Food Science and Technology Research
Online ISSN : 1881-3984
Print ISSN : 1344-6606
ISSN-L : 1344-6606
Notes
The Vitro Fermentation of Six Functional Oligosaccharides by Clostridium butyricum TK2 and Clostridium butyricum CB8
Haikuan WangYan ShiShuli ZhangXueqin GaoFeifei LiuHuitu ZhangYujie DaiYue WangFuping Lu
Author information
JOURNAL FREE ACCESS FULL-TEXT HTML

2014 Volume 20 Issue 5 Pages 1005-1011

Details
Abstract

In vitro fermentation of six functional oligosaccharides including fructooligosaccharides (FOS), galactooligosaccharides (GOS), xylooligosaccharides (XOS), isomaltooligosaccharides (IMOS), raffinose and stachyose was performed by using Clostridium butyricum TK2 and Clostridium butyricum CB8. Among the six oligosaccharides, IMOS revealed the strongest fermentability for both strains, which was supported by the highest viable cells (10.1 ± 1.05 and 9.8 ± 0.41 log (CFU / mL)) and the highest levels of SCFA or butyrate (11.77 ± 0.37 and 14.47 ± 1.05 mM) from fermentation with this functional oligosaccharide. The fermentability of GOS to two strains was better than raffinose or stachyose. The last groups were FOS and XOS. However, every oligosaccharide showed no significant difference in promoting the growth of both of C. butyricum (p > 0.05), and exerted some differences in the production of SCFA between two strains. This study provides a rational basis for establishing synbiotics with C. butyricum to improve the gut health.

Introduction

In the human large intestine, the bacterial population is composed by several genera among which Clostridium butyricum is identified as a butyric acid producing beneficial bacterium and plays a crucial role in host health and maintaining gut function. They can decreases ammonia, amine and benzopyrrole from abnormal fermentation in intestinal tract and inhibit some pathogenic bacteria. The bacterium show remarkable stability but can be modulated by many endogenous and exogenous factors, especially prebiotics. Resistant starch type 3 (RS3, a potential prebiotic) is retrograded starch which is not digested by human starch degrading enzyme, and will thus undergo bacterial degradation in the colon. Current research suggests that C. butyricum have the capability to ferment RS3 with featured regarding short chain fatty acids (SCFA) released in the human colon with potential health implication (Purwani et al., 2011).

Prebiotics are defined as ‘'non-digestible food substances that beneficially affect the host by selectively stimulating the growth and/or activity of a limited number of bacterial species already resident in the colon and thus attempt to improve the host health” (Gibson and Roberfroid, 1995). In brief, prebiotics have better effect on normal intestinal flora. Many prebiotics belong to the group of nondigestible oligosaccharides, which resist digestion and absorption in the human small intestine and are fermented into SCFA in the large intestine. The SCFA (acetate, propionate and butyrate) have an beneficial influence on colonic health (Mortensen and Clausen, 1996; Scheppach et al., 2001; Williams et al., 2001; Wong et al., 2006; Macfarlane and Macfarlane, 2012). In particular butyrate is the prime energy substrates for the colonic mucosa and has an effect on immune function (Roediger, 1980; Fleming and Floch, 1986; Wong et al., 2006; McOrist et al., 2008; Serpa et al., 2010). Furthermore, in vitro studies as well as animal studies indicate that butyrate may reduce risk factors of gut inflammation (Roediger, 1990; Cummings and Englyst, 1991; Segain et al., 2000; Galvez et al., 2005). It also has been observed to reverse the resistance of colorectal cancer cells to apoptosis (Caderni et al., 1998; Leng et al., 2001; Brouns et al., 2002; Hinnebusch et al., 2002; Blottiere et al., 2003; Kien et al., 2006; McOrist et al., 2008; Serpa et al., 2010).

Because of the multiple beneficial health effects attributed to functional oligosaccharides, they are used widely in food products. As nutraceuticals, oligosaccharides are used in beverages and milk products. Also, the functional oligosaccharides are widely used in confectionary. Other current applications include desserts, table spreads, bakery products, breakfast cereals, meat products and food for infants, elders and diabetics (Crittenden and Playne, 1996; Nakakuki, 2002; Patel and Goyal, 2011).

