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- Materials and methods
Aims: Considering the significant rise in the probiotic market in Columbia, and given the lack of reports concerning the microbial population and strain performance in products from different producers, this study aims at determining the number of viable starter bacteria and probiotics in bio-yoghurts available at the Columbian market, identifying the species and analysing the performance of the isolated strains in bile acid resistance, antagonistic activity against pathogens, and adherence capacity to human intestinal epithelial cells.
Methods and Results: Seven bio-yoghurts were analysed for the bacterial species present. Species identification was carried out using 16S rRNA gene targeted PCR. The cultured bacteria were tested for bile acid resistance, adherence to a human intestinal epithelial cell line, and antagonism against the pathogen Salmonella enterica serovar Typhimurium. A total of 17 different strains were identified. Based on plate counting, all bio-yoghurts have at least total viable cells of ∼107 CFU ml−1. Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus were the most frequently isolated bacteria. Viable Bifidobacterium was only recovered from one product. However, after PCR analysis, DNA of this genus was confirmed in five out of seven products. Major differences were found for S. typhimurium antagonism. The adherence capacity to Caco-2 cells was observed in 10 of the isolated strains. In general, low survival to simulated gastric juice was observed.
Conclusions: Some of the isolated strains have probiotic potential, although not all of them were present in the advised amount to exert beneficial health effects. However, the full correct scientific name of the isolated bacteria and their viable counts were not included on the product label.
Significance and Impact of the Study: This is the first report describing the identification and functionality of starter bacteria and probiotics present in dairy products on the Columbian market.
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- Materials and methods
Probiotics are defined as ‘live microorganisms which when administered in adequate numbers confer a health benefit on the host’ (FAO/WHO 2001). To date, probiotics have been mainly selected from the genera Lactobacillus and Bifidobacterium, because of their long history of safe use in dairy industry, and their natural presence in the human intestinal tract (Saxelin 1997).
The term starter lactic acid bacteria (SLAB) is used to describe bacterial strains capable of growth under the selective conditions of dairy production. A starter culture of traditional yoghurt normally includes a mixture of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. However, although L. delbrueckii ssp. bulgaricus was reported to survive the gastric transit (Mater et al. 2005), it is not native to humans in contrast to most probiotic bacteria for human consumption. Consequently, in order to improve yoghurt’s health value, the traditional L. delbrueckii ssp. bulgaricus in many products has been replaced by or combined with well-known probiotics like Lactobacillus acidophilus, Lactobacillus casei and Lactobacillus rhamnosus, which are frequently present in the human gastro-urogenital tract (Heller 2001; Vaughan et al. 2002; Reid and Bruce 2003).
In general, the selection criteria for starter cultures are their acidification rate and flavour-producing characteristics. For probiotics, selection is based on a detectable health effect for the host (Saarela et al. 2000; Teusink and Smid 2006). The essential characteristics for lactic acid bacteria (LAB) to be used as probiotics during manufacturing include the following: (i) recognition as safe (GRAS; generally recognized as safe); (ii) viability during processing and storage; (iii) antagonistic effect against pathogens; (iv) tolerance to bile acid challenge; and (v) adherence to the intestinal epithelium of the host among others (MacFarlane and Cummings 2002; Begley et al. 2005; Vesterlund et al. 2005; Lin et al. 2006). It is generally considered that minimum numbers required for a probiotic to provide a health benefit are 107 CFU ml−1 (Ross et al. 2005; Jayamanne and Adams 2006). Therefore, the viability and survival of probiotic bacteria during dairy processing and storage are important parameters for providing a health benefit (Corcoran et al. 2005; Lin et al. 2006). Fermented milk products containing probiotics were introduced on the Columbian dairy market at the beginning of this century. However, until now, there are no reports in scientific literature concerning the content and viability of probiotic and starter bacteria of these products. For this reason, we identified the bacterial species in seven yoghurts available on the Columbian market representing the seven bigger dairy food companies, and determined their in vitro performance. In addition, the viable counts of the starter bacteria and probiotics identified in the dairy products analysed, were determined.
