Isolation and characterization of a native isolate of Leuconostoc for functional attributes

Shobharani, P. (2008) Isolation and characterization of a native isolate of Leuconostoc for functional attributes. Doctoral thesis, University of Mysore.

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Abstract

During the past several years, the focus of nutritional sciences has shifted from deficiency disease prevention to optimizing health and prevention of chronic diseases. Accordingly the research has encompassed the health effects of bioactive food components. In this regard, probiotic therapy is being used increasingly in humans and veterinary medicine due to their apparent high index of safety and public perception about natural or alternative therapies (Gherty 1995; Sander 1998; FAO Report 2002). Probiotics are a category of ‘nutraceuticals’ i.e., viable cultures added to food with the intension of maintaining or improving the nutritional health of consumers. These bacteria favorably alter the intestinal microflora, inhibit growth of harmful bacteria, promote good digestion, boost immune function and increase resistance to infection (Malin et al 1996; Haudault et al 1997). Lactic acid bacteria (LAB) are in the focus of extensive research because of their probiotic nature. One important way in which they affect health of the host is by providing enzymatic activities that improve the utilization of nutrients within the intestine. Today, probiotic market promises the disease prevention and better health for all as a natural alternative therapy. One of the important challenges to the present day probiotic industry is the eradication of lactose intolerance problem which is quite common all over the world. It is estimated that 70-90% of adults are lactose intolerant (Swegerty 2002), suffering from intestinal discomfort with the symptoms like nausea, cramps and gas (Stiles and Holzapfel 1997). In the present scenario, with increase desire of consumer for natural food products as source of providing nutrition and other desirable benefits, research work towards 2 the selection of strain with functional properties has become very important especially for treating lactose intolerance. Considering all these aspects, in the present work a strain of LAB was isolated from milk and milk products, characterized and studied for its probiotic functional attributes to be used in food formulation. Objectives 1) Isolation and characterization of Leuconostoc from milk products 2) Properties of the isolated bacterium in relation to functional significance 3) Colonization of LAB in relation to homoserine lactone Chapter 1 Isolation and screening of lactic acid bacteria from milk and milk product The problem for choosing a culture to be used in health promoting probiotic ingredients in food and pharmaceutical preparations was apparent even in the original work of Metchnikoff (1906). Reid (1999) and Sobel (1999) specify certain properties like ability to adhere intestinal cell wall, exclude/ reduce pathogenic microorganisms, produce antimicrobial compound, resist microbicides, non-carcinogenic and non-pathogenic character to be present in the selected strain for their use as probiotic culture. Now a days, a consensus is emerging for selection criteria of LAB to achieve positive probiotic effect (Collins et al 1998). Therefore, in the present work a screening procedure is performed to select a potential probiotic culture with an ultimate aim of using the culture in functional food for beneficial effect. In the present work, 45 isolates were screened for basic LAB characteristics from milk and milk products. As the most critical characteristics of probiotic strain are tolerance gastrointestinal condition (Ammor and Mayo 2007), the isolated strains were initially screened for their resistance in this harsh environment and further adapted to grow at low pH and high bile salt concentration. The strains were further analyzed for their survival under simulated gastrointestinal condition. The strains that were able to survive under 3 such environment were characterized and identified through biochemical assays and molecular techniques. The identified cultures Leuconostoc mesenteroides (Lsr-1(W)) and Lactobacillus plantarum (Lsr-12(Cu)) were further studied for their resistance to digestive enzymes pepsin and trypsin. Leuconostoc mesenteroides (Lsr-1(W)) that was better resistant to these enzymes was selected for further studies and was coded as PLsr-12(Cu). Chapter 2 Probiotic functional properties of culture isolate Probiotics have health promoting effects including inhibition of pathogens, antimutagenic, anticarcinogenic activity, prevention of diarrhea, stimulation of immune response and ability to reduce serum cholesterol levels (Tannock 1999). The development of new applications such as life vaccines and probiotic foods reinforces the need for these characteristics. In this regard, the selected culture of Leuconostoc mesenteroides (PLsr-1(W)) was evaluated for its functional properties. According to the data obtained, the culture PLsr-1(W) shows antimicrobial activity against 7 toxic food pathogens such as E. coli, S. typhi, S. dysenteriae, P. aeroginosa, V. cholerae, Y. enterocolitica and S. aureus. The isolate was found resistant to 3 antibiotics tested. The inhibitory activity of intracellular cell free extract of culture to ascorbate autooxidation, ferrous ion chelating ability and scavenging ability of oxygen radical represent the antioxidative property of culture isolate. The culture was also able to assimilate 28 μg/ml of cholesterol from media which shows the anticholesterol activity of the strain. Analysis of β-galactosidase in culture indicate the ability of the culture to hydrolyze lactose into simple sugar for easy absorption. From the results of cell surface hydrophobicity and intestinal