This article was downloaded by: [Instituto Politécnico Nacional][MRST Consortium] On: 23 November 2009 Access details: Access Details

: [subscription number 916239063] Publisher Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 3741 Mortimer Street, London W1T 3JH, UK

Food Biotechnology

Publication details, including instructions for authors and subscription information: http://www.informaworld.com/smpp/title~content=t713597251

Probiotics: An emerging food supplement with health benefits

Renu Agrawal a a Department of Food Microbiology, Central Food Technological Research Institute, Mysore, Karnataka, India

To cite this Article Agrawal, Renu'Probiotics: An emerging food supplement with health benefits', Food Biotechnology, 19:

3, 227 — 246

To link to this Article: DOI: 10.1080/08905430500316474 URL: http://dx.doi.org/10.1080/08905430500316474

PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf This article may be used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.

Food Biotechnology, 19:227–246, 2005 Copyright © 2005, Taylor & Francis ISSN: 0890-5436 print DOI: 10.1080/08905430500316474

Downloaded By: [Instituto Politécnico Nacional][MRST Consortium] At: 13:04 23 November 2009

Probiotics: An emerging food supplement with health benefits
Food Biotechnology 0000-0000 0890-5436 LFBTBiotechnology, Vol. 19, No. 03, September 2005: pp. 0–0 R. Agrawal Probiotics

Renu Agrawal
Department of Food Microbiology, Central Food Technological Research Institute, Mysore, Karnataka, India Probiotics are among the important functional foods. They comprise approximately 65% of the world functional food market. Probiotic products are foods, which improve intestinal microflora and support good health of the consumer. The live bacteria present in the probiotic products are lactic acid bacteria, including Lactobacilli, Bifidobacteria and Enterococci. Apart from health claims and maintenance of intestinal microflora, they protect against infections, alleviate lactose intolerance, reduce blood cholesterol levels and also stimulate the immune system. The interactive research between physiology, microbiology, food technology and molecular biology followed by clinical trials may produce a multi-functional probiotic strain for human consumption. Key Words: Probiotic; lactic acid bacteria; functional food

INTRODUCTION
The world population is becoming more conscious of the relation between nutrition and good health. This has stimulated increased research of the identification of food and food components that have special benefits to the consumer. With these efforts, probiotic products have come into the market, which are identified as functional foods. These include foods containing phytochemicals, dietary fibre, structural lipids, bioactive peptides, polyunsaturated fattyacids, etc. Prebiotics, probiotics and synbiotics (Holzapfel and Schillinger, 2002) are included in this category. The probiotics are related to beneficial microorganisms. The term probiotic has a number of definitions but widely accepted is that of Fuller (1989). According to him, “probiotics” are live microbial food supplements that beneficially affect the host animal by improving the intestinal microbial balance.

Address correspondence to Renu Agrawal, Department of Food Microbiology, Central Food Technological Research Institute, Mysore, Karnataka, 570 013, India. E-mail: renuagrawal46@rediffmail.com

228

R. Agrawal

The usefulness of lactic acid bacteria was first proposed by Metchnikoff (1908), who suggested that longevity was due to their high intake.

TAXONOMY OF PROBIOTIC MICROORGANISMS
Downloaded By: [Instituto Politécnico Nacional][MRST Consortium] At: 13:04 23 November 2009

Phylogenetic and genetic criteria
It is early to make generalizations about probiotic performance on a scientific basis, but it is generally assumed that probiotic properties are strain specific. Hence, the need for strain identification and detection is very important (Brandt and Alatossava, 2003). Important genera that are employed for probiotic purpose are generally Lactobacilli, Bifidobacteria and Enterococci. Lactic acid bacteria (LAB) are gram positive, non-sporing, catalase negative organisms that are devoid of cytochrome C and are non-aerobic, but are aerotolerant and acid tolerant. LAB can be divided into two physiological groups: the heterofermentative LAB, which produce CO2, lactic acid, acetic acid, ethanol and mannitol from hexoses, and the homofermentative LAB, which produce primarily lactic acid from hexose (Kandler, 1983). Traditionally, LAB has been classified on the basis of phenotypic properties such as mode of glucose fermentation, growth at different temperatures, configuration of lactic acid produced (L/D)and fermentation of carbohydrates. Holzapfel et al. (1997) and Klein et al. (1998) have dealt in detail about the taxonomy and physiology of probiotic lactic acid bacteria. According to international standards, the probiotic cultures used in food should be well defined and correctly named according to valid taxonomic systems. The selection criteria for probiotic microorganisms have been reviewed by Holzapfel et al. (2001). Phylogenetic analysis was found not sufficient for a better characterization up to the strain level. Here genetic methods play a very important role for further characterization. Holzapfel (1997) has dealt in detail with systematic identification of probiotic lactic acid bacteria with reference to phenotypic and genomic methods. These genomic methods have been applied with a good amount of success. In genetic analysis, plasmid profiling was formerly preferred, but it was found that extra chromosomal DNA was unstable. Therefore, methods based on chromosomal DNA were developed. These include restriction enzyme analysis (Ferrero et al., 1996), ribotyping (Rodtong, 1993), Randomly amplified polymorphic DNA analysis (Duplessis, et al., 1995; Klijn et al., 1995; Collins et al., 1991; Erlandson and Bhatt, 1997) and gradient gel electrophoresis (Alatossava, 2000; Walters et al., 2000). These workers have shown the efficacy of the genotypic methods in strain identification. Pot et al. (1997) have studied taxonomy of microorganisms used as probiotics with the help of the above methods for species of Enterococci, Lactococci and Lactobacilli. Charteris et al. (1997) and Simpson et al. (2001) demonstrated the selective details, enumeration and identification of potential probiotic lactic acid

Probiotics

229

bacteria, Bifidobacterium bifidum and other species. For a correct identification of lactic acid bacterial strain, it is essential to employ both phenotypic and genetypic methods. Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus are mainly used as probiotics, whereas strains of Enterococci are mainly used in nutrition. For phenotypic based criteria, important functions for consideration are carbohydrate fermentation pattern, resistance to different NaCl concentrations, growth on different nutrient media, growth at different temperatures and resistance against antibiotics. Recently, molecular-based identification has been done by ribotyping (Zhong et al., 1998). In modern taxonomic phenotypic methods analysis of cell wall composition (peptidoglycan) and fermentation pathways of pentoses and hexoses are undertaken. Lactobacillus and Bifidobacterium constitute a significant proportion of probiotic cultures used in developed countries. Although these two species have been isolated from different parts of the GI tract, the terminal ileum and colon appear to be the preferred sites. Species of Bifidobacterium are generally characterized as gram positive, non-spore forming, non-motile, catalase negative and anaerobes. The enzyme fructose-6-phosphate phospho- ketolase, the key enzyme of the glycolytic pathway, serves as a taxonomic character in identifying genera but does not enable inter-specific differentiation. At present, 29 species of Bifidobacteria genera are recognized, of which 10 are of human origin. Differential detection and enumeration can be performed in a number of ways. Different plating media are employed which permit separate cultivation of each genus. In another method, a single plating medium is used that supports the growth of designated genera. Final characterization consists of sugar tolerance, nutritional requirement and antibiotic susceptibility. More details are given by Charteris et al (1997), including a number of probes that have been designed for Lactobacillus and Bifidobacterium species. Klein et al. (1998) have used both phenotypic and genotypic methods for detailed analysis of various probiotic bacteria. In order to see that probiotics should not have any negative effect on the host, careful selection of the strain is very important. They show the importance of proper identification with the help of phenotypic and genotypic methods of application to differentiate various probiotic species.

