Functional activity of commercial prebiotics

ARTICLE IN PRESS

International Dairy Journal 17 (2007) 770–775 www.elsevier.com/locate/idairyj

Functional activity of commercial prebiotics J. Huebner, R.L. Wehling, R.W. Hutkins Department of Food Science and Technology, 338 Food Industry Complex, University of Nebraska-Lincoln, Lincoln, NE 68583-0919, USA Received 13 June 2006; accepted 3 October 2006

Abstract This work established a quantitative score to describe the extent to which prebiotics (fructooligosaccharides, inulin, and galactooligosaccharides) support selective growth of lactobacilli and bifidobacteria. The prebiotic activity assay was based on the change in cell biomass after 24 h of growth of the probiotic strain on 1% prebiotic or 1% glucose relative to the change in cell biomass of a mixture of enteric strains grown under the same conditions. From the biomass data, a prebiotic activity score was calculated for five lactobacilli and five bifidobacteria. In general, the scores were dependent on the probiotic bacterial strain tested and the type of prebiotic carbohydrate utilized. The highest score was obtained for Lactobacillus paracasei 1195 grown on inulin (1.17), and the lowest score was for Bifidobacterium bifidum NCI grown on galactooligosaccharides (1.24). Results reported here provide a basis for evaluating and optimizing combinations of probiotics and prebiotics for applications as synbiotics. r 2006 Elsevier Ltd. All rights reserved. Keywords: Fructooligosaccharides; Galactooligosaccharides; Inulin; Prebiotics; Probiotics

1. Introduction Fructooligosaccharides (FOS), inulin, galactooligosaccharides, and other related carbohydrates have received considerable attention due to the health benefits they are believed to confer on the host. These so-called prebiotic carbohydrates are defined as ‘‘nondigestible food ingredient(s) that beneficially affects host health by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon’’ (Gibson & Roberfroid, 1995). Among the resident intestinal bacteria that are stimulated by prebiotics are various lactobacilli and bifidobacteria. Some strains of these bacteria are also added to yogurt and other fermented dairy foods in the form of probiotics. Ultimately, it is their ability to metabolize prebiotic sugars, in vivo, that provides for their selective enrichment in the gastrointestinal tract and that result in the formation of lactic, acetic, and other short chain organic acids that may be antagonistic to their intestinal competitors (Wang & Gibson, 1993). Thus, prebiotics, alone or combined with probiotic bacteria in Corresponding author. Tel.: +1 402 472 2820; fax: +1 402 472 1693.

E-mail address: [email protected] (R.W. Hutkins). 0958-6946/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2006.10.006

the form of synbiotics, are now recognized as having the ability to influence and improve the gastrointestinal health of humans (Tuohy, Probert, Smejkal, & Gibson, 2003). Several studies have shown that the ability of lactobacilli and bifidobacteria to ferment prebiotic carbohydrates is both strain and substrate specific (Kaplan & Hutkins, 2000; Schrezenmeir & de Vrese, 2001). In addition, it is not clear which prebiotic carbohydrates are the most suitable substrates for selective growth of specific strains. Recently, several quantitative approaches were devised to determine the functional activity of prebiotics during in vitro fermentation conditions (Olano-Martin, Gibson, & Rastall, 2002; Palframan, Gibson, & Rastall, 2003; Vulevic, Rastall, & Gibson, 2004; Sanz, Gibson, & Rastall, 2005; Sanz, Polemis et al., 2005). In general, these methods provide indices that reflect the relative ability of a given prebiotic to produce specific effects, and are based on measurement of microbial populations, growth rates, substrate assimilation, and/or short-chain fatty acid production. The indices were then used to rank various carbohydrates for their potential to stimulate growth of specific members of a mixed microflora. However, because fermentation of prebiotics is dependent on the bacterial strain, rather than based on the species or genera, it is

