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1 From the Departments of Biochemistry and Food Chemistry and Pediatrics, University of Turku, Turku, Finland, and Food Science Australia, Melbourne Laboratory, Highett, Australia.
2 Presented at the symposium Probiotics and Prebiotics, held in Kiel, Germany, June 11–12, 1998. 3 Supported by The Academy of Finland. 4 Address reprint requests to S Salminen, Department of Biochemistry and Food Chemistry, University of Turku, 20014 Turku, Finland. E-mail: seppo.salminen{at}utu.fi.
ABSTRACT
Acid and bile stability and intestinal mucosal adhesion properties are among the criteria used to select probiotic microbes. The quality control of probiotic cultures in foods traditionally has relied solely on tests to ensure that an adequate number of viable bacteria are present in the products throughout their shelf lives. 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 the human gastrointestinal tract. 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. Molecular techniques are also available to examine strain stability. 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 (eg, Caco-2) and human intestinal mucus. This article outlines the types of in vitro testing that can be used to ensure quality control of functional probiotic strains.
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.
Examples of how each of these criteria can be unstable are most abundant in the area of acid stability, which can vary considerably by strain depending on how the strains are used in different food products. This illustrates the necessity for ongoing quality control of probiotic bacteria during manufacture and use and for continual monitoring of the effectiveness of probiotics in humans. It also indicates the need for selection of more stable probiotic strains for commercial use.
THE QUALITY OF METHODOLOGY USED FOR SELECTION
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, mucus-secreting 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 colonic 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 immune-stimulating 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. Lactobacillus 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). 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.
CRITERIA FOR FUNCTIONAL QUALITY ASSURANCE
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 tests have been applied to lactic acid bacteria and Bifidobacterium strains used in the dairy industry and proposed as probiotics. As shown in Figure 1, the results of these tests can predict the ability of the strains to survive in acidic products. In vivo validation of survival through the human stomach is more difficult to obtain. In vitro assays examining the inhibitory effect of bile acids on the growth of probiotic strains are also relatively simple to perform, although again, quantitative extrapolation to probiotic performance in vivo is difficult. Intraspecies variation in the ability to grow in the presence of bile is often observed between potential probiotic strains (Figure 2), and in vitro tests can be used to select the best strains on a relative basis.
FIGURE 1. . In vitro tests of acid tolerance (A) can be predictive of survival of probiotic strains in yogurt (B). In A, the viable counts of 3 strains of bifidobacteria were measured at time 0 () and after incubation for 105 min at 37°C in 0.2 mol HCl-KCl/L buffer, pH 2.0, plus 0.1% peptone (). This correlated well with the ability of the strains to survive in yogurt, as shown in B. The data for B were supplied by L Tran and M Harvey from Gist-brocades Australia, Moorebank, Sydney, Australia.
FIGURE 2. . The effect of bile on the growth of Lactobacillus amylovorus CSCC 5442 (A) and Lactobacillus amylovorus CSCC 5197 (B). •, control (MRS broth); , treatment (MRS broth containing 0.3% ox bile).
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 quality-control method for assessing the surface structure of probiotic bacteria and related gut barrier effects. In 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). Adhesion of some common probiotic strains was studied by using a human colon carcinoma cell line (Caco-2) and human ileostomy glycoproteins as in vitro models for intestinal epithelium and mucus, respectively (Figure 3). Of 6 probiotic strains tested, only Lactobacillus johnsonii LJ-1 and Lactobacillus GG were adhesive in both models. The most adhesive strain to Caco-2 cells [Lactobacillus casei (Fyos)] adhered poorly to ileostomy glycoproteins, indicating that the surface properties needed for adhesion to epithelial cells and mucus may be different. Therefore, possible changes in adhesion stability should be examined by using more than one model.
FIGURE 3. . Adhesion of commercial probiotic strains in 2 in vitro models of the intestinal mucosa. , adhesion to a differentiated Caco-2 cell monolayer; , adhesion to intestinal mucins.
Some reports on the stability of adhesion properties are available in the literature. Elo et al (16) tested the stability of Lactobacillus GG from different production lots and products by comparing the original strain with cultures used for a longer period in industrial processes. Only slight variation in adhesion properties was observed. However, a more significant drop was reported in the adhesion properties of a culture that had been maintained in MRS broth for 3.5 y with a weekly transfer. Lactobacillus GG isolated from the fecal samples of subjects consuming a fermented whey drink containing Lactobacillus GG had adherence properties equal to those of the original strain (16; Table 1).
