Structural elucidation and in vitro fermentation of extracellular α-d -glucan from Lactobacillus reuteri SK24.003

Bioactive Carbohydrates and Dietary Fibre 6 (2015) 109–116

Contents lists available at ScienceDirect

Bioactive Carbohydrates and Dietary Fibre journal homepage: www.elsevier.com/locate/bcdf

Structural elucidation and in vitro fermentation of extracellular α-D-glucan from Lactobacillus reuteri SK24.003 Ming Miao a,n, Yajun Ma a, Bo Jiang a, Steve W. Cui a,b, Shengfang Wu a, Tao Zhang a a State Key Laboratory of Food Science & Technology, Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu 214122, PR China b Guelph Food Research Centre, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ont., Canada N1G 5C9

art ic l e i nf o

a b s t r a c t

Article history: Received 1 September 2015 Received in revised form 25 September 2015 Accepted 26 September 2015

The molecular structure and its relationship to in vitro fermentation properties of α-D-glucan from Lactobacillus reuteri SK24.003 were investigated in this study. The chain structure was elucidated by 2D NMR spectroscopy and a possible repeating unit was deduced. During in vitro fecal fermentation, the relatively low initial bacterial population was observed during first 6 h incubation and small increase for α-D-glucan for 12–48 h incubation. A significant decrease in pH (approximately 2.0) was observed for fecal microbiota exposed to α-D-glucan. Acetic acid, propionic acid, n-butyric acid along with total shortchain fatty acid (SCFA) increased upon incubation, and D-glucan was initially fermented well resulting in the higher production of SCFA at 12 h. The n-butyric acid value at 48 h fermentation (1.29 mM) was higher than that of some commercial prebiotic oilosaccharides. These results suggested that α-D-glucan might be efficiently utilized by gut bacteria and may have a positive effect for gastrointestinal health. & 2015 Elsevier Ltd. All rights reserved.

Keywords: α-d-glucan Lactobacillus reuteri SK24.003 Molecular fine structure In vitro fecal fermentation Short-chain fatty acid

1. Introduction The gut microbiota has become a hot research topic due to its importance to human health and well-being (Rastall & Gibson, 2015). It is known that there are 1013–14 bacteria inside the adult colon, consisting of 41000 different species, which not only digest the materials coming from the small intestine for growth and proliferation and to generate short-chain fatty acids (SCFA) that are important for the health of host, but also contribute to postnatal gut development and the maturity of the host innate immune system (Hooper, 2004; Kaur, Rose, Rumpagaporn, Patterson & Hamaker, 2011; Zhang & Hamaker, 2010). To date, functional carbohydrates have been developed to modulate the large intestine environment for the maintenance of the colon ecosystem. Among them, dietary prebiotics has been found to be effective in specific changes in the composition and activity of the gastrointestinal microbiota, such as increasing the number of bifidobacteria and lactobacilli (Gibson et al., 2010). Fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), inulin and lactulose have been reported to possess prebiotics activity, due to their efficacy, in vivo studies and history of safe commercial applications (Cui, 2005; Gibson et al., 2010; Hamaker & Tuncil, 2014). In recent years, some novel carbohydrates have shown n

Corresponding author. Fax: þ 86 510 859 19161. E-mail address: [email protected] (M. Miao).

http://dx.doi.org/10.1016/j.bcdf.2015.09.007 2212-6198/& 2015 Elsevier Ltd. All rights reserved.

emerging prebiotics potential, including isomalto/malto-oligosaccharide, α-glucooligosaccharide and dextran (Leemhuis et al., 2014; Sarbini et al., 2011; Sarbini, Kolida, Gibson &Rastall, 2013). These gluco-oligosaccharides were selectively fermented by bifidobacteria, lactobacilli and bacteroids, but poorly metabolized by potentially detrimental strains such as enterobacteria and clostridia (Djouzi et al., 1995). Chung and Day (2002) also reported that branched-chain gluco-oligosaccharides from L. mesenteroides B-742 were readily utilized by bifidobacteria and lactobacilli but not by Salmonella spp. or Escherichia coli . Furthermore, Grimoud and others demonstrated that α-1,6 gluco-oligosaccharide or oligodextran were metabolised by several lactic acid bacteria and promoted bacterial growth more efficiently than alternate α-1,3/α1,6 gluco-oligosaccharide (Grimoud et al., 2010). According to Gibson (2004), even though those glucans exhibit promising fermentation characteristics, which is un-sufficient to categorize them as prebiotics. Thus, further investigations focusing on the structure–bioactivity relationship of novel polymers will add to the current knowledge of their functionality and potential application. In our previous studies, a probiotic strain Lactobacillus reuteri SK24.003 was isolated from a traditional Chinese fermented dairy product and produced a neutral extracellular α-D-glucan with weight-average molecular weight of 4.31
107 g/mol (Miao et al., 2014). This bioengineered glucan had an α-1,4 backbone with an α-1,6 branch at every fourth residue, and its solution exhibited an opalescent, milky-white color and non-Newtonian pseudoplastic