Downloaded By: [Instituto Politécnico Nacional][MRST Consortium] At: 13:04 23 November 2009

In vitro studies
The site of action of lactic acid bacteria and Bifidobacteria is the gastrointestinal tract. Before the organism reaches the gut, it has to pass through the stomach having approximately 2.5 litres of gastric juice (pH 2.0) secretion per day (Charteris et al., 1998). This usually causes destruction of most of the microorganisms ingested. Therefore, the acid resistance property of probiotic bacteria is of utmost importance and consideration. At the same time, the

230

R. Agrawal

Downloaded By: [Instituto Politécnico Nacional][MRST Consortium] At: 13:04 23 November 2009

organism has to resist bile acids in the intestine and should adhere to the intestinal walls for its effectiveness. In order to achieve probiotic lactic acid bacteria with all these properties scientists are developing a keen interest in inducing these properties in lactic acid bacteria. Towards this, Jacobson et al. (1999) studied 47 strains of Lactobacilli, observing that 29 of them could survive a low pH (pH 2.5) for four hours and 16 strains grew well with bile salts and oxgall. Only four strains were found to have strong adhesion to Caco-2 cells, whereas the rest exhibited mild adherence. The ability of each bacterium was found to be different and was strain specific (Kimoto et al., 1999). Hamilton- Miller (2004) and Vinderola et al. (2003) made a comparative study on probiotic characteristics and biological barrier resistance in yogurt. Bernet et al. (1994) studied the mechanism involved in the adherence of microorganism to intestinal epithelial cells. Gerriste et al. (1990) studied the properties by oral administration of TNP-Lactobacillus conjugates in mice and evaluated mucosal and systemic immune responses and memory formation. Greene and Klaenhammer (1994) studied the factors involved in the adherence of Lactobacilli to human Caco-2 cells.

PROBIOTIC PRODUCTS
Probiotic organisms require a vehicle to reach the site of action in an active form, which is the G.I. tract of the human body. The vehicle is generally a food product, which contains these live bacteria. Scientific evidence suggests that probiotic bacteria consumed at a level of 109-1011 cfu/day can decrease the incidence and severity of some intestinal illnesses (Zubillaga et al., 2001). The products should have a good shelf life and should have a cell count higher than 106 cfu/ml till the end. The product should also go through the harsh conditions of gastric acid and bile salts before it reaches the G.I. tract, which is the site of action.At present, most of the individual probiotic foods belong to dairy products like yogurt, fermented milk and cheese (Table 1). A number of new products based on cereals, fruits, vegetables and meat are in the development stages. These products have to undergo human trials before
Table 1: Probiotic products available in the market with lactic acid bacteria

supplementation.
Product Bacteria

Yogurt Kefir Acidophilus milk Fermented milk Fermented vegetables

Lactobacillus bulgaricus, Streptococcus thermophilus Lactobacillus casei and Bifidobacterium sp. Lactobacillus sp. Lactococcus sp. Leuconostoc sp. Lactobacillus acidophilus Enterococcus faecium Streptococcus thermophilus Lactobacillus sp.

Adopted from E.R. Farmworth (2000).

Probiotics

231

they are medically accepted. Gilliland et al. (2002) studied the viability of L. casei and Bifidobacterium in yogurt-like products along with L. casei and B. longum. It was observed that pH influenced the survival rate in the products when stored at 5° C for 35 days. Similar studies have been done by Schillinger (1999). Vinderola et al. (2003) studied the behavior of Argentina yogurt and observed that full fat probiotic yogurt (pH 4.5) was inhibitory when stored at 5° C for 4 weeks. A thorough study has been done by Ostlie et al. (2003) towards the growth and metabolism of selected strains of probiotic bacteria in milk. In a detailed study of different probiotic milk products, Hattingh and Viljoen (2001) noticed that the survival of microbes was dependent on various factors like the strain used, interaction between species present in the product and culture conditions. Among milk products, the main probiotic carriers in foods are yogurt, fermented milk and cheese. A number of reviews are available on the health benefits of the consumption of fermented dairy products (Dave and Shah, 1997; Kurman and Rasie 1991). It is generally assumed that consumption of probiotic yogurt should be more than 100 g/day containing more than 106 Cfu/ml (Rybka and Kailaspathy, 1995). In recent years, there has been a significant increase in the popularity of yogurt emphasizing the incorporation of L. casei and B. bifidum. The conventional yogurt starter bacteria L. bulgaricus and St. thermophilus lack the ability of surviving the passage through the G.I. tract and consequently do not play a role in the human gut. Hattingh and Viljoen (2001) reviewed the role of yogurt as a probiotic carrier food. The consumption of probiotic products is helpful in controlling intestinal diseases (Mittal and Garg, 1992). Besides yogurt, other probiotic products are fermented milk (Nighswonger et al., 1996), beverages and probiotic cheese (Gomes et al., 1995), Cotton cheese (Blanchette et al., 1995), powdered milk, cookies and ice creams (Hekmat and Mc Mohan, 1992) and dairy desserts (Laroia and Martin, 1991; Anandan et al., 1999). Cheddar cheese has been shown to be an effective vehicle for the delivery of some probiotic organisms, especially L. paracasei. Cheese has a higher pH than yogurt and fermented milk and hence can support survival of probiotic bacteria for a longer duration (Gardiner et al., 1998). Dinaker and Mistry (1994) studied the growth and viability of Bifidobacterium bifidum in cheddar cheese. According to them, viability of this organism in products over a long shelf life at refrigeration temperature is not satisfactory. The low pH of fermented milk and the aerobic conditions of packaging are not conducive for their survival. In their study the organisms were viable during 24 weeks of study.

Downloaded By: [Instituto Politécnico Nacional][MRST Consortium] At: 13:04 23 November 2009

Non-milk probiotic products
Besides milk-based probiotic products, attempts are being made to develop non-milk probiotic products (Molin, 2001) mainly for the treatment of

232

R. Agrawal

Downloaded By: [Instituto Politécnico Nacional][MRST Consortium] At: 13:04 23 November 2009

lactose intolerance and control of cholesterol levels which is a drawback in milk based products. Arihara and Itoh (2000) have isolated UV induced L. gasseri mutants that are resistant to NaCl and sodium nitrite. These can be used as starter culture for the preparation of probiotic meat products. As this organism is dominantly found in the human intestinal tract it may be an appropriate probiotic starter culture for meat fermentations. Papamanoli et al. (2003) isolated lactic acid bacterial strains from fermented sausages, which can be used as starter cultures for the preparation of meat fermentations. They found that traditionally prepared sausages with long ripening time have better sensory qualities than when a UV induced mutant strain was used. After the fermentation they isolated the culture strains, and found that 90% belonged to Lactobacillus.

Fermented Milk Products
According to Nighswonger et al. (1996), L. casei and L. acidophilus survive better in cultured buttermilk than in yogurt. Ostlie et al. (2003) studied the cell count in milk which was above 8.7–9.18 log cfu/ml after 6–16 h of incubation. According to Sodini et al. (2002), the addition of casein hydrolysate to a milk product gave it a good taste and improved the flavor. Hattingh and Viljoen (2001) made a detailed study of milk products with probiotics, concluding that the survival of microbes depends on various factors such as strains used, interaction between species, culture conditions, growth promoters and inhibitors, etc. When children were fed with probiotic curd, the increase in body weight was found to be much higher at the end of 90 days as compared with controls. Also, the incidence of diarrhea was found to be reduced.

