Wageningen Academic  P u b l i s h e r s

Beneficial Microbes, 2015; 6(4): 451-455

http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0118 - Thursday, September 21, 2017 10:46:03 PM - Göteborgs Universitet IP Address:130.241.16.16

Consumption of partially hydrolysed guar gum stimulates Bifidobacteria and butyrateproducing bacteria in the human large intestine Y. Ohashi1*, K. Sumitani1, M. Tokunaga2, N. Ishihara2, T. Okubo2 and T. Fujisawa1 1Laboratory of Food Hygiene, Department of Food Science and Technology, Nippon Veterinary and Life Science University,

1-7-1, Kyonan-cho, Musashino, Tokyo 180-8602, Japan; 2Central Research Laboratories, Taiyo Kagaku Co., Ltd., 1-3, Takara-cho, Yokkaichi, Mie 510-844, Japan; [email protected] Received: 20 August 2014 / Accepted: 1 October 2014 © 2014 Wageningen Academic Publishers

RESEARCH ARTICLE Abstract Partially hydrolysed guar gum (PHGG) is a water-soluble dietary fibre that is non-digestible in the upper gastrointestinal tract. It is believed that PHGG benefits the health of hosts by altering the colonic microbiota and stimulating shortchain fatty acid (SCFA) production. However, it remains unclear which bacteria ferment PHGG in the human large intestine. In this study, the effect of PHGG on faecal bacteria was analysed to specify the bacteria that contribute to the fermentation of PHGG in the human large intestine. Ten healthy volunteers consumed PHGG (6 g/day) for 2 weeks. Faeces were collected at 2 weeks prior to consumption, at the end of 2 weeks of consumption, and 2 weeks after consumption of PHGG. Bacterial DNA was extracted from these collected faeces and subjected to real-time PCR using bacterial group- or species-specific primers. The copy number of the butyryl-CoA CoA-transferase gene and the 16S rRNA gene copy numbers of Bifidobacterium, the Clostridium coccoides group, the Roseburia/ Eubacterium rectale group, Eubacterium hallii, and butyrate-producing bacterium strain SS2/1 were significantly increased by the intake of PHGG. Other bacteria and bacterial groups were not significantly influenced by the intake of PHGG. It was believed that the Roseburia/E. rectale group bacteria, Bifidobacterium, the lactate-utilising, butyrate-producing bacteria, E. hallii and bacterium strain SS2/1, would contribute to the fermentation of PHGG in the human large intestine. PHGG may benefit health by stimulating Bifidobacterium and butyrate-producing bacteria in the human large intestine. Keywords: water-soluble dietary fibre, prebiotics, short-chain fatty acid

1. Introduction A large number of bacteria inhabit the human colon, constructing a complex microbial ecosystem. These intestinal bacteria significantly affect the host’s health, not only beneficially, but also harmfully. Therefore, it has been considered that the stimulation of beneficial bacteria, such as Bifidobacterium and Lactobacillus, in the gastrointestinal tract contributes to maintaining and improving the host’s health (Fuller and Gibson, 1997). Prebiotics, non-digestible food ingredients, are selectively fermented by beneficial bacteria in the large intestine and modulate microbial composition and activity. This leads to stimulated production of short-chain fatty acids (SCFAs) of which health-promoting effects have been reported (Sakata,

1997; Topping and Clifton, 2001). Recently, it was reported that activation of the SCFA receptor GPR43 suppressed insulin-mediated fat accumulation (Kimura et al., 2013). The ability of SCFAs to regulate body energy utilisation has been expected. In addition, SCFAs regulate the function of colonic regulatory T-cells (Smith et al., 2013). Thus, through improvement of microbiota in the large intestine, prebiotics exert beneficial health effects on the host. Guar gum is a water-soluble dietary fibre derived from the seeds of Cyamopsis tetragonoloba, and is used in the food industry as a stabiliser and a thickener. Partially hydrolysed guar gum (PHGG) is a water-soluble dietary fibre that is prepared from guar gum by microbial endoβ-mannanase to reduce the high viscosity of guar gum.

ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2014.0118451

http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0118 - Thursday, September 21, 2017 10:46:03 PM - Göteborgs Universitet IP Address:130.241.16.16