110

M. Miao et al. / Bioactive Carbohydrates and Dietary Fibre 6 (2015) 109–116

behavior (Miao et al., 2015). However, little is known about prebiotics properties of this polymer. In this study, we investigated the molecular structure of α-D-glucan from L. reuteri using 2D NMR spectroscopy, and studied in vitro fermentation of α-D-glucan was carried out with human fecal cultures. Changes in bacterial population, pH and SCFA were also evaluated. Moreover, dextran, galacto-oligosaccharide, fructose–oligosaccharide and inulin were selected as the reference substrates for the in vitro fermentation experiments, because these are one of the most frequently described prebiotic oligosaccharides and much is already known about their in vitro fermentation behavior.

2. Materials and methods 2.1. Materials L. reuteri SK24.003 was screeded in our lab and deposited in the China Center for Type Culture Collection (CCTCC) under the accession number M 2011397. Dextran (Cat. no. D5501), acetic acid (Cat. no. 71251), propionic acid (Cat. no. 94425), isobutyric acid (Cat. no. 58370), n-butyric acid (Cat. no. 19215), isovaleric acid (Cat. no. 129542) and n-valeric acid (Cat. no. 75054) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Galacto-oligosaccharide (GOS57) and fructose–oligosaccharide (FOS95) were kindly provided by Baolingbao Biology Co. (Yucheng, Shandong, China). Long chain inulin (ORFTI™ HP) was purchased from BENEO-Orafti (Tienen, Belgium). All other chemicals were reagent grade and obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2.2. Production of water soluble

α-D-glucan from L. reuteri

The strain L. reuteri SK24.003 was inoculated into the optimised MRS medium containing 100 g sucrose, 10 g yeast extract, 1 ml Tween 80, 20 g K2HPO4, 0.02 g CaCl2, 0.2 g MgSO4  7H2O, 0.01 g NaCl, 0.01 g MnSO4  H2O, 0.01 g FeSO4  7H2O per liter, pH 6.8–7.0 and grown at 37 °C for 48 h in an anaerobic incubator. The culture was heated in a boiling water bath for 30 min, and then centrifuged at 10,000g and 4 °C for 20 min to separate the cells. The crude polysaccharide was precipitated in three volumes of 95% (v/v) ethanol at room temperature. The alcohol solution was stored overnight at 4 °C and the sediments were collected through centrifugation. The resulting sample was then dissolved in deionised water, dialyzed at 4 °C to remove small molecules. The process of precipitation and re-dissolution was repeated twice. The solution was freeze-dried and obtained water soluble polysaccharide was further purified using DEAE-Sepharose Fast Flow and Sepharose CL-2B columns as described in a previous study (Miao et al., 2014). 2.3. NMR analysis The dried α-D-glucan (30 mg) was dissolved in 99.99% deuterium oxide (D2O) and lyophilization. The process was repeated three times to completely replace H with D and the final sample was dissolved in 0.5 ml of D2O for 6 h before NMR analysis. The spectra of 1H, 13C, 1H–1H correlated spectroscopy (COSY), 1H–1H total correlated spectroscopy (TOCSY), and 1H–13C heteronuclear multiple-bond correlation (HMBC) were conducted at 70 °C on an AVANCE III NMR spectrometer (Brucker Co., Billerica, MA, USA). Chemical shifts were referenced to acetone (δH 2.225) and 1, 4-dioxan (δC 66.50), respectively.

2.4. In-vitro fecal fermentation 2.4.1. Fecal slurry preparation The fresh fecal samples was obtained from four healthy male volunteers, aged 23–29, who had not received antibiotic treatment for 6 months before participation in the study and had no history of gastrointestinal diseases. The collected samples were mixed, kept in an anaerobic jar before use. The fecal samples was diluted in phosphate buffer solution (1.0 M, pH 7.0) in the ratio of 1:5 (w/ v), and homogenized for 5 min to obtain the fecal slurry. 2.4.2. In vitro fermentation The growth medium, comprised of 2.0 g of peptone water, 2.0 g of yeast extract, 2.0 g of NaHCO3, 0.1 g of NaCl, 0.5 g of L-cysteine HCl, 0.04 g of KH2PO4, 0.04 g of K2HPO4, 0.01 g of MgSO4  7H2O, 0.01 g of CaCl2, 2.0 g NaHCO3, 0.5 g of bile salt, 2.0 ml of Tween 80, and 4.0 ml of resazurin solution (0.025%, w/v) per liter (pH 7.0) was used for static batch culture fermentation. The test carbohydrate (1%, w/w) was added into the autoclaved medium just before the addition of fecal slurry (10%, w/w). The incubation was carried out inside an anaerobic cabinet at 37 °C. After 0, 6, 12 or 48 h of fermentation, the liquid sample was removed from each vessel for metabolite analysis.

