CRITERII DE ASIGURARE A CALITĂŢII PENTRU BACTERIILE PROBIOTICE

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1 Lucrări ştiinţifice Zootehnie şi Biotehnologii, voi. 39 (1) (2006), Timişoara QUALITY ASSURANCE CRITERIA FOR PROBIOTIC BACTERIA ORLICD.*, ŢIBRUI. ** *Veterinary Scientific Institute "Novi Sad", Novi Sad, Serbia and Montenegro **Faculty of Veterinary Medicine, Timişoara România CRITERII DE ASIGURARE A CALITĂŢII PENTRU BACTERIILE PROBIOTICE Acid and bile stability and intestinal mucosal adhesion properties are among the criteria used to select probiotic microbes. Viability is an important factor, but not the only criterion for quality assurance. To be effective, probiotic strains must retain the functional health characteristics for which they were originally selected. Such characteristics include the ability to survive transit through the stomach and small intestine and to colonize gastrointestinal tract. Adhesion characterization may be an important quality-control method for assessing gut barrier effects. Adhesion has been related to shortening the duration of diarrhea, immunogenic effects, competitive exclusion, and other health effects. Adhesion properties should be carefully monitored, including, adhesion to intestinal cells Key words: probiotics, quality control, adhesion, acid stability, viability Introduction Probiotics are viable bacteria that beneficially influence the health of the host (1, 2). Probiotic bacteria selected for commercial use in foods and in therapeutics must retain the characteristics for which they were originally selected (1-3). These include characteristics for growth and survival during manufacture and, after consumption, during transit through the stomach and small intestine. Importantly, probiotics must retain the characteristics that give rise to their health effects. Consequently, it is necessary to test the stability of these characteristics during manufacture and storage and to ensure that they are retained in different types of foods (3, 4). The initial screening and selection of probiotics includes testing of the following important criteria: phenotype and genotype stability, including plasmid stability; carbohydrate and protein utilization patterns; acid and bile tolerance and survival and growth; bile metabolism; intestinal epithelial adhesion properties; production of antimicrobial substances; antibiotic resistance patterns; ability to inhibit known gut pathogens, spoilage organisms, or both; and immunogenicity. 311

2 Materials and Methods Many in vitro tests are performed when screening for potential probiotic strains. The ability to adhere to the intestinal mucosa is one of the more important selection criteria for probiotics because adhesion to the intestinal mucosa is considered to be a prerequisite for colonization (5). As substrata, enterocyte-like Caco-2 tissue culture cells and intestinal mucus are currently used. However, these represent only a distinct part of the intestinal mucosa. In this respect, mucussecreting HT29-MTX tissue culture cells would come closer to the true situation in the intestine. In addition to these models, human ileostomy glycoproteins have been used to study adhesion to the small-intestinal mucosa (6). All of these in vitro systems provide valuable information on the ability of probiotics to adhere and colonize the intestine. Adhesion to colonie or intestinal biopsy samples, if possible, should be considered as a final in vitro adhesion test that would be most like the in vivo situation. Not only would this be a better approximation of the in vivo situation, it would allow for the study of adhesion to different parts of the intestine. This is especially important regarding immune stimulation by oral administration of probiotics. Adhesion is also considered important for stimulation of the immune system. Adhesion to M cells or Peyer's patches may therefore be an important determinant of possible immunestimulating properties of probiotic microorganisms. Fecal samples have been used in most colonization studies with probiotic bacteria. These, however, reflect only the bacteriologic situation in the fecal material and do not give an accurate picture of the different parts of the gastrointestinal tract or the mucosal layer of the gut. The use of biopsies from the intestinal mucosa is a more accurate means of determining colonization. Gactobacillus strains were found to adhere to rectal mucosa obtained from volunteers who had consumed a fermented oatmeal soup (7). When tested in vitro, probiotics are usually grown in laboratory media. With many probiotics, the aim is at least transient colonization, in which case the probiotics may need to grow or at least metabolize in the intestine. The adhesive properties, metabolism, and morphology of probiotics grown in intestinal contents or intestinal mucus have been shown to be different from those of probiotics grown in laboratory media. These differences may affect the health effects of the probiotics. By using culture media more resembling the nutrients available in the intestine, one may obtain a more accurate understanding of the properties of probiotics in vivo. One of the selection criteria for probiotics is the production of antimicrobial substances, and many probiotic bacteria have been shown to produce them (8). 312

