International Journal of Food Sciences and Nutrition
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Variations in prebiotic oligosaccharide fermentation by intestinal lactic acid bacteria Akihito Endo, Saki Nakamura, Kenta Konishi, Junichi Nakagawa & Takumi Tochio To cite this article: Akihito Endo, Saki Nakamura, Kenta Konishi, Junichi Nakagawa & Takumi Tochio (2016): Variations in prebiotic oligosaccharide fermentation by intestinal lactic acid bacteria, International Journal of Food Sciences and Nutrition, DOI: 10.3109/09637486.2016.1147019 To link to this article: http://dx.doi.org/10.3109/09637486.2016.1147019
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Date: 23 February 2016, At: 17:38
INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION, 2016 http://dx.doi.org/10.3109/09637486.2016.1147019
RESEARCH ARTICLE
Variations in prebiotic oligosaccharide fermentation by intestinal lactic acid bacteria Akihito Endoa, Saki Nakamurab, Kenta Konishib, Junichi Nakagawaa and Takumi Tochiob
International Journal of Food Sciences and Nutrition
a Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, Abashiri, Japan; b Food Science Co, Ltd, Aichi, Japan
ABSTRACT
ARTICLE HISTORY
Prebiotic oligosaccharides confer health benefits on the host by modulating the gut microbiota. Intestinal lactic acid bacteria (LAB) are potential targets of prebiotics; however, the metabolism of oligosaccharides by LAB has not been fully characterized. Here, we studied the metabolism of eight oligosaccharides by 19 strains of intestinal LAB. Among the eight oligosaccharides used, 1-kestose, lactosucrose and galactooligosaccharides (GOSs) led to the greatest increases in the numbers of the strains tested. However, mono- and disaccharides accounted for more than half of the GOSs used, and several strains only metabolized the mono- and di-saccharides in GOSs. End product profiles indicated that the amounts of lactate produced were generally consistent with the bacterial growth recorded. Oligosaccharide profiling revealed the interesting metabolic manner in Lactobacillus paracasei strains, which metabolized all oligosaccharides, but left sucrose when cultured with fructooligosaccharides. The present study clearly indicated that the prebiotic potential of each oligosaccharide differs.
Received 25 August 2015 Revised 18 January 2016 Accepted 24 January 2016 Published online 18 February 2016
Introduction Prebiotics were originally defined as ‘‘a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health’’ (Gibson & Roberfroid 1995). This definition was recently revised by FAO as ‘‘a non-viable food component that confers a health benefit on the host associated with modulation of the microbiota’’ (Pineiro et al. 2008). Oligosaccharides are mainly used as prebiotics. Several intervention studies reported the beneficial effects of prebiotic oligosaccharides in human health, including reductions in the incidence and alleviation of the severity of atopic dermatitis in infants (Moro et al. 2006; Shibata et al. 2009; Gruber et al. 2010). Oligosaccharides have also been shown to be effective for prevention or treatment of infantile diarrhea (Bruzzese et al. 2009; Passariello et al. 2011). Prebiotics have been therefore added to infant formulas and several food products for several years, especially in Japan and Westernized countries. As described in the definition, prebiotic oligosaccharides are metabolized by intestinal microbiota. The main
KEYWORDS
Lactobacillus; metabolic profiles; organic acid production
target microorganisms for prebiotics are usually intestinal bifidobacteria, and several in vitro and in vivo studies have shown the bifidogenic activities of prebiotics (Kajiwara et al. 2002; Paineau et al. 2014; Scott et al. 2014). Lactic acid bacteria (LAB) are also beneficial intestinal microbes and potential targets for prebiotic approaches, and some intestinal LAB have been shown to metabolize prebiotic oligosaccharides (Saulnier et al. 2007). Intestinal LAB are generally more important in infants than in adults and several studies demonstrated that the population of intestinal lactobacilli was linked to infantile health (Johansson et al. 2011; Partty et al. 2012). Environmental factors, including diet and hygiene, have been identified as factors influencing the LAB microbiota in infants (Aakko et al. 2015). Despite their importance, the metabolism of prebiotics by LAB has not been characterized as extensively as that by bifidobacteria. Therefore, the metabolic properties of oligosaccharides in LAB need to be studied in more detail for the adequate selection of synbiotics. Fructooligosaccharides (FOSs) and galactooligosaccharides (GOSs), which include a mixture of oligosaccharides with different degrees of polymerization, are
CONTACT Akihito Endo [email protected] Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, Abashiri 099-2493, Japan Supplemental data for this article can be accessed 10.3109/09637486.2016.1147019. ! 2016 Taylor & Francis
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A. ENDO ET AL.
Table 1. Oligosaccharides used in this study. Oligosaccharides
Obtained from (product namea)
Abbreviation
Oligosaccharides profile (ratio)
1-Kestose Nystose Fructooligosaccharides
Food Science Co. Ltd. Wako Chemical Co. Ltd Meiji Food Materia Co. Ltd. (Meioligo P)
Kes Nys FOSs
Galactooligosaccharides
Yakult Pharmaceutical Industry Co., Ltd.
GOSs
Xylooligosaccharides
Food Science Co. Ltd. (Xylooligosaccharide 95P)
XOSs
Isomaltooligosaccharides
Showa Sangyo Co. Ltd. (IMO900P)
IMOSs
Raffinose Lactosucrose
Meiji Food Materia Co. Ltd. (Beet Oligo FP) Ensuiko Sugar Refining Co. Ltd. (LS-90P)
Raf LS
Kestose (99%) Nystose (98%) Kestose (28%), nystose (59%), fructosylnystose (9%), sucrose (2%), glucose (1%), fructose (1%) Galactosyllactose (35%), lactose (29%), glucose (23%), galactose (7%) Xylobiose (33%), xylotriose (29%), xylotetraose (17%), xylopentose (9%), xylohexaose (10%) Isomaltose (44%), isomaltotriose (28%), isomaltotetraose (15%), isomaltopentaose (5%), isomaltohexaose (1%), glucose (7%) Raffinose (98%) Lactosucrose (89%), sucrose (3%), glucose (1%)
International Journal of Food Sciences and Nutrition
a
Product name if available.
mostly used in prebiotic studies (Shoaf et al. 2006; Saulnier et al. 2007; Hughes et al. 2011; Paineau et al. 2014; Sivieri et al. 2014). In addition to these two oligosaccharides, a few oligosaccharides, including raffinose (Martinez-Villaluenga et al. 2005), have also been studied based on their prebiotic abilities and have been incorporated into infant formula. However, these studies used limited numbers of oligosaccharides and broad evaluations in a single system were not conducted to study the metabolism of prebiotics by LAB. Different evaluation systems, e.g. variations in the strains, media and culture conditions used, sometimes lead to inadequate conclusions, and, thus, the metabolic profiles of prebiotics in a single system are essential. In the present study, the metabolism of eight potential prebiotics was tested using 19 strains of intestinal LAB classified into 12 bacterial species. The metabolic profiles of the oligosaccharides were also characterized.