adherence test, the adhesion ability of the culture was confirmed. Analysis of S-layer adhesion protein by SDS-PAGE showed a prominent protein band of 60 KD. Volatile compounds analyzed by GC and GCMS of culture extract confirmed the presence of therapeutically important compounds. Stress response of PLsr-1(W) to low pH and high bile salt indicate the increased content of stress 4 proteins and membrane fatty acids (saturated and unsaturated). All these properties make the present isolate a potent probiotic and will stand out as a natural cure to many diseases. Chapter 3 Leuconostoc as a source for β-galactosidase enzyme Lactose is a non-reducing disaccharide which provides almost half the total energy required by infants. In presence of lactase/ β-galactosidase, lactose gets hydrolyzed into galactose and glucose for easy absorption. But in persons deficient in lactase show symptoms of intestinal discomfort known as lactose intolerance (Sieber et al 1997). Hence studies were carried out to enhance the enzyme activity in the present culture isolate. Leuconostoc mesenteroides PLsr-1(W) that had the ability to survive the harsh conditions of GIT was studied for its ability to increase lactose tolerance. Primarily strain was improved by UV irradiation and chemical mutagenesis for enhanced enzyme activity. UV mutant strain showed 2 folds higher activity than the parent strain and hence was selected for all further studies (mutant strain was coded as M7-PLsr-1(W)). Cell permeabilization method was optimized for maximum release of enzyme. Response surface methodology (RSM) studies were undertaken for optimization of chemical and physical parameter. The enzyme produced under optimum condition of pH 7.5 with 1.25 % lactose was partially purified and studied for kinetics. The results indicate a 25 fold increase in the activity of partially purified enzyme as compared to the crude extract. Chapter 4 Enhancement of culture shelf life on storage LAB in food biotechnology or for any probiotic formulations are strictly concerned for the preservation techniques employed to ensure stable culture in terms of viability and bacterial metabolism. It is very important to use suitable technology or process to enhance and maintain the viability of the culture on storage for a beneficial effect. 5 As the present isolate M7-PLsr-1(W) is very important culture due to its characteristic multifunctional probiotic properties, it becomes necessary to keep the culture viable for a longer time. Hence the aim of the present work was to preserve the culture for longer shelf life. In this regard, the culture was subjected to different preservation techniques i.e., oven, spray, vacuum and freeze drying. The viability and resistance of culture to these methods was tested and it was found that the culture showed maximum survival to freeze drying condition. The viability under freeze drying condition was further enhanced with supplementation of different cryoprotective agents (PEG, lactose and sucrose). Enhanced membrane fatty acid composition and cellular protein confirmed the adoptive nature of the culture to freeze drying. In the present work, we also report the viability of the freeze dried culture for 6 months. It showed resistance to low pH (2.0) and high bile salt (4%). Even after 6 months of freeze drying, culture showed antimicrobial activity against 6 toxic food pathogens. Data determines that the culture retains the β-galactosidase enzyme even after 6 months of storage. The above study determines the capability of culture to remain viable even after 6 months of storage. It also shows the importance of cryoprotectants in enhancing the viability and beneficial attributes of culture during storage. This determines the capability of the culture to be utilized in the form of capsules or any functional food. Chapter 5 Functional food with Leuconostoc: a native isolate The demand for probiotic foods is increasing all over the world reflecting the heightened awareness among the public relationship between diet and health. Fermented dairy products are the most widely used food vehicles for these probiotic bacteria because of their healthy image. During the past twenty years there has been a tremendous increase in the world wide sales of cultured products containing probiotic bacteria because of their health effects (Ostlie 2005; Maltila-Sandholm 1999). 6 In this regard, the culture M7-PLsr-1(W) that has the potent probiotic properties was used as starter culture in the preparation of fermented milk beverage. The product was analyzed to be rich in protein, fat, sugar and minerals like iron, zinc and magnesium. Hence the viability of culture in the product was further enhanced with supplementation of different adjuvants (tryptone, casein hydrolysate, cysteine hydrochloride and ascorbic acid). The results conclude that the culture maintained maximum viability on supplementation of tryptone (100 mg/L) after 5 days of storage at 4°C. Fatty acid composition of the product also confirmed the nutritional property of the product. Chapter 6 Preservation of fermented milk over shelf storage Spoilage causes a significant loss to dairy industries and also disease outbreaks. In this concern, research work was carried out to investigate and preserve fermented milk beverage prepared with M7-PLsr-1(W). In this regard, the functional fermented milk beverage prepared by M7-PLsr-1(W) was studied for the predominant bacterial cultures responsible for spoilage of the product. Pseudomonas sp was identified as dominant spoilage bacteria and through TLC, GC and GCMS, the signal molecule for spoilage was investigated as hexanoyl homoserine lactone (HHSL). This culture releases HHSL and forms a quorum when high cell density is reached. Inhibition of bacterial growth in this biofilm by using biocides, antibiotic and bacteriophages has many obstacles such as cell permeability, specificity and efficacy in mode of delivery. Hence the natural furanones which are non-toxic are used in present study because of its small size and ease of delivery. Because of its structural similarity they specifically interfere with signal molecule without any adverse effect on the beneficial bacterial consortia. Results show that 2(5H)-furanone tested was having better inhibitory activity against Pseudomonas than bromofuranone. This was also in concurrence with reduction in rhamnolipid content, reduced motility and exoprotease enzyme activity. Using 2(5H)-furanone about 5-6 log of Pseudomonas culture was reduced in the fermented beverage. 