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International Dairy Journal 17 (2007) 1278–1283 www.elsevier.com/locate/idairyj
In vitro assessment of probiotic bacteria: From survival to functionality L. Morelli Istituto di Microbiologia, U.C.S.C., Via Emilia Parmense 84, 29100 Piacenza, Italy Received 25 September 2006; accepted 17 January 2007
Abstract Selection by means of in vitro tests of new probiotic bacteria presents a challenge for scientists as well as for dairy companies. Presently, most probiotics are assessed using assays focused on their ability to survive in, and subsequently colonise, the gastrointestinal environment. This approach has led to selection of strains with conﬁrmed in vivo probioticity, but the science underlying these assays still needs to be reﬁned. As a consequence, we see a different approach emerging that uses functional properties and/or genomic evaluation to select appropriate probiotic strains. r 2007 Elsevier Ltd. All rights reserved. Keywords: Probiotics; Selection; Adhesion: Gastric tolerance; Bile tolerance; Genomic
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ecological criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Survival during gastric transit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Tolerance to bile salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Assessing adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional genomic selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The consumption of dairy products containing bacterial strains claimed to promote well-being has been steadily increasing over the last 10 years. Beneﬁcial effects, such as improved balance among different components of the intestinal ﬂora, regulation of the intestinal immune system or reinforcement of the natural barrier, are claimed for each of these products (for recent reviews see Floch et al., 2006; Reid, Kim, & Kohler, 2006; Sazawal et al., 2006). The dairy food industry is therefore under considerable pressure to scientiﬁcally validate both already existing and new probiotic food products. Corresponding author. Tel.: +39 523 599248; fax: +39 523 599246.
E-mail address: [email protected]
0958-6946/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2007.01.015
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There is a large scientiﬁc consensus that, in order to assess the properties of probiotic bacterial strains, it is mandatory to perform a preliminary in vitro assessment (FAO/WHO, 2001, 2002). This assessment has traditionally paid special attention to the ecological origin of bacteria, their tolerance to the hostile conditions of the stomach and the small intestine, and their ability to adhere to intestinal surfaces. Several papers (Acharya & Shah, 2002; Collado & Sanz, 2006; Mishra & Prasad, 2005) suggest that a probiotic bacterial strain should be assessed according to the following (or a very similar) scheme: (i) it must be of human origin, (ii) it must survive during gastric transit,
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(iii) it has to tolerate bile salts, (iv) it has to adhere to gut epithelial tissue. The ﬁrst criterion assumes that strains belonging to bacterial species that are present in the intestinal ﬂora could have a better chance of survival in their native environment. The remaining criteria provide an assessment of the potential of putative probiotic strain to overcome the gastric environment, the presence of bile salts and to resist, through adhesion, the ﬂux of the intestinal content. It is important to point out, however, that the ultimate criterion to select a successful probiotic strain is the ability to confer to consumers a health beneﬁt and that the above criteria represent the ﬁrst steps in the selection scheme. These criteria, based on ecological considerations, have been used to select strains that have been successfully tested in vivo (Dunne et al., 2001) and are currently the active ingredients of probiotic products that have been proven to perform well in contributing to the well-being of consumers. More recently, a number of papers (Dogi & Perdigon, 2006; McCartney, 2002) have suggested two additional approaches in probiotic bacteria selection: the in vitro assessment of functionality and genomics analysis. The ﬁrst is related to the activities exerted by probiotics towards what is termed gut-associated lymphoid tissue (GALT), while the second is the outcome of the availability of the complete genomic sequences of lactobacilli and biﬁdobacteria. These approaches are, however, strictly linked to the approach of selection against ecological criteria, because both of them hold that the bacterium has to survive and colonise after ingestion. New tools are therefore emerging to select probiotics, but they probably need to be validated by in vivo human trials, while the old tools still need to be reﬁned. 2. Ecological criteria Early studies using in vitro assays (Barrow, Brooker, Fuller, & Newport, 1980; Mayra-Makinen, Manninen, & Gyllenberg, 1983; Morishita, Mitsuoka, Kaneuchi, Yamamoto, & Ogata, 1971; Suegara, Morotomi, Watanabe, Kawai, & Mutai, 1975; Tannock, Szylit, Duval, & Raibaud, 1982) suggested that bacterial adhesion to epithelial tissues could be ‘‘host-speciﬁc’’, i.e., lactobacilli isolated from mammals adhere only to cells obtained from mammals, etc. However, differences noticed in these studies could be, at least partly, explained in terms of incorrect taxonomic identiﬁcation of the species used. As an example, after reclassiﬁcation of Lactobacillus acidophilus into six different species, humans appeared to harbour L. gasseri (Zoetendal et al., 2002) while L. amylovorus was the most frequently isolated L. acidophilus from pigs (Pryde, Richardson, Stewart, & Flint, 1999). L. gasseri is also a dominant Lactobacillus in calves and there are no data about the potential of this species to colonise the human gut (Morelli, 2000).