ENCAPSULATION OF PROBIOTICS
Encapsulation is a new technology being applied to increase the storage life of the probiotic product. Betoret et al. (2003) have developed apple cylinders impregnated with apple juice containing L. casei. Samples were air dried at 40° C to a water content of 0.037 Kg water/kg product and stored at ambient temperature for two months. At the end of this period viability of cells in the product was 106 Cfu/g. Cruce and Goulet (2001) have also suggested microencapsulation as affording protection to products containing LAB. Certain protective agents like sugars, sugar alcohols, milk, starch, maltodextrin and N2 flushing are known to enhance the ability of microorganisms to survive. According to Hansen et al. (2002), however, encapsulation was not very successful as it resulted in off-flavor in some cases. Fito et al. (2001) have tried to use the methodology for incorporation of active components such as prebiotics,

Probiotics

233

dietary fibres and plant sterols. In their study Cruce and Goulet (2001) reported that in non-fat dry milk the stability after a shelf life of 4 months at 30° C of microencapsulated P. acidilactici was 69%, while that of a standard freeze-dried product (probiotic powder) was only 15%. This technology promises to be of great help in increasing the survival rate of probiotics and should see much more developments in the coming years.
Downloaded By: [Instituto Politécnico Nacional][MRST Consortium] At: 13:04 23 November 2009

CLINICAL STUDIES
This is an area of probiotic therapy that requires detailed attention. If the studies are carried out systematically, they will prove useful in the treatment of various clinical disorders such as different types of diarrhea, gastrointestinal disorders and cholesterol levels in the blood. This aspect is different from the one where probiotics are used for the well being; the latter does not require a medical prescription. Probiotics are also used for their immunological and anti-microbial properties. There are many reports in the literature regarding clinical trials but most of the studies did not have proper controls like blindfold studies and therefore could not be taken ahead. Farmworth (2000), Tamboli et al. (2003), Bengmark (2003), Surawicz (2003) and Marteau (2003) have reviewed the medical studies carried out recently with proper controls.

STUDIES WITH DIFFERENT TYPES OF DIARRHOEA 1. Infantile diarrhea
The most common cause of diarrhea in children is rotavirus infection. Savedra et al. (1994) randomized 55 children in the age group of two years treated with B. bifidum and St. thermophilus in milk fermentations. The overall rate of diarrhea was reduced in the treated group. Other workers have also confirmed the usefulness of probiotics in infantile diarrhea (Isolauri et al., 1991; Oberhelman et al., 1999).

2. Studies with traveler’s diarrhea
These studies have not been equivocal (Hilton et al., 1997; Black et al., 1989). It is not possible to compare the results of one strain to another, as mechanisms underlying clinical effects are not clearly understood.

3. Antibiotic associated diarrhea (AAD)
Siitonen et al. (1990) reviewed the use of probiotics like Lactobacillus rhamnosus G.G and the yeast Saccharomyces boulardii in antibiotic-associated

234

R. Agrawal

Downloaded By: [Instituto Politécnico Nacional][MRST Consortium] At: 13:04 23 November 2009

diarrhea. Both microorganisms were found effective. It is felt that more controlled studies and safety trials are required before this treatment is accepted. Mc Farland et al. (1995) and Tankanow et al. (1990) found positive results in patients suffering with AAD, although Barlett et al. (1980) did not notice any encouraging results. It is therefore felt that much more work has to be done with travelers’ and antibiotic associated diarrhea except infantile diarrhea, which is caused by rotavirus.

Inflammatory Bowel Disease (IBD)
Tamboli (2003) and Martin (2005) reviewed the effect of probiotics in IBD. The pathogenesis of IBD is not clearly understood. Intestinal microorganisms as mediator of this inflammation are receiving increasing attention. Studies of host-microbe interactions have provided support for probiotic therapy in IBD. Many workers (Kruis et al., 1997) have studied the effect of mesalamine and compared it with an E. coli strain. After 12 weeks of therapy about 11–16% patients had relapse in both groups. In a randomized trial, non-pathogenic E. coli treatment was compared with mesalamine therapy for treating ulceritis colitis. Remission in both the groups was around 70% (Rembaekan et al., 1999). The numbers of trials conducted over a period do not give a clear-cut picture about the utility of probiotics in ulceritis colitis treatment. Reid et al. (1995, 1998) have observed the prevention of urinary tract microbial infections when Lactobacilli strains were instilled.

Critically ill patients
Probiotics and prebiotics have been shown to reduce the rate of infection in sick and postoperative patients. Compounds such as antibiotics and pharmacological agents have detrimental effects on mucosal flora and immune systems. Recent studies in patients with severe and acute pancreatitis abdominal surgery and liver transplant patients have shown some beneficial effects with the use of probiotic Lactobacillus plantarum 299V along with oral fibre as a prebiotic (Olah et al., 2002). In another study of abdominal surgery patients, when L. plantarum 299V was fed with oat fibre, no significant differences were found as compared with the control possibly due to the low level of lactic acid bacteria and oat fibre employed in the study.Bengmark (2003) and Edgar (2005) has reviewed the use of probiotics in many other surgical cases.

Immune Systems
Recently, Gill et al. (2000) have shown that probiotics enhance natural immune functions by dietary consumption of L. lactis. According to Schiffrin et al. (1995), it appears that the cultured strain may adhere transiently and colonize the GI tract, ultimately increasing the IgA levels. Isolauri et al.

Probiotics

235

(2001) proposed that many probiotic effects are modulated through immune regulation of pro and anti-inflammatory cytokines. Similarly probiotic ingestion is reported to stimulate cytokine production in blood cells.

Antimicrobial Properties
Downloaded By: [Instituto Politécnico Nacional][MRST Consortium] At: 13:04 23 November 2009

With the emergence of antibiotic resistant microorganisms, the concept of probiotic therapy is gaining ground. The lowering of pH by acids like lactic acid and acetic acid has bacteriocidal and bacteriostatic effects. In addition, they also produce H2O2 and bacteriocins. Kasper (1998) demonstrated antimutagenic properties by acids produced against a number of mutagenic compounds. Guarner (2003) and Tuohy et al. (2003) have shown that LAB ingestion reduced the activity of enzymes like β-glucoronidase, nitro reductase and azoreductase which transform precarcinogen into active form. Lactic acid bacteria are useful in the promotion of human health. According to medical experts correct identification of the strains with medical evidence along with clinical trials will allow the authorities to recommend them for therapeutic purposes.

SAFETY ASPECTS OF PROBIOTICS
Most probiotics are marketed as ingredients of foodstuff or drugs, while others are sold in the live form as freeze dried powders, tablets or capsules. Hence, consideration of safety of these microorganisms or products is of utmost importance, as they are being used for human consumption. According to Gasser (1994) many lactic acid bacterial strains have been isolated from bloodstream infections and local infections. Aguirre and Collins (1993) reported lactic acid bacteria from infection sites; these reports ensure the safety of lactic acid bacteria as probiotics. Adams and Marteau (1995) have tested the safety of lactic acid bacteria by in vitro methods, animal studies and human clinical studies.