There have been several reports on the utilization of soybean oligosaccharides (stachyose and raffinose as the main components), FOS, and GOS by C. butyricum (Hayakawa et al. 1990; Sako et al., 1999). However, the results only showed the efficacy of oligosaccharides in increasing the number of C. butyricum. In fact, the functional oligosaccharides varying in monomeric composition and the diverse structure may not only influence intestinal microbiota, but also the formation of SCFA (Nilsson and Nyman, 2005). Therefore, the current study aimed to evaluate and compare the fermentation characteristics of various functional oligosaccharides depending on the growth of two types of C. butyricum and the production of SCFA, especially butyrate.

Materials and Methods

Functional oligosaccharide The functional oligosaccharides used in this study are food material and described in Table 1.

Table 1. Various types of functional oligosaccharides used in this study (Sako et al., 1999; Patel and Goyal, 2011).
Type Nomenclature Monosaccharides Bonds indicative of functions source
Fructooligosaccharides FOS Sucrose, fructose β-1,2 Jiangxi Ouliduo biotechnology Co . Ltd .
Galactooligosaccharides GOS Galactose β-1,4;α-1,6 Xi'an Deshipu biotechnology Co . Ltd .
Xylooligosaccharides XOS Xylose β-1,4 Shangdong Longli biotechnology Co . Ltd .
Isomaltooligosaccharides IMOS Glucose α-1,6 Baolingbao biotechnology Co . Ltd .
Raffinose Galactose, fructose, glucose α-1,6 Shanghai Ruji biotechnology Co . Ltd .
Stachyose Galactose, fructose, glucose α-1,6 Xi'an Deshipu biotechnology Co . Ltd .

Biochemical characterization Strain TK2 and CB8 used in this study were originally isolated from the soil and human feces, respectively. Fermentation tests of carbohydrate and traditional biochemical tests for the two strains were performed according to Berger's Manual of Systematic Bacteriology (Sneath et al., 1986).

16S rDNA gene sequencing analysis Strain TK2 and CB8 were identified through 16S ribosomal DNA (rDNA) analysis. The 16S rDNA sequences of the isolated strains were determined by Beijing Liuhe Huada gene technology Co., Ltd. A homology search using the reference strains registered in DDBJ/EMBL/GenBank was performed using NCBI BLAST.

Growth conditions C. butyricum TK2 and C. butyricum CB8 were stored at −80°C in 20% sterile glycerol. Before use, the strains were propagated twice in 20 mL test tube with 10 mL liquid medium (peptone 20 g/L, glucose 5 g/L, K2HPO4 5 g/L, MnSO4 0.2 g/L, MgSO4 0.2 g/L, pH 7.4) under anaerobic gas phase (H2:CO2:N2, 10:10:80, v/v/v) at 37°C for 16 h. The cells were then harvested and resuspended in sterilized physiological saline and adjusted to 7.30 log (CFU / mL). The cells suspensions were ready to serve as inoculums for in vitro fermentation.

In vitro fermentation Fermentation studies were carried out in 50 mL bottom flasks with 40 mL fermentation medium (peptone 20 g/L, functional oligosaccharides 5 g/L, K2HPO4 5 g/L, MnSO4 0.2 g/L, MgSO4 0.2 g/L, pH 7.4). Each flask was sterilized at 115°C for 20 min. Then the cells suspensions of C. butyricum TK2 or C. butyricum CB8 (5%, v/v) were inoculated into the flask. It was then incubated under anaerobic gas phase (H2:CO2:N2, 10:10:80, v/v/v) at 37°C. Fermentation was carried out for 48 h and performed in triplicate.

Viable count method The number of viable cells was determined using the Semi-solid AGAR method. And the counting medium is consisted of the following (in g/L): peptone, 20; glucose, 5; agar, 10 g.

The viable cells were counted as follows. Serial 10-fold dilutions were performed in sterile phosphate-buffered saline. Then 1 mL of appropriate dilutions were injected into anaerobic tubes, which had been added into counting medium (9 mL), and incubated under anaerobic gas phase at 37°C for 36 h.

Preparation of samples Culture medium was centrifuged at 10000 rpm for 10 min and the supernatant remove 2.0 mL, diethyl ether (2 mL) and about 50% H2SO4 (0.4 mL) were then added. The sample was centrifuged at 3000 rpm for 5 min at room temperature after mixed for 45 min with an orbital shaker, and the organic phase was filtered through a 0.22 μm filter into a 1.5 mL eppendorf tube for detecting (Schneider et al., 2006).