Materials and methods Oligosaccharides used Eight oligosaccharides: 1-kestose, nystose, FOSs, GOSs, xylooligosaccharides (XOSs), isomaltooligosaccharides (IMOSs), raffinose and lactosucrose, were used in the present study. The characteristics of these oligosaccharides are summarized in Table 1. The composition of each oligosaccharide was determined by HPLC on a Shodex KS802 column with a refractive index detector. Degassed water was used as an eluent. The chemical structures of the oligosaccharides were available elsewhere (Gorin et al. 1964; Berman 1970; Kohmoto et al. 1988; Crittenden & Playne 2002; Golan et al. 2004; Han et al. 2009). Tested strains and pre-culturing conditions The LAB species and strains used in the present study are shown in Table 2. The inclusion criteria of the LAB
species were that they have been found in the stools of both human infants and adults. These strains were obtained from the Japan Collection of Microorganisms (JCM), the NODAI Culture Collection Center, Tokyo University of Agriculture (NRIC), and the American Type Culture Collection (ATCC). They were precultured in MRS broth supplemented with 0.05% L-cysteine-HCl at 37 C for 24 h and were incubated in a CO2 generating kit (AnaeroPack, Mitsubishi Gas Chemical Ltd., Tokyo, Japan). Growth on prebiotics The pre-cultured cells were washed twice and diluted 10 times with PBS buffer supplemented with 0.05% L-cysteine-HCl. Fifteen microliters of the cell suspension was inoculated into 1.5 ml of the tested broth and incubated for 48 h in the gas generating kit. The tested broth was composed of (l1) 5 g yeast extract, 5 g polypeptone, 5 g tryptone, 5 g lab-lemco powder, 0.5 g L-cysteine-HCl, 0.5 g Tween80, 2 g K2HPO4, 0.2 g MgSO4–7H2O, 0.01 g MnSO4–4H2O, 0.01 g FeSO4– 7H2O, 0.01 g NaCl and 5 g oligosaccharide (pH 6.8). Glucose (98% purity) was used as a control and sugarfree medium served as a negative control. Growth was monitored at 660 nm with a spectrophotometer (Model U-2,800A, Hitachi, Tokyo, Japan). Growth percentages were also determined by the following calculation. ð%Þ ¼ 100 ðO:D:sample O:D:free Þ=ðO:D:gluc O:D:free Þ
Organic acid compositions produced from the metabolism of oligosaccharides and glucose and the composition of each oligosaccharide before/after culturing were determined by HPLC and by high performance anion exchange chromatography coupled with a pulsed amperometric detection (HPAEC-PAD) method (Model ICS-3000, Dionex Ltd., United Kingdom), respectively, as described elsewhere (Saulnier et al. 2007) with minor modifications. All experiments were performed in duplicate.
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Table 2. Growth (O.D.660nm) of LAB strains on prebiotics. Lactobacillus acidophilus NRIC 1547T Lactobacillus acidophilus NRIC 0525 Lactobacillus delbrueckii JCM 1012T Lactobacillus gasseri JCM 1639T Lactobacillus johnsonii NRIC 0220T Lactobacillus plantarum NRIC 1067T Lactobacillus plantarum NRIC 1068 Lactobacillus rhamnosus JCM 1136T Lactobacillus rhamnosus GG Lactobacillus paracasei NRIC 0625 Lactobacillus paracasei NRIC 1028 Lactobacillus paracasei NRIC 1044 Lactobacillus sakei NRIC 1071T Lactobacillus sakei NRIC 0126 Lactobacillus fermentum NRIC 1752T Lactobacillus fermentum NRIC 1047 Lactobacillus reuteri JCM 1112T Leuconostoc citreum NRIC 1776T Weissella confusa NRIC 0207T
Glu
Kes
Nys
FOSs
GOSs
XOSs
IMOSs
Raf
LS
Free
0.58 0.43 2.13 0.45 0.40 1.83 1.75 2.92 2.80 2.08 2.28 2.51 2.12 1.09 1.49 1.48 1.97 0.92 2.00
0.59 0.31 0.81 0.42 0.20 1.92 1.90 0.73 0.54 1.59 0.43 2.26 0.85 1.31 0.97 1.38 1.16 1.05 0.22
0.48 0.24 0.60 0.38 0.27 0.60 0.51 0.67 0.54 1.53 0.37 2.10 0.65 0.67 0.42 0.43 0.64 0.22 0.13
0.57 0.30 0.74 0.35 0.21 1.02 0.88 0.70 0.72 1.69 0.40 2.28 0.72 1.25 0.57 0.68 0.79 0.49 0.11
0.56 0.41 1.51 0.36 0.36 1.59 1.45 2.23 1.07 1.35 0.84 1.66 1.69 0.97 0.96 1.28 2.03 0.29 0.49
0.28 0.16 0.43 0.25 0.31 0.40 0.49 0.74 0.49 0.46 0.41 0.62 0.81 0.60 0.24 0.28 0.71 0.18 1.69
0.37 0.39 0.95 0.31 0.28 0.70 0.98 1.64 0.60 1.52 0.59 0.97 1.54 0.65 0.84 0.45 1.09 0.50 0.42
0.33 0.17 1.30 0.21 0.16 1.50 1.56 0.54 0.35 0.30 0.26 0.46 0.48 0.58 1.07 0.95 1.30 0.13 0.08
0.63 0.24 0.31 0.28 0.26 1.75 1.67 1.37 0.33 1.75 0.32 2.12 0.40 0.48 0.91 1.12 1.58 0.16 0.09
0.16 0.17 0.39 0.15 0.11 0.41 0.36 0.43 0.35 0.29 0.23 0.40 0.36 0.27 0.20 0.26 0.47 0.06 0.06
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Growth percentage (%) was determined with the following calculation: 100 (O.D.sample – O.D.free)/(O.D.gluc – O.D.free).
Table 3. Growth ratio (%) of LAB strains on prebiotics to glucose. Lactobacillus acidophilus NRIC 1547T Lactobacillus acidophilus NRIC 0525 Lactobacillus delbrueckii JCM 1012T Lactobacillus gasseri JCM 1639T Lactobacillus johnsonii NRIC 0220T Lactobacillus plantarum NRIC 1067T Lactobacillus plantarum NRIC 1068 Lactobacillus rhamnosus JCM 1136T Lactobacillus rhamnosus GG Lactobacillus paracasei NRIC 0625T Lactobacillus paracasei NRIC 1028 Lactobacillus paracasei NRIC 1044 Lactobacillus sakei NRIC 1071T Lactobacillus sakei NRIC 0126 Lactobacillus fermentum NRIC 1752T Lactobacillus fermentum NRIC 1047 Lactobacillus reuteri JCM 1112T Leuconostoc citreum NRIC 1776T Weissella confusa NRIC 0207T
Glu
Kes
Nys
FOSs
GOSs
XOSs
IMOSs
Raf
LS
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
101 52 24 89 32 106 110 12 8 73 10 88 28 127 59 92 46 115 8
75 27 12 77 55 14 10 10 8 69 7 81 16 49 17 14 11 18 4
99 51 20 68 35 43 37 11 15 79 8 89 21 119 29 34 22 49 3
96 91 64 72 86 83 78 72 29 59 30 60 76 85 59 83 104 26 22
28 0 2 34 69 0 9 13 6 10 9 11 26 41 2 1 16 14 84
49 85 33 55 59 20 44 49 10 69 18 27 67 47 50 15 41 51 18
40 0 52 21 20 77 86 4 0 1 1 3 7 39 68 56 56 7 1
113 25 0 43 54 94 94 38 0 82 4 81 3 26 55 70 74 11 2
Growth percentage (%) was determined with the following calculation: 100 (O.D.sample – O.D.free)/(O.D.gluc – O.D.free).