Some of species of lactobacilli that are present in the gut, e.g., L. rhamnosus, have also been isolated from the urogenital tract (Giorgi, Torriani, & Dellaglio, 1987). It seems, therefore, worthwhile to ﬁnd out if there are some differential phenotypes between one L. rhamnosus isolated from the vagina and another strain of L. rhamnosus isolated from the gut. A clear-cut answer is not yet available, but several pieces of evidence seem to suggest that the human body should, probably, be considered as single environmental niche. Some of these strains, when orally administered, were shown to be able to reach the urogenital tract, suggesting that the gut and urogenital tract may be a single ecological niche for lactobacilli. Reid and co-workers have also demonstrated in a number of papers (review: Reid, Kim & Kohler, 2006) that lactobacilli originally isolated from the vagina could be excellent human gut coloniser; these data have been conﬁrmed by an independent study (Morelli, Zonenenschain, Del Piano, & Cognein, 2004). The complexities encountered in providing sound science to the apparently simple deﬁnition of ‘‘human origin’’ were also pointed out in results reported by Nielsen et al. (2003) showing that mucosa-associated lactobacilli and biﬁdobacteria were different from those recovered from faecal samples. Recently, we demonstrated the presence of a mucosa-speciﬁc Lactobacillus population (Morelli & Bessi, 2006). A further puzzling set of data about the need for selecting probiotics for humans from the ‘‘human environment’’ was provided by Jacobsen et al. (1999). These authors assessed, both in vitro and in vivo, the ability of 47 Lactobacillus strains isolated from different sources to survive and colonise the human gut. All these strains were examined for resistance to pH 2.5% and 0.3% oxgall as well as for their adhesion to Caco-2 cells. After these tests the strains were used for an in vivo trial with healthy volunteers. The overall conclusion was that among the three strains frequently isolated from faecal samples of healthy subjects fed with the assayed strains, two were of human origin but the third was of porcine origin. FAO/WHO (2001) also suggested that human origin as a mandatory trait for probiotic bacteria should be reconsidered. In this document the speciﬁcity of the action, and not the source, of the microorganism was proposed to be important. In addition, authors of the Expert Consultation pointed out that, at the moment, it is impossible to identify the original source of a microorganism. This statement also pointed out that, at the moment, there are no analytical tools to discriminate the original environmental source of a strain after its primary isolation. This inability may also have legal ramiﬁcations, i.e., misleading or false claims. Selection of new probiotic strains from human isolates is a straightforward way towards development of a new product, albeit more for ‘‘practical reasons’’ (i.e., an easy way to avoid strains unable to survive into the gut) and ‘‘common sense’’ (i.e., seems reasonable to use a strain
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naturally present in the selected environment) than for scientiﬁcally based reasons, which are yet to be provided. 2.1. Survival during gastric transit To reach the intestine, strains must ﬁrst pass through the stomach, which provides a powerful barrier to the entrance into the gut. The ﬁrst studies to assess the survival of lacticacid bacteria to gastric environment were devoted to identify strains able to survive across several species. Conway, Gorbach, and Goldin (1987) used gastric juice obtained from human volunteers to demonstrate that strains of Streptococcus thermophilus and L. bulgaricus (L. delbrueckii subsp. bulgaricus) are extremely sensitive, while enteric species of lactobacilli are more resistant, with signiﬁcant strain-to-strain variations. Quite interestingly, recent papers have shown that the sensitivity of yoghurt cultures to acid conditions could reduce the recovery of these bacteria from faecal samples of consumers, but there was still a number of cells that were able to remain viable (Elli et al., 2006; Mater et al., 2005) and this has prompted some scientist to suggest that fermented milks containing viable cultures could have a real probiotic action (Guarner et al., 2005). Strains belonging to species normally inhabiting the human gut have been shown to behave better when assayed for their in vitro resistance to low pH or to simulated gastric juice (Conway et al., 1987). Among these enteric species of lactobacilli there was also a puzzling situation: strains with a well documented ability to colonise the human gut scored poorly in the in vitro assay used by some authors (Charteris, Kelly, Morelli, & Collins, 1998; Jacobsen et al., 1999; Mishra & Prasad, 2005); this discrepancy between in vitro and in vivo results points out the need to reﬁne these kinds of tests. As an example, a limited tolerance to acid of L. paracasei strains in vitro has been demonstrated (Charteris et al., 1998; Mishra & Prasad, 2005), but the same strains have been shown to have given excellent in vivo results (Fonden, Bjorneholm, & Ohlson, 2000). As a conclusion, it could be stated that in vitro evaluation of acid tolerance of probiotics seems to be a poor predictor of the in vivo behaviour of these strains. Only strains that are extremely sensitive to acid in vitro seem to be unable to survive gastric transit, while a moderate resistance to low pH seems necessary. However, it is still to be proven that high resistance assessed in vitro correlates with better colonisation ability. 2.2. Tolerance to bile salts