2.5. Metabolite analysis 2.5.1. Bacterial population The optical density (OD) value of fermented products was measured at 600 nm on a UV/visible Spectrophotometer (UV2102PC, Unico Instrument Co., Ltd., Shanghai, China). 2.5.2. Change of pH value The pH value was measured using a standard pH meter (Mettler-Toledo International Inc., Shanghai, China). 2.5.3. Short-chain fatty acid production The fermentation culture was centrifuged at 10,000 rpm for 10 min, and 1.0 μl of the supernatant was injected onto a gas chromatograph (GC-2010 Plus, Shimadzu Co., Ltd., Kyoto, Japan) system equipped with a flame ionization detector and a column (DB-WAX, 30 m
0.32 mm i.d.; film thickness 0.5 μm, Agilent Technologies Inc., USA). The temperatures of injection port and detector were set at 250 °C. The column temperature was programmed as follows: the initial temperature was set at 50 °C, held for 3 min, and ramped at 6 °C/min to 120 °C, where it was held for 1 min and again ramped at 10 °C/min to 220 °C and held for 5 min. Nitrogen was used as the carrier gas at 3 ml/min with a split ratio of 1:3. The flow rates of hydrogen and air were 47 and 400 ml/min, respectively. The running time for each sample was 35 min. Sample quantification was carried out using the standard SCFA sample (acetic, propionic or butyric acid) at concentration between 0.5 and 100 mM.

2.6. Statistical analysis All numerical results are the average of at least three independent replicates. The mean differences were determined by Tukey's HSD test (p o0.05) using analysis of variance (ANOVA) by Sigma Stats version 2.0 software (Jandel Scientific/SPSS Inc., Chicago, IL, USA).

M. Miao et al. / Bioactive Carbohydrates and Dietary Fibre 6 (2015) 109–116

3. Results and discussion 3.1. Structure analysis of

α-D-glucan

In the molecular structure analysis of polysaccharides, the 1H and 13C NMR spectra have become the most powerful and noninvasive physicochemical technique (Cui, 2005; Leeuwen, Leeflang, Gerwig &Kamerling 2008). It can provide detailed structural information of polysaccharide, including identification of monosaccharide composition, elucidation of α- or β-anomeric configurations, establishment of linkage patterns, and sequences of the sugar units. Recently, van Leeuwen and coworker also developed a 1 H structural-reporter-group concept for the primary structural analysis of α-D-glucan, resulting in a detailed composite model (van Leeuwen et al., 2008). Thus, the extracellular α-D-glucan from L. reuteri SK24.003 was purified using the chromatography method and its structure was further analyzed by NMR spectroscopy (Fig. 1). In our recent study (Miao et al., 2014), this polymer produced from sucrose was composed exclusively of glucose. The inter-glycosidic linkages determined by methylation analysis were composed of α-1,4-linked D-glucopyranose (A), α-1,6-linked Dglucopyranose (B), α-1,4,6-linked D-glucopyranose (C) and nonreducing terminal residue (D) with the relative contents of 63.4%,15.3%, 12.6% and 8.7%, respectively. The date suggested that

111

α-D-glucan from L. reuteri SK24.003 was a highly branched soluble glucan that possessed predominantly α-1,4 bonds, with fewer α1,6 linkages and α-1,4,6 branching points. The chemical shifts of individual glucosyl residues (A, B, C, D) assigned based on a series of NMR experiments are listed in Table 1. The chemical shifts at 5.37 ppm was assigned to the anomeric proton of the α-1,4-linked D-glucopyranose (A). The signal at 4.96 ppm was attributed to the signals of anomeric protons of α-1,6 D-glucopyranose (B). Moreover, the broad peak of α-1,4 linkage was split into two overlapping peaks, which were assigned to the α-1,4,6-linked D-glucopyranose (C) resudes and non-reducing terminal residue (D), respectively. In the 13C NMR spectrum (Fig. 1b), the chemical shift at around 100.12 ppm were identified as the C1 of α-1,4-linked Dglucopyranose, α-1,6-linked D-glucopyranose and α-1,4,6-linked Dglucopyranose, respectively. The signals at 98.45 ppm were linked to the C1 of non-reducing terminal residue, whereas the signal at 77.78 ppm corresponded to the C4 of α-1,4-linked D-glucopyranose and α-1,4,6-linked D-glucopyranose, and those at 61.10 ppm were attributed to the C-6 of non-reducing terminal residues and α-1,4-linked D-glucopyranose. Some major signals in the nonanomeric region (65–75 ppm) were attributed to the C2, C3, C4 and C5 substituted glucose residues. These results agreed with some previous literatures (Nie et al., 2011; van Leeuwen et al., 2008).