Results Of the 19 strains tested, eight recorded O.D.660nm greater than 1 when supplemented with 1-kestose, whereas only two and four strains did with nystose and FOSs, respectively (Table 2). Since five strains recorded O.D.660nm less than 1 when supplemented with glucose, growth on oligosaccharides was also evaluated by the growth ratio to glucose. Eight strains recorded more than 80% when supplemented with 1-kestose, while one and three strains did with nystose and FOSs, respectively (Table 3). Raffinose and lactosucrose, which are trisaccharides and well characterized for their bifidogenic activities, enhanced (O.D.660nm 41) the growth of five and seven of the tested strains, respectively (Table 2), and scored a
greater than 80% growth ratio to glucose by one and five strains, respectively (Table 3). Ten and four strains recorded O.D.660nm greater than 1 when supplemented with GOSs and IMOSs, respectively (Table 2), and seven and one strain(s) recorded greater than 80% growth ratio with GOSs and IMOSs, respectively (Table 3). XOSs were actively fermented (O.D.660nm 41) by only one of the tested strains, Weissella confusa. Oligosaccharide profiles in oligosaccharide mixtures, i.e. FOSs, GOSs, XOSs and IMOSs, consumed by bacterial metabolism are determined with HPAECPAD and summarized in Figure 1. After being cultured with FOSs, Lactobacillus plantarum, L. sakei, L. fermentum and Leuconostoc citreum strains utilized almost all 1-kestose available, whereas most parts of nystose and
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A. ENDO ET AL. (%) 200
405%
(A) 180 160 140 120 100 80 60 40 20 0
International Journal of Food Sciences and Nutrition
control NRIC 1547
NRIC 0525
JCM 1012
NRIC 1639
NRIC 0220
NRIC 1067
1-kestose
Sucrose
NRIC 1068
JCM 1136
GG
Nystose
NRIC 0625
NRIC 1028
NRIC 1044
NRIC 1071
NRIC 0126
NRIC 1752
NRIC 1047
JCM 1112
NRIC 1776
NRIC 0207
Fructosyl-nystose
(%) 200
(B) 180 160 140 120 100 80 60 40 20 0 control NRIC 1547
NRIC 0525
Galactose + glucose
JCM 1012
NRIC 1639
NRIC 0220
lactose
NRIC 1067
NRIC 1068
JCM 1136
GG
NRIC 0625
NRIC 1028
NRIC 1044
NRIC 1071
RT13.2 (isomer of lactose or galactosyllactose)
NRIC 0126
NRIC 1752
NRIC 1047
JCM 1112
NRIC 1776
NRIC 0207
Galactosyllactose (containing isomer)
Figure 1. Ratio of each oligosaccharide remaining after bacterial metabolism of (a) FOSs, (b) GOSs, (c) XOSs and (d) IMOSs. Medium without the inoculation of bacteria served as the control and each oligosaccharide concentration contained in the control was set at 100%.
fructosylnystose remained in the cultured broth (Figure 1a). Lactobacillus acidophilus and L. paracasei strains showed strain-dependent manner. L. paracasei NRIC 0625 and NRIC 1044 were only the strains that
metabolized all 1-kestose, nystose and fructosylnystose available, resulting in the accumulation of large amounts of sucrose after culturing. The two strains grew well on nystose (Table 2). Lactobacillus delbrueckii and L. gasseri
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5
(%) 200
(C) 180 160 140 120 100 80 60 40 20 0
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control NRIC 1547
NRIC 0525
xylobiose
JCM 1012
NRIC 1639
NRIC 0220
xylotriose
NRIC 1067
NRIC 1068
JCM 1136
GG
NRIC 0625
NRIC 1028
RT18.4 (possible xylotetraose)
NRIC 1044
NRIC 1071
NRIC 0126
NRIC 1752
NRIC 1047
JCM 1112
NRIC 1776
NRIC 0207
NRIC 1776
NRIC 0207
RT21.2 (possible xylopentaose)
(%) 200
(D) 180 160 140 120 100 80 60 40 20 0 control NRIC 1547
Glucose
NRIC 0525
JCM 1012
NRIC 1639
NRIC 0220
Isomaltose
NRIC 1067
NRIC 1068
JCM 1136
GG
NRIC 0625
NRIC 1028
Isomaltotriose (containing isomers)
NRIC 1044
NRIC 1071
NRIC 0126
NRIC 1752
NRIC 1047
JCM 1112
RT23.8 (possible isomaltotetraose )
Figure 1. Continued.
metabolized 65% and 35% of 1-kestose available, respectively, whereas the ratios of metabolized nystose and fructosylnystose in the strains were within 5%. Large amounts of 1-kestose, nystose and fructosylnystose
remained in the cultured broths of Lactobacillus johnsonii, L. rhamnosus, L. reuteri and W. confusa. Of the 14 actively grown strains with GOSs (O.D.660nm 41 and/or greater than 80% growth ratio to glucose)
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A. ENDO ET AL.
(Tables 2 and 3), L. delbrueckii JCM 1012T, two L. plantarum strains, L. fermentum NRIC 1047 and L. reuteri JCM 1112T metabolized almost all amounts of mono-, di- and trisaccharides in GOSs (Figure 1b). Two L. acidophilus strains metabolized galactosyllactose and monosaccharides or lactose. Lactobacillus johnsonii NRIC 0220T consumed only galactosyllactose, whereas 45% of galactosyllactose available remained after culturing. The remaining six actively grown strains with GOSs, including two L. rhamnosus strains, L. paracasei NRIC 0625 and NRIC 1044, and two L. sakei strains mainly used monosaccharides and lactose, and the residue of unidentified carbohydrate named RT13.2 (an isomer of lactose or galactosyllactose) and galactosyllactose ranged from 63 to 114% and from 61 to 121%, respectively. Carbohydrates in XOSs, which did not enhance the growth of most of the tested strains, were only metabolized well by W. confusa. This strain consumed all amounts of xylobiose and xylotriose and half of unidentified carbohydrate named RT18.4 (possible xylotetraose) available, whereas xylobiose is a disaccharide. A few strains metabolized 15–20% of sugars in XOSs. IMOSs actively enhanced the growth of five of the tested strains (O.D.660nm 41 and/or greater than 80% growth ratio to glucose) (Tables 2 and 3). Two of the five strains (L. paracasei NRIC 0625 and L. reuteri JCM 1112) metabolized large parts of isomaltose, isomaltotriose and unidentified carbohydrate named RT23.8 (possible isomaltotetraose) (Figure 1d). Another three strains: L. rhamnosus JCM 1136T, L. sakei NRIC 1071T, and L. acidophilus NRIC 0525, mainly consumed monoand disaccharides present. Organic acid production from the metabolism of oligosaccharides is summarized in Supplementary Table S1. The tested strains produced large amounts of lactate with small amounts of acetate, except for W. confusa NRIC 0207T, which produced almost equal amounts of lactate and acetate from XOSs. L. fermentum NRIC 1752T produced lactate and acetate with a molar ratio of 2:1 from 1-kestose, whereas other strains, which actively metabolized 1-kestose at least five-fold more lactate than acetate. The amounts of lactate produced were generally consistent with the bacterial growth recorded (Table 2).