tantly, the bile preparation used by Gilliland et al. (1984) to assay strains in vitro was ‘‘oxgall’’, a product derived from bovine bile. The same bile preparation was used by several authors to assess the bile resistance of probiotic bacteria, lactobacilli or biﬁdobacteria. Very few data are available about the behaviour of strains when challenged with human bile or with bile originating from other sources. Further, no data are available about a possible correlation between the original animal host of a bacterium and the bile resistance of that bacterium. In other words, is it possible that a Lactobacillus isolated from a pig could be more resistant to porcine bile than another strain belonging to the same species, but isolated from calves? To address this question, we have recently challenged 10 strains of L. johnsonii isolated from humans (three strains), calves (ﬁve isolates) as well as pigs and poultry, pigs (one isolate each) not only with oxgall but also with commercially available porcine bile. To also address the problem of a species related bile resistance, ﬁve strains of L. acidophilus (two isolated from humans and three from pigs) were also included into this assay. Strains were plated on MRS agar containing a bile preparation and plate counts were compared with those obtained using the same dilution plated on MRS without any bile. The percentage of reduction in colony counts was then calculated. The results shown in Figs. 1 and 2 show that: (i) porcine bile had an higher inhibitory power than bovine bile, (ii) the role of species was far more relevant than the ecological origin, (iii) a certain strain-speciﬁc behaviour was also noticed, and (iv) only at an extreme, unphysiological concentration could an inﬂuence of the original habitat be noticed.
2.3. Assessing adhesion To resist the intestinal ﬂow of digesta, probiotics have to adhere to the intestinal surface. To evaluate in vitro the 120 100 Resistance (%)
80 60 40 20
The ability to survive the action of bile salts is generally included among the criteria used to select potentially probiotic strains. In a pioneering study, Gilliland, Staley, and Bush (1984) showed that bile resistance could differ among strains of one single species of enteric lactobacilli and that this difference could account for differences in ability to colonise the intestinal tract of calves. Impor-
Strains Fig. 1. Resistance to bovine bile of selected L. acidophilus (~) and L. johnsonii (&) strains. Strains were plated in MRS agar with and without the presence in the medium of 1.5% (w/v) bovine bile (Sigma).
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presence of such a phenotype, some authors have suggested the use of pieces of intestinal tissues (Vesterlund, Paltta, Karp, & Ouwehand, 2005a), cell lines (Greene & Klaenhammer, 1994), or mucus (Ouwehand, Tuomola, Tolkko, & Salminen, 2001). Results obtained for probiotic used in animal feeding have suggested for Lactobacillus the presence of a species-dependent mechanism, strains isolated from one kind of animal were the best-adhering organisms to tissues obtained from animals of the same species (Barrow et al., 1980; Fuller, 1975, 1978; Savage, 1984). However, these studies did not provide clear-cut results. Savage (1984) showed that a Lactobacillus strain isolated from pig and a strain isolated from mouse gut, adhered in vitro to mouse tissues in equally high numbers. In addition, Fuller (1978) assayed strains, both isolated from poultry, which scored for adhesion positively in vitro and in vivo. The strain that performed best in vitro, however, performed the poorest in vivo.
100 90 Resistance (%)
80 70 60 50
The potential of adhesion in vivo of probiotic strains intended for human use has been assessed mainly by means of tumoural, intestine-derived cell lines. However, since the beginning, methodologies used were imperfect and strongly affected by the procedures used (Greene & Klaenhammer, 1994). As the need of reﬁnement for these assays has been already reviewed (Blum et al., 1999) it seems worthwhile here to simply compare results obtained by different authors with a range of strains. Results compiled in Table 1 show that adhesion index was a strain-speciﬁc feature, but there was no apparent relationship with the original environment from which the strain was isolated. Similar observations were reported by Mishra and Prasad (2005) for Lactobacillus casei, with a strain of dairy origin showing the highest adhesion index to CaCo2 cells. Cell line models are therefore able to provide only a preliminary discrimination between adhering and nonadhering strains, but the relative scorings seem to be unreliable for further differentiate strongly or weakly adhering strains. Beside cell lines human mucous glycoproteins have also been used to assess adhesion. New data obtained at the genetic level as regards proteins secreted by lactobacilli and able to adhere to mucous (for a review see Vesterlund, Paltta, Karp, & Ouwehand, 2005b) give promise of a breakthrough in this ﬁeld in the near future.