Fig. 1. 1D and 2D 1H, 13C NMR spectra of α-D-glucan from L. reuteri SK24.003. (a) 1D 1H NMR spectrum, (b) 1D 13C NMR spectrum, (c) 2D 1H–1H COSY spectrum, (d) 2D 1H–1H TOCSY spectrum, (e) 2D 1H–13C HMBC spectrum.

112

M. Miao et al. / Bioactive Carbohydrates and Dietary Fibre 6 (2015) 109–116

1.5

Table 1 NMR signals assignment of α-D-glucan from L. reuteri SK24.003. Glucosyl residue

Assigned position

1

13

A: -4)-Glcp-(1-

1 2 3 4 5 6

5.37 3.61 3.93 3.65 3.83 3.82, 3.94

100.12 71.79 72.08 77.78 71.97 61.10

1 2 3 4 5 6

5.33 3.57 3.70 3.42 4.01 3.82, 3.95

100.10 72.07 73.52 73.46 72.2 67.13

B: -6)-Glcp-(1-

C (ppm)

1.2

0.9

OD

H (ppm)

blank inulin GOS57 FOS95 dextran α-D-glucan

0.6

0.3

0.0 0

10

20

30

40

50

Time (h) C:-4,6)-Glcp-(1-

D: Glcp-(1-

1 2 3 4 5 6

5.36 3.60 3.82 3.65 3.97 3.85, 3.95

100.39 72.12 73.50 77.89 71.26 67.19

1 2 3 4 5 6

4.96 3.57 3.70 3.41 3.72 3.75, 3.85

98.45 72.19 73.24 70.07 72.39 61.10

The COSY spectrum enables us to assign the chemical shifts of the anomeric protons from the α-1,4-linked D-glucopyranose (A), α-1,6-linked D-glucopyranose (B), α-1,4,6-linked D-glucopyranose (C) and non-reducing terminal residue (D) coupling with their respective H-2 s, as shown in Fig. 1(c). Also, the TOCSY spectrum provided all intra-residue connectivity of all protons of four individual residues (Fig. 1(d)). As described in Table 1, the C1 signals at 100.12, 100.10, 100.39 and 98.45 ppm were assigned to α-1,4linked D-glucopyranose, α-1,6-linked D-glucopyranose, α-1,4,6linked D-glucopyranose and non-terminal residue, respectively. These C1 signals cross-linked to the H signals at chemical shifts 5.37, 5.33, 5.36 and 4.96 ppm, respectively. The HMBC spectrum shows long-range connectivity between carbon atoms and their coupled protons through two or three bonds, which allows us to further analyse the chain sequence (Cui, 2005). The H1 signal of 1,4-linked D-glucopyranose correlated with the C4 of 1,4-linked Dglucopyranose and 1,4,6-linked D-glucopyranose. Besides, the C1 of 1,4-linked D-glucopyranose correlates with the H4 of 1,4-linked Dglucopyranose and 1,4,6-linked D-glucopyranose (Fig. 1(e)). Combined the results from 1D and 2D NMR spectroscopy, the schematic proposed structure of α-D-glucan from L. reuteri SK24.003 is shown in Fig. 2. 3.2. Change of bacterial population in fermentation culture The extracellular α-D-glucan from L. reuteri SK24.003 was used as carbon source in anaerobic fermentations. The bacterial population after 0, 6, 12 and 48 h of incubation of test carbohydrate using small-scale batch cultures is shown in Fig. 3. The largest

D B

A

C

Fig. 2. Schematic structure of α-D-glucan from L. reuteri SK24.003.

Fig. 3. Change of OD600 of fermented cultures added with α-D-glucan at different time during in vitro fecal fermentation.

changes in bacterial population took place during the first 24 h of incubation. Compared to the blank, a strong increase in OD600 was observed, which can be explained by the fermentable carbon source of test carbohydrate. It is known that the human gut microbiota possesses an enormous diversity of carbohydrate-active enzymes for degradation of the complex dietary polysaccharides (Kaoutari, Armougom,Gordon, Raoult &Henrissat, 2013). In a recent review of Hamaker and Tuncil (2014), variation in chemical structure of dietary fiber affects its utilization by gut microbiota, and the glycosidic linkage in discrete structure of complex carbohydrate was cleaved into simple sugar for selective metabolism. As shown in Fig. 3, the faster increase in OD600 with FOS95, inulin and GOS57 in the first 6 h of fermentation, confirmed the prebiotics property of these carbohydrates reported in earlier studies (Rycroft, Jones, Gibson & Rastall, 2001). They found these prebiotics increased the numbers of bifidobacteria, but decreased clostridia. For α-D-glucan and dextran, the relatively low initial OD600 was observed during 6 h fermentation; and OD600 for α-Dglucan increased at 12–48 h fermentation. This was similar to the studies of Sarbini et al. (2011) who showed the role of chain structure in fermentation selectivity. In our previous study, α-Dglucan from L. reuteri SK24.003 exhibited an α-(1-4) backbone with an α-(1-6) branch at every fourth residue (Miao et al., 2014), compared to dextran with only α-1,6 glycosidic linkages. According to Sanz, Gibson and Rastall (2005), the prebiotic index of α-1,6 linkage was higher than that of α-1,4 linkage, resulting in higher bacterial populations as observed in Fig. 3. 3.3. Change of pH of fermentation culture Fig. 4 shows the pH value of the fermentation cultures at the beginning and end of the incubation period. The pH ranged approximately from 6.7 to 6.8 before fermentation. At 48 h fermentation of test carbohydrate, a drop in the pH was observed compared to the blank (no substrate), which could be related to greater bacterial population (Fig. 3) and higher SCFA concentration (Table 2) in batch fecal fermentation. In microbiota fed with inulin, GOS57 and FOS95, a significantly sharper drop in pH during the first 6 h of fermentation was experienced, compared to the bacteria receiving α-D-glucan or dextran. Also, a significant decrease in pH was observed for bacteria exposed to α-D-glucan between 6 and 12 h of fermentation. After 12 h of fermentation, the pH change of fermentation of all the substrates was negligible, except for dextran with continuing drop in the pH. Similar observations of pH decrease in the polysaccharides fermentation were shown in