Discussion Prebiotics have been applied for several health promotion purposes in infants and adults, and the prebiotics consumed are generally considered to be metabolized by beneficial microbes including lactobacilli. Despite the importance of the metabolism of prebiotics in lactobacilli
both in researches and industrial fields, few studies have compared prebiotic potentials among oligosaccharides using multiple Lactobacillus spp. We herein clearly demonstrated that the fermentation abilities of oligosaccharides varied not only at the species level, but also at the strain level. Moreover, certain oligosaccharides contained large amounts of mono- and disaccharides, resulting in markedly fewer prebiotic properties since these mono- and disaccharides are known to be metabolized by host metabolic systems. The ratio of lactate and acetate produced did not appear to be related to the oligosaccharides used. Among the prebiotics used, 1-kestose and GOSs were the most promising oligosaccharides in terms of growth stimulation of lactobacilli (Tables 2 and 3). However, mono- and disaccharides accounted for 59% of the GOSs used in the present study (Table 1), and six of the 14 strains actively grown with GOSs mainly metabolized these mono- and disaccharides (Figure 1b). GOSs are generally produced from lactose using bacterial or fungal -galactosidase (Rodriguez-Colinas et al. 2011; Song et al. 2013; Yu & O’Sullivan 2014); however, mono- or disaccharides account for approximately half of its components (Shoaf et al. 2006; Quintero et al. 2011). Therefore, the prebiotic activities of GOSs were overestimated in the present study and further studies are needed in order to see their activities following the removal of these simple saccharides. As compared with 1-kestose, few strains metabolized nystose, which is fructosylated 1-kestose. Although a previous study suggested that nystose was specifically metabolized by L. acidophilus in lactobacilli (Endo et al. 2012), our study indicated that nystose metabolism in L. acidophilus was in a strain-dependent manner (Table 3). Two L. paracasei strains grew well with nystose and completely metabolized nystose in FOSs (Table 2 and Figure 1a). -Fructosidase activity was previously suggested to be linked with the nystose metabolism (Endo et al. 2012). The growth of the strains tested was better with FOSs than with nystose, but was less or similar to that with 1-kestose. Carbohydrate profiles after fermentation indicated that three out of the five strains that actively grew with FOSs (Tables 2 and 3), mainly metabolized 1-kestose in FOSs (Figure 1a), suggesting that 1-kestose content was a key for the prebiotic activity in FOSs. Similar results have been reported by Munoz et al. (2012). Interestingly, L. paracasei NRIC 0625 and NRIC 1044 metabolized all oligosaccharides present in FOSs, but left larger amounts of sucrose in the medium than in the original content (Figure 1a). This result suggested that these two strains preferentially metabolized fructose residue of oligosaccharides and excreted sucrose in the medium. Certain dairy LAB are known to
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excrete galactose after the lactose metabolism (Gunnewijk & Poolman 2000). The metabolic profile of lactosucrose was similar to that of 1-kestose, suggesting that both were metabolized in a similar metabolic manner. A previous study suggested that 1-kestose was degraded by the sucrose metabolic system in L. plantarum (Saulnier et al. 2007). XOSs were actively metabolized by only one of the tested 19 strains, W. confusa NRIC 0207T, suggesting that XOSs stimulate only specific intestinal LAB. The bifidogenic activities of XOSs without the stimulation of lactobacilli have been reported in adults (Finegold et al. 2014). NRIC 0207T produced almost equal amounts of lactate and acetate from XOSs (Table S1). This was expected since heterofermentative LAB produce acetate instead of ethanol from pentoses (Zaunmuller et al. 2006). Similar finding, XOSs metabolism in W. confusa/cibaria strains, has been recently reported (Patel et al. 2013). This study also showed cell-associated -xylosidase activity in W. confusa/cibaria strains. W. confusa has been found in various niches, including human and animal guts, fermented food and soils. This specific trait would be beneficial for living in such broad habitats. Among the strains used, L. rhamnosus GG, one of the most widely studied and commercialized probiotic strains, was a non-fermenter of prebiotics. This is consistent with previous studies that have reported poor carbohydrate/oligosaccharide metabolic ability in L. rhamnosus GG (Kaplan & Hutkins 2000; Douillard et al. 2013).
Conclusion The present study clearly demonstrated that the prebiotic potential of each oligosaccharide differs. In GOSs and IMOSs, mono- and disaccharides but not oligosaccharides in their mixtures were mainly metabolized by several LAB strains. Metabolic manner of oligosaccharides are highly strain dependent. The results presented here are important for selection of an adequate combination of probiotics and prebiotics for synbiotic usage. Future researches are needed to study impacts of prebiotics on growth of other bacterial species in gut microbiota, including pathogens and opportunistic pathogens, since such microorganisms would compete with beneficial microbes, including LAB, to obtain nutrition.
Acknowledgements We thank to Ryosuke Nomura, Department of Food and Cosmetic Science, Tokyo University of Agriculture, for his technical assistance.
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Disclosure statement Takumi Tochio, Kenta Konishi and Saki Nakamura are employees of Food Science Co., which is the producer of 1-kestose and XOSs used in this study. The remaining authors declare no conflicts of interest.
Funding information This study was partly funded by Food Science Co.
References Aakko J, Endo A, Mangani C, Maleta K, Ashorn P, Isolauri E, Salminen S. 2015. Distinctive intestinal Lactobacillus communities in 6-month-old infants from rural Malawi and Southwestern Finland. J Pediatr Gastroenterol Nutr. 61:641–648. Berman HM. 1970. The crystal structure of a trisaccharide, raffinose pentahydrate. Acta Crystallogr B. 26:290–299. Bruzzese E, Volpicelli M, Squeglia V, Bruzzese D, Salvini F, Bisceglia M, Lionetti P, Cinquetti M, Iacono G, Amarri S, et al. 2009. A formula containing galacto- and fructooligosaccharides prevents intestinal and extra-intestinal infections: an observational study. Clin Nutr. 28:156–161. Crittenden RG, Playne MJ. 2002. Purification of food-grade oligosaccharides using immobilised cells of Zymomonas mobilis. Appl Microbiol Biotechnol. 58:297–302. Douillard FP, Ribbera A, Jarvinen HM, Kant R, Pietila TE, Randazzo C, Paulin L, Laine PK PK, Caggia C, von Ossowski I, et al. 2013. Comparative genomic and functional analysis of Lactobacillus casei and Lactobacillus rhamnosus strains marketed as probiotics. Appl Environ Microbiol. 79:1923–1933. Endo H, Tamura K, Fukasawa T, Kanegae M, Koga J. 2012. Comparison of fructooligosaccharide utilization by Lactobacillus and Bacteroides species. Biosci Biotechnol Biochem. 76:176–179. Finegold SM, Li Z, Summanen PH, Downes J, Thames G, Corbett K, Dowd S, Krak M, Heber D. 2014. Xylooligosaccharide increases bifidobacteria but not lactobacilli in human gut microbiota. Food Funct. 5:436–445. Gibson GR, Roberfroid MB. 1995. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr 125:1401–1412. Golan G, Shallom D, Teplitsky A, Zaide G, Shulami S, Baasov T, Stojanoff V, Thompson A, Shoham Y, Shoham G. 2004. Crystal structures of Geobacillus stearothermophilus alphaglucuronidase complexed with its substrate and products: mechanistic implications. J Biol Chem. 279:3014–3024. Gorin PAJ, Spencer JFT, Phaff HJ. 1964. The structures of galactosyl-lactose and galactobiosyl-lactose produced from lactose by Sporobolomyces singularis. Can J Chem. 42:1341–1344. Gruber C, van Stuijvenberg M, Mosca F, Moro G, Chirico G, Braegger CP, Riedler J, Boehm G, Wahn U. 2010. Reduced occurrence of early atopic dermatitis because of immunoactive prebiotics among low-atopy-risk infants. J Allergy Clin Immunol. 126:791–797. Gunnewijk MG, Poolman B. 2000. Phosphorylation state of HPr determines the level of expression and the extent of
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phosphorylation of the lactose transport protein of Streptococcus thermophilus. J Biol Chem. 275:34073–34079. Han WC, Byun SH, Kim MH, Sohn EH, Lim JD, Um BH, Kim CH, Kang SA, Jang SA. 2009. Production of lactosucrose from sucrose and lactose by a levansucrase from Zymomonas mobilis. J Microbiol Biotechnol. 19:1153–1160. Hughes C, Davoodi-Semiromi Y, Colee JC, Culpepper T, Dahl WJ, Mai V, Christman MC, Langkamp-Henken B. 2011. Galactooligosaccharide supplementation reduces stressinduced gastrointestinal dysfunction and days of cold or flu: a randomized, double-blind, controlled trial in healthy university students. Am J Clin Nutr. 93:1305–1311. Johansson MA, Sjogren YM, Persson JO, Nilsson C, Sverremark-Ekstrom E. 2011. Early colonization with a group of Lactobacilli decreases the risk for allergy at five years of age despite allergic heredity. PLoS One. 6:e23031. Kajiwara S, Gandhi H, Ustunol Z. 2002. Effect of honey on the growth of and acid production by human intestinal Bifidobacterium spp.: an in vitro comparison with commercial oligosaccharides and inulin. J Food Prot. 65:214–218. Kaplan H, Hutkins RW. 2000. Fermentation of fructooligosaccharides by lactic acid bacteria and bifidobacteria. Appl Environ Microbiol. 66:2682–2684. Kohmoto T, Fukui F, Takaku H, Machida Y, Arai M, Mitsuoka T. 1988. Effect of Isomalto-oligosaccharides on Human Fecal Flora. Bifidobacteria Microflora. 7:61–69. Martinez-Villaluenga C, Frias J, Vidal-Valverde C, Gomez R. 2005. Raffinose family of oligosaccharides from lupin seeds as prebiotics: application in dairy products. J Food Prot. 68:1246–1252. Moro G, Arslanoglu S, Stahl B, Jelinek J, Wahn U, Boehm G. 2006. A mixture of prebiotic oligosaccharides reduces the incidence of atopic dermatitis during the first six months of age. Arch Dis Child. 91:814–819. Munoz M, Mosquera A, Almeciga-Diaz CJ, Melendez AP, Sanchez OF. 2012. Fructooligosaccharides metabolism and effect on bacteriocin production in Lactobacillus strains isolated from ensiled corn and molasses. Anaerobe. 18:321–330. Paineau D, Respondek F, Menet V, Sauvage R, Bornet F, Wagner A. 2014. Effects of short-chain fructooligosaccharides on faecal bifidobacteria and specific immune response in formula-fed term infants: a randomized, double-blind, placebo-controlled trial. J Nutr Sci Vitaminol (Tokyo). 60:167–175. Partty A, Kalliomaki M, Endo A, Salminen S, Isolauri E. 2012. Compositional development of Bifidobacterium and Lactobacillus microbiota is linked with crying and fussing in early infancy. PLoS One. 7:e32495. Passariello A, Terrin G, De Marco G, Cecere G, Ruotolo S, Marino A, Cosenza L, Tardi M, Nocerino R, Berni Canani R. 2011. Efficacy of a new hypotonic oral rehydration solution containing zinc and prebiotics in the treatment of childhood acute diarrhea: a randomized controlled trial. J Pediatr. 158:288–292.