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- Materials and methods
For commercial-, industrial- and health-related reasons, probiotic and starter cultures used during the manufacturing of bio-yoghurts must be functional in order to promote human health at the site of action (Sanders and Huis in’t Veld 1999; Saarela et al. 2000). Although recent progress in probiotic research has been accomplished, not all of the probiotic bacteria available in the market are well characterized, or have adequate technological performance (Ross et al. 2005). Additionally, recent reports have clearly highlighted the poor quality of many probiotic products in terms of their contents and label information (Hamilton-Miller et al. 1996, 1999; Yeung et al. 2002; Temmerman et al. 2003a,b). This study showed that this is also the case for some of the probiotic products available in the Columbian market. During recent years, commercialization of new probiotic products in the Columbian market has witnessed a strong increase. However, the label information of the purchased products, analysed in this study, was rather vague, and did not indicate the full scientific name of the probiotic micro-organism present in the product. Indeed, although in this study, the isolation procedure was carried out before the expiry date, and a broad range of selective media was used, the diversity of the different identified bacteria was lower than that claimed on the product labels. Only one of the seven evaluated products actually contained one bacterium stated on the label (C-1). Moreover, one product (E) mentioned on its label to contain ‘lactic ferment’. Surprisingly, besides the starter bacteria L. delbrueckii ssp. bulgaricus (E-2) and S. thermophilus (E-3), we unexpectedly isolated the gram-negative acetic acid bacterium Gluconobacter oxydans (E-1), which is mostly associated with vinegar production, sorbose fermentation, and wine and beer spoilage (Gupta et al. 2001). Moreover, the number of viable cells present in the product was not stated on the label. To confer any health effect, it is generally believed that probiotic bacteria should be present in a product to a minimum of ∼107 CFU ml−1 or g−1, and should remain viable until they reach the intestinal tract (Ross et al. 2005). This requires their survival in the food vehicle and their resistance to the acidic conditions of the stomach and bile salts in the small intestine. The low recovery in this study could be related to the presence of nonviable cells in the product as a consequence of either low bacterial tolerance to processing conditions, or antagonistic microbial interactions between the microbial populations in the products (Saarela et al. 2000). Nevertheless, although some probiotics can exert their beneficial health effects even if they are dead (Jijon et al. 2004), these findings are raising questions about the initial selection of the probiotics by the companies. There is a need for adequate microbial quality control before claims by the manufacturers of functional food are made. Although the overall findings in this study do not deny the beneficial effect of the product itself, the increasing consumer’s knowledge of the relation between diet and health creates the necessity of having the correct information of the strains present in a product at the time of consumption, and their corresponding benefits.
Different probiotic characteristics were determined in this study, such as antimicrobial capacity of the isolated strains (Table 3). Antagonistic properties of probiotic strains are essential in order to prevent the infection and/or invasion of pathogenic bacteria. In this preliminary screening of the antimicrobial capacity of the strains present in the products, two different strains of the gastrointestinal pathogen S. typhimurium were selected as indicator strains. Interestingly, the two Lactobacillus paracasei isolated (A-1 and F-3), Leuconostoc mesenteroides (B-1) and S. thermophilus (D-1) have strong antagonistic activity against both Salmonella strains. Unexpectedly, the only L. casei isolated (C-1) showed full inhibition against strain SL1344, but no inhibition against ATCC 14028. These differences in susceptibility could be explained by reported differences in gene expression of some membrane-related Salmonella proteins (Lee et al. 2000; Feng et al. 2003). Interestingly, the only Bifidobacterium spp. isolated (G-2) did not show any antagonist effect against the indicator strains, although some Bifidobacterium strains have been previously reported to antagonize different intestinal pathogens (Servin 2004). The results observed when using neutralized SCS suggested that the antagonistic activity exerted by the tested strains is related to the acidic nature of their SCS. We have previously reported that the strong antimicrobial activity exerted by L. rhamnosus GG against Salmonella is mediated by lactic acid (De Keersmaecker et al. 2006b). However, it is important to consider that the production of antimicrobial compounds, such as organic acids, can be dependent on medium composition. Moreover, the in vitro results cannot be merely extrapolated to in vivo situations without further testing.