M. Miao et al. / Bioactive Carbohydrates and Dietary Fibre 6 (2015) 109–116

3.4. Chang of SCFA concentration in fermentation culture

7.0 6.5 blank inulin GOS57 FOS95 dextran α-D-glucan

6.0

pH

113

5.5 5.0 4.5 4.0 0

10

20

30

40

50

Time (h) Fig. 4. Change of pH value in fermented cultures added with α-D-glucan at different time during in vitro fecal fermentation.

several literatures (Campbell, Fahey &Wolf, 1997; Rose et al., 2009; Hu, Nie, Li & Xie, 2013). Rose et al. (2009) reported that pH was lowered from 6.2 to nearly 4.8 during fermentation of starch entrapped alginate microspheres. Starch-entrapped microspheres ferment more slowly than the waxy corn starch control. Hu et al. (2013) also showed that a pH drop (6.1–5.1) for the polysaccharide from Plantago asiatica L. by intestinal bacteria with significantly increased SCFA levels, due to the fermentation of glucuronic acid, arabinose and xylose. Moreover, a decrease in rat colon pH was observed after intake of oligosaccharides and polysaccharide (Cambell et al., 1997; Hu, Nie, Min & Xie, 2012). A lowering pH could be used as a marker for reducing colonic cancer incidence, inhibiting the proliferation of pathogens, and improving colon health (Zhang & Hamaker, 2010). Therefore, the reduction of approximately 2.0 for pH value in this study might be beneficial for gastrointestinal health.

According to two previous reviews (Topping & Clifton, 2001; Zhang & Hamaker, 2010), SCFA are colonic fermentation products of non-starch cereal carbohydrates and beneficial for the colon health, particular important for human physiology. In this study, SCFA was determined by GC method and the typical profiles of SCFA in standard solution and fermentation sample are given in Fig. 5(a and b). The DB-WAX column successfully separated the SCFA (acetic acid, propionic acid, isobutyric acid, n-butyric acid, isovaleric acid and n-valeric acid) within 20 min. Total SCFA concentration at different time during in vitro fecal fermentation of αD-glucan from L. reuteri SK24.003 is present in Fig. 5(c). It could be seen that total SCFA increased along with the increase of fermentation time. Inulin, GOS57, FOS95 and α-D-glucan were initially fermented well by the human fecal microbiota resulting in the higher production of SCFA at 12 h. Fermentation of dextran resulted in the lower SCFA production during this initial fermentation period. This is in accordance with the OD600 and pH data (Figs. 3 and 4), indicates a slower initial fermentation. For α-Dglucan, total SCFA reached the maximum level of 60.0 mM after 48 h fermentation, which was approximately 3.0–3.5 fold lower than of inulin, GOS57 or FOS95 at the same time. The concentration acetic acid, propionic acid, isobutyric acid, n-butyric acid, isovaleric acid and n-valeric acid are shown in Table 2. Among the SCFA, acetic acid was the most abundant SCFA, followed by propionic acid and considerably lower concentration of n-butyric acid. Acetic acid is oxidized by brain, heart and peripheral tissues; propionic acid attenuates hepatic cholesterol synthesis and improves liver metabolism, and butyric acid serves as an energy source for intestinal mucosa and provides protection against colorectal cancer. Those beneficial physiological effects have been reported by a number of groups (Topping & Clifton, 2001; Wolever, 1995; Ximenes, Hirata, Rocha, Curi & Carpinelli, 2007; Yu, Waby, Chirakkal, Staton & Corfe, 2010). The SCFA production might be related with some structural characterizations of

Table 2 SCFA concentrations in batch fermentation cultures at 0, 6, 12, 48 h using α-D-glucan from L. reuteri SK24.003. Sample