Short chain fatty acid analysis Samples of 1 uL were injected into a high-resolution gas chromatography (Agilent 7890A GC System), which is equipped with an HP-Innowax 19091 N-213 column (30 m × 0.32 mm × 0.5 μm) and a flame ionization detector. The flow rate of carrier gas was 1.8 mL/min, and the split ratio was 40:1. Injector and detector temperatures were 275°C. The column temperature was held at 90°C for 0.5 min, increased to 110°C at 10°C min−1, and then increased to 170°C at 5°C min−1 and maintained for 5 min. Concentration of SCFA was determined used external standards.

Statistical analysis Data were expressed as the mean values ± standard deviation (SD) for each measurement. The data were also analyzed by one-way analysis of variance (one-way ANOVA). Tukey's procedure was used for significance of difference (p<0.05). Analysis was performed with SPSS 16.0 (SPSS, Inc., Chicago, IL).

Results

Description of C. butyricum TK2 and C. butyricum CB8 Table 2 shows the results of the tests usually applied for characterization of strain TK2 and CB8. And the two strains were identified as C. butyricum based on an analysis of 16S rDNA sequences, which revealed 99% and 100% homology, respectively. The 16S rDNA gene sequences of strain TK2 and CB8 have been deposited in GenBank under accession number KJ558432 and KJ558433, respectively.

Table 2. Biochemical characters of C. butyricum TK2 and C. butyricum CB8 by comparing the type strain of C. butyricum MIYAIRI 588 (Ikeda et al., 1988)
C. butyricum TK2 C. butyricum CB8 C. butyricum MIYAIRI 588
Gas from glucose + + +
nitrate reduction
Gelatin liqueed
Milk coagulation
Fermentation of:
glucose + + +
lactose + + +
starch + + +
maltose + + +
mannose + + +
rafnose + + +
ribose + + +

Explanation of the symbols:+, Test result is positive;−, Test result is negative.

Effect of different functional oligosaccharides on the growth of C. butyricum TK2 and C. butyricum CB8 The capacity for microbial degradation of the functional oligosaccharides during fermentation of C. butyricum TK2 and C. butyricum CB8 can be indicated by the number of the viable cells (Fig.1). Overall, the six functional oligosaccharides were able to be fermented by both of C. butyricum and every oligosaccharide exerted no significant difference on the propagation of the two cells from different source (p > 0.05). However, the cell concentrations of C. butyricum TK2 or C. butyricum CB8 were different depending on the different carbon source. Among the six oligosaccharides, the highest number of C. butyricum TK2 and C. butyricum CB8 were noticed in the group of fermenting with IMOS, 10.1 ± 1.05 log (CFU / mL) and 9.8 ± 0.41 log (CFU / mL), respectively. GOS resulted in higher populations of the two types of C. butyricum than raffinose or stachyose. Fig.1 showed that FOS and XOS caused a much lower concentration than the other oligosaccharides fermented by C. butyricum TK2 and C. butyricum CB8 (p < 0 .05).

Fig. 1.

Effect of different functional oligosaccharides on the growth of C. butyricum TK2 and CB8. IMOS: Isomaltooligosaccharides; GOS: Galatooligosaccharides; FOS: Fructooligosaccharides; XOS: Xylooligosaccharides. Various types of functional oligosaccharides (0.5%) were as only carbon source. All groups were incubated under anaerobic gas phase (H2:CO2:N2, 10:10:80, v/v/v) at 37°C for 48 h. Data were expressed as mean ± SD from three independent experiments. Indices above data bars represent significant differences within effects of six functional oligosaccharides on the growth of C. butyricum TK2 or C. butyricum CB8. Different letters indicate significantly different results (P <0.05).

The changes of pH of medium during fermentation Fig. 2 shows the changes of pH of medium fermented by C. butyricum TK2 and C. butyricum CB8. The pH of both medium with IMOS decreased more sharply than that with other functional oligosaccharides during the initial 24 h of fermentation, and then tended towards stability. It is founded that IMOS was preferentially utilized by C. butyricum used in the study. In addition, the pH of the medium with IMOS, GOS, stachyose and raffinose were apparently lower than that with FOS and XOS.

Fig. 2.

Changes of pH of culture media during fermentation by C. butyricum TK2 and C. butyricum CB8. (A) Changes of pH of culture media during fermentation by C. butyricum TK2. (B) Changes of pH of culture media during fermentation by C. butyricum CB8. —□—, changes of pH of media with IMOS; —▲—, changes of pH of media with GOS; —▼—, changes of pH of media with raffinose; —■—, changes of pH of media with stachyose; —△—, changes of pH of media with FOS; —◆—changes of pH of media with XOS. Various types of functional oligosaccharides (0.5%) were as only carbon source. Data were expressed as mean ± SD from three independent experiments.