The determination of the acid tolerance of starter and probiotic bacteria is important in order to predict strain performance during gastric transit. The results in the present study showed that the survival rates of the isolated bacteria were low in comparison with the high survival rate of L. rhamnosus GG. Bifidobacteria have been reported to be highly sensitive to acid exposure (pH 3–5 for 3 h) with the exception of B. lactis and B. animalis (Matsumoto et al. 2004). However, in our hands, B. animalis ssp. lactis did not survive to the acid challenge, and no viable cells were observed after 30-min incubation in simulated gastric juice. Interestingly, in this study, all S. thermophilus strains were highly sensitive towards the simulated gastric juice, although it has been reported that S. thermophilus survives the gastrointestinal transit (Mater et al. 2005). The reason for different abilities to tolerate acidity is uncertain. Overall, our results show that the survival of the cultures varies among species (e.g. A-1 and F-3), and those differences are also apparent at the strain level (e.g. F-1 and F-2), thereby confirming previous reports (Corcoran et al. 2005). However, it must be noted that the tolerance of strains in bile acid broth systems may not truly reflect their ability to tolerate bile in vivo. Like other physiological stresses, it is difficult to simulate exact in vivo conditions in a laboratory setting, and not always all parameters that affect survival can be taken into account.
Adhesion to intestinal surfaces and subsequent colonization of the human gastrointestinal tract is considered to be one of the main criteria for selecting probiotics for human use. The adherent strains of probiotic bacteria are expected to persist longer in the intestinal tract and thus have better possibilities of showing metabolic and immunomodulatory effects than nonadhering strains (Saarela et al. 2000). Adhesion may also provide competitive exclusion of pathogenic bacteria from the intestinal epithelium (Coconnier et al. 1993). Our results showed that almost 60% of the isolated strains were able to adhere to Caco-2 cells in comparison with the well-recognized adherent strain L. rhamnosus GG (Elo et al. 1991; Tuomola et al. 2001). Remarkably, although L. acidophilus JMC1132 has been reported as a nonadherent strain (Kimoto et al. 1999), in our test, the strain showed adhesion to Caco-2 cells, although less than L. rhamnosus GG. These differences might be explained by differences in the adhesion assays applied.
Until now, there are no reports in the literature describing either the strain content of Columbian dairy products, or the specific probiotic characteristics of the present strains. Table 5 shows a summary of the bacterial performance from the isolated strains. In this study, four of the tested strains (A-1, B-1, F-2 and F-3) showed, in general, satisfactory in vitro probiotic characteristics. Nonetheless, F-3 was present in the product at a number lower than the one suggested being necessary to confer health benefit effects. Additionally, it became clear that probiotic behaviour varies among species and strain level, as exemplified by the seven different S. thermophilus isolated.
Table 5. Summary of bacterial performance from the isolated strains
|Product and species identified||Antimicrobial activity||Gastric juice tolerance||Adhesion to Caco-2 cells|
|A-1 Lactobacillus paracasei||+||+||+|
|A-2 Streptococcus thermophilus||+||−||−|
|B-1 Leuconostoc mesenteroides||+||+||+|
|B-2 S. thermophilus||−||−||+|
|C-1 Lactobacillus casei||+||+||−|
|C-2 S. thermophilus||+||−||+|
|D-1 S. thermophilus||+||−||+|
|E-1 Gluconobacter oxidans||+||−||−|
|E-2 L. delbrueckii ssp. bulgaricus||+||+||−|
|E-3 S. thermophilus||+||−||+|
|F-1 L. delbrueckii ssp. bulgaricus||−||−||+|
|F-2 L. delbrueckii ssp. lactis||+||+||+|
|F-3 L. paracasei||+||+||+|
|F-4 Streptococcus thermophilus||−||−||−|
|G-1 L. delbrueckii ssp. bulgaricus||+||−||−|
|G-2 Bifidobacterium animalis ssp. lactis||−||−||+|
|G-3 S. thermophilus||+||−||−|
This study aimed to determine the microbial composition and strain characteristics present in probiotic dairy products available in the Columbian market. Although molecular techniques are highly sensitive and specific for the detection of bacterial DNA, classical culture techniques remain essential for the assessment of cell viability. This application could, therefore, serve as a valuable tool for the microbial quality control of probiotic preparations in order to provide reliable and safe products to the Columbian consumers. According to Euromonitor International (Chicago-USA), who speculated on growing competition in the probiotics market, the Latin America dairy industry is set to increase nearly 11% between 2002 and 2007 (http://www.euromonitor.com).