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A STUDY ON ENZYMATIC ACTIVITIES WITH A REFERENCE OF Abstract SCREENING PROCEDURE CHANDRAMANI KUMAR RESEARCH SCHOLAR OF OPJS UNIVERSITY, CHURU (RAJ.) Increasing interest in health foods in recent years has stimulated innovation and new product development in food industry around the world. Also there is increased demand of products that are able to enhance health beyond providing basic nutrition. Probiotics is one of the fastest growing category with in food for which scientific research has demonstrated therapeutic evidence. Probiotics are defined as live microorganisms that when ingested in adequate amounts, are expected to confer health benefits to the host. The current paper describes the screening of different enzymatic activities. Keywords: Enzyme, Micro organism, Screening Introduction Probiotics are substances secreted by one microorganism that stimulate the growth of another and thus were contrasted with antibiotic. Probiotics are found both in dairy and non-dairy products. Most of the studies till date are carried out on strains of human or animal internal cavities with the rationale that these would be better adapted to colonize the human/animal gastrointestinal tract. Studies on probiotic-like strains from dairy origin are interesting as these strains are better adapted to dairy products in which they are intended to be used. On the other hand, some strains from yeast and dairy origin like Saccharomyces cerevisiae, subtype boulardii and Bifidobacterium animalis/lactis have internationally recognized probiotics status. According to the guidelines for evaluation of probiotics in food reported jointly by FAO/WHO, probiotic bacteria should be 2
able to survive in acidic conditions in stomach and resist bile acids at the beginning of small intestine. The survival of probiotics in gut depends on bacterial properties like cell surface hydrophobicity and autoaggregation. Autoaggregation of probiotic strains appeared to be necessary for adhesion to intestinal epithelial cells which forms a barrier to prevent colonization by pathogenic microorganisms. Probiotics mediate health benefits on the host by several mechanisms which are broadly classified into three categories: (i) antimicrobial activity against pathogens; (ii) enhanced intestinal epithelial barrier to protect intestinal wall from pathogenic bacteria and (iii) modulation of host immune response. Additionally a good probiotic should be non-pathogenic and nontoxic. Probiotic also confer benefits by providing enzymes including proteases, lipases and antibacterial enzymes/proteins. Some of them are also indigenous to healthy digestive system. Production of bioactive peptides from enzymatic hydrolysis of whey proteins has also been reported. Proteolytic activity of probiotic bacteria convert milk protein into bioactive peptides with anthihypertensive effect. Antihypertensive effect can also be exerted through inhibition of release of endothelin-1 by endothelial cells, stimulation of bradykinin activity, enhancement of endothelium-derived nitric oxide production and enhancement of vasodilatory action of binding to opiate receptors. The probiotic microorganisms mostly belong to strains of genera Bacillus, Bifidobacterium, Enterococcus, Lactobacillus, Lactococcus, Streptococcus and numerous yeasts. The dominant members of probiotics are Bifidobacteria (Bifidobacterium bifidum), Lactobacteria (Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus), Propionobacteria, Peptostreptococci and Enterococci. A therapeutic dose of probiotics can help by preventing and treating conditions like auto immune diseases (e.g. rheumatoid arthritis), inflammatory bowel disease (e.g. ulcerated colitis and Crohn's disease), colic disease and lactose intolerance, ear infections, throat 3
infections, irritable bowel syndrome (IBS), vaginal infections (e.g. candida or thrush) and eczema. Probiotic consumption also help to reduce diarrhea and allergies. Probiotics found in dairy and meats e.g. Lactobacillus reuteri lowered low density lipoprotein (LDL) levels and killed the bacteria that caused tooth decay and also lessened the harmful effects of gingivitis. Probiotics are available in foods and probiotic supplements i.e. capsules and powders. Probiotic cultures have been associated with milk culture and dairy products and have positive effects on human health and general well-being. Screening of proteases on gelatin plate Presence of protease activity on plates was checked by Gelatin Clearing Zone (GCZ) technique which was later standardized. One hundred microlitre of dialyzed extracellular sample was loaded in well and zone was visible after 24 h of incubation. A clear zone of hydrolysis on gelatin agar plate confirmed gelatinase activity. Depending on the zone of diameter and clearance, bacteria can be selected as good protease producer and used for further investigation. The results were further substantiated by gelatin zymography. Figure 1: Gelatin agar plate showing zone of hydrolysis. Zymographic analysis of protease Zymography (in-gel activity staining) involves two stage technique with protein separation by electrophoresis followed by 4
