spread plate of indicator (pathogen) and incubated for 24 h. The zone of inhibition was recorded after 24 h. Finally, bacterial strain F14 was selected on the basis of its strongest antagonistic potential for further probiotic study. The sequence analysis of 16S ribosomal RNA gene technique (16S rRNA) was employed for identification of isolate F14. Then the sequence homologies were analysed by comparative studies using “The National Centre for Biotechnology Information (NCBI) using web link (http://www.ncbi.nlm.nih.gov/) and Basic Alignment Search Tool (BLAST). L. acidophilus F14 registered under the accession number KT865225.
The tolerance of the strains to simulated gastric juices was tested as described by Gotcheva et al. (2002). The isolate was grown on MRS broth and incubated for 24 h at 37oC. The cells were harvested by centrifugation at 5,000 rpm for 10 min at 4oC, washed twice in sterile phosphate buffer saline PBS, the cells were resuspended in PBS by lowering pH to 1, 2 and 3 followed by incubation at 37oC in each set of pH for 30, 60 and 90 min. Total viable count was determined before and after incubation period at pH 1, 2 and 3, under aerobic conditions. Control samples without acidification were also prepared. The percent survival of cells was calculated using formula as given below:
% Survival = (log cfu 3rd h / log cfu 0th h) × 100
Bile Salt Tolerance
The resistance of the strains to bile was performed according to Lindgren and Dobrogosz (1990). L. acidophilus F14 cells were inoculated into sterilized 10 ml of MRS broth containing 0.3 % Ox-bile (Hi-media) and incubated at 37oC for 60 h. The optical density (OD) at 620 nm followed by viable count was measured and compared to a control culture. The percent survival of cells was calculated using formula as given below:
Survival (%) = (∆OD0% BS - ∆OD0.3, 1 or 2% BS/ ∆OD0% BS) × 100.
The autoaggregation capacity of L. acidophilus F14 was determined according to Kos et al. (2003). The culture was grown in MRS Broth for 18 h at 37oC. The pellet was washed twice in phosphate-buffered saline (PBS) and re-suspended in similar solution to reach 108cfu/ml number of cells. Autoaggregation was determined by measuring their absorbance at 0 h (A0) and after 5 h (At) and calculated using the following formula:
Autoaggregation (%) = [1- (At/A0)] x 100.
Cell surface hydrophobicity
Bacterial adhesion to hydrocarbons, was determined by the method of Mishra and Prasad (2005). L. acidophilus F14 was harvested after growing for 18 h at 37oC followed by centrifugation for 15 min at 5000 rpm. The cells were washed twice in Phosphate Urea Magnesium Sulphate (PUM) buffer and suspended separately in the same buffer at the level of 108cfu/ml. The absorbance of the suspension was measured at 600 nm (A). Five ml of cell suspension was mixed with 1 ml of different hydrocarbon viz. xylene, toluene, ethyl acetate and chloroform. The mixture was vortexed for 1 min and the phases were allowed to separate for 1 h at 37oC. The lower aqueous phase was carefully removed with a sterile Pasteur pipette and final absorbance (A0) was recorded at 600 nm to calculate cell hydrophobicity:
Hydrophobicity % = [(A-A0)/A] x 100.
Antibiotic sensitivity test
Antibiotic sensitivity of isolated strain was determined on solid MRS medium by the use of 10 different discs of antibiotics (HiMedia®, India) (Table 1) and sensitivity
was measured in term of zone of inhibition (Halami et al., 1999).
Results and Discussion
Physicochemical and Nutritional properties of Lassi:
The samples collected from the local village of Solan i.e. Nauni was evaluated for their physicochemical and nutritional properties (Table 2). The total soluble solids in the lassi were 3.0 oB. And pH (mean acidity) of the sample was 3.5. The low pH of lassi may be due to fermenting microbes present during fermentation or this may be due to prolong heating lactose degrade to organic acid rather than lactic acid. Secondly, the precipitation of calcium phosphate and dephosphorylation of casein also lead to increase in titratable acidity (Fox and McSweeney, 1998). Moisture content was found to be of 79.3%. Nutritional studies have demonstrated potential benefits of lassi (Buttermilk). In present study, lassi has been found to be rich in proteins, carbohydrates and minerals. DPPH is a free- radical generating compound and has been widely used to evaluate the free-radical scavenging ability of various antioxidant compounds. The methanolic extracts of lassi showed 40 % of scavenging ability. The antioxidant activity of lassi can be attributed to their potential to act as reducing agents, hydrogen donars, free radical scavengers, and metal chelators which are based on the number and location of hydroxyl groups present. Colorimetry of total phenolics, flavonoides showed that phenolics are abundant in lassi.
