Feruloyl oligosaccharides stimulate the growth of Bifidobacterium bifidum

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Anaerobe 11 (2005) 225–229 www.elsevier.com/locate/anaerobe

Food microbiology

Feruloyl oligosaccharides stimulate the growth of Bifidobacterium bifidum Xiaoping Yuan, Jing Wang, Huiyuan Yao School of Food Science and Technology, Southern Yangtze University, 170 Huihe Road, Wuxi, 214036, Jiangsu Province, PR China Received 12 June 2004; received in revised form 3 February 2005; accepted 25 February 2005 Available online 19 April 2005

Abstract Insoluble dietary fiber from wheat bran contains some feruloyl groups linked to the arabinose residues in the cell wall arabinoxylan. Treatment of wheat bran insoluble dietary fiber with xylanase from Bacillus subtilis yielded feruloyl oligosacchairdes, which were purified with Amberlite XAD-2. Saponification of the feruloyl oligosaccharides released ferulic acid and arabinoxylan oligosaccharides which consist of arabinose and xylose. The effect of the feruloyl oligosacchairdes on the growth of Bifidobacterium bifidum F-35 was investigated in vitro. The B. bifidum produced acid when cultivated anaerobically in TPY broth with 0.5% feruloyl oligosacchairdes as the carbohydrate source. The biomass yield of the B. bifidum increased with increasing the concentration of feruloyl oligosaccharides in TPY broth. The maximum cell growth was increased by 50% in TPY broth supplemented with 0.1% feruloyl oligosaccharides compared to TPY broth. These results indicated that the growth of B. bifidum F-35 was promoted by the feruloyl oligosaccharides from wheat bran insoluble dietary fiber, and not suppressed by the ferulic acid moiety of them. r 2005 Elsevier Ltd. All rights reserved. Keywords: Bifidobacterium bifidum; Dietary fiber; Feruloyl oligosaccharides; Growth stimulation; Xylanase

1. Introduction Wheat bran as an important by-product of the cereal industry is produced worldwide in enormous quantities and recognized as a good source of dietary fiber [1,2]. Studies on animal model or on human volunteers have indicated that the protective effect against carcinogenesis is the greatest for the insoluble fibers and that the protective effect is modified by fermentation [3,4]. Wheat bran is rich in hemicellulose, among which arabinoxylan represent 40% of dry matter [5]. It is well known that wheat bran arabinoxylan is associated with ferulic acid via an ester bond. Feruloyl oligosaccharides were released from wheat bran by treatment with endoxylanase [6,7]. The interest in these oligosaccharides is motivated by their biological activities and their Corresponding author. Tel.: +86 510 5884277; fax: +86 510 5807976. E-mail address: [email protected] (H. Yao).

1075-9964/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2005.02.002

functional applications. They can act as regulators of growth and development in plants [8], and are potential sources of antioxidants with a capacity of inhibiting the peroxidation of low density lipoproteins [9,10]. In the food industry, such oligosaccharides are useful due to their unique gelating properties induced by oxidative crosslinking [8,11]. Many commercial carbohydrates and oligosaccharides have been reported to be bifidogenic [12–14]. Most of the studies have been made with inulin, fructooligosaccharides or transgalactooligosaccharides. However, the bifidogenic property of the feruloyl oligosaccharides on Bifidobacterium bifidum, which contain arabinoxylan oligosaccharides moiety, has not been previously reported. The feruloyl oligosaccharides used in the present study were prepared from wheat bran insoluble dietary fiber by treatment with xylanase from Bacillus subtilis. The effect of the feruloyl oligosacchairdes on the growth of B. bifidum F-35 was investigated in vitro.