3. Functional genomic selection
20 10 0
Strains Fig. 2. Resistance to porcine bile of selected L. acidophilus (~) and L. johnsonii (&) strains. Strains were plated in MRS agar with and without the presence in the medium of 0.25% (w/v) bovine bile (Sigma).
Genomes of some probiotic bacteria have been sequenced and some others are in progress, but after the whole chromosomal sequence of a strain is made available, the effort of data mining begins. These data could provide information on the genetic potential of bacteria to survive in the intestinal tract and, what is more important, to predict some probiotic activity such as immune stimulation.
Table 1 Adhesion index of at least one bacterium per Caco-2 cell; data have been rearranged from Blum et al. (1999) and Morelli (2000) Strain
L. L. L. L. L. L. L. L. L. L. L. L. L. L. L.
2.3 2.1 0.66 0.18 0.23 0.008 0.23 1.5 0.009 1.55 2.0 2.1 1.9 2.39 1.5
Coconnier, Klaenhammer, Kerne´is, Bernet, and Servin (1992) Chauvie`re, Coconnier, Kerne´is, Fourniat, and Servin (1992) Bernet, Brassart, Neeser, and Servin (1994); Bernet et al. (1994)
Chauvie`re et al. (1992)
Chauvie`re et al. (1992)
Sarem et al. (1996)
acidophilus BG2FO4 acidophilus LB acidophilus La3 acidophilus La10 acidophilus La18 acidophilus NCK 88 acidophilus CIP 6218 acidophilus C7 acidophilus Ki johnsonii LA1 helveticus CNRZ 32 helveticus CNRZ 240 delbrueckii subsp. lactis CNRZ 239 delbrueckii subsp. lactis ATCC 7830 delbrueckii subsp. lactis LY
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As regards probiotics, there are several chromosomal sequences completed. A ﬁrst example of a probiotic functional characterisation was provided by the functional analysis of Bifidobacterium longum NCC2705 genome and proteome (Schell et al., 2002; Yuan et al., 2006). Bioinformatics analysis provides an explanation of the well-known capacity of biﬁdobacteria to ferment the non-digestible oligosaccharides, as a large number of the predicted proteins appeared to be specialised for catabolism of a variety of oligosaccharides, including novel glycosyl hydrolases not detected by fermentation assays. This genomic wealth of hydrolases could readily contribute to the competitiveness and persistence of biﬁdobacteria in the colon. In the same genome authors have also identiﬁed peptides homologous to proteins needed for production of glycoprotein-binding ﬁmbriae, suggesting that biﬁdobacteria could use these structures to adhere to intestinal tissues in a manner similar to that of, and therefore competitive with, Enterobacteriaceae. Using sequence data from B. longum NCC2705 we have also shown the presence of the gene encoding ﬁmbriae in another strain of B. longum but this gene was absent in all B. breve strains that we have assayed, showing a difference between the two species for adhesion and persistence in the GIT (Morelli et al., unpublished). Further, in L. johnsonii NCC 533, a commercially exploited probiotic Lactobacillus, genomic analysis showed the presence of more than 12 large and unusual cell-surface proteins, including ﬁmbrial subunits, possibly involved in adhesion to intestinal mucins (Pridmore et al., 2004). In L. plantarum WCFS1 genomic analysis has provided a worth of data about the behaviour of this strains under bile stress (Bron et al., 2004), and in B. longum NCC2705 an eukaryotic-type serine protease inhibitor (serpin) was found that was possibly involved in the reported immunomodulatory activity of biﬁdobacteria (Schell et al., 2002). Genomic analysis is therefore a promising and fast growing new tool for probiotic strain selection, which could be used not only to provide solid science to the already used ecological selection criteria but also to provide an in vitro insight to the functional activities of probiotic strains (Vaughan, Mollet, & deVos, 1999). Probiotic strains of the future will be probably selected by means of a combination between a preliminary genomic-based step followed by in vitro assays, but in vivo assessment will remain mandatory for a ﬁnal selection step. Most in vitro assays are yet to be conﬁrmed by in vivo trials, and this will be a major effort for the future. From the basic research point of view, it is also the case that strains scoring poorly in vitro should be used in some in vivo trials, to conﬁrm the evaluation procedures used. An additional selection scheme could be also developed in the near future, based on in vitro or ex vivo assays assessing the potential of bacteria to modulate the GALT response; in this way pro-inﬂammatory or anti-inﬂamma-
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