Isolation and Identification of potential probiotic microorganism
F14 lactic acid bacterial strain was isolated from lassi and its morphological and biochemical characteristics were noted down. Isolate F14 was indentified upto genera level by morphological and biochemical characteristics. Morphologically F14 appeared white and pin pointed colonies on MRS and of smooth texture (Figure 1). Biochemical tests viz. Gram’s staining, catalase test, oxidase test, citrate utilization test, gas production from glucose, casein hydrolysis and H2S production and sugar fermentation. Isolate F14 was found to be catalase positive, gram positive. H2S production was found to be negative for the isolate whereas, it was found to utilize different sugars viz. Sucrose, trehalose, xylose, maltose, lactose and dextrose. On the basis of above mentioned characteristics F14 was identified as lactobacillus as per Bergey’s Mannual of Determinative Bacteriology (Breed et al. 1957). The identified genus was further identified using 16S rRNA gene technique. The determined sequence of the isolate was compared directly with the Genbank database. The higher level of homology i.e., (96% of matches) of F14 was observed with sequence of L. acidophilus. The 16S rRNA sequence of L. acidophilus F14 is registered under accession number KT865225 in NCBI.
Antagonistic Potential of L. acidophilus F14 isolated from Lassi
Antagonistic potential of L. acidophilus F14 was tested against selected food borne/spoilage causing bacteria viz., S. aureus IGMC, E. faecalis MTCC 2729, L. monocytogens MTCC 839, C. perfringens MTCC 1739, L. mesenteroids MTCC 107 and B. cereus CRI. The data on inhibitory spectrum of the isolate by bit/disc method is shown in Table 3. Among all isolates, L. acidophilus F14 showed broadest and strongest antagonism ranging from (12 – 25 mm) against all the test indicators, thus selected for further studies. The wide spectrum inhibitory activity against challenging food borne pathogens make this isolate desirable for exploring their potential for health benefits in production of functional food.
L. acidophilus F14 was studied for its probiotic properties viz. bile tolerance, acid tolerance, autoaggregation capacity, hydrophobic capacity and antibiotic sensitivity. Before reaching the intestinal tract, probiotic bacteria have to survive in the transit through the stomach where pH can be as low as 1.5 to 2 (Dunne et al. 2001). L. acidophilus F14 has been found to tolerate 1 % bile salt, and was able to survive at 1.0 pH for 3 hours. The adherence to gut is an important criterion as far as probiotic bacteria are concerned. Indeed, the probiotic ability to adhere to the intestinal epithelium is regarded as a prerequisite to colonize the human GIT (Gastrointestinal Tract) for exerting beneficial effects of probiotics such as the exclusion of enteropathogenic bacteria (Collado et al. 2007; Litch and Wilcks, 2005). L. acidophilus F14 was found highly hydrophobic as it showed 44.8% adhesion towards xylene and 60% of autoaggregation capacity. L. acidophilus F14 found sensitive towards all antibiotics tested. The antibiotic susceptibility of strain is crucial from the safety point of view for their use as potential probiotics because probiotic bacteria may act as potential reservoir of antimicrobial resistance genes and which can be transferred to Gastointestinal tract.
Cumulative probiotic score of L. acidophilus F14
The probiotic potential of the bacterial strains is largely assessed by cumulative probiotic score (Tambekar and Bhutada, 2010). Cumulative probiotic potential is the sum of score of bile tolerance, acid tolerance, autoaggregation capacity, hydrophobic capacity, antibiotic sensitivity and antimicrobial activity. The probiotic potential of L. acidophilus F14 was adjudged very high i.e., 100% as shown in Table 4 which is far high as compared to most of the commercially available probiotic preparations have probiotic score in the range of 75 to 85%. Thus, the present study revealed that this strain meets criteria of FAO/WHO, (2002) for assigning it the status of a safe probiotic. Similar studies has been done by Tambekar and Bhutada (2010) where they isolated the strains of L. rhamnosus G119b and L. plantarum G95 form fermented milk and evaluated probiotic potential. They reported that the strains have equal potential as that of commercially available probiotics.
The present study clearly showed that Indians indigenous Lassi is indispensible item of diet since immemorial. Lassi is essential in every religious and cultural occasion in India which is nutritionally and therapeutically superior to milk. Beyond its nutritional properties, it has been found to be a good source of probiotic bacteria which can helpful in various ways upon consumption. The traditional knowledge with its holistic and systematic approach supported by research finding can serve as an innovative and useful tool for the development of functional drinks like Lassi.