Factors involved in safety considerations
Strains of LAB found in the intestinal microflora are preferentially used as probiotics. Knowledge of their reactions in the intestinal microbial association has to be increased (Darla et al., 2005). It is necessary to understand the ecological performance of these probiotic strains. Prebiotics A prebiotic is defined as non-digestable food ingredient that beneficially affects the host by selectively stimulating the growth and activity of a number of bacteria in the colon, improving the host’s health. Commercially available

236

R. Agrawal

prebiotics are fructooligosaccharides (FOS), insulin, lactulose and galactooligosaccharides. Prebiotics mainly help the individuals with low levels of Bifidobacteria in the elderly (Tuohy, 2001). He observed the beneficial effects in the modulation of gut microflora. The prebiotic effect has been studied in human clinical trials by feeding biscuits containing partially hydrolysed guar gum and fructooligosaccharides. Although there is no recommended daily dose of prebiotics, a report by Roberfroid et al. (1988) suggests a daily dose of 4g/day of insulin or FOS to increase gut Bifidobacteria. Rastall and Gibson (2002) have shown the natural presence of prebiotics in breast milk and vegetables such as Jerusalem artichokes and onions. Hamilton (2004) has written a review of clinical trials for benefits from prebiotics and probiotics to elderly populations helpful in malnutrition, lactose intolerance, calcium absorption and constipation. Pool-zobel et al. (2002) found that insulin and FOS reduced the number and size of precancerous lesions as well as tumor incidence in carcinogen-treated rats. Roberfroid (2000) linked prebiotics to an enhancement of mineral absorption in the large bowel. A synbiotic approach (a mixture of probiotics and prebiotics) has shown beneficial results. Kiebling et al. (2002) have shown that long-term (7 weeks) consumption of synbiotic yogurt (L. acidophilus 145, B. longum 193 + FOS) led to significant effects in LDL/HDL cholesterol ratios in 29 healthy women. Despite the numerous studies and human clinical trials, there is still a gap in our knowledge of the metabolic pathways taken by prebiotics and probiotics. Safety is also required for prebiotics, which are frequently used with probiotics. If the prebiotic is a traditional food or a food component, there is no concern. Hammes and Hertel (2002) have given some details about the safety aspects of LAB used as probiotics. According to them, the intestinal microflora may exhibit properties in foods that are not revealed in the intestinal tract. It is possible that LAB may have pathogenic potential, as reports in the literature indicates for their involvement in human infection. According to Link et al. (1994), changes in intestinal flora with intake of fermented milk have been reported. It is recorded that strains of Enterococci are involved in human infections. Because of these observations, doubts have been raised as to whether Enterococci can be classified as safe probiotics (Aguirre and Collins, 1993). Ishibashi and Yamaguchi (2001) have written an excellent review on probiotics and safety, in which they covered various aspects such as absence of pathogenicity and infectivity as a requisite for probiotic safety and enzymatic activity associated with production of harmful substances.

Downloaded By: [Instituto Politécnico Nacional][MRST Consortium] At: 13:04 23 November 2009

MARKET POTENTIAL OF PROBIOTICS
Like acceptance of any product in the market, it should be realized that increased consumption is dependent on many factors. As more and more people are becoming health conscious, the sales of probiotics is increasing.

Probiotics

237

Downloaded By: [Instituto Politécnico Nacional][MRST Consortium] At: 13:04 23 November 2009

Manufacturers are aware of this and are trying to get newer products into the market. Since these are edible products, the foremost requirement is that the probiotic product should be sensorily acceptable and liked by people. The concept of probiotics for improving the health status of an individual was developed in Japan in the 1980s. In the last ten years the probiotic products mainly being dairy products have established themselves in Europe and in the USA. There is not much literature on probiotic consumption in developing countries. The problem with probiotic preparations is that in many products although the addition of lactic acid bacteria is mentioned, the particular strain used is not mentioned. According to Klein (1999), it is very important to know whether all the strains of LAB or bifidobacteria are useful as probiotics.

PROBIOTIC CONSUMPTION AROUND THE WORLD
Japan: A product “Yakult” consisting of bifidobacterial strains, whey, minerals and dietary fibre had sales of more than 11 trillion yen. In addition, fermented milk drinks are in great demand (Arunachalam, 1999). According to the literature, the market in Japan for probiotics and other functional foods is approximately $ 3.5 billion. Europe: In Europe (1997), 65% of functional food market consists of probiotic products of $890 million. The yogurt market is worth $600 million. Yogurt with live lactic acid bacteria is on the forefront with its acceptable taste and flavor. Consumers are mostly concerned with health problems such as osteoporosis, cholesterol level in serum, heart diseases and general well being. Hence, products should be available for treating them. A survey in Europe (1997) indicated that more than 97% of consumers look for healthier food. U.S.A.: The labeling requirement of probiotic food products is less as compared with Japan and Europe. However, strong medical claims are not allowed without strong clinical data. “Acidophilus yogurt” is on the forefront among probiotic products. Overall, enough evidence is available to indicate beneficial effects of probiotic products on good health. The increase in sales in this direction shows the growing health consciousness of people. More data are necessary to determine the medical benefits and claims for better health. It is expected that in the first decade of the twenty-first century the global market for probiotic foods will be around $17 billion. Stanton et al. (2001) discussed about the various probiotic products and the different companies dealing with it. Currently, most of the products are milk products; new products are being investigated with base as fruit juices or fermented whey (Calvo et al., 2002). Arihara and Itoh (2000) studied the importance of probiotics in meat products to be utilized for large-scale preparation.

238

R. Agrawal

LABELING OF COMMERCIAL PROBIOTICS
In veterinary and human medicine, probiotic therapy is becoming very important, and many probiotic products are available commercially. Although probiotics are mainly food supplements they are not regulated for efficacy and quality control. In a survey examining the labels of 44 probiotic products, Weese (2003) observed that labeling on 44% of the products was improper, as most of them did not contain the stated number of organisms (Weese, 2002). According to Weese (2003), a probiotic label should state the organism upto strain level, correctly spelled, and also state the number of live organism until the expiry of the shelf life. To date, none of the products in the market fulfill all these criteria. Also, the product should be scrutinized by the veterinary practitioners like any other pharmaceutical products. There is much scope for research and development in this line, as proper labeling will help in the prevention and treatment of many diseases in a natural way.

Downloaded By: [Instituto Politécnico Nacional][MRST Consortium] At: 13:04 23 November 2009

GENERAL HEALTH BENEFITS BY PROBIOTICS
Probiotics have shown their potential in the prevention of certain diseases and also promoting specific aspects of health. They are helpful in improving problems of malnutrition, lactose intolerance, calcium absorption and constipation (Hamilton-Miller, 2004; Groote et al., 2005). The gut microbiota plays an important role in human health and disease by preventing the colonization of pathogenic microorganisms (Tuohy et al., 2003). A range of probiotic strains have been evaluated for their antidiarrheal capabilities (Isolauri, 1991), prevention of colorectal cancer (CRC) and chronic mucosal inflammation (IBD) by a non-pathogenic strain of E. coli (Rembacken et al., 1999). Many probiotics have shown to lower the activity of β-glucuronidase, β-glycosidase, azoreductase and nitroreductase, which are known to convert precarcinogens to carcinogens (Burns and Rowland, 2000). Probiotics have also been shown to liberate low molecular weight peptides in G.I tract and trigger the immune system (Isolauri et al., 2001). There is currently a concerted effort in Europe to study the gaps in knowledge concerning the medical efficacy and mechanistic principles of microbiota using probiotics, prebiotics and synbiotics (Mattila-Sandholm et al., 2003). All these studies have enhanced the general health of human beings and reduced the onset of diseases.