ARTICLE IN PRESS J. Huebner et al. / International Dairy Journal 17 (2007) 770–775

important to understand the extent to which metabolism of prebiotics occurs by specific strains of bacteria, especially for those organisms whose intended use are as probiotics. Therefore, the aims of this work were to: (1) establish a prebiotic activity assay based on specific substrates and strains and (2) use this assay to determine the prebiotic activity score of various commercial prebiotics for selected strains of lactobacilli and bifidobacteria. Ultimately, these results may be useful in identifying combinations of probiotics and prebiotics that could be added into dairy and other foods. 2. Materials and methods 2.1. Bacterial strains Bifidobacterium adolescentis 15706, B. breve 15698, B. infantis 17930, B. longum 15708, Lactobacillus acidophilus 33200, L. plantarum 4008 (American Type Culture Collection, Rockville, MD, USA); B. bifidum NCI (Nebraska Cultures, Inc., Walnut Creek, CA, USA); L. acidophilus NCFM (North Carolina State University, Raleigh, NC, USA); L. paracasei 1195 (University of Nebraska, Lincoln, NE, USA); L. plantarum 12006 (Institute for Fermentation, Osaka, Japan); Escherichia coli ECOR 1, E. coli ECOR 2, and E. coli ECOR 22 (E. coli Reference Collection, University of Rochester, Rochester, NY, USA) were used for this study. The specific test strains of lactobacilli and bifidobacteria were selected because they are either already established as probiotics and are used in dairy products or they have potential probiotic properties. The Lactobacillus and Bifidobacterium cultures were maintained at 80 1C in MRS Broth (Difco Laboratories, Sparks, MD, USA) containing 15% (wt/vol) glycerol, and E. coli cultures were maintained at 80 1C in Tryptic Soy Broth (TSB; Difco Laboratories) containing 15% (wt/vol) glycerol. B. bifidum NCI required 0.05% L-cysteine HCl for growth in MRS medium. For the prebiotic activity assays, frozen cultures were streaked onto MRS agar, for the Lactobacillus and Bifidobacterium cultures or Tryptic Soy Agar (TSA), for E. coli, followed by incubation at 37 1C for 24–48 h. Then, one colony from each plate was transferred into 10 mL of MRS broth or TSB and incubated overnight. For the E. coli strains, an additional transfer of 1%

771

(vol/vol) was made from a TSB overnight culture into 10 mL of M9 Minimal Medium broth (Atlas, 1993) and incubated overnight. 2.2. Commercial prebiotics The commercial prebiotics used in this study are described in Table 1. The two fructooligosaccharide (FOS) products, NutraFlora P-95 and Raftilose P95, were obtained from GTC Nutrition (Golden, CO, USA) and Orafti Group (Tienen, Belgium), respectively. The two inulin products, inulin from chicory (referred to as Inulin-S) and Raftiline HP, were obtained from SigmaAldrich (St. Louis, MO, USA) and Orafti Group (Tienen, Belgium), respectively. Oligomate 55, a galactooligosaccharide (GOS)—containing product, was obtained from Yakult Pharmaceutical Ind. Co., Ltd. (Minatoku, Japan). Because the latter material contained appreciable levels of mono- and disaccharides (about 45%), compared with the other commercial products, the Oligomate 55 was purified by size exclusion chromatography. Briefly, a 30% solution was applied to a 98.5  3 cm column containing Sephadex G-10 (Sigma-Aldrich). Fractions were eluted with water and analyzed by refractive index measurement (Bausch & Lomb, Rochester, NY, USA). Portions (5 mL) of the carbohydrate-containing fractions were spotted onto silica gel thin layer chromatography plates (Fisher Scientific, Pittsburgh, PA, USA), along with standard solutions of glucose, galactose, lactose, and the unpurified GOS. The plates were developed in 1-butanol:2-propanol:ddH2O (3:12:4), dried, sprayed with a 50% ethanolic sulfuric acid solution, and then heated at 135 1C to visualize the dark grey, carbohydrate-containing spots. The fractions containing only GOS (corresponding to Rf values of 0.30–0.67) were pooled and lyophilized. 2.3. Prebiotic activity assay Prebiotic activity, as it relates to this study, reflects the ability of a given substrate to support the growth of an organism relative to other organisms and relative to growth on a non-prebiotic substrate, such as glucose. Therefore, carbohydrates have a positive prebiotic activity score if they are (1) metabolized as well as glucose by probiotic

Table 1 Commercial prebiotic carbohydrates Commercial prebiotics

Chemical structurea

Degree polymerization (DP)

Purity (%)

NutraFlora P-95 Raftilose P95 Inulin-S Raftiline HP Purified GOS

Glua1-2-[bFru-1-2]n Glua1-2-[bFru-1-2]n & [bFru1-2]n Glua1-2-[bFru-1-2]n Glua1-2-[bFru-1-2]n Glua1-4-[bGal-1-4]n