Production of short chain fatty acids during the vitro fermentation The short chain fatty acids were produced by C. butyricum TK2 and C. butyricum CB8 during vitro fermentation of varied oligosaccharides, as shown in Table 3, and the total SCFA is considered as a symbol of functional oligosaccharides fermentability (Li et al., 2012). Although SCFA were composed of acetate, propionate and butyrate, propionate was not detected for the two strains. Acetate and butyrate were the major end products and butyrate was the dominant SCFA during the fermentation of six functional oligosaccharides by both strains. In contrast to other oligosaccharides, fermentation of IMOS by the two types of C. butyricum resulted in the highest concentration of SCFA and butyrate (11.77 ± 0.37 mmol/L and 14.47 ± 1.05 mmol/L). Moreover, the yield of SCFA and butyrate from fermentation of FOS and XOS were the least for C. butyricum TK2 and C. butyricum CB8.

Table 3. Production of short chain fatty acids during the vitro fermentation of C. butyricum TK2 and C. butyricum CB8.
Treatment Type of Clostridium butyricum Total SCFA(mM) Acetate(mM) Butyrate(mM)
IMOS TK2 13.99 ± 0.44c 2.22 ± 0.13d 11.77 ± 0.37b
CB8 19.23 ± 1.03d 4.77 ± 0.19d 14.47 ± 1.05d
Stachyose TK2 10.18 ± 1.83b 2.11 ± 0.59b 8.07 ± 1.24b
CB8 10.27 ± 0.42b 2.88 ± 0.18c 7.39 ± 0.30b
Rafnose TK2 12.33 ± 1.18c 2.59 ± 0.96c 9.74 ± 0.71b
CB8 10.93 ± 0.07bc 2.80 ± 0.12bc 8.13 ± 0.11bc
GOS TK2 13.48 ± 1.22c 2.16 ± 0.61d 11.32 ± 0.50b
CB8 11.45 ± 0.96c 2.94 ± 0.16c 8.51 ± 0.79c
FOS TK2 2.81 ± 0.17a 0.86 ± 0.21a 1.95 ± 0.19a
CB8 8.60 ± 0.17a 2.60 ± 0.10ab 6.00 ± 0.15a
XOS TK2 3.54 ± 0.46a 1.12 ± 0.08a 2.42 ± 0.39a
CB8 7.86 ± 0.18a 2.41 ± 0.09a 5.44 ± 0.11a

Concentration of SCFA was determined by high-resolution gas chromatography used external standards. Data were presented as the mean of triplicate measurement ± SD. IMOS, Isomaltooligosaccharides; GOS, Galatooligosaccharides; FOS, Fructooligosaccharides; XOS, Xylooligosaccharides. Indices above data bars represent significant differences within effects of six functional oligosaccharides on the levels of SCFA producted by C. butyricum TK2 or C. butyricum CB8. Different letters indicate significantly different results (P <0.05).

However, the two types of C. butyricum exerted some differences in the production of SCFA and butyrate. In terms of the production of SCFA, IMOS, FOS and XOS resulted in significant differences between C. butyricum TK2 and C. butyricum CB8 (p<0.05), while statistically distinctions of the output of butyrate between the two strains were observed from fermentation with IMOS, GOS and raffinose (p < 0.05).

Discussion

At the present study, the six functional oligosaccharides were utilized by C. butyricum TK2 and C. butyricum CB8 at different levels. However, there were some differences in the fermentation characteristics of the six oligosaccharides between the two types of C. butyricum, which may be in relation with the specificity of two strains. It has been reported that Clostridium species can degrade polysaccharide (Rockova et al., 2011; Nakajima et al., 2002). Montoya et al. (2001) have demonstrated that new solvent-producing Clostridium sp. strains, which are closely related to C. butyricum, can also hydrolyze a wide range of polysaccharides. It has been found that C. butyricum utilizes both soybean oligosaccharides and FOS and can not utilize GOS (Hayakawa et al., 1990; Sako et al., 1999). However, in our study, the fermentability of GOS to two strains was better than raffinose or stachyose. Compared with the other oligosaccharides, IMOS revealed the strongest fermentability for both strains, which was supported by the highest viable cells and the highest levels of SCFA or butyrate from fermentation with this functional oligosaccharide. Varying oligosaccharides showed different structure which may affect on the fermentation characteristics of C. butyricum TK2 and C. butyricum CB8. The monosaccharides of IMOS and GOS are glucose and galactose, respectively. In contrast to the two oligosaccharides, disaccharides and pentaose as monosaccharides units present in FOS and XOS, respectively. In addition, raffinose and stachyose are both composed with galactose, fructose and glucose. In our study, C. butyricum grew preferentially on the oligosaccharides with hexanose units. It seems likely that both of C. butyricum have a specific transport system for hexanose. In fact, there has been little research about the structure-function relationships of functional oligosaccharides recently.