Patel A, Falck P, Shah N, Immerzeel P, Adlercreutz P, Stalbrand H, Prajapati JB, Holst O, Nordberg Karlsson E. 2013. Evidence for xylooligosaccharide utilization in Weissella strains isolated from Indian fermented foods and vegetables. FEMS Microbiol Lett. 346:20–28. Pineiro M, Asp NG, Reid G, Macfarlane S, Morelli L, Brunser O, Tuohy K. 2008. FAO Technical Meeting on prebiotics. J Clin Gastroenterol. 42:S156–S159. Quintero M, Maldonado M, Perez-Munoz M, Jimenez R, Fangman T, Rupnow J, Wittke A, Russell M, Hutkins R. 2011. Adherence inhibition of Cronobacter sakazakii to intestinal epithelial cells by prebiotic oligosaccharides. Curr Microbiol. 62:1448–1454. Rodriguez-Colinas B, de Abreu MA, Fernandez-Arrojo L, de Beer R, Poveda A, Jimenez-Barbero J, Haltrich D, Ballesteros Olmo AO, Fernandez-Lobato M, Plou FJ. 2011. Production of galacto-oligosaccharides by the -galactosidase from Kluyveromyces lactis: comparative analysis of permeabilized cells versus soluble enzyme. J Agric Food Chem. 59:10477–10484. Saulnier DM, Molenaar D, de Vos WM, Gibson GR, Kolida S. 2007. Identification of prebiotic fructooligosaccharide metabolism in Lactobacillus plantarum WCFS1 through microarrays. Appl Environ Microbiol. 73:1753–1765. Scott KP, Martin JC, Duncan SH, Flint HJ. 2014. Prebiotic stimulation of human colonic butyrate-producing bacteria and bifidobacteria, in vitro. FEMS Microbiol Ecol. 87:30–40. Shibata R, Kimura M, Takahashi H, Mikami K, Aiba Y, Takeda H, Koga Y. 2009. Clinical effects of kestose, a prebiotic oligosaccharide, on the treatment of atopic dermatitis in infants. Clin Exp Allergy. 39:1397–1403. Shoaf K, Mulvey GL, Armstrong GD, Hutkins RW. 2006. Prebiotic galactooligosaccharides reduce adherence of enteropathogenic Escherichia coli to tissue culture cells. Infect Immun. 74:6920–6928. Sivieri K, Morales ML, Saad SM, Adorno MA, Sakamoto IK, Rossi EA. 2014. Prebiotic effect of fructooligosaccharide in the simulator of the human intestinal microbial ecosystem (SHIME(R) model). J Med Food. 17:894–901. Song J, Imanaka H, Imamura K, Minoda M, Yamaguchi S, Nakanishi K. 2013. The discoidin domain of Bacillus circulans -galactosidase plays an essential role in repressing galactooligosaccharide production – plays essential in galactooligosaccharide. Biosci Biotechnol Biochem. 77:73–79. Yu L, O’sullivan DJ. 2014. Production of galactooligosaccharides using a hyperthermophilic -galactosidase in permeabilized whole cells of Lactococcus lactis – in whole of lactis. J Dairy Sci. 97:694–703. Zaunmuller T, Eichert M, Richter H, Unden G. 2006. Variations in the energy metabolism of biotechnologically relevant heterofermentative lactic acid bacteria during growth on sugars and organic acids. Appl Microbiol Biotechnol. 72:421–429.
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Variations in prebiotic oligosaccharide fermentation by intestinal lactic acid bacteria Akihito Endo, Saki Nakamura, Kenta Konishi, Junichi Nakagawa & Takumi Tochio To cite this article: Akihito Endo, Saki Nakamura, Kenta Konishi, Junichi Nakagawa & Takumi Tochio (2016): Variations in prebiotic oligosaccharide fermentation by intestinal lactic acid bacteria, International Journal of Food Sciences and Nutrition, DOI: 10.3109/09637486.2016.1147019 To link to this article: http://dx.doi.org/10.3109/09637486.2016.1147019
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Date: 23 February 2016, At: 17:38
INTERNATIONAL JOURNAL OF FOOD SCIENCES AND NUTRITION, 2016 http://dx.doi.org/10.3109/09637486.2016.1147019
RESEARCH ARTICLE
Variations in prebiotic oligosaccharide fermentation by intestinal lactic acid bacteria Akihito Endoa, Saki Nakamurab, Kenta Konishib, Junichi Nakagawaa and Takumi Tochiob
International Journal of Food Sciences and Nutrition
a Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, Abashiri, Japan; b Food Science Co, Ltd, Aichi, Japan
ABSTRACT
ARTICLE HISTORY
Prebiotic oligosaccharides confer health benefits on the host by modulating the gut microbiota. Intestinal lactic acid bacteria (LAB) are potential targets of prebiotics; however, the metabolism of oligosaccharides by LAB has not been fully characterized. Here, we studied the metabolism of eight oligosaccharides by 19 strains of intestinal LAB. Among the eight oligosaccharides used, 1-kestose, lactosucrose and galactooligosaccharides (GOSs) led to the greatest increases in the numbers of the strains tested. However, mono- and disaccharides accounted for more than half of the GOSs used, and several strains only metabolized the mono- and di-saccharides in GOSs. End product profiles indicated that the amounts of lactate produced were generally consistent with the bacterial growth recorded. Oligosaccharide profiling revealed the interesting metabolic manner in Lactobacillus paracasei strains, which metabolized all oligosaccharides, but left sucrose when cultured with fructooligosaccharides. The present study clearly indicated that the prebiotic potential of each oligosaccharide differs.