7 Chapter 7 In-vivo studies using Leuconostoc for functional attributes The health benefits described for probiotic lactic acid bacteria make them a good agent for preparation of functional food and hence a number of bacterial strains are being identified and incorporation into these foods. As more probiotic organisms are discovered, it is important to carefully document the efficacy of the strain for its potential application and safety. Looking into the probiotic characteristic of the present isolate M7-PLsr-1(W) of being resistant to GIT condition, adherence ability, antimicrobial, β-galactosidase activity and anticholesterol activity through in-vitro assays the present study was aimed to assess its safety and functions in in-vivo model using albino rats. According to the result there was no deleterious effect on probiotic feeding for 3 months. No bacterial translocation was observed and hence it is likely to be safe for human consumption. Increase in the general body weight and serum urea concentration provides a potential proof for the health promoting effect of the culture. High LAB count in feces and ceacum shows the ability of the culture to resist the GIT condition and adhere to exert beneficial effect. Results of cholesterol assay confirmed the anticholesterol effect of the culture. The ability of the isolate to protect against pathogens was determined by decrease in E. coli count. Lactose intolerance, a clinical problem associated with unpleasant abdominal discomfort is due to undigested lactose. According to a survey, about 70-80% of the world population are lactose intolerant (Swagerty 2002). Innovative approaches have been tried as alternative to antibiotics in treating lactose intolerance because of the growing antibiotic resistance problem. Alternative methods such as exogenous β-galactosidase administration in functional foods or in pharmaceutical preparation is advised but the draw back is the inactivation of the enzyme in gastrointestinal transit. The other approach is gene therapy or exclusion of milk and dairy products with lactose from diet which may cause nutritional disadvantages. 8 Considering this, the present culture M7-PLsr-1(W) was tested for its ability to reduce lactose intolerance problem by in-vivo experiments. Single dose study and long term (3 months) experiments were carried out with albino Wister rats. From the study, the effective dose of culture was determined to be 108 cfu/ml. Disappearance of diarrhea in lactose intolerant induced rats after culture feeding confirmed the positive impact of culture in treating lactose intolerance. From the report presented here, the culture M7-PLsr-1(W) was found to have probiotic characters in terms of resistance to GIT, antimicrobial activity, anticholesterol activity and adherence ability. The culture was also able to reduce the symptoms of lactose intolerance and hence can be used as an alternative source to treat the problem. Achievement of the work The culture is native, isolated in laboratory and shows potential probiotic characteristics along with high β-galactosidase activity. The in-vivo experiments conducted with albino Wister rats conclude the potential probiotic functional properties and its importance in reducing lactose intolerance problem. The culture preserved by freeze drying shows viability even after 6 months of storage. The fermented milk beverage prepared with the present isolate is having a high nutritive value so that it can be used by all ages for its beneficial effects. This fermented milk beverage was preserved over a storage time by interrupting the signal molecules produced by of spoilage bacteria using 2(5H)-furanone which is a natural compound produced by an algae and known to be safe. Social and scientific relevance With the rise in the consumer’s awareness of individual health, nutrition and well being, the interest and demand for value added foods and beverages has expanded. Although some companies have marketed the probiotic products, they are either expensive or are specified for one specific cure. Most of these products 9 are not indigenous in our country and the viable count of the culture is questionable or not to the mark as labeled. Today WHO and Indian five year plans both have a common priority to replace antibiotics with natural means of cure. In this regard, the present isolate having probiotic functional properties promises to be as a source of natural alternative cure. The fermented beverage prepared by Leuconostoc mesenteroides (M7-PLsr-1(W)) has many functional attributes.

Item Type:	Thesis (Doctoral)
Uncontrolled Keywords:	Milk and milk products; Lactic acid bacteria; Probiotic functional properties; beta-galactosidase enzyme
Subjects:	600 Technology > 08 Food technology > 27 Dairy products 500 Natural Sciences and Mathematics > 07 Life Sciences > 04 Microbiology 600 Technology > 08 Food technology > 18 Processed foods > 04 Functional foods
Divisions:	Food Microbiology
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	15 Mar 2010 05:41
Last Modified:	17 Oct 2018 06:55
URI:	http://ir.cftri.res.in/id/eprint/9395

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