in-situ assay of enzyme. It has proved to be extremely useful for detection of wide range of microbial, animal and plant enzymes. It is sensitive and rapid assay method for analyzing protease activity. Gelatin zymography was performed in polyacrylamide slab gels containing SDS and gelatin (0.1%) as copolymerised substrate. Zymogram activity staining revealed clear zone of proteolytic activity against blue background. play a key role in food productions such as cheese ripening. Thus proteolytic activities help in investigating the strain as functional starter culture for production of various products. High proteolytic activity also contributes to sensorial and textural properties to food. Hydrolysed proteins exhibit biofunctional activities like antioxidant properties. But the contribution of these enzyme(s) in imparting health benefits is yet to be explored. Proteolytic enzymes are not only important for nitrogen metabolism of LAB but also Figure 2: Zymography analysis on 15% SDS-PAGE show zone of hydrolysis with gelatin as substrate. 5
Screening of DPP-II (exopeptidase) activity In microbes protein degradation is initiated by endopeptidases followed by some peptide transporters that take up the peptides into cell for further hydrolysis by exopeptidases and degrade the peptide into shorter peptides and amino acids which are used by microbe for nutrition and growth. Different extracellular activities were screened in Pediococcus acidilactici. Exopeptidase activity was screened using synthetic substrtate Lys-Ala-4mȕNA by measuring absorbance at 5β0 nm. The enzyme was further purified and characterized for Lys- Ala-4mȕNAas substrate. Exopeptidases have dual advantage (i) release free amino acid (nutritional importance) and (ii) release of bioactive compounds by trimming oligopeptides (therapeutic and thus probiotic significance). Due to limited synthesis of amino acids growth of LAB depends on amino acids released from organic nitrogen source. Zone of hydrolysis Reserarch is now emphasized to study role of dipeptidyl peptidases in physiological processes and different pathologies of immune system. Changes in DPP-II activity has been observed in a number of pathologies, such as neurogenerative dsorders, myopathies, cancer and gastrointestinal disorders. So it holds a great potential as targets for treatment of certain inflammatory disorders. DPP-II activity is isolated and characterized from extracellular fluid of a probiotic bacteria i.e. Pediococcus acidilactici. Screening of esterase/lipase activity The production of lipase/esterase by P. acidilactici was verified by tributyrin agar plate method at 37 o C. Tributyrin is a very convenient substrate for the first screening tests because most bacterial strains hydrolyse tributyrin more rapidly than any other simple triglycerides. Seventy microlitre of dialysed extracellular fluid was loaded in wells and a clear zone of hydrolysis was observed after 24 hours of incubation. It confirmed extracellular lipase/esterase activity in Pediococcus acidilctici. Lipolysis is a technologically relevant property that is usually determined to evaluate the strain suitability as starter culture in fermentation due to its role in flavor development of fermented meat products and in cheese making. So P. acidilactici can be recommended as a good 6
starter culture in sausage production. This is even advantageous because strain is better adapted to fermented foods and dairy products in which it is intended to be used. Benefit of being a probiotic lipolytic strain, this activity produce short chain fatty acids in intestine which inhibit cholesterol synthesis in liver thereby resulting in a decrease in blood lipid level. Probiotics and prebiotics have potential to decrease serum lipid levels but their mechanisms are yet to be elucidated. Conjugated fatty acids from LAB have attracted much attention as a novel type of biologically beneficial functional lipid. Figure 3: Tributyrin agar plate with zone of hydrolysis Conclusion Pediococcus acidilactici was studied for probiotic attributes and the ultimate performance criterion is to confer health benefits to host along with some enzyme activities viz proteolytic activities, lipase and peptidoglycan hydrolase activities which are also believed to play a beneficial role in efficient utilization of ingested nutrients. Metabolic end products vary according to the conditions provided. A wide variety of exopeptidase activities, lipase, peptidoglycan hydrolase and endoprotease activities were screened. Endoprotease activity was screened using 7
gelatin clear zone technique and gelatin zymography. Exopeptidase activity was screened with synthetic substrates Lys-Ala-4mȕNA which is dipeptidyl peptidase activity. Peptidoglycan hydrolase activity was detected by turbidometric method with Bacillus cereus and Staphylococcus albus as substrates. These enzyme activities also confer industrial importance to the strain in addition to health benefits. Its association with immune-related diseases should be investigated. Studies on enzyme activities on probiotic bacteria in general and Pediococcus acidilactici in specific are limited so they need to be explored. Moreover probiotic studies are strain specific so these activities are further needed to be purified and characterized for better understanding. References Acta (BBA)-Lipids Lipid Metabol, 1214: 43-53. Thomas D (2008). J Am Oil Chem Soc, 75, 691-695. Toldra F (2008). Journal of Agricultural and Food Chemistry, 46(10), 3977 3984. Sentandreu (2010). Journal of Agricultural and Food Chemistry, 48(10), 5014 5022. Daniels (2011). Food Chemistry, 75(2), 159 168. Vander (2011). Meat Science, 57(1), 93 103. Veken P, et al (2013).J Comb Chemistry, 5(3), 336 44. Augustyns K (2012). Bioorg Med Chem Lett, 12(20), 2825-2828. Schlegel HG (2013). General Microbiology. 7th edition. Cambridge University Press, Cambridge, UK, 2013. Dannert C, Sztajer H, Stocklein W, Menge U and Schmid RD (2014). Biochemical Biophysica 8