This work was carried out in Microbiology section of Dr. Y. S. Parmar University of Horticulture and Forestry Nauni, Solan, H. P. India. Authors wish to thank Department of Biotechnology, Govt. of India, New Delhi, India for giving financial support to carry out this work.
1. Aneja KR, 2003. Experiments in Microbiology, Plant Pathology and Biotechnology. 4th ed. New Delhi: New Age International Publishers.
2. AOAC, 2007. Official methods of analysis of association of official analytical chemists. Arlington, Virginia, USA: Association of Official Analytical Chemists.
3. Barefoot SF, Klanhammer TR (1983) Detection and activity of Lactacin B, a bacteriocin produced by Lactobacillus acidophilus. Appl Env Microbiol 45(6): 1808-1815
4. Brand WW, Cuvelier ME, Berset C (1995) Use of free radical method to evaluate antioxidant activity. Food Sci Technol 28: 25-30
5. Bray HG, Thorpe WV (1954) Analysis of phenolic compounds of interest in metabolism. Methods Biochem Analysis 52: 1-27
6. Breed RS, Murray EGD, Smith NR (1957) Bergey’s Manual of Determinative Bacteriology, the Williams and Wilkins Co., Baltimore: American Society of Microbiology.
7. Campbell-Platt G (1987) Fermented foods of the world - a dictionary and guide. London, Butterworths.
8. Collado M, Meriluoto J, Salminen S (2007) In vitro analysis of probiotic strain combinations to inhibit pathogen adhesion to human intestinal mucus. Food Research Int 40: 629-639
9. Collins JK, Thornton G, Sullivan GO (1998) Selection of probiotic strains for human applications. Int J Sys Bacteriol 8: 487-490
10. Dunne C, O’Mahony L, Murphy L, Thornton G, Morrissey D (2001) In vitro selection criteria for probiotic bacteria of human origin: Correlation with in vivo findings. Am J Clin Nutr 73: 386S-392S
11. FAO/WHO (2002) Guidelines for the evaluation for the probiotics in food: report of a joint FAO/WHO Working Group on Drafting Guidelines for the evaluation of probiotics in food.
12. Folch J, Lees M, Sloane-Stanley G (1957) A simple method for isolation and purification of total lipids from animal tissues. J Bio Chem 226: 497-509
13. Fox PF, McSweeney PLF (1998) Dairy chemistry and biochemistry: Physical properties of milk, Blackie Academic and professional Publication London
14. Fuller R (1989) Probiotics in man and animals: A review. J App Bacteriol 66: 365-378
15. Gotcheva V, Hristozova E, Hristozova T, Guo M, Roshkova Z, Angelov A (2002) Assessment of potential probiotic properties of lactic acid bacteria and yeast strains. Food Biotechnol 16: 211-22
16. Halami PM, Chandrashekar A, Joseph R (1999) Characterization of bacteriocinogenic strains of lactic acid bacteria in fowl and fish intestines and mushroom. Food Biotechnol 13(2): 121-136
17. Hofrieter BT, Hedge JE (1962) In: Methods in carbohydrate chemistry, (Academic press, New York) pp-10-12
18. Kos B, Suškovic´ J, Vukovic´ S, Sˇimpraga M, Frece J, Matošic´ S (2003) Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92. J Appl Microbiol 94: 981–987
19. Litch TR, Wilcks A (2005) Conjugative gene transfer in the gastrointestinal environment. Adv Appl Microbiol 58: 77-95
20. Lindgren SE, Dorbogosz (1990) Antagonistic activities of lactic acid bacteria in food and feed fermentation. FEMS Microbiol Rev 87: 149-163
21. Madaan R, Bansal G, Kumar S, Sharma A (2012) Estimation of total phenols and flavonoids in extract of Acta easpicata roots and antioxidant studies. Indian J Pharm Sci 73(6): 666-669
22. Mishra V, Prasad D (2005) Application of in vitro methods for selection of Lactobacillus casei strains as potential probiotics. Indian J Food Microbiol 103: 109-115
23. Ranganna S (1997) Handbook of analysis and quality control for fruit and vegetable products, (Tata McGraw Hill Publishing Comapany Ltd., New Delhi, India), 1109 p.
24. Sadasivam S, Manickam A (1992) Biochemical methods for agricultural science, (Willey eastern limited, New Delhi) pp-10-11
25. Tambekar DH, Bhutada SA (2010) An evaluation of probiotic potential of Lactobacillus sp. from milk of domestic animals and commercial available probiotic preperations in prevention of enteric bacterial infections. Recent Research Sci Technol 2(10): 82-88