Received 25 August 2015 Revised 18 January 2016 Accepted 24 January 2016 Published online 18 February 2016
Introduction Prebiotics were originally defined as ‘‘a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health’’ (Gibson & Roberfroid 1995). This definition was recently revised by FAO as ‘‘a non-viable food component that confers a health benefit on the host associated with modulation of the microbiota’’ (Pineiro et al. 2008). Oligosaccharides are mainly used as prebiotics. Several intervention studies reported the beneficial effects of prebiotic oligosaccharides in human health, including reductions in the incidence and alleviation of the severity of atopic dermatitis in infants (Moro et al. 2006; Shibata et al. 2009; Gruber et al. 2010). Oligosaccharides have also been shown to be effective for prevention or treatment of infantile diarrhea (Bruzzese et al. 2009; Passariello et al. 2011). Prebiotics have been therefore added to infant formulas and several food products for several years, especially in Japan and Westernized countries. As described in the definition, prebiotic oligosaccharides are metabolized by intestinal microbiota. The main
KEYWORDS
Lactobacillus; metabolic profiles; organic acid production
target microorganisms for prebiotics are usually intestinal bifidobacteria, and several in vitro and in vivo studies have shown the bifidogenic activities of prebiotics (Kajiwara et al. 2002; Paineau et al. 2014; Scott et al. 2014). Lactic acid bacteria (LAB) are also beneficial intestinal microbes and potential targets for prebiotic approaches, and some intestinal LAB have been shown to metabolize prebiotic oligosaccharides (Saulnier et al. 2007). Intestinal LAB are generally more important in infants than in adults and several studies demonstrated that the population of intestinal lactobacilli was linked to infantile health (Johansson et al. 2011; Partty et al. 2012). Environmental factors, including diet and hygiene, have been identified as factors influencing the LAB microbiota in infants (Aakko et al. 2015). Despite their importance, the metabolism of prebiotics by LAB has not been characterized as extensively as that by bifidobacteria. Therefore, the metabolic properties of oligosaccharides in LAB need to be studied in more detail for the adequate selection of synbiotics. Fructooligosaccharides (FOSs) and galactooligosaccharides (GOSs), which include a mixture of oligosaccharides with different degrees of polymerization, are
CONTACT Akihito Endo [email protected] Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, Abashiri 099-2493, Japan Supplemental data for this article can be accessed 10.3109/09637486.2016.1147019. ! 2016 Taylor & Francis
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Table 1. Oligosaccharides used in this study. Oligosaccharides
Obtained from (product namea)
Abbreviation
Oligosaccharides profile (ratio)
1-Kestose Nystose Fructooligosaccharides
Food Science Co. Ltd. Wako Chemical Co. Ltd Meiji Food Materia Co. Ltd. (Meioligo P)
Kes Nys FOSs
Galactooligosaccharides
Yakult Pharmaceutical Industry Co., Ltd.
GOSs
Xylooligosaccharides
Food Science Co. Ltd. (Xylooligosaccharide 95P)
XOSs
Isomaltooligosaccharides
Showa Sangyo Co. Ltd. (IMO900P)
IMOSs
Raffinose Lactosucrose
Meiji Food Materia Co. Ltd. (Beet Oligo FP) Ensuiko Sugar Refining Co. Ltd. (LS-90P)
Raf LS
Kestose (99%) Nystose (98%) Kestose (28%), nystose (59%), fructosylnystose (9%), sucrose (2%), glucose (1%), fructose (1%) Galactosyllactose (35%), lactose (29%), glucose (23%), galactose (7%) Xylobiose (33%), xylotriose (29%), xylotetraose (17%), xylopentose (9%), xylohexaose (10%) Isomaltose (44%), isomaltotriose (28%), isomaltotetraose (15%), isomaltopentaose (5%), isomaltohexaose (1%), glucose (7%) Raffinose (98%) Lactosucrose (89%), sucrose (3%), glucose (1%)
International Journal of Food Sciences and Nutrition
a
Product name if available.
mostly used in prebiotic studies (Shoaf et al. 2006; Saulnier et al. 2007; Hughes et al. 2011; Paineau et al. 2014; Sivieri et al. 2014). In addition to these two oligosaccharides, a few oligosaccharides, including raffinose (Martinez-Villaluenga et al. 2005), have also been studied based on their prebiotic abilities and have been incorporated into infant formula. However, these studies used limited numbers of oligosaccharides and broad evaluations in a single system were not conducted to study the metabolism of prebiotics by LAB. Different evaluation systems, e.g. variations in the strains, media and culture conditions used, sometimes lead to inadequate conclusions, and, thus, the metabolic profiles of prebiotics in a single system are essential. In the present study, the metabolism of eight potential prebiotics was tested using 19 strains of intestinal LAB classified into 12 bacterial species. The metabolic profiles of the oligosaccharides were also characterized.
Materials and methods Oligosaccharides used Eight oligosaccharides: 1-kestose, nystose, FOSs, GOSs, xylooligosaccharides (XOSs), isomaltooligosaccharides (IMOSs), raffinose and lactosucrose, were used in the present study. The characteristics of these oligosaccharides are summarized in Table 1. The composition of each oligosaccharide was determined by HPLC on a Shodex KS802 column with a refractive index detector. Degassed water was used as an eluent. The chemical structures of the oligosaccharides were available elsewhere (Gorin et al. 1964; Berman 1970; Kohmoto et al. 1988; Crittenden & Playne 2002; Golan et al. 2004; Han et al. 2009). Tested strains and pre-culturing conditions The LAB species and strains used in the present study are shown in Table 2. The inclusion criteria of the LAB
species were that they have been found in the stools of both human infants and adults. These strains were obtained from the Japan Collection of Microorganisms (JCM), the NODAI Culture Collection Center, Tokyo University of Agriculture (NRIC), and the American Type Culture Collection (ATCC). They were precultured in MRS broth supplemented with 0.05% L-cysteine-HCl at 37 C for 24 h and were incubated in a CO2 generating kit (AnaeroPack, Mitsubishi Gas Chemical Ltd., Tokyo, Japan). Growth on prebiotics The pre-cultured cells were washed twice and diluted 10 times with PBS buffer supplemented with 0.05% L-cysteine-HCl. Fifteen microliters of the cell suspension was inoculated into 1.5 ml of the tested broth and incubated for 48 h in the gas generating kit. The tested broth was composed of (l1) 5 g yeast extract, 5 g polypeptone, 5 g tryptone, 5 g lab-lemco powder, 0.5 g L-cysteine-HCl, 0.5 g Tween80, 2 g K2HPO4, 0.2 g MgSO4–7H2O, 0.01 g MnSO4–4H2O, 0.01 g FeSO4– 7H2O, 0.01 g NaCl and 5 g oligosaccharide (pH 6.8). Glucose (98% purity) was used as a control and sugarfree medium served as a negative control. Growth was monitored at 660 nm with a spectrophotometer (Model U-2,800A, Hitachi, Tokyo, Japan). Growth percentages were also determined by the following calculation. ð%Þ ¼ 100 ðO:D:sample O:D:free Þ=ðO:D:gluc O:D:free Þ
Organic acid compositions produced from the metabolism of oligosaccharides and glucose and the composition of each oligosaccharide before/after culturing were determined by HPLC and by high performance anion exchange chromatography coupled with a pulsed amperometric detection (HPAEC-PAD) method (Model ICS-3000, Dionex Ltd., United Kingdom), respectively, as described elsewhere (Saulnier et al. 2007) with minor modifications. All experiments were performed in duplicate.
