Elsevier

Anaerobe

Volume 18, Issue 5, October 2012, Pages 489-497
Anaerobe

Clinical microbiology
Prebiotic-non-digestible oligosaccharides preference of probiotic bifidobacteria and antimicrobial activity against Clostridium difficile

https://doi.org/10.1016/j.anaerobe.2012.08.005Get rights and content

Abstract

Bifidobacterium breve 46, Bifidobacterium lactis 8:8 and Bifidobacterium longum 6:18 and three reference strains B. breve CCUG 24611, B. lactis JCM 10602, and Bifidobacterium pseudocatenulatum JCM 1200 were examined for acid and bile tolerance, prebiotic utilization and antimicrobial activity against four Clostridium difficile (CD) strains including the hypervirulent strain, PCR ribotype NAP1/027. B. lactis 8:8 and B. lactis JCM 10602 exhibited a high tolerance in MRSC broth with pH 2.5 for 30 min. B. breve 46 and B. lactis 8:8 remained 100% viable in MRSC broth with 5% porcine bile after 4 h. All six strains showed a high prebiotic degrading ability (prebiotic score) with galactooligosaccharides (GOS), isomaltooligosaccharides (IMOS) and lactulose as carbon sources and moderate degradation of fructooligosaccharides (FOS). Xylooligosaccharides (XOS) was metabolized to a greater extent by B. lactis 8:8, B. lactis JCM 10602, B. pseudocatenulatum JCM 1200 and B. longum 6:18 (prebiotic score >50%). All strains exhibited extracellular antimicrobial activity (AMA) against four CD strains including the CD NAP1/027. AMA of B. breve 46, B. lactis 8:8 and B. lactis JCM 10602 strains was mainly ascribed to a combined action of organic acids and heat stable, protease sensitive antimicrobial peptides when cells were grown in MRSC broth with glucose and by acids when grown with five different prebiotic-non-digestible oligosaccharides (NDOs). None of C. difficile strains degraded five prebiotic-NDOs. Whole cells of B. breve 46 and B. lactis 8:8 and their supernatants inhibited the growth and toxin production of the CD NAP1/027 strain.

Highlights

► Probiotic bifidobacteria were tested for stress tolerance, prebiotic degradation and AMA against CD. ► Bifidobacterium breve 46 and Bifidobacterium lactis 8:8 were robust to acid and porcine bile. ► B. breve 46, B. lactis 8:8 and Bifidobacterium longum 6:18 degraded GOS, IMOS and lactulose. ► These strains exhibited high AMA against CD when grown in medium with and without prebiotics. ► B breve 46 and B. lactis 8:8cells and their CFS inhibited growth of CD NAP1/027 and toxin production.

Introduction

Bifidobacteria is a major beneficial bacterial group in the healthy human gut microbiota, colonising the intestinal mucosa [1], [2]. They exhibit several health-promoting effects such as antimicrobial activity (AMA) against various enteric pathogens, immune-modulating properties decrease the serum cholesterol levels, reduce the incidence of colon cancer, and effective against gut disorders on a regular consumption as fermented food products [2], [3], [4], [5], [6]. They metabolize many complex carbohydrates that escape hydrolysis by digestive enzymes in the gut and reach the colon unabsorbed [2], [5], [7], [8]. Many non-digestible oligosaccharides (NDOs) in the gut act as prebiotics enhancing the growth of bifidobacteria, some lactic acid bacteria (LAB) and other beneficial gut microbes [9], [10], [11]. The two most studied prebiotic-NDOs are fructooligosaccharides (FOS) and galactooligosaccharides (GOS). Other prebiotic-NDOs such as xylooligosaccharides (XOS); isomaltooligosaccharides (IMOS), glucooligosaccharides, pectin oligosaccharides (POS) mannanooligosacharides (MOS), gentiooligosaccharides (GTO), chitooligosaccharides (CHOS), soy bean oligosaccharides (SOS) and polydextrose have also been evaluated but these substances, with high purity, are not commercially available [10], [11], [12].

To develop a synbiotic formulation, containing a single or a multi-strain probiotic and prebiotic mix, it is essential to screen probiotic strains based on a prebiotic index i.e. ability of strains to degrade several prebiotics and subsequently determine the prebiotic score i.e. the ability of each strain to degrade specific prebiotic-NDOs and explore how each prebiotic increase cell yields when grown in a defined medium. Some in vitro studies showed that a number of NDO's stimulate the growth of bifidobacteria and lactobacilli [13], [14], [15].