3 Among these substances are not only growth-inhibiting metabolites, eg, organic acids and hydrogen peroxide, but also bacteriocins, adhesion inhibitors, and a range of small antimicrobial substances. These substances have been shown to be produced in laboratory media but their production and efficacy in vivo remain uncertain (8). It has not been tested whether administration of purified bacteriocins alone has effects, eg, on diarrheal disease. Nor has it been tested whether bacteriocins are produced in vivo. If bacteriocins are produced and active in vivo, it may be necessary to assess their effects on the indigenous microflora. There is the potential risk that beneficial strains in the indigenous microflora are also affected by the presence of a bacteriocin and that the bacteriocins may thereby alter the natural resistance of the indigenous microflora to colonization. Because the production of antimicrobial substances is regarded as an important selection criterion for probiotics, it must be confirmed whether these substances are indeed produced in vivo and exert a beneficial effect. Intestinal or fecal microflora studies are needed to confirm these properties. Results and Discussions The criteria currently used to select probiotics define the optimal quality control of probiotic strains in industrial practice. Probiotics are often used in fermented foods and fermentation acts to retain and optimize microbial viability and productivity while simultaneously preserving the probiotic's properties. During fermentation, several metabolic products appear in the food product, including acetic acid, lactic acid, and possibly bacteriocins, and the ph of the product is lowered. Such changes may affect the stability of probiotic bacteria and may alter the bacteria's functional properties. Also, long-term industrial use of the starter culture for production purposes may influence both viability and functional properties. Important quality-control properties that must be constantly controlled and optimized are the following: adhesive properties; bile and acid stability; viability and survival throughout the manufacturing process; effects on carbohydrate, protein, and fat utilization; and, especially, colonization properties and immunogenicity. Most of these properties are related to the physiologic properties of the strain, but long-term industrial processing and storage conditions may influence probiotic properties. Thus, in addition to technologic properties, functional properties should be considered in quality-control measures. Acid and bile stability To survive passage through the stomach and small intestine, probiotic strains must tolerate the acidic and protease-rich conditions of the stomach, and survive and grow in the presence of bile acids. Acid tolerance is also important for the probiotics' survival in food (3). The dominant food vehicles for probiotics remain to be yogurts and fermented milks, both of which provide a relatively low-ph environment in which the probiotic bacteria must survive. Hence, acid tolerance is one of the first properties screened for when selecting probiotic strains. Simple in vitro tests can be used to assess acid tolerance. Such 313

4 tests have been applied to lactic acid bacteria and Bifidobacterium strains used in the dairy industry and proposed as probiotics. These in vitro tests for selection of acid- and bile-tolerant strains can readily be applied to ensuring the quality of probiotic cultures during manufacture and storage and throughout the shelf life of the product. Both short-term environmental factors affecting gene regulation (eg, culture growth phase and stress leading to the production of shock proteins), and selection of variants through long-term subculturing may produce changes in culture performance (3, 9). The former of these is the most likely to have the greatest influence on probiotic performance (potentially positively) because appropriate culture maintenance procedures limiting the number of passages should prevent the selection of genetic variants in industrial processes. Acid tolerance is likely to be a relatively intrinsic property of bacteria and acidification of culture broth during fermentation would also make selection of less acid-tolerant variants unlikely if serial subculturing was practiced. However, data showing the long-term stability of acid and bile tolerance in probiotics during subculturing are lacking. Adhesion stability Adhesion characterization may be an important qualitycontrol method for assessing the surface structure of probiotic bacteria and related gut barrier effects, hi several studies, adhesion was related to a shortening of duration of diarrhea, immunogenic effects, competitive exclusion, and other health effects (2, 5, 10-12). Adhesion of probiotic strains is variable. Adhesion in different in vitro models varies even within the same strain and differences between strains can be significant (13-15). Conclusions It is important to ensure that the specific properties of a probiotic used originally as selection criteria are also targets for quality assurance. This is an important prerequisite for any health claims and should be continuously controlled if the health claims are approved for use. Thus, functional food regulations should take into account strain properties and their stability during industrial processing and use. On the other hand, not all selection criteria are always necessary for in vivo functional effects. Each important strain property and its influence on health should be assessed in relation to the clinical effects observed in various studies. In vitro test protocols can be readily adopted to examine the maintenance of a strain's ability to tolerate acidic conditions, survive and grow in the presence of bile, and metabolize selective substrates. Adhesion characterization may be an important quality-control method for assessing the surface structure of probiotic bacteria and related gut barrier effects. Adhesion has been related to immunogenic effects, shortening the duration of diarrhea, and other health effects. If adhesion is modified during industrial 314