FUTURE DEVELOPMENTS
If probiotics are to represent a real and effective alternative to antibiotics and chemotherapeutics, much more work is required to select LAB strains with strong probiotic effects, and methods should be developed to ensure maximum efficacy of probiotic at the time of consumption. Food companies will continue

Probiotics

239

to research new functional food products with health claims. Product launches in areas like probiotic food products, fortified foods and drinks see a lot of future. Of course, the market growth will depend on scientific substantiation. The rate of increase in resistance to antibiotics is a major public health problem all over the world (Neu, 1994; Bengmark, 1998). Hence, natural alternatives are becoming more attractive. In fact, the World Health Organization recommends a global programme to reduce the use of antibiotics. The clinical use of probiotics showed suppression of pathogens, giving evidence for their consumption in inhibiting the growth of enteropathogens (Gousalez et al., 1993, Drago et al., 1997; Hudault et al., 1997). In addition, the data collected suggest the potential use of probiotics in preventing urino genital tract microbial infections (Reid et al., 1995, 1998).There is a need to work out the use of probiotics as alternatives to antibiotics in animals and poultry. With the advances in sequencing and bioinformatics technology, genomesequencing projects will be looked into in the near future for probiotic microorganisms. According to Tamboli (2003), the development of probiotics capable of effectively delivering cytokinin therapy is a major advancement in biological therapy to IBD. The use of genetically modified organisms in humans may take some time for their application. Bengmark (2003) has emphasized the use of a probiotic consortium. It is expected that strong clinical efficiency can be achieved in the future by the use of probiotic LAB consortiums with several bioactive properties. According to Surawiz (2003), the population of high risk AAD should be identified before widespread use of probiotics is undertaken. Also, there is need for well-controlled trials of efficacy with adequate safety studies. In spite of numerous feeding studies showing positive effects on health, there is limited understanding concerning the mechanism of probiotic activity in vivo. Modern high-resolution molecular techniques based on the phylogenetic information encoded by 16S rRNA gene are being applied to characterize the gut microflora within different disease strains. This is likely to bypass the inherent limitations of lack of selective growth media and uncontrollable bacteria. Recently, in Europe, a number of European Union projects have been funded to identify the mechanisms through which pro-, pre- and synbiotics can improve host health. According to Steidler (2003), it is possible through genetic engineering to improve the existing strains and also to create completely new probiotics. This is possible with thorough knowledge of metabolic reactions in various probiotics. The metabolism of microorganisms can be modified through the integration of foreign enzymes. Paton et al. (2001) have already constructed an E. coli strain that could answer the need for therapeutics to counteract intestinal infection with Shigella toxin producing bacteria. It must be realized that for current genetically modified (GM) probiotics, biological safety has to be assured. Also, the uninhibited spread of GM

Downloaded By: [Instituto Politécnico Nacional][MRST Consortium] At: 13:04 23 November 2009

240

R. Agrawal

microorganisms in the environment is highly undesirable. Thus, biosafety of the strains produced has to be thoroughly investigated. Steilder (2003) in his review has given details of application of GM probiotics under various clinical conditions.

Downloaded By: [Instituto Politécnico Nacional][MRST Consortium] At: 13:04 23 November 2009

CONCLUSIONS
Since there is substantial demand for natural treatments with respect to various diseases, probiotics are gaining importance. Moreover, improved functional characteristics of milk products could be potentially achieved by altering the aminoacid sequence. Targets for modification include α-lactoalbumin, β-interferon, γ- interferon, factor IX, protein C, serum albumin superoxidase, lactoferrin, lyzozyme and immunoglobulins (Mathur et al., 2003). To capitalize on this emerging knowledge better molecular and metabolic studies of bacteria from infection sites and the ones used for probiotics are essential (Saxelin et al., 1996). Further understanding the expression of novel disease-protective proteins in probiotic systems could be an attractive solution for disease management through the diet.

ACKNOWLEDGEMENT
I express my gratitude to Dr. V. Prakash, Director, CFTRI. I thank Dr. S. Umesh, HOD, Food Microbiology for encouragement. I thank Dr. M.V. Patwardhan for discussions and Shobha Rani. P for assistance.

REFERENCES
Adams, M.R., Marteau, P. (1995). On the safety of lactic acid bacteria from food. Int. J. Fd. Microbiol. 27:263–264. Anandan, S., Dey, A., Deb, S.M., Kumar, S., Harbola, P.C. (1999). Effect of curds as a probiotic supplement on performance of ehenghu cross bred kids. Small ruminant Res 32:93–96. Arihara, K., Itoh, M. (2000). UV induced Lactobacillus gasseri mutants resisting sodium chloride and sodium nitrite for meat fermentation. Int. Microbiol. 56:227–230. Arunachalam, K.D. (1999). Role of Bifidobacteria in nutrition, medicine and technology. Nutr. Res. 19 (10):1559–1597. Augirre, M., Collins, M.D. (1993). Lactic acid bacteria and human clinical infection. J. Appl. Bacteriol. 75:95–107. Barlett, J.G., Tedesco, F.J., Shull, S., et al. (1980). Symptometric relapse after oral vancomycin therapy of antibiotic associated pseudomembranous colitis. Gastroenterol 78:434–434. Bengmark, S. (1998). Ecological control of gastrointestinal tract: The role of probiotic flora. Gut 42:2–7.