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2. Materials and methods 2.1. Materials Wheat bran was obtained from a local milling plant in Wuxi, China. The bran was milled and passed through a 0.5 mm sieve. Xylanase from B. subtilis was kindly provided by Dr. Tao Liu of the Sunhy Biology Company (Wuhan, the People’s Republic of China). Amberlite XAD-2 was obtained from Rohm and Haas Company (Philadelphia, USA). Trans-ferulic acid, arabinose, xylose, and birch wood xylan were purchased from Sigma Chemical Co. (St. Louis, USA). All other chemicals and solvents used were of analytical grade.

(10 000  g, 20 min). The supernatant solution was passed through 0.45 mm filter and concentrated to 100 ml by rotary evaporation. The concentrated solution was applied to an open column (80  2.5 cm i.d.) packed with Amberlite XAD-2 (previously washed with 95% ethanol and then water). Elution was successively carried out with 2 column volumes of distilled water, 3 column volumes of 50% (v/v) methanol/water and 2 column volumes of methanol. The fraction eluted by methanol/water was concentrated and lyophilized with a freeze dry system (AlPHA1-4, Christ, Germany) to get feruloyl oligosaccharides for further analysis. 2.5. Analysis of feruloyl oligosaccharides by paper chromatography

2.2. Organism and culture conditions B. bifidum F-35 was obtained from the laboratory of food microbiology of the SYTU, Wuxi, China. The B. bifidum was revived from frozen stock cultures stored at
80 1C by culturing in TPY broth [15] which contained, in gram per liter: trypticase, 10; phytone, 5; glucose, 5; Tween 80, 1; yeast extract 2.5, cysteine, 0.5; dipotassium phosphate, 2; magnesium chloride hexahydrate, 0.5; zinc sulphate heptahydrate, 0.25; calcium chloride, 0.15; ferric chloride, trace, and the final pH of the medium was adjusted to 6.5070.1, using 4 M NaOH or HCl. The organism was incubated in the broth at 37 1C for 3 days in an anaerobic atmosphere of 5% CO2, 10% H2 and 85% N2 (YQX-1 type anaerobic incubator, Yuejing Medical Instrument Co., Ltd, Shanghai, China), and transferred weekly. 2.3. Growth experiments Cells were cultured anaerobically in 10 ml TPY for 48 h at 37 1C, then inoculated (5% v/v) into triplicate tubes of TPY containing feruloyl oligosaccharides with different concentrations. Cells were incubated anaerobically at 37 1C, and growth was determined by measuring the absorbance at 540 nm (A540) of appropriate dilutions of cells using a UV-1000 UV/VIS Recording Spectrophotometer (Rayleigh Analytical Instruments, Beijing, China). All experiments were performed in triplicate. 2.4. Preparation of feruloyl oligosaccharides Wheat bran insoluble dietary fiber was prepared according to the method of Bunzel et al. [16]. One hundred grams of wheat bran insoluble dietary fiber were incubated in 2000 ml of 0.5% xylanase (in 50 mM acetate buffer pH 5.0) at 50 1C in the dark for 60 h with constant stirring. After heat inactivation of the enzyme (100 1C, 10 min), the hydrolysate of wheat bran insoluble dietary fiber was obtained by centrifugation