2–4 2–7 2–60 423 (average) 2–4

97% FOSb 95% FOSb 499% Inulinb 499% Inulinb 499% GOSc

a

Glu, Glucose; Fru, Fructose; Gal, Galactose; FOS, fructooligosaccharide; GOS, galactooligosaccharide. Based on manufacturer’s analysis. c Approximate composition after purification (see text). b

ARTICLE IN PRESS J. Huebner et al. / International Dairy Journal 17 (2007) 770–775

772

strains and (2) are selectively metabolized by probiotics but not by other intestinal bacteria. The assay was performed by adding 1% (vol/vol) of an overnight culture of each probiotic strain to separate tubes containing MRS Broth with 1% (wt/vol) glucose or 1% (wt/vol) prebiotic. The cultures were incubated at 37 1C under anaerobic conditions (85% N2, 10% CO2, and 5% H2) in an anaerobic chamber (Thermo Forma, Marietta, OH, USA) for Bifidobacterium and L. acidophilus strains and at ambient atmosphere for all other strains. After 0, 24, and 48 h of incubation, samples were enumerated on MRS agar. In addition, overnight cultures of E. coli strains, ECOR 1, ECOR 2, and ECOR 22 were mixed in a 1:1:1 ratio (enteric mixture), then added at 1% (vol/vol) to separate tubes containing M9 broth with 1% (wt/vol) glucose or 1% (wt/vol) prebiotic. The cultures were incubated at 37 1C at ambient atmosphere, and enumerated on TSA after 0, 24, and 48 h of incubation. Each assay was replicated three times. 2.4. Prebiotic activity score The prebiotic activity score was determined using the following equation: Prebiotic activity score  ¼ ðprobiotic log cfu mL1 on the prebiotic at 24 h  probiotic log cfu mL1 on the prebiotic at 0 hÞ= ðprobiotic log cfu mL1 on glucose at 24 h

probiotic log cfu mL1 on the glucose at 0 hÞ   ðenteric log cfu mL1 on the prebiotic at 24 h  enteric log cfu mL1 on the prebiotic at 0 hÞ= ðenteric log cfu mL1 on glucose 24 h

enteric log cfu mL1 on the glucose at 0 hÞ . By definition, substrates with a high prebiotic activity score support good growth of the probiotic bacteria, with cell densities (cfu mL1) comparable with that when grown on glucose. However, the cell densities of the enteric strains grown on the prebiotics should, in theory, be very low relative to growth on glucose. Using this equation, the prebiotic activity score of a particular oligosaccharide can be determined relative to any given strain.

General Linear Model with a two-way factorial experimental design (probiotic and prebiotic treatments are fixed) followed by the Least Squares Means comparison procedure (po0.05). 3. Results 3.1. Growth of lactobacilli, bifidobacteria, and enteric mixture on prebiotic carbohydrates The increases in cell densities for five strains of Lactobacillus and five strains of Bifidobacterium following growth (24 h) on 1% (wt/vol) glucose or 1% (wt/vol) prebiotic sugars are shown in Table 2. Cell densities were also measured after 48 h, but no further increases were observed, except for L. plantarum 12006 grown with purified GOS and B. bifidum NCI grown with Raftilose P95. For a given sugar to have prebiotic activity, that sugar should be metabolized by a test strain as well, or nearly as well, as glucose is metabolized. For most of the test strains, growth (as cfu mL1) on the prebiotics was less than on glucose, with 37 of the 50 possible combinations having a significantly lower (po0.05) increase in cell density on the prebiotic compared to glucose. In contrast, the increase in cell density of L. paracasei 1195 grown on Raftilose P95, Inulin-S, and Raftiline HP were significantly higher (po0.05) than for glucose. B. bifidum NCI had a significantly higher (po0.05) increase in cell density when grown on NutraFlora P-95 and Raftilose P95 than on glucose. Also, the increase in cell densities of L. plantarum 4008 and L. acidophilus 33200 were significantly larger (po0.05) for purified GOS than for glucose. For the remaining strain-prebiotic combinations, there were no significant differences compared with growth on glucose. The other characteristic property of a prebiotic substrate is that it should be selective and not fermented by commensal organisms. Therefore, growth on each prebiotic was also determined for a mixture of three enteric bacteria, E. coli ECOR 1, ECOR 2, and ECOR 22, chosen to represent the enteric portion of the commensal flora