Probiotics Bengmark, S. (2003). Use of some pre, pro and symbiotics in critically ill patients. Best Practice and Res. Clin. Gastroenterol. 17 (5):833–846. Bernet, M.F.D., Brassart, J.R., Nesser Servin, A.L. (1994). Lactobacillus acidophilus LAI binds to cultured human intestinal cell lines and inhibits cell attachment and cell invasion by enterovirulent bacteria. Gut. 35:483. Betoret, N., Puente, L., Diaz, M.J., Pagan, M.J., Garcia, M.L., Gras, J., Martinez, M., Fito, P. (2003). Development of probiotic enriched dried fruits by vaccum impregnation. J. Fd. Eng. 56:273–277. Black, F.T., Anderson, P.L., Orskow, J., Orskow, F., Gaarslev, K., Laudlund, S. (1989). Prophylactic efficacy of Lactobacillus in traveller’s diarrhoea. In: Travel Medicine Conference on International Travel Medicine. Steffen, R. (ed.). Berlin: Springer, pp. 335–333. Blanchette, L., Roy, D., Gautheir, S.F. (1995). Production of cultured cottage cheese dressing by Bifidobacteria. J. Dairy Sci. 78:1421–1429. Brandt, K., Alatossava, T. (2003). Specific identification of certain probiotics. L. rhamnosus strain with PCR primers based on phage related sequences. Int. J. Fd. Microbiol. 84:189–196. Burns, A.J., Rowland, I. R. (2000). Anticarcinogenicity of probiotics and prebiotics. Curr. Iss. Intest. Microbiol. 1:13–24. Calvo, M.M., Diez, O., Cobos, A. (2002). Use of rectified grape juice in yogurt edulcoration. J. Fd. Sci. 67 (8):3140–3143. Charteris, W.P., Kelley, P.M., Morelli, L., Collins, J.K. (1997). Selective detection, enumeration and identification of potentially probiotic Lactobacillus and Bifidobacterium sp. in mixed bacterial population. Int. J. Fd. Microbiol. 35:1–27. Charteris, W.P., Kelley, P.M., Morelli, L., Collins, J.K. (1998). Antibiotic susceptibility of potentially probiotic Lactobacillus species. J. Fd. Protection. 61:1636–1643. Collins, M.D., Rodrigues, V., Ash, C. (1991). Phylogenetic analysis of the genus Lactobacillus and related lactic acid bacteria as determined by reverse transcriptase sequencing of 16s. rRNA. FEMS Microbiol. Lett. 77:5–12. Cruce, P.S., Goulet, J. (2001). Improving probiotic survival rates. Fd. Technol. 55:36–43. Darla, V., Grimes, C.A, Lee Giles, C. (2005). Farm to table: A situation awareness model for food safety assurance for porous borders. Comprehensive reviews. Fd.Sci.Fd. Safety. 4 (2):31–33. Dave, R.I., Shah, N.P. (1997). Viability of yogurt and probiotic bacteria in yogurts made from commercial starter cultures. Int. Dairy J. 7:31–41. Dinakar, P., Mistry, V.V. (1994). Growth and viability of Bifidobacteria trifidacemin in cheddar cheese. J. Dairy Sci. 77:2854–2864. Drago, L., Gismondo, M.R., Lombardi, A., de Haen, C., Gozzini, L. (1997). Inhibition of in vitro growth of enteropathogens by Lactobacillus isolates of human intestinal origin. FEMS Microbiol. Lett. 153:455–463. Drouanlt, S., Juste, C., Marteau, P. (2002). Oral treatment with Lactococcus lactis expressing Staph. lyicus lipase enhances lipid digestion in pigs with induced pancreatic insufficiency. Appl. Environ. Microbiol. 68:3166–3168. Du Plessis, E.M., Dicks, L.M.T. (1995). Evaluation of randomly amplified polymorphic DNA (RAPD) PCR as a method to differentiate L. acidophilus, L. Crispatus, L. amylovorus, L. gallinarum, L. gasseri and L. Johnsonii. Curr. Microbiol. 31:114–118. Edgar, B. (2005). Enteric infections. Curr. Opinion. Gast. 21 (1):1–3.

241

Downloaded By: [Instituto Politécnico Nacional][MRST Consortium] At: 13:04 23 November 2009

242

R. Agrawal Erlandson, K., Batt, C. (1997). Strain specific differentiation of Lactococci in mixed starter culture population using randomly amplified polymorphic DNA derived probes. Appl. Environ. Microbiol. 63:2702–2707. Farmworth, E.R. (2000). Designing a proper control for testing the efficacy of a probiotic product. J. Nutr. Func. Med. Fds. 2 (4):55–63. Ferrero, M., Cesena, C., Morelle, L., Scolari, G., Vesecovo, M. (1996). Molecular characterization of L. casei strains. FEMS Microbiol. Lett. 140:215–219.

Downloaded By: [Instituto Politécnico Nacional][MRST Consortium] At: 13:04 23 November 2009

Fito, P., Chiralt, A., Bardt, J.M., Andre, A., Martine, Z.M., Monzo, J., MartinezNavevvete, N. (2001). Vacuum impregnation for development of new dehydrated products. J. Fd. Eng. 49:297–302. Fuller, R. (1989). Probiotics in man and animals. J. Appl. Bacteriol. 66:365–378. Gardiner, G., Ross, R.P., Collins, J.K., Fitzgerald, G., Stanton, C. (1998). Development of probiotic cheese containing human derived L. paracasei strain. Appl. Environ. Microbiol. 64:2192–2199. Gasser, F. (1994). Safety of lactic acid bacteria and their occurrence in human clinical infections. Bull. Inst. Pasteur, 92:45–67. Gerriste, K., Posno, M., Schellekens, M.M., Boersma, W.J.A., Cleassen, E. (1990). Oral administration of TNP. Lactobacillus conjugates in mice: a model for evaluation of mucosal and systemic immune responses and memory formation elicited by transformed lactobacilli. Res. Microbiol. 141:955. Gill, H.S., Rutherfurd, K.J., Prasad, J., Gopal, P.K. (2000). Enhancement of natural and acquired immunity by Lactobacillus rhamnosus (HN001), Lactobacillus acidophilus (HN017) and Bifidobacterium lactis (HN019). Brit. J. Nutr. 83:167–176. Gilliland, S.E., Reilly, S.S., Kim, G.B., Kim, H.S. (2002). Viability during storage of selected probiotic Lactobacilli and Bifidobacteria in a yogurt like product. J. Fd. Sci. 67 (8):3091–3095. Gomes, A.M.P., Malcata, F.X., Klaver, F.A.M., Granade, H.G. (1995). Incorporation and Survival of Bifidobacterium Sp. stain Bo and Lactobacillus acidophilus stain ki in a cheese product. Neth. Milk Dairy. 49:71–95. Gousalez, S.N., Appella, M.C., Romero, N.C., Nader de Macias, M.E., Oliver, G. (1993). Inhibition of enteropathogens by Lactobacilli strains used in fermented milk. J. Fd. Prot. 56:773–76. Greene, J.D., Klaenhammer, T.R. (1994). Factors involved in adherence of Lactobacilli to human Caco-2 cells. Appl. Environ. Microbiol. 60 (12):4487–4494. Groote, De., Mary, A., Daniel, F., Elaine, D., Mary, G., Norman, P. (2005). Lactobacillus rhamnosus GG bacteremia associated with probiotic use in a child with short gut syndrome. Ped. Inf. Dis. J. 24 (3):278–280. Guarner, F., Malagelada, J.R. (2003). Gut flora in health and disease. The Lancet 360:512–519. Hamilton-Miller, J.M.T. (2004). Probiotics and prebiotics in the elderly. Post graduate Med. J. 80:447–451. Hammes, W.P., Hertel, C. (2002). Research approaches for pre and probiotics: Challenges and Outlook. Fd. Res. Int. 35:165–170. Hansen, L.T., Wojtas, P.M., Jin, Y.L., Paulson, A.T. (2002). Survival of Ca-alginate microencapsulated Bifidobacterium sp in milk & simulated gastrointestinal conditions. Fd. Microbiol. 19:135–145.