The feruloyl oligosaccharides were analysed by paper chromatography, which was performed on Whatman No.1 filter paper by the descending method with nbutanol/acetic acid/water (12:3:5) as the mobile phase. The separated feruloyl oligosaccharides were located by UV radiation (before and after exposure to NH3) [17], and the spots were visualized with an oxalate/aniline reagent (2 volumes of 2% aniline in ethanol and 3 volumes of 2.5% oxalic acid) by heating in an oven at 105 1C for 10–20 min [6]. 2.6. Analysis of characterization of feruloyl oligosaccharides 2.6.1. Identification of ferulic acid Feruloyl oligosaccharides (100 ml, 1 mg/ml) were sapponified with NaOH (100 ml, 0.4 M) for 2 h in the dark at room temperature. The reaction was stopped by adding H3PO4 (150 ml, 0.4 M). This solution was analysed for ferulic acid by HPLC using a C18 Symetry column (150  3.9 mm i.d., 5 mm particle size, Waters, USA). The column was maintained at 30 1C. A sample volume of 10 ml was injected into the HPLC column, and the phenolic acid was eluted with methanol /water/acetic acid (50:50:0.5) at a flow rate of 0.8 ml/min in an isocratic program for 15 min. The absorbance of the eluate was monitored continuously at 320 nm. 2.6.2. Analysis of sugar by high-performance anionexchange chromatography Feruloyl oligosaccharides (100 ml, 1 mg/ml) were deesterified with NaOH (100 ml, 0.4 M) for 2 h in the dark at room temperature. The reaction was stopped by adding H3PO4 (150 ml, 0.4 M). The deesterified oligosaccharides were hydrolysed in CF3COOH (2 M, 1 h, 121 1C). The resulting solution was diluted 50-fold, filtered, and analysed by high-performance anionexchange chromatography with pulse amperometric detection (HPAEC-PAD) on a Dionex BioLC system using a CarbopacTM PA1 column (250  4 mm i.d.,

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3. Results and discussion 3.1. Fractionation of feruloyl oligosaccharides The enzymatic degradation of wheat bran insoluble dietary fiber was performed at 50 1C and pH 5.0. The hydrolysate was centrifuged and passed through 0.45 mm filter to remove non-hydrolysed wheat bran insoluble dietary fiber and other insoluble contaminants. The resulting solution was concentrated to a final volume of 100 ml and applied to an open column (80  2.5 cm i.d.) packed with Amberlite XAD-2, which is a polymeric adsorbent binding aromatic compounds [18]. Initially, all of the oligosaccharides were retained on the column. Application of distilled water to the column led to the elution of oligosaccharides that do not contain esterlinked ferulic acid. The feruloyl oligosaccharides were eluted using aqueous 50% methanol. The fraction eluted by methanol/water was subjected to paper chromatography. These separated compounds fluoresced blue in UV radiation and their color into green on exposure to NH3, indicating they were feruloyl oligosaccharides [19]. These spots with fluorescence occurred reddish color when they were stained with an oxalate/aniline reagent (Fig. 1), and the oligosaccharide moieties of the feruloyl oligosaccharides were branched polymers ranging in size from DP (degree of polymerization) 1–5 or more.

3.2. Characterization of feruloyl oligosaccharides The HPLC analysis of saponified product of feruloyl oligosaccharides showed the presence of ferulic acid (Fig. 2). The glycosyl residue composition of deesterified products were arabinose and xylose by HPAEC (Fig. 3). These results demonstrated that feruloyl oligosaccharides were released from wheat bran insoluble dietary fiber by treatment with xylanase from B. subtilis. 3.3. Effect of carbon substrates on B. bifidum growth A lowering of culture pH as a result of short-chain fatty acids production for certain bacterial species has often been used as broad index of the fermentability of various carbohydrates in pure culture [20]. The B. bifidum F-35 was examined for its ability to produce acid when cultivated in TPY broth with 0.5% glucose, arabinose, xylose or feruloyl oligosaccharides as carbon sources. It was discovered that the strain produced acid only with glucose, or feruloyl oligosaccharides as carbohydrate sources; DpH value were 2.28 and 1.08, respectively. Free arabinose and xylose were not fermented by the B. bifidum F-35. Although biomass yield of the strain, using feruloyl oligosaccharides as a substrate, was lower than using glucose (data not shown), the results revealed that the B. bifidum F-35

[mAU]

Dionex, Sunnyvale, CA, USA). The column was maintained at 25 1C and eluted with 100 mM NaOH at a flow rate of 0.7 ml/min.

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Fig. 2. HPLC of the saponified product of feruloyl oligosaccharides from wheat bran insoluble dietary fiber. Trans: trans-ferulic acid; Cis: cis-ferulic acid.