Most of functional oligosaccharides are not digested by humans for lack of relevant enzymes in the human body, which reaching colon as they have been eaten and are further fermented by anaerobic bacteria (Crittenden and Playne, 1996). Such metabolic process, it produces short chain fatty acids. The amounts and types of SCFA produced depend on the type of non-digestible oligosaccharides as well as on the composition of the intestinal flora (Sako et al., 1999). The production of SCFA results in decrease of pH in the colon to inhibit the growth of certain pathogenic bacterium and stimulate the growth of the beneficial bacteria (Cherrington et al., 1991; Van Immerseel et al., 2002; Van Immerseel et al., 2004; Defoirdt et al., 2006; Woo et al., 2011). In particular butyrate promotes apoptosis and inhibits growth of cancer cells in vitro (Hague et al., 1995) and plays a protective role against colorectal cancer in vivo (Avivi-Green et al., 2001; Perrin et al., 2001). Based on these experimental observations, it is hypothesized that functional oligosaccharides as an indirect source of butyrate to the gut may be beneficial to reduce risk factors for colorectal cancer and other diseases.

Bifidobacteria and Lactobacilli, which considered as the target species for prebiotic stimulation in the colon with the aim of improving host health (Durieux et al., 2001), but do not produce butyrate. However, nowadays, there is an increasing interest in the use of prebiotics that are capable of improving the yield of butyrate or the number of butyrate-producing bacterium because of the beneficial effects of butyrate on the gut. In our study, the effect of IMOS and GOS was found on the number and the butyrate production of C. butyricum, moreover, it has been confirmed that IMOS and GOS are selectively increase Bifidobacteria and Lactobacilli in the gut (Panesar et al., 2011). Bifidobacterium spp hydrolysis polymeric α-(1,6) and α-(1,4) with extracellular enzymes (Ryan et al., 2006). In addition, α- or β-glucanase can also been secreted to degrade polysaccharides by some Clostridium strains (Montoya et al., 2001; Nakajima et al., 2002). In terms of C. butyricum, the enzymes for IMOS and GOS metabolism are not clear. It can be hypothesized C. butyricum have some kinds of extracellular or intracellular glycosyl hydrolases which metabolize oligosaccharides with hexanose units preferentially. In future, more study is needed to fully elucidate the roles of cell-associated glycosidases. Nevertheless, the present study showed that there was no significant effect of FOS and XOS on the fermentation characteristics of two types of strains.

It is a very promising direction that the development of synbiotics in the area of functional food ingredients. Synbiotic describes a combination of a prebiotic and a probiotic. The fermentation characteristics of the bacterium with various prebiotics in this study reflect the extent to which functional oligosaccharides would promote the growth and butyrate production of C. butyricum. Therefore, this study provides a rational basis for establishing synbiotics with C. butyricum to maintain intestinal health towards healthier community.

Conclusion

The present study determined which functional oligosaccharides could show the better fermentability by promoting the growth and SCFA or butyrate production of C. butyricum TK2 and C. butyricum CB8. The largest increase in the two types of C. butyricum and the highest butyrate level were seen on IMOS. While the viable cell concentration and the butyrate level from the fermentation of FOS and XOS were much lower than the other treatments. On the other hand, the different fermentation properties between the two strains were also studied. For the same functional oligosaccharide, the numbers of both bacterium and the production of SCFA were different. This study provides a rational basis for establishing synbiotics with C. butyricum to ensure the human health.

Acknowledgement The authors wish to acknowledge the financial support provided by the National Natural Science Foundation of China (No.30900961) and TianJin Social Science Planning Program (No.TJGLWT11-08).

References
 
© 2014 by Japanese Society for Food Science and Technology
feedback
Top