Probiotics Hattingh, A.L., Viljoen, B.C. (2001). Yogurt as probiotic carrier food. Int. Dairy J. 11:1–17. Hekmat, S., Mc Mohan, D.J. (1992). Survival of L. acidophilus and B. bifidum in ice cream for use as a probiotic food. J. Dairy Sci. 75:1415–1422. Hilton, E., Kolakowski, P., Singer, C., Smith, M. (1997). Efficacy of Lactobacillus G.G. as a diarrhoeal preventive in travellers. J. Travel. Med. 4:41–43.
Downloaded By: [Instituto Politécnico Nacional][MRST Consortium] At: 13:04 23 November 2009

243

Holzapfel, W.H., Schillinger, V. (2002). Introduction to pre and probiotics. Fd. Res. Int. 35:109–116. Holzapfel, W.H., Heburer, P., Geisen, R., Bjorkroth, J., Schillinger, U. (2001). Taxonomy and important features of probiotic microorganisms in food and nutrition. Amer. J. Clin. Nutr. 73 Suppl: 365s–373s. Holzapfel, W.H., Shillinger, U., Du Toit, M., Dicks, L. (1997). Systematics of probiotic lactic acid bacteria with reference to modern phenotypic and genomic methods. Microecol. Therapy. 26:1–10. Hudault, S., Lievin, V., Bernot Camcard, M. F., Servin, A. (1997). Antagonistic activity exerted in vitro and in vivo by L. casei (strain GG) against Salmonellatyphmurium C5 infection. Appl. Environ. Microbiol. 63:513–518. Ishibashi, N., Yamaguchi, S. (2001). Probiotics and safety. Amer. J. Clin. Nutr. 73 (3):465(S)–470(S). Isolauri, E., Arvilommi, H., Salminen, S. (1999). Gastrointestinal infections. In: Colonic microbiota, nutrition and health. Dordrecht: Kluwer Academic Publishers.: 267–279. Isolauri, E., Juntunen, M., Rautanen, T., Sillanaykee, P., Koivula, T. (1991). A human Lactobacillus strain (L. casei strain G.G.) promotes recovery from acute diarrhoea in children. Pediatrics 88:90–97. Isolauri, E., Sutas, Y., Kankaanpaa, P., Arvillonin, H., Salminen, S. (2001). Probiotics: Effect on immunity. Amer. J. Clin. Nutr. 73 (2 Supplement):444s–450. Jacobsen, C.N., Nielson, V.R., Hayford, A.E., Moller, P.L., Michaelsen, K.F., Perregaard, A., Sandstorm, B., Tvede, M., Jakobsen, M. (1999). Screening of probiotic activities of forty seven strains of Lactobacillus spp. by in vitro techniques and evaluation of colonization ability of five selected strains of humans. Appl. Environ. Microbiol. 65 (11):4949–4956. Kandler, O. (1983). Carbohydrate metabolism of lactic acid bacteria. Antonie Van Leeuwenhoek. 49:2099–2224. Kasper, H. (1998). Protection against gastrointestinal diseases – present facts and future developments. Int. J. Fd. Microbiol. 4:127–131. Kiebling, G., Schneider, J., Jahreis, G. (2002). Long term consumption of fermented dairy products over 6 months increases HDL cholesterol. Eur. J. Clin. Nutr. 56:843–849. Kimoto, H., Kurisak, J., Tsuji, N.M., Ohmomo, S., Okamoto, T. (1999). Lactococci as probiotic strains: adhesion to human enterocyte like Caco-2 cells and tolerance to low pH and bile. Lett. Appl. Microbiol. 29:313–316. Klaenhammer, T.R. (1998). Functional activities of Lactobacillus probioticals: genetics mandate. Int. Dairy J. 49:7–505. Klein G. (1999) Probiotische and technologisch eingesctzte Mikroorganisnen in lebensmitteln, Arzneimitteln und in Tierfutter phano-und genotypische untersuchungen zur identifizierung und zur biologischen sicherheit. Habilitation thesis, Faculty of Veterinary Medicine, Free University of Berlin.

244

R. Agrawal Klein, G., Pack, A., Bonaparte, C., Reuter, G. (1998). Taxonomy and physiology of probiotic lactic acid bacteria. Int. J. Fd. Microbiol. 41:103–125. Klijn, N., Weerkamp, A.H., de Vos, W.M. (1995). Genetic marking of Lactobacilluslactis shows its survival in the human gastrointestinal tract. Appl. Environ. Microbiol. 61 (7):2771–2774. Kruis, W., Schutz, E., Fric, P. (1997). Double blind comparison of an oral E. coli preparation and mesalamine in maintaining remission of ulcerative colitis. Alimentary Pharma. and Therapeutics. 11:853–858. Kurman, J.A., Rasie, J.L. (1991). The health potential of products containing Bifidobacteria. In: Therapeutic properties of fermented milks R.K., Robinson, ed. London, UK, pp. 117–158. Laroia, S., Martin, S.H. (1991). Effect of pH on survival of B. bifidum and L. acidophilus in frozen fermented dairy desserts. Cultured Dairy Prodts. J. 2:13–21. Link-Amster, H., Rochat, F., Saudan, K.Y., Mignot, O., Aeschlimann, J.M. (1994). Modulation of a specific humoral response and change in intestinal flora mediated through fermented milk intake. FEMS Immunol. Med. Microbial. 10:55–63. Marteau, P. (2003). Basic aspects and pharmacology of probiotics: an overview of pharmacokinetics, mechanism of action and side effects. Best practice and Res. Clin. Gastroenterol. 17 (5):725–740. Martin, F. (2005). Use of diet and probiotic therapy in the irritable bowel syndrome: Analysis of the literature. J. Clin. Gastroenterol. 39 (43s):s243–s246. Mathur, B.N., Rao, K.H., Shruthe, Sethi. (2003). Recent trends in processing of genetically modified dairy foods. Ind. Dairy Man. 55 (6):29–35. Mattila-Sandholm, T., Blaut, M., Daly, C., De Vuyst, L., Doré, J., Gibson, G., Goossens Knorr, D., Lucas, J., Lähteenmaki, L., Mercenier, A., Saarela, M., Shanahan, F., de Vos, W.M. (2002). Food, GI-tract functionality and human health cluster: PROEUHEALTH. Micro.Ecol.Health Dis. 14:65–74. Mc Farland, L.V., Surawicz, C.M., Greenberg, R.N. (1995). Prevention of B-lactum associated diarrhoea by Saccharomyces boulardii compared with placebo. Amer. J. Gastroenterol. 90:439–448. Mechnikoff, E. (1908). The prolongation of life. London: Putnam’s sons. Meijun, Z., Min, D., Joseph, C., Dong UK, A. (2005). Control of Listeria monocytogenes contamination in ready to eat meat products. Comprehensive reviews in Fd. Sci. Fd. Safety. 4 (2):34–42. Mittal, B.K., Garg, S.K. (1992). Acidophilus milk products: Manufacture and therapeutics. Fd. Res. Int. 8 (3):347–389. Molin, G. (2001). Probiotics in food not containing milk or milk constituents with special reference to L. plantarum 299V. Amer. J. Clin. Nutr. 73:3805–3816. Neighswonger, B.D., Brashears, M.M., Gilliland, S.E. (1996). Survival of cells of L. acidophilus and L. casei during refrigerated storage in fermented milk products. J. Dairy Sci. 79:212–219. Neu, H.C. (1994). The crisis in antibiotic resistance. Science, 257:1064–1073. Oberhelman, R.A., Gilman, R.H., Sheen, P. (1999). A placebo-controlled trial of Lactobacillus G.G. to prevent diarrhoea in undernourished Peruvian children. J. Pediatr. 134:15–20. Olah, A., Belagyi, T., Issekutz, A. (2002). Early enteral nutrition with specific Lactobacillus and fibre reduces sepsis in severe acute pancreatitis. Brit. J. Surgery. 89:1103–1107.