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Minutes Fig. 1. Paper chromatography of feruloyl oligosaccharides. The sugars were stained with an oxalate/aniline reagent. DP, degree of polymerization.

Fig. 3. HPAEC-PAD of hydrolysis products of deesterified feruloyl oligosaccharides treated with 2 M CF3COOH.

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was able to utilize the feruloyl oligosaccharides as the sole source of carbon and energy. Bifidobacteria can utilize or ferment a great variety of carbohydrates [21], and different species of bifidobacterium differ in fermentation profiles when tested on different monosaccharides [22]. Interestingly, in the present study, the B. bifidum F-35 was able to utilize the feruloyl oligosaccharides but did not utilize arabinose and xylose. This suggests the strain has a specific uptake system for the utilization of feruloyl oligosaccharides over the monomers. In other words, the organism imports feruloyl oligosaccharides before hydrolysing them and does not possess efficient membrane transport mechanisms for free arabinose and xylose. Similar phenomena have been observed in bifidobacteria for other nondigestible oligosaccharides and their constituent monosaccharides [23–25]. 3.4. Effect of feruloyl oligosaccharides on B. bifidum growth In order to investigate the effect of feruloyl oligosacchairdes on the growth of the B. bifidum F-35, the organism was cultured in TPY broth supplemented with different concentrations of feruloyl oligosaccharides (0–0.5%) at 37 1C for 24 h. As shown in Fig. 4, feruloyl oligosacchairdes had a potential to stimulate growth of B. bifidum F-35. The amount of cell growth increased as the concentration of feruloyl oligosaccharides rose to 0.5%. Rate of growth was determined for the B. bifidum F-35 in TPY broth supplemented with 0.1% feruloyl oligosaccharides. Fig. 5 showed that strain B. bifidum grew rapidly after 6 h, with maximum growth observed at 36 h. Also, the maximum cell growth was increased by 50% in TPY broth supplemented with 0.1% feruloyl oligosaccharides compared to TPY broth.

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Fig. 5. Growth of B. bifidum F-35 in TPY broth (J) or supplemented with 0.1% feruloyl oligosaccharides (K) as a function of time. Results are mean values of three determinations 7standard deviation (N ¼ 3).

It has been reported that the xylooligosaccharides from wheat bran can promote the growth of B. bifidum [26,27]. The characterization of the feruloyl oligosaccharides demonstrated that they contained arabinoxylan oligosaccharides moiety which was also investigated for their bifidogenic property. The feruloyl oligosaccharides were deesterified with 0.4 M NaOH for 2 h at 35 1C, and then acidified to pH 2.0 with 0.4 M HCl. After extraction of ferulic acid with ether, the pH of the mixture was adjusted to 7.0. Samples were freeze-dried to get arabinoxylan oligosaccharides. As shown in Fig. 4, the arabinoxylan oligosaccharides stimulated growth of strain B. bifidum more rapidly than the feruloyl oligosaccharides, even so, the growth of strain B. bifidium was not suppressed by the ferulic acid moiety of feruloyl oligosacchairdes. Certain of the phenolic monomers, including ferulic acid, have been reported to be toxic to many species of ruminal microorganisms [28–31]. Feruloyl esterase has been isolated from a human typical intestinal bacterium, Lactobacillus acidophilus [32], which is probably involved in the release of ferulic acid in the human colon. Although the results show that the ferulic acid moiety of feruloyl oligosacchairdes has not a negative effect on the strain in vitro, the possible negative effect of the released ferulic acid in vivo on the microflora clearly merits further investigation.

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Concentration (%) Fig. 4. Effect of different concentrations of feruloyl oligosaccharides (J) or arabinoxylan oligosaccharides (K) on B. bifidum F-35 growth. The strain was cultured anaerobically in TPY broth supplemented with various concentration of feruloylated oligosaccharides or arabinoxylan oligosaccharides for 24 h at 37 1C. Results are given as means7standard deviation (N ¼ 3).

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