Time (h)

Acetic acid (mM)

Propionic acid (mM)

Isobutyric acid (mM)

α-D-glucan

0 6 12 48

9.99 7 0.21a 34.747 0.65b 32.577 0.09a 31.96 7 0.50a

1.26 7 0.34a 17.747 0.75a 26.98 7 0.08a 25.93 7 0.45a

0.04 70.00a 0.0770.01a 0.0770.03a 0.05 70.01a

Dextran

0 6 12 48

2.137 0.19a 3.34 7 0.23a 8.23 7 1.45b 17.477 1.36b

0.23 7 0.01a 1.107 0.13b 4.20 7 0.00b 8.767 0.04b

FOS 95

0 6 12 48

10.727 1.22a 45.767 2.05b 48.507 1.27b 59.517 0.89b

GOS 55

0 6 12 48

Inulin

0 6 12 48

Isovaleric acid (mM)

n-Valeric acid (mM)

0.46 70.07a 0.81 70.10a 0.97 70.09b 1.29 70.05b

0.09 7 0.02a 0.157 0.02a 0.107 0.04a 0.167 0.00a

1.36 7 0.45a 1.23 7 0.33a 0.94 7 0.15a 0.65 7 0.07b

0.21 70.02a 0.04 70.02a 0.21 70.05a 0.09 70.01a

0.2470.08a 0.21 70.00a 0.28 70.01a 0.64 70.01b

0.34 7 0.05a 0.067 0.00b 0.277 0.05a 0.107 0.03a

0.29 7 0.11a 0.067 0.00a 0.247 0.05a 0.08 7 0.02a

1.767 0.51a 95.23 7 0.32ab 146.56 7 3.82a 146.517 1.95a

0.0770.04a 0.14 70.03a 0.18 70.00a 0.19 70.05a

0.66 70.05a 0.68 70.00a 0.72 70.12a 0.95 70.10b

0.05 7 0.01a 0.137 0.06b 0.127 0.03b 0.05 7 0.00a

0.26 7 0.08a 0.447 0.04a 0.247 0.04a 0.25 7 0.00a

34.34 7 0.68a 54.377 1.02a 78.08 7 1.17a 78.777 2.58a

2.577 0.32a 58.56 7 0.93b 111.22 7 2.37b 107.60 7 2.05a

0.69 70.12a 0.36 70.10b 0.12 70.00a 0.18 70.05a

1.05 70.31a 1.08 70.09a 1.09 70.15a 0.94 70.04a

1.05 7 0.09a 0.34 7 0.11b 0.28 7 0.08b 0.20 7 0.02b

1.05 7 0.32a 2.277 0.25a 2.29 7 0.47a 2.187 0.21a

31.357 0.33a 37.26 7 0.42ab 83.84 7 3.05a 103.87 7 2.12ab

2.95 7 0.00a 10.277 0.56a 74.747 1.44a 72.687 1.39a

0.49 70.13a 1.3370.21a 0.28 70.02a 0.25 70.02a

1.15 70.00a 2.73 70.29b 5.54 70.45ab 10.6770.38b

1.007 0.35a 1.917 0.42b 0.42 7 0.13a 0.447 0.10a

1.357 0.06a 1.86 7 0.12a 1.127 0.09a 0.69 7 0.14a

Mean 7 standard deviations of duplicate analysis. Significant difference in each column are expressed as different superscript letters (p o 0.05).

n-Butyric acid (mM)

114

M. Miao et al. / Bioactive Carbohydrates and Dietary Fibre 6 (2015) 109–116

Total SCFA concentration (mM)

250

200

150 inulin GOS57 FOS95 dextran α -D-glucan

100

50

0 0

10

20

30

40

50

Time (h) Fig. 5. Gas chromatograms of SCFA in standard solution (a), α-D-glucan fermentation culture 48 h, and change in total SCFA concentrations at different time during in vitro fecal fermentation (c).