A long term antibiotic therapy may cause intestinal overgrowth of Clostridium difficile (CD), extended spectrum of β-lactamase producing Escherichia coli, methicillin-resistant Staphylococcus aureus, vancomycin resistant Enterococcus and other multiple resistant bacteria and Candida albicans [16]. The epidemic CD NAP1/027 strain could produce up to 16 times higher levels of toxin A and B and a binary toxin, than most hospital outbreak associated strains [17], [18]. Thus, there is an urgent need to develop alternative non-antibiotic based therapies such as probiotic and synbiotic dietary supplements, to stimulate and restore a healthy indigenous gut microbiota after various antimicrobial therapies [8], [9]. Several synbiotic supplements are reported to reduce inflammation, infection and promote immune modulation and alleviate other gastrointestinal disorders [8]. However, knowledge on the compatibility of strains in multi-strain synbiotic combinations, minimum effective dose to impart the desired health benefit without any side effects and the appropriate biomarkers in the in vivo trials are lacking, hence there is a need to address such issues. In the present study, potential probiotic properties of three enteric human bifidobacterial strains were analysed for (i) acid and bile tolerance, (ii) in vitro fermentation of selected prebiotic-NDOs i.e. FOS, GOS, IMOS, XOS, and lactulose to design optimal synbiotic combination and (iii) antimicrobial activity (AMA) against four clinical C. difficile strains including the virulent CD NAP1/027 strain. These properties were compared with three reference strains from two international type culture collections.

Section snippets

Chemicals

Proteinase K, pronase E, p-nitrophenol β-d-galactopyranoside, p-nitrophenol β-d-xylopyranoside and bromocresol purple were purchased from Sigma–Aldrich, St. Louis, Mo, USA. deMan Rogosa Sharp with l-cysteine-HCl (MRSC) agar was purchased from Oxoid Ltd, Basingstoke, UK. Native porcine bile (PB) was prepared as described previously [19]. Prebiotic oligosaccharides and polysaccharides studied are listed in Table 1.

Bacterial strains and culture conditions

All strains (Table 2) were maintained at −110 °C in Trypticase soy broth (TSB,

Acid and bile tolerance

The six bifidobacterial strains showed significant variations in the acid and bile tolerance tests (Fig. 1A). B. lactis strains showed the highest survival rate at pH 2.5 with no significant reduction (P > 0.05) compared with the other five strains. All strains were viable in MRSC broth at pH 2.5 after 30 min incubation (Fig. 1A). Four of the six tested strains retained 100% viability when grown in MRSC broth with 5% porcine bile for 4 h, except Bifidobacterium longum 6:18 and Bifidobacterium

Discussion

In the present study, acid and bile stress tolerance tests of bifidobacterial strains provided the first-level relevant strain selection criteria (Fig. 1). Two B. lactis strains were highly resistant to acid stress as reported by Masco et al., [24]. This acid tolerance could be due to an over expression of the F1Fo-ATPase as shown in the wild type and mutant strains for B. longum [25].

All strains except B. longum 6:18 showed ≥90% viability in the bile tolerance assay. The porcine bile simulate

Conclusions

B. breve 46 and B. lactis 8:8 strains were the robust as determined by acid and bile stress tolerance assays. Although B. longum 6:18 exhibited a high AMA against CD and a high prebiotic-NDO metabolizing ability, it did not survive bile stress, i.e. a less robust strain. These three in-house strains and three reference strains are highly active in degrading GOS, FOS, IMOS and lactulose. B. lactis 8:8 and B. longum 6:18 also metabolized XOS. All the bifidobacterial strains exhibited high AMA

Acknowledgements

Authors thank Ellen Ertmann at Friesland Campina for expert advice on the choice of GOS for this study. Authors also thank Prof A. Weintraub (Karolinska Institute, Stockholm, Sweden) for kindly providing the CD NAP1/027 strain, Dr. Torbjörn Norén (Clinical Microbiology Laboratory, University hospital, Örebro, Sweden) for performing PCR ribotyping of the CD strains and Sweet Town Biotech (Taiwan) for providing the XOS. This study was supported by a grant from the European community's seventh

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K. K. K & P. A contributed equally to the experimental design, laboratory work and manuscript writing and are the first authors of this manuscript.

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