View this table:
TABLE 1. Adhesion properties of Lactobacillus rhamnosus strain GG (LGG) from different sources1
With use of the human intestinal mucus glycoprotein adhesion model, the adhesion properties of Lactobacillus GG were studied by using different production lots as well as the strain recovered from feces. The adhesion tests were conducted as described earlier (6). The production lots included the original Lactobacillus GG (ATCC 53103; a gift from SL Gorbach, Tufts University, Boston), a Lactobacillus GG pharmaceutical grade starter culture (Valio Ltd, Helsinki), a Lactobacillus GG starter culture provided by Valio Ltd, and frozen samples of freeze-dried production lots 1089 (Valio Ltd, 1987) and WA IV (Valio Ltd, 1987). In addition, a fecal isolate of Lactobacillus GG recovered after consumption of a whey drink containing Lactobacillus GG was studied.
A less adhesive lot of Lactobacillus GG was found in Boston in the course of clinical studies (B Goldin, unpublished observations, 1996). This isolate was also found to colonize human subjects less frequently than the original culture when assessed according to fecal counts. Later, the adhesion properties of 3 different production lots of Lactobacillus GG were studied in humans by observing the persistence of the strain in fecal samples (16). It was observed that at an intake of 1 x 1010 colony-forming units/d, differences between production lots were reported, some of which could be related back to the adhesion study with the Caco-2 cell line. At higher intakes the colonizing properties of different production lots were similar (17).
Some production lots were tested for their ability to adhere to human ileal cells (B Goldin, unpublished observations, 1990). When compared with the original Lactobacillus GG isolate, adhesion of Lactobacillus GG from lyophilized powder found to colonize human subjects poorly was reduced. Binding of lot 1089 to human ileal cells was lower than the binding of the original Lactobacillus GG although they both adhered similarly to intestinal mucus glycoproteins, indicating that the same lots express different adhesion characteristics in different models.
In another test, we studied a probiotic strain from a commercial French Lactobacillus acidophilus yogurt; the first isolate was obtained in 1995 and the second was isolated in 1996. These isolates were analyzed both in the Food Science Australia laboratories (Highett, Australia) and at the University of Turku laboratories in Finland. In these studies, the strains isolated 1 y apart had significantly different adhesion properties. The first samples adhered well in both the Caco-2 model and the mucus model. However, the second sample taken 1 y later adhered significantly poorer in both models (Figure 4). The identities of both isolates were compared by using the sugar fermentation method (API, Biomerieux, France) and the Riboprint method (DuPont Qualicon, Wilmington, DE) and the strains were identical in both tests systems. Thus, changes in the adhesion capacity apparently occurred during industrial production.
FIGURE 4. . Adhesion of a commercial Lactobacillus strain isolated at different times (A and B) from the same product line. , adhesion to a differentiated Caco-2 cell monolayer; , adhesion to intestinal mucins.
If adhesion is modified during industrial processes, other probiotic traits may also be altered. Adhesion properties, including adhesion to intestinal cells (eg, Caco-2) and human intestinal mucus preparations, should be monitored carefully. These control measurements form the basis of human intestinal colonization, health effects, and future monitoring of production procedures.
Viability and properties during processing and storage
Consumption of probiotics may aid lactose digestion, control intestinal infections, and balance the intestinal mucosal barrier. However, most such studies were conducted with viable bacterial preparations, and the definition of a probiotic includes viability as an important factor (1, 18). The viability of several strains in fermented milks is dependent on both the production method and the strain. In one study, 5 strains of L. acidophilus and Lactobacillus GG (ATCC 53103) were tested to determine the effect of refrigeration on the viability of the strains in cultured buttermilk and yogurt (19). In cultured buttermilk, 3 of the strains showed no significant loss of viability during storage, but 2 strains had significantly decreased viability. Results were similar in yogurt. It is possible that cultures producing organic acids, diacetyl, or other inhibitory compounds in the fermented milk may influence the survival of some probiotic cultures. The baceriocins produced by different dairy cultures were reviewed by Ouwehand and Salminen (20). L. casei GG showed no loss of viability during storage of any of the cultured products. Thus, the results indicate that the production method for fermented milk needs to be carefully evaluated to offer consumers the right amount of viable cultures to obtain the reported health effects.
Studies of a defined probiotic preparation for the prevention of antibiotic-associated diarrhea produced conflicting results. In 2 studies, Clements et al (21) reported that 1 of 2 batches of a lyophilized lactobacillus preparation reduced the volume and duration of neomycin-associated diarrhea. A second batch had no effect, although the question of differences in viability between the preparations may be raised. It is important to take viability into account because many strains exert effects in the nonviable form as well (21, 22). Further studies on viability and health effects are clearly needed.
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 processes, probiotic effects may consequently also be altered. Adhesion properties and their dependence on processes and process changes should be monitored. Suitable models include adhesion to intestinal cells, such as Caco-2 cells, and binding to human intestinal mucus preparations. At least 2 models should be used for routine quality control of adhesion of probiotic microbes because this would allow different adhesion characteristics to be measured.
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