Y. Ohashi et al.

It is reported that PHGG can reduce the incidence of diarrhoea, enhance mineral absorption, and reduce blood cholesterol (Slavin and Greenberg, 2003, Yasukawa et al., 2012). PHGG is not digested in the upper gastrointestinal tract and fermented by colonic bacteria. Therefore, PHGG stimulates SCFA-production – butyrate in particular – in the in vitro human faecal fermentation (Ohashi et al., 2012; Stewart and Slavin, 2006; Velázquez et al., 2000). In our in vitro study, PHGG stimulated not only Bifidobacterium, but also butyrate-producing bacteria (Ohashi et al., 2012). These bacteria should contribute to the fermentation of PHGG in the human large intestine. Through alteration of colonic microbiota and stimulation of SCFA production, PHGG should exert the beneficial health effects on the host. However, the effect of PHGG on microbiota and SCFA production is also analysed by the in vitro study. Only the in vivo study was reported by Okubo et al. (1994). In this report, an increase of faecal Bifidobacterium and Lactobacillus with the intake of PHGG was indicated using the culture method. However, the butyrate-producing bacteria were not analysed because these bacteria could not be analysed by the culture method due to the absence of an appropriate selective medium for these bacteria. Therefore, we investigated the effect of PHGG on faecal microbiota, butyrate-producing bacteria in particular, in the in vivo study using a molecular biological technique to estimate which bacteria contribute to PHGG fermentation.

2. Materials and methods Subjects Ten healthy female volunteers (Nippon Veterinary and Life Science University, Tokyo, Japan) between 21 and 24 years of age consumed normal nonspecific Japanese diets during the experiment. They had no probiotics, prebiotics, and antibiotics for 1 month prior to and during the experiment. Subjects consumed their normal diets for 2 weeks (before period). Subsequently, in addition to the normal diet, they consumed 6 g of commercially available PHGG (Sunfiber, Taiyo Kagaku Co., Ltd., Mie, Japan) once daily for 2 weeks (intake period), followed by their normal diets for 2 weeks (after period). This study was approved by Taiyo Kagaku Co. Ltd., Ethics Committee and performed in accordance with the ethic guidelines. All volunteers were explained the detail of this study. An informed consent agreement was obtained from all volunteers before the experiment.

Collection of faeces Fresh faeces of volunteers were collected on the final day in each period. Faeces were immediately transported at 4 °C to the laboratory under anaerobic conditions using Anaero Pack (Mitsubishi Gas Chemical Co., Ltd., Tokyo, Japan). The faeces were treated within 1 h for the analysis of faecal bacteria and SCFA concentration. 452

Analysis of faecal bacteria Bacterial genomic DNA was extracted from 0.1 g of faeces according to Godon et al. (1997). The 16S rRNA gene of Bifidobacterium, the Clostridium coccoides group, the Clostridium leptum group, the Clostridium cluster I, the Clostridium cluster XI, the Atopobium cluster, Prevotella, the Bacteroides fragilis group, Enterococcus, the Lactobacillus group, Faecalibacterium prausnitzii, the Roseburia/Eubacterium rectale group, Eubacterium hallii and the butyrate-producing bacterium SS2/1, as well as the butyryl-CoA CoA-transferase gene were quantified by realtime PCR using the MyiQ real-time PCR system (Bio-Rad, Tokyo, Japan). The reaction mixture (20 µl) contained 10 µl of the iQ SYBER Green Supermix (Bio-Rad, Hercules, CA, USA), 0.5 µl of faecal DNA, and 400 µmol/l of each primer. The primers that were described previously (Matsuki et al., 2004; Ohashi et al., 2012; Rinttilä et al., 2004; Song et al., 2004) were used in this study. The thermal program consisted of an initial denaturation at 95 °C for 3 min, followed by 40 cycles of at 94 °C for 10 s, primer annealing at optimum temperature for 30 s and at 72 °C for 40 s, and final elongation at 72 °C for 5 min. Fluorescent products were detected at the last step of each cycle. Melting curve analysis of the product was performed after completion of the amplifications to determine the specificity of the PCR. A plasmid containing a partial sequence of the 16S rRNA gene or the butyryl-CoA CoA-transferase gene identical to the targeted bacteria was constructed in our laboratory and used as a standard DNA for the real-time PCR.

Analysis of faecal organic acids A portion of the faeces (0.5 g) was suspended with 1 ml distilled water, and 150 µl of perchloric acid (70%) was added to eliminate protein. After being centrifuged at 20,000×g at 4 °C for 10 min, the supernatant was filtered through a cellulose acetate membrane filter with a 0.45 µm pore size (Minisart RC4, Sartorius Stedim Biotech, Goettingen, Germany), and then its organic acid content was analysed by ion-exclusion high performance liquid chromatography as described by Ushida and Sakata (1988).

Statistical analyses The copy number of targeted genes calculated by real-time PCR and the concentration of organic acids were analysed using the Steel-Dwass method after the Friedman test.

3. Results Faecal bacteria Numbers of faecal bacteria are given in Table 1. The 16S rRNA gene copy numbers of Bifidobacterium (P

Consumption of partially hydrolysed guar gum stimulates Bifidobacteria and butyrate-producing bacteria in the human large intestine.

Partially hydrolysed guar gum (PHGG) is a water-soluble dietary fibre that is non-digestible in the upper gastrointestinal tract. It is believed that ...
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