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Table 2. Growth (O.D.660nm) of LAB strains on prebiotics. Lactobacillus acidophilus NRIC 1547T Lactobacillus acidophilus NRIC 0525 Lactobacillus delbrueckii JCM 1012T Lactobacillus gasseri JCM 1639T Lactobacillus johnsonii NRIC 0220T Lactobacillus plantarum NRIC 1067T Lactobacillus plantarum NRIC 1068 Lactobacillus rhamnosus JCM 1136T Lactobacillus rhamnosus GG Lactobacillus paracasei NRIC 0625 Lactobacillus paracasei NRIC 1028 Lactobacillus paracasei NRIC 1044 Lactobacillus sakei NRIC 1071T Lactobacillus sakei NRIC 0126 Lactobacillus fermentum NRIC 1752T Lactobacillus fermentum NRIC 1047 Lactobacillus reuteri JCM 1112T Leuconostoc citreum NRIC 1776T Weissella confusa NRIC 0207T
Glu
Kes
Nys
FOSs
GOSs
XOSs
IMOSs
Raf
LS
Free
0.58 0.43 2.13 0.45 0.40 1.83 1.75 2.92 2.80 2.08 2.28 2.51 2.12 1.09 1.49 1.48 1.97 0.92 2.00
0.59 0.31 0.81 0.42 0.20 1.92 1.90 0.73 0.54 1.59 0.43 2.26 0.85 1.31 0.97 1.38 1.16 1.05 0.22
0.48 0.24 0.60 0.38 0.27 0.60 0.51 0.67 0.54 1.53 0.37 2.10 0.65 0.67 0.42 0.43 0.64 0.22 0.13
0.57 0.30 0.74 0.35 0.21 1.02 0.88 0.70 0.72 1.69 0.40 2.28 0.72 1.25 0.57 0.68 0.79 0.49 0.11
0.56 0.41 1.51 0.36 0.36 1.59 1.45 2.23 1.07 1.35 0.84 1.66 1.69 0.97 0.96 1.28 2.03 0.29 0.49
0.28 0.16 0.43 0.25 0.31 0.40 0.49 0.74 0.49 0.46 0.41 0.62 0.81 0.60 0.24 0.28 0.71 0.18 1.69
0.37 0.39 0.95 0.31 0.28 0.70 0.98 1.64 0.60 1.52 0.59 0.97 1.54 0.65 0.84 0.45 1.09 0.50 0.42
0.33 0.17 1.30 0.21 0.16 1.50 1.56 0.54 0.35 0.30 0.26 0.46 0.48 0.58 1.07 0.95 1.30 0.13 0.08
0.63 0.24 0.31 0.28 0.26 1.75 1.67 1.37 0.33 1.75 0.32 2.12 0.40 0.48 0.91 1.12 1.58 0.16 0.09
0.16 0.17 0.39 0.15 0.11 0.41 0.36 0.43 0.35 0.29 0.23 0.40 0.36 0.27 0.20 0.26 0.47 0.06 0.06
International Journal of Food Sciences and Nutrition
Growth percentage (%) was determined with the following calculation: 100 (O.D.sample – O.D.free)/(O.D.gluc – O.D.free).
Table 3. Growth ratio (%) of LAB strains on prebiotics to glucose. Lactobacillus acidophilus NRIC 1547T Lactobacillus acidophilus NRIC 0525 Lactobacillus delbrueckii JCM 1012T Lactobacillus gasseri JCM 1639T Lactobacillus johnsonii NRIC 0220T Lactobacillus plantarum NRIC 1067T Lactobacillus plantarum NRIC 1068 Lactobacillus rhamnosus JCM 1136T Lactobacillus rhamnosus GG Lactobacillus paracasei NRIC 0625T Lactobacillus paracasei NRIC 1028 Lactobacillus paracasei NRIC 1044 Lactobacillus sakei NRIC 1071T Lactobacillus sakei NRIC 0126 Lactobacillus fermentum NRIC 1752T Lactobacillus fermentum NRIC 1047 Lactobacillus reuteri JCM 1112T Leuconostoc citreum NRIC 1776T Weissella confusa NRIC 0207T
Glu
Kes
Nys
FOSs
GOSs
XOSs
IMOSs
Raf
LS
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
101 52 24 89 32 106 110 12 8 73 10 88 28 127 59 92 46 115 8
75 27 12 77 55 14 10 10 8 69 7 81 16 49 17 14 11 18 4
99 51 20 68 35 43 37 11 15 79 8 89 21 119 29 34 22 49 3
96 91 64 72 86 83 78 72 29 59 30 60 76 85 59 83 104 26 22
28 0 2 34 69 0 9 13 6 10 9 11 26 41 2 1 16 14 84
49 85 33 55 59 20 44 49 10 69 18 27 67 47 50 15 41 51 18
40 0 52 21 20 77 86 4 0 1 1 3 7 39 68 56 56 7 1
113 25 0 43 54 94 94 38 0 82 4 81 3 26 55 70 74 11 2
Growth percentage (%) was determined with the following calculation: 100 (O.D.sample – O.D.free)/(O.D.gluc – O.D.free).
Results Of the 19 strains tested, eight recorded O.D.660nm greater than 1 when supplemented with 1-kestose, whereas only two and four strains did with nystose and FOSs, respectively (Table 2). Since five strains recorded O.D.660nm less than 1 when supplemented with glucose, growth on oligosaccharides was also evaluated by the growth ratio to glucose. Eight strains recorded more than 80% when supplemented with 1-kestose, while one and three strains did with nystose and FOSs, respectively (Table 3). Raffinose and lactosucrose, which are trisaccharides and well characterized for their bifidogenic activities, enhanced (O.D.660nm 41) the growth of five and seven of the tested strains, respectively (Table 2), and scored a
greater than 80% growth ratio to glucose by one and five strains, respectively (Table 3). Ten and four strains recorded O.D.660nm greater than 1 when supplemented with GOSs and IMOSs, respectively (Table 2), and seven and one strain(s) recorded greater than 80% growth ratio with GOSs and IMOSs, respectively (Table 3). XOSs were actively fermented (O.D.660nm 41) by only one of the tested strains, Weissella confusa. Oligosaccharide profiles in oligosaccharide mixtures, i.e. FOSs, GOSs, XOSs and IMOSs, consumed by bacterial metabolism are determined with HPAECPAD and summarized in Figure 1. After being cultured with FOSs, Lactobacillus plantarum, L. sakei, L. fermentum and Leuconostoc citreum strains utilized almost all 1-kestose available, whereas most parts of nystose and
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A. ENDO ET AL. (%) 200
405%
(A) 180 160 140 120 100 80 60 40 20 0
International Journal of Food Sciences and Nutrition
control NRIC 1547
NRIC 0525
JCM 1012
NRIC 1639
NRIC 0220
NRIC 1067
1-kestose
Sucrose
NRIC 1068
JCM 1136
GG
Nystose
NRIC 0625
NRIC 1028
NRIC 1044
NRIC 1071
NRIC 0126
NRIC 1752
NRIC 1047
JCM 1112
NRIC 1776
NRIC 0207
Fructosyl-nystose
(%) 200
(B) 180 160 140 120 100 80 60 40 20 0 control NRIC 1547
NRIC 0525
Galactose + glucose
JCM 1012
NRIC 1639
NRIC 0220
lactose
NRIC 1067
NRIC 1068
JCM 1136
GG
NRIC 0625
NRIC 1028
NRIC 1044
NRIC 1071
RT13.2 (isomer of lactose or galactosyllactose)
NRIC 0126
NRIC 1752
NRIC 1047
JCM 1112
NRIC 1776
NRIC 0207
Galactosyllactose (containing isomer)
Figure 1. Ratio of each oligosaccharide remaining after bacterial metabolism of (a) FOSs, (b) GOSs, (c) XOSs and (d) IMOSs. Medium without the inoculation of bacteria served as the control and each oligosaccharide concentration contained in the control was set at 100%.
fructosylnystose remained in the cultured broth (Figure 1a). Lactobacillus acidophilus and L. paracasei strains showed strain-dependent manner. L. paracasei NRIC 0625 and NRIC 1044 were only the strains that
metabolized all 1-kestose, nystose and fructosylnystose available, resulting in the accumulation of large amounts of sucrose after culturing. The two strains grew well on nystose (Table 2). Lactobacillus delbrueckii and L. gasseri
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(%) 200
(C) 180 160 140 120 100 80 60 40 20 0
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control NRIC 1547
NRIC 0525
xylobiose
JCM 1012
NRIC 1639
NRIC 0220
xylotriose
NRIC 1067
NRIC 1068
JCM 1136
GG
NRIC 0625
NRIC 1028
RT18.4 (possible xylotetraose)
NRIC 1044
NRIC 1071
NRIC 0126
NRIC 1752
NRIC 1047
JCM 1112
NRIC 1776
NRIC 0207
NRIC 1776
NRIC 0207
RT21.2 (possible xylopentaose)
(%) 200
(D) 180 160 140 120 100 80 60 40 20 0 control NRIC 1547
Glucose
NRIC 0525
JCM 1012
NRIC 1639
NRIC 0220
Isomaltose
NRIC 1067
NRIC 1068
JCM 1136
GG
NRIC 0625
NRIC 1028
Isomaltotriose (containing isomers)
NRIC 1044
NRIC 1071
NRIC 0126
NRIC 1752
NRIC 1047
JCM 1112
RT23.8 (possible isomaltotetraose )
Figure 1. Continued.