M. Miao et al. / Bioactive Carbohydrates and Dietary Fibre 6 (2015) 109–116

polymer, such as monomer, glycosidic linkage, molecular weight, branch chain, surface area, particle size, solubility and viscosity (Guillon & Champ, 2000; Sanz et al., 2005; Zhang & Hamaker, 2010; Hu et al., 2012). As shown in Table 2, acetic acid and propionic acid concentrations increased upon α-D-glucan incubation with fecal microbiota, with a rapidly increasing rate in the first 6 h of fermentation. Also, n-butyric acid concentration increased with incubation time (Table 2). Moreover, the n-butyric acid value at 48 h fermentation (1.29 mM) was higher than that of dextran, FOS95 or GOS57, probably indicating a more efficient utilization of the former substrate by the human gut microbiota, which may be essential for the maintenance of the colon ecosystem. Moreover, in vitro fermentation of inulin showed highest acetic acid and n-butyric acid production at 48 h. This was familiar to the in vitro evaluation of fermentation properties of prebiotic oilosaccharides where a highest butyric acid production from inulin was monitored over 24 h (Rycroft et al., 2001). Sanz et al. (2005) determined the influence of different glycosidic linkages and monosaccharide compositions of disaccharides on the selectivity of microbial fermentation. They found glucosyl-glucose disaccharides with linkages of 1–2, 1–6, and 1–4 generated a high prebiotic index score. Sarbini et al. (2011) reported that α-1,2 branching increased the dietary fiber content, which induced selective effect on the gut microbiota and stimulated short-chain fatty acids. Also, low molecular mass dextan was the best substrate for the growth of beneficial bacteria. Therefore, it is suspected that the significant amount of butyrate following the fermentation of α-D-glucan was due to the complex structure, such as long chain length and highly branch. Hopefully, structure–function study will be addressed in our future study. These data certainly would help us to understand the specific structure modulating the gut microflora in depth.

4. Conclusion In this study, the linkage pattern of extracellular α-D-glucan from L. reuteri SK24.003 was measured by 2D NMR analysis and the molecular fine structure was then proposed. During in vitro fecal fermentation, this glucan could be well-utilized by human microbiota, and bacterial population and SCFA concentration increased along with the incubation time. It was found that production of n-butyric acid was higher than that of some commercial prebiotic oilosaccharides. These results showed that α-D-glucan from L. reuteri SK24.003 was demonstrated to be a potential prebiotics that was physiologically active for the gut microbiota homeostasis and host health. Additional research is undergoing to investigate the metabolism and prebiotic functionality. The experimental results gained would help design and development of novel functional foods.

Acknowledgements This study is financially supported by the National Natural Science Foundation of China (31000764, 31230057), the International Cooperative Program of Jiangsu Province (BZ2012031) and the Science & Technology Pillar Program of Jiangsu Province (BE2012613, BE2013647, BE2014703).

References Campbell, J. M., Fahey, G. C., & Wolf, B. W. (1997). Selected indigestible oligosaccharides affect large bowel mass, cecal and fecal short-chain fatty acids, pH and microflora in rats. Journal of Nutrition, 127, 130–136.

