Probiotic potential of Lactobacillus strains isolated from dairy products

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Abstract

Twenty-nine Lactobacillus strains of dairy origin were examined in vitro for their probiotic potential. Only a few strains were able to survive at pH 1 or in the presence of pepsin, while all were unaffected by pH 3, pancreatin and bile salts. Strains exhibited variable bile salt hydrolase activity. None was haemolytic. The majority of strains were resistant to vancomycin and teicoplanin, but sensitive to chloramphenicol and tetracycline. A few strains were able to adhere to Caco-2 cells. Although no bacteriocin activity was detected in vitro, strains L. casei Shirota ACA-DC 6002, L. plantarum ACA-DC 146 and L. paracasei subsp. tolerans ACA-DC 4037 were able to inhibit the adhesion of Escherichia coli and Salmonella typhimurium to Caco-2 cells. They also induced the secretion of pro- and anti-inflammatory cytokines by human peripheral blood mononuclear cells. These three strains were therefore found, in vitro, to possess desirable probiotic properties.

Introduction

The genus Lactobacillus has a long history of safe use, especially in the dairy industry, and plays a major role in the production of fermented milk products. Over the past few decades, an increased drive has existed for the isolation of novel Lactobacillus strains that exert a beneficial health effect when ingested by humans. Such strains are termed probiotic. According to Guarner and Shaafsma (1998) probiotics are “living micro-organisms, which upon ingestion in certain numbers, exert health benefits beyond inherent basic nutrition”.

Beneficial effects conferred by lactobacilli include inhibition of pathogenic organisms, such as Salmonella, Shigella and Helicobacter (Bernet-Camard et al., 1997; Hudault, Lievin, Bernet-Camard, & Servin, 1997; Aiba, Suzuki, Kabir, Tagaki, & Koga, 1998; Hammilton-Miller, 2003; Sgouras et al., 2004). Furthermore, lactobacilli have been associated with numerous other health benefits, such as reduction of lactose intolerance (Gilliland & Kim, 1984) and increased immune response (Matsuzaki, Yamazaki, Hashimoto, & Yokokura, 1998). A beneficial role for lactobacilli has also been implied in cancer (Hirayama & Rafter, 2000; Kopp-Hoolithan, 2001), and especially in the case of colon cancer (Mercennier, Pavan, & Pot, 2002; Guarner & Malagelada, 2003).

In order for a probiotic strain to exert its beneficial effect on the host, it has to be able to survive passage through the host's digestive tract. So far, research has mainly focused on strains sensitivity towards low pH, proteolytic enzymes and bile salts (Conway, Gorbach, & Goldin, 1987; Charteris, Kelly, Morelli, & Collins, 1998; Du Toit et al., 1998; Jacobsen et al., 1999). Another relevant property is the ability of probiotic bacteria to assimilate cholesterol (Dashkevicz & Feighner, 1989; Buck & Gilliland, 1994; Du Toit et al., 1998; Franz, Specht, Haberer, & Holzapfel, 2001). This has been linked to the bile salt deconjugation activity of some strains because of the enzyme bile salt hydrolase (BSH).

The ability of Lactobacillus strains to adhere to the mucosal surfaces of the intestine and the subsequent long or short-term colonization has long been one of the most commonly encountered criteria for the selection of probiotic strains. Adhesive probiotic lactobacilli have been reported to have beneficial health effects, especially related to the inhibition of pathogen adhesion to intestinal cell lines (Hudault et al., 1997; Lievin-Le Moal, Amsellem, Servin, & Coconnier, 2002), although the underlying mechanism has not been clearly elucidated yet. The immunostimulatory effect of probiotic lactobacilli has also been examined in the past. Lactobacillus strains are generally good inducers of the pro-inflammatory IL-12, TNF-α and IFN-γ, with fewer strains able to induce IL-10 production by immune system cells, such as peripheral blood mononuclear cells (PBMC) (Miettinen, Vuopio-Varkila, & Varkila, 1996; Miettinen et al., 1998; Hessle, Hanson, & Wold, 1999; Maassen et al., 2000).

The aim of this study was to apply established in vitro tests to evaluate the probiotic potential of Lactobacillus strains isolated from dairy sources, and to select candidate probiotic strains that fulfill the established criteria and could therefore be potentially used as novel probiotic strains in the food industry.

Section snippets

Bacterial strains and growth conditions

A total of 29 Lactobacillus strains isolated from dairy products (and held at the ACA-DC Collection at the Agricultural University of Athens, Athens, Greece) were included in the present study (Table 1). Strains were stored at −80 °C in MRS broth (Biokar Diagnostics, Beauvais, France), supplemented with 20% (v/v) glycerol. For routine analysis, strains were subcultured twice in MRS broth (Biokar Diagnostics), for 18 h at 30 °C.

For the antimicrobial production screening and the inhibition of

Results and discussion

The in vitro criteria used in our study for the selection of candidate probiotics have been described in previous studies and are referred to as selection guidelines by the FAO/WHO committee (Joint FAO/WHO Working Report, 2002). The in vitro screening of the survival of lactobacilli in simulated GI tract conditions may only have value in predicting the actual in vivo survival of a strain when consumed in a non-protected way. Strains embedded in a food matrix, for example, or consumed in an

Acknowledgements

The present work was financially supported by the Greek General Secretariat of Research and Technology (EPET II, Project 97-DIATRO-26). Petros Maragkoudakis would like to express his thanks to the Marie Curie Programme (Project type PHD20, HPMT-GH-01-00275-02) of the Research-Directorate General of the European Commission for financial support.

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