metabolized 65% and 35% of 1-kestose available, respectively, whereas the ratios of metabolized nystose and fructosylnystose in the strains were within 5%. Large amounts of 1-kestose, nystose and fructosylnystose
remained in the cultured broths of Lactobacillus johnsonii, L. rhamnosus, L. reuteri and W. confusa. Of the 14 actively grown strains with GOSs (O.D.660nm 41 and/or greater than 80% growth ratio to glucose)
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(Tables 2 and 3), L. delbrueckii JCM 1012T, two L. plantarum strains, L. fermentum NRIC 1047 and L. reuteri JCM 1112T metabolized almost all amounts of mono-, di- and trisaccharides in GOSs (Figure 1b). Two L. acidophilus strains metabolized galactosyllactose and monosaccharides or lactose. Lactobacillus johnsonii NRIC 0220T consumed only galactosyllactose, whereas 45% of galactosyllactose available remained after culturing. The remaining six actively grown strains with GOSs, including two L. rhamnosus strains, L. paracasei NRIC 0625 and NRIC 1044, and two L. sakei strains mainly used monosaccharides and lactose, and the residue of unidentified carbohydrate named RT13.2 (an isomer of lactose or galactosyllactose) and galactosyllactose ranged from 63 to 114% and from 61 to 121%, respectively. Carbohydrates in XOSs, which did not enhance the growth of most of the tested strains, were only metabolized well by W. confusa. This strain consumed all amounts of xylobiose and xylotriose and half of unidentified carbohydrate named RT18.4 (possible xylotetraose) available, whereas xylobiose is a disaccharide. A few strains metabolized 15–20% of sugars in XOSs. IMOSs actively enhanced the growth of five of the tested strains (O.D.660nm 41 and/or greater than 80% growth ratio to glucose) (Tables 2 and 3). Two of the five strains (L. paracasei NRIC 0625 and L. reuteri JCM 1112) metabolized large parts of isomaltose, isomaltotriose and unidentified carbohydrate named RT23.8 (possible isomaltotetraose) (Figure 1d). Another three strains: L. rhamnosus JCM 1136T, L. sakei NRIC 1071T, and L. acidophilus NRIC 0525, mainly consumed monoand disaccharides present. Organic acid production from the metabolism of oligosaccharides is summarized in Supplementary Table S1. The tested strains produced large amounts of lactate with small amounts of acetate, except for W. confusa NRIC 0207T, which produced almost equal amounts of lactate and acetate from XOSs. L. fermentum NRIC 1752T produced lactate and acetate with a molar ratio of 2:1 from 1-kestose, whereas other strains, which actively metabolized 1-kestose at least five-fold more lactate than acetate. The amounts of lactate produced were generally consistent with the bacterial growth recorded (Table 2).
Discussion Prebiotics have been applied for several health promotion purposes in infants and adults, and the prebiotics consumed are generally considered to be metabolized by beneficial microbes including lactobacilli. Despite the importance of the metabolism of prebiotics in lactobacilli
both in researches and industrial fields, few studies have compared prebiotic potentials among oligosaccharides using multiple Lactobacillus spp. We herein clearly demonstrated that the fermentation abilities of oligosaccharides varied not only at the species level, but also at the strain level. Moreover, certain oligosaccharides contained large amounts of mono- and disaccharides, resulting in markedly fewer prebiotic properties since these mono- and disaccharides are known to be metabolized by host metabolic systems. The ratio of lactate and acetate produced did not appear to be related to the oligosaccharides used. Among the prebiotics used, 1-kestose and GOSs were the most promising oligosaccharides in terms of growth stimulation of lactobacilli (Tables 2 and 3). However, mono- and disaccharides accounted for 59% of the GOSs used in the present study (Table 1), and six of the 14 strains actively grown with GOSs mainly metabolized these mono- and disaccharides (Figure 1b). GOSs are generally produced from lactose using bacterial or fungal -galactosidase (Rodriguez-Colinas et al. 2011; Song et al. 2013; Yu & O’Sullivan 2014); however, mono- or disaccharides account for approximately half of its components (Shoaf et al. 2006; Quintero et al. 2011). Therefore, the prebiotic activities of GOSs were overestimated in the present study and further studies are needed in order to see their activities following the removal of these simple saccharides. As compared with 1-kestose, few strains metabolized nystose, which is fructosylated 1-kestose. Although a previous study suggested that nystose was specifically metabolized by L. acidophilus in lactobacilli (Endo et al. 2012), our study indicated that nystose metabolism in L. acidophilus was in a strain-dependent manner (Table 3). Two L. paracasei strains grew well with nystose and completely metabolized nystose in FOSs (Table 2 and Figure 1a). -Fructosidase activity was previously suggested to be linked with the nystose metabolism (Endo et al. 2012). The growth of the strains tested was better with FOSs than with nystose, but was less or similar to that with 1-kestose. Carbohydrate profiles after fermentation indicated that three out of the five strains that actively grew with FOSs (Tables 2 and 3), mainly metabolized 1-kestose in FOSs (Figure 1a), suggesting that 1-kestose content was a key for the prebiotic activity in FOSs. Similar results have been reported by Munoz et al. (2012). Interestingly, L. paracasei NRIC 0625 and NRIC 1044 metabolized all oligosaccharides present in FOSs, but left larger amounts of sucrose in the medium than in the original content (Figure 1a). This result suggested that these two strains preferentially metabolized fructose residue of oligosaccharides and excreted sucrose in the medium. Certain dairy LAB are known to
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excrete galactose after the lactose metabolism (Gunnewijk & Poolman 2000). The metabolic profile of lactosucrose was similar to that of 1-kestose, suggesting that both were metabolized in a similar metabolic manner. A previous study suggested that 1-kestose was degraded by the sucrose metabolic system in L. plantarum (Saulnier et al. 2007). XOSs were actively metabolized by only one of the tested 19 strains, W. confusa NRIC 0207T, suggesting that XOSs stimulate only specific intestinal LAB. The bifidogenic activities of XOSs without the stimulation of lactobacilli have been reported in adults (Finegold et al. 2014). NRIC 0207T produced almost equal amounts of lactate and acetate from XOSs (Table S1). This was expected since heterofermentative LAB produce acetate instead of ethanol from pentoses (Zaunmuller et al. 2006). Similar finding, XOSs metabolism in W. confusa/cibaria strains, has been recently reported (Patel et al. 2013). This study also showed cell-associated -xylosidase activity in W. confusa/cibaria strains. W. confusa has been found in various niches, including human and animal guts, fermented food and soils. This specific trait would be beneficial for living in such broad habitats. Among the strains used, L. rhamnosus GG, one of the most widely studied and commercialized probiotic strains, was a non-fermenter of prebiotics. This is consistent with previous studies that have reported poor carbohydrate/oligosaccharide metabolic ability in L. rhamnosus GG (Kaplan & Hutkins 2000; Douillard et al. 2013).
Conclusion The present study clearly demonstrated that the prebiotic potential of each oligosaccharide differs. In GOSs and IMOSs, mono- and disaccharides but not oligosaccharides in their mixtures were mainly metabolized by several LAB strains. Metabolic manner of oligosaccharides are highly strain dependent. The results presented here are important for selection of an adequate combination of probiotics and prebiotics for synbiotic usage. Future researches are needed to study impacts of prebiotics on growth of other bacterial species in gut microbiota, including pathogens and opportunistic pathogens, since such microorganisms would compete with beneficial microbes, including LAB, to obtain nutrition.
Acknowledgements We thank to Ryosuke Nomura, Department of Food and Cosmetic Science, Tokyo University of Agriculture, for his technical assistance.
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Disclosure statement Takumi Tochio, Kenta Konishi and Saki Nakamura are employees of Food Science Co., which is the producer of 1-kestose and XOSs used in this study. The remaining authors declare no conflicts of interest.
Funding information This study was partly funded by Food Science Co.
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