5 processes, probiotic effects may consequently also be altered. Adhesion properties and their dependence on processes and process changes should be monitored. Bibliography 1. Salminen S, Bouley C, Boutron-Ruault MC.- Functional food science and gastrointestinal physiology and function. Br J Nutr 1998; nr. 80. p Salminen S, Isolauri E, Salminen E.- Clinical uses of probiotics for stabilizing the gut mucosal. barrier: successful strains and future challenges. Antonie Van Leeuwenhoek 1996, nr. 70, p Lee YK, Salminen S. The coming of age of probiotics. Trends Food Sci Technol 1995, nr. 6, p Huis Int Veld JHJ, Shorn C. Selection criteria for probiotic microorganisms. London: The Royal Society of Medicine Press Ltd; International Congress and Symposium Series, 1996, nr. 21. p Salminen S, Laine M, von Wright A, Vuopio-Varkila J, Korhonen T, Matrila-Sandholm T. - Development of selection criteria for probi.otic strains to assess their potential in functional foods: a Nordic and European approach. Biosci Microflora 1996, nr. 15: p Ruseler van Ebmden JHG, van Lieshout L, Gosselink M, Marteau P. Inability of Lactobacillus casei strain GG, Lncrobacillus acidophilus, and Bifidobacterium bifidum to degrade intestinal mucosa glycoproteins; clearing the way for mucosaprotective therapy. Scand J Gastroenterol 1995, nr.30, p Alander M, Korpela R, Saxelin M, Vilpponen-Salmila T, MattilaSandholm T, von Wright A. Recovery of Lactobacillus rhamnosus GG from human colonie biopsies. Lett Appl Microbiol 1997, nr. 24, p Ouwehand AC. Antimicrobial components from lactic acid bacteria. In: Salminen S, von Wright A, eds. Lactic acid bacteria, microbiology and functional aspects. New York: Marcel Dekker Inc, p Lee YK, Wong S-F. Stability of lactic acid bacteria in fermented milk. In: Salminen S, von Wright A, eds. Lactic acid bacteria, microbiology and functional aspects. New York: Marcel Dekker Inc, 1998, p Malin M, Verronen P Korhonen H, et al. Dietary therapy with Lactobacillsts GG, bovine colostrum or bovine immune col.ostrum in patients with juvenile chronic arthritis: evaluation of effect on gut defence mechanisms. Inftammopharmacology 1997, nr. 5, p Saavedra JM, Bauman N, Oung I, Perman J, Yolken R. Feeding of Bifidobacterium bifidum and Streptococcus thermophilus to infants in hospital for prevention of diarrhea and shedding of rotavirus. Lancet 1994, nr. 344, p Isolauri E, Juntunen M, Rautanen T, Sillanaukee P, Koivula T. A human Lacrobacillus strain (Lactobacillus GG) promotes recovery from acute diarrhea in children. Pediatrics 1991, nr. 88, p Lehto E, Salminen S. Adhesion of two Lncrobncillus strain.s, one Lactococcus strain and one Propionibacrerium strain to cultured human intestinal Caco-2 cell line. Biosci Microflora 1997, nr. 16, p Lehto E, Salminen S. Adhesion of twelve different Lncrobacillus strains to 315

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