Downloaded By: [Instituto Politécnico Nacional][MRST Consortium] At: 13:04 23 November 2009

Probiotics Ostlie, H.M., Helland, M.H., Narvhus, J.A. (2003). Growth and metabolism of selected strains of probiotic bacteria in milk. Int. J. Fd. Microbiol. 87:17–27. Papamanoli, E., Tzanetakis, N., Tzanetali, E.L., Kotzekidon, P. (2003). Characterization of lactic acid bacteria isolated from Greek dry fermented sausage in respect of their technological and probiotic properties. Meat Sci. 65:859–867. Paton, A.W., Marona, R., Paton, J.C. (2001). Neutralization of Shiga toxins Stxl, Stx2e and Stx2e by recombinant bacteria expressing mimics of globatriose and globotetrose. Inf. Immun. 69:1967–1970. Pool-Zobel, B., Van Loo, J., Rowland, I., Roberfroid, M.B. (2002). Experimental evidences on the potential of prebiotic fructans to reduce the risk of colon cancer. Br.J. Nutr. 87:s273–s281. Pot, B., Coenye, T., Kersters, K. (1997). The taxonomy of microorganism used as probiotics with special focus on Enterococci, Lactococci and Lactobacilli. Microecol. Ther. 26:11–25. Rastall, R.A., Gibson, G.R. (2002). prebiotic oligosaccharides: evaluation of biological activities and potential future developments. In: Tannock, G.W., ed. probiotic and prebiotics: where are we going?. Wymondham: Caister Academic press, pp. 107–148. Reid, G., Bruce, A.W., Smeianov, V. (1998). The role of Lactobacilli in prevention of urogenital and intestinal infections. Int. Dairy J. 8:555–562. Reid, G., Bruce, A.W., Taylor, M. (1995). Instillation of Lactobacilli strain and stimulation of indigenous organisms to prevent recurrence of urinary tract infections. Microecol. Ther. 23:32–45. Rembaekan, D.J., Snelling, A.M., Hawkey, P.M., et al. (1999). Non-pathogenic E. coli Vs mesalamine for the treatment of ulcerative colitis: a randomised trial. Lancet 354:635–639. Roberfroid, M.B. (2000). Prebiotic and probiotics are they functional foods?. Am. J.Clin.Nutr. 71:s1682–s1687. Roberfroid, M.B., Van Loo, J.A., Gibson, G.R. (1998). The bifidogeni nature of chicory inulin and its hydrolysis products. J. Nutr. 128 (1):11–19. Rodtong, S., Tannock, G.W. (1993). Differentiation of Lactobacillus strains by ribotyping. Appl. Environ. Microbiol. 59:3480–3484. Rybka, S., Kailaspathy, K. (1995). The survival of culture bacteria in fresh and freeze dried AB yogurts. The Austr. J. Dairy Tech. 50 (2):51–57. Savedra, J.M., Bauman, N.A., Oung, I., Perman, J.A., Yolken, R.H. (1994). Feeding of Bifidobacterium bifidum and Streptococcus thermophilus to infants in hospitals for prevention of diarrhoea and shedding of rotavirus. Lancet 344:1046–1049. Saxelin, M., Chuang, N.H., Chassy, H.R., Makela, P.H., Salminen, S., Gorbach, L. (1996). Lactobacilli and Bacteremia in Southern Finland. Clin. Infect. Dis. 22:564–566. Schiffrin, E.J., Rochat, F., Linkmster, H.L., Aeschlimann, J.M., Donnet, H.A. (1995). Immuno-modulation of human blood cells following the ingestion of lactic acid bacteria. J. Dairy Sci. 78:491–497. Schillinger, U. (1999). Isolation and identification of Lactobacillus from novel type probiotic and mild yogurts and their stability during refrigerated storage. Int. J. Fd. Microbiol. 47:79–87. Shah, N.P. (2000). Probiotic bacteria, selective enumeration and survival in dairy foods. J. Dairy Sci. 83:894–907.

245

Downloaded By: [Instituto Politécnico Nacional][MRST Consortium] At: 13:04 23 November 2009

246

R. Agrawal Siitonen, S., Vapaatalo, H., Salmonin, S., et al. (1990). Effect of Lactobacillus G.G. yogurt in prevention of antibiotic associated diarrhoea. Ann. Med. 22:57–59. In: Tuohy K.M. et al., (2003) Therapeutic focus. 8(15): 692-700. Simpson, P.J., Ross, R.D., Stanton, C. (2001) The selective enumeration and genetic finger printing of Pediococcus and Bifidobacterium strains recovered from animal feed. Eurolab. Conf. Cork Ireland, 112.

Downloaded By: [Instituto Politécnico Nacional][MRST Consortium] At: 13:04 23 November 2009

Sodini, I., Lucas, A., Oliveira, M.N., Remenf, F., Corrieu, G. (2002). Effect of milk base and starter culture on acidification, texture and probiotic cell counts in fermented milk processing. J. Dairy. Sci. 85 (10):2479–2488. Stanton, C., Gardiner, G., Meehan, H., Collins, K., Fitzgerald, G., Lynch, P.B., Ross, R.P. (2001). Market potential for probiotics. Amer. J. Clin. Nutr. 73 (S):476(S)– 483(S). Steer, T., Carpenter, H., Tuohy, K., Gibson, G.R., Steer, T. E. (2000). Perspectives on the role of the human gut microbiota and its modulation by pro and prebiotics. Nutr. Res. Revs. 13:229–254. Steidler, L. (2003). Genetically engineered probiotics. Best Practice and Res. Clin. Gastroenterol. 17 (5):861–876. Surawicz, C.M. (2003). Probiotics, antibiotic associated diarrhoea and Clostridium difficile diarrhoea in humans. Best Practice and Res. Clin. Gastroenterol. 17 (5):775–785. Tamboli, C.P., Caucheteux, C., Cortot, A., Colombel, J.F., Desreumaux, P. (2003). Probiotics in inflammatory Bowel disease. A critical Review. Best Practice and Res. Clin. Gastroenterol. 17 (5):805–820. Tankanow, R.M., Ross, M.B., et al. (1990). A double blind placebo controlled study of the efficiency of Lactinex prophylaxis of ammoxicillin induced diarrhoea. DICP Ann. Pharmacother. 24:382–384. Tuohy, K.M., Kolida, S., Lustenberger, A., Gibson, G.R. (2001). The prebiotic effects of biscuits containing partially hydrolysed guar gum and fructo-oligosaccharides- a human volunteer study. Br. J. Nutr. 86:341–348. Tuohy, K.M., Probert, H.M., Smejkal, C.W., Gibson, G.R. (2003). Using probiotics and prebiotics to improve gut health. Therapeutic focus 8 (15):692–700. Vinderola, C.G., Bailo, N., Reinheimer, J.A. (2003). Survival of probiotic microflora in Argentinian yohurts during refrigerated storage. Fd. Res. Int. 36:97–102. Walter, J., Tannock, G.W., Tilsala-Timisjarvi, A., Rodtong, S., Loach, D.M., Munrok, Alatossava, T. (2000). Detection and identification of gastrointestinal Lactobacillus sp. by using denaturing gradient gel electrophoresis and species specific PCR primers. Appl. Env. Microbiol. 66:297–303. Weese, J.S. (2003). Evaluation of deficiencies in labeling of commercial probiotics. The Can.Vet. J. 44 (12):982–983. Weese, J.S. (2002). Microbiologic evaluation of commercial probiotics. J. Am. Vet. Med.Assoc. 220:794–797. Zhong, W., Millsap, K., Bialkowska-Hobrazauska, H., Reid, G. (1998). Differentiation of Lactobacillus species by molecular typing. Appl. environ. Microbiol. 64:2418–23. Zubillaga, M., Weill, R., Postaire, E., Goldman, C., Caro, R., Boccio, J. (2001). Effect of probiotics and functional foods and their use in different diseases. Nutr. Res. 21:569–579.