115

Chung, C. H., & Day, D. F. (2002). Glucooligosaccharides from Leuconostoc mesenteroides B-742 (ATCC 13146): a potential prebiotic. Journal of Industrial Microbiology and Biotechnology, 29, 196–199. Cui, S. W. (2005). Food carbohydrates: chemistry, physical properties, and applications. Boca Raton: CRC Press. Djouzi, Z., Andrieux, C., Pelenc, V., Somarriba, S., Popot, F., Paul, F., … Szylit, O. (1995). Degradation and fermentation of α-gluco-oligosaccharides by bacterial strains from human colon: in vitro and in vivo studies in gnotobiotic rats. Journal of Applied Bacteriology, 79, 117–127. Gibson, G. R. (2004). Fibre and effects on probiotics (the prebiotic concept). Clinical Nutrition Supplements, 1, 25–31. Gibson, G. R., Scott, K. P., Rastall, R. A., Tuohy, K. M., Hotchkiss, A., Dubert-Ferrandon, A., … Buddington, R. (2010). Dietary prebiotics: current status and new definition. Food Science and Technology Bulletin: Functional Foods, 7, 1–19. Grimoud, J., Durand, H., Courtin, C., Monsan, P., Ouarné, F., Theodorou, V., & Roques, C. (2010). In vitro screening of probiotic lactic acid bacteria and prebiotic glucooligosaccharides to select effective synbiotics. Anaerobe, 16, 493–500. Guillon, F., & Champ, M. (2000). Structural and physical properties of dietary fibres, and consequences of processing on human physiology. Food Research International, 33, 233–245. Hamaker, B. R., & Tuncil, Y. E. (2014). A perspective on the complexity of dietary fiber structures and their potential effect on the gut microbiota. Journal of Molecular Biology, 426, 3838–3850. Hooper, L. V. (2004). Bacterial contributions to mammalian gut development. Trends in Microbiology, 12, 129–134. Hu, J.-L., Nie, S.-P., Li, C., & Xie, M.-Y. (2013). In vitro fermentation of polysaccharide from the seeds of Plantago asiatica L. by human fecal microbiota. Food Hydrocolloids, 33, 384–392. Hu, J.-L., Nie, S.-P., Min, F.-F., & Xie, M.-Y. (2012). Polysaccharide from seeds of Plantago asiatica L. increases short-chain fatty acid production and fecal moisture along with lowering pH in mouse colon. Journal of Agricultural and Food Chemistry, 60, 11525–11532. El Kaoutari, A., Armougom, F., Gordon, J. I., Raoult, D., & Henrissat, B. (2013). The abundance and variety of carbohydrate-active enzymes in the human gut microbiota. Nature Reviews Microbiology, 11, 497–504. Kaur, A., Rose, D. J., Rumpagaporn, P., Patterson, J. A., & Hamaker, B. R. (2011). In vitro batch fecal fermentation comparison of gas and short-chain fatty acid production using “slowly fermentable” dietary fibers. Journal of Food Science, 76, 137–142. Leemhuis, H., Dobruchowska, J. M., Ebbelaar, M., Faber, F., Buwalda, P. L., van der Maarel, M. J. E. C., … Dijkhuizen, L. (2014). Isomalto/malto-polysaccharide, a novel soluble dietary fiber made via enzymatic conversion of starch. Journal of Agricultural and Food Chemistry, 62, 12034–12044. van Leeuwen, S. S., Kralj, S., van Geel-Schutten, I. H., Gerwig, G. J., Dijkhuizen, L., & Kamerling, J. P. (2008). Structural analysis of the α-D-glucan (EPS35-5) produced by the Lactobacillus reuteri strain 35-5 glucansucrase GTFA enzyme. Carbohydrate Research, 343, 1251–1265. van Leeuwen, S. S., Leeflang, B. R., Gerwig, G. J., & Kamerling, J. P. (2008). Development of a 1H NMR structural-reporter-group concept for the primary structural characterization of α-D-glucans. Carbohydrate Research, 343, 1114–1119. Miao, M., Ma, Y., Huang, C., Jiang, B., Cui, S. W., & Zhang, T. (2015). Physicochemical properties of a water soluble extracellular homopolysaccharide from Lactobacillus reuteri SK24.003. Carbohydrate Polymers, 131, 377–383. Miao, M., Ma, Y., Jiang, B., Huang, C., Li, X., Cui, S. W., & Zhang, T. (2014). Structural investigation of a neutral extracellular glucan from Lactobacillus reuteri SK 24.003. Carbohydrate Polymers, 106, 384–392. Nie, S.-P., Cui, S. W., Phillips, A. O., Xie, M.-Y., Phillips, G. O., Al-Assaf, S., & Zhang, X.L. (2011). Elucidation of the structure of a bioactive hydrophilic polysaccharide from Cordyceps sinensis by methylation analysis and NMR spectroscopy. Carbohydrate Polymers, 84, 894–899. Rastall, R. A., & Gibson, G. R. (2015). Recent developments in prebiotics to selectively impact beneficial microbes and promote intestinal health. Current Opinion in Biotechnology, 32, 42–46. Rose, D. J., Keshavarzian, A., Patterson, J. A., Venkatachalam, M., Gillevet, P., & Hamaker, B. R. (2009). Starch-entrapped microspheres extend in vitro fecal fermentation, increase butyrate production, and influence microbiota pattern. Molecular Nutrition and Food Research, 53, S121–S130. Rycroft, C. E., Jones, M. R., Gibson, G. R., & Rastall, R. A. (2001). A comparative in vitro evaluation of the fermentation properties of prebiotic oligosaccharides. Journal of Applied Microbiology, 91, 878–887. Sanz, M. L., Gibson, G. R., & Rastall, R. A. (2005). Influence of disaccharide structure on prebiotic selectivity in vitro. Journal of Agricultural and Food Chemistry, 53, 5192–5199. Sarbini, S. R., Kolida, S., Gibson, G. R., & Rastall, R. A. (2013). In vitro fermentation of commercial α-gluco-oligosaccharide by faecal microbiota from lean and obese human subjects. British Journal of Nutrition, 109(11), 1980–1989. Sarbini, S. R., Kolida, S., Naeye, T., Einerhand, A., Brison, Y., Remaud-Simeon, M., … Rastall, R. A. (2011). In vitro fermentation of linear and α-1,2-branched dextrans by the human fecal microbiota. Applied and Environmental Microbiology, 77, 5307–5315. Topping, D. L., & Clifton, P. M. (2001). Short-chain fatty acids and human colonic function: roles of resistant starch and nonstarch polysaccharides. Physiological Reviews, 81(3), 10311–10364. Wolever, T. (1995). Physiological and clinical aspects of short-chain fatty acids. New York: Cambridge University Press.

116

M. Miao et al. / Bioactive Carbohydrates and Dietary Fibre 6 (2015) 109–116

Ximenes, H. M., Hirata, A. E., Rocha, M. S., Curi, R., & Carpinelli, A. R. (2007). Propionate inhibits glucoseinduced insulin secretion in isolated rat pancreatic islets. Cell Biochemistry and Function, 25, 173–178. Yu, D. C., Waby, J. S., Chirakkal, H., Staton, C. A., & Corfe, B. M. (2010). Butyrate

suppresses expression of neuropilin I in colorectal cell lines through inhibition of Sp1 transactivation. Molecular Cancer, 9, 276. Zhang, G., & Hamaker, B. R. (2010). Cereal carbohydrates and colon health. Cereal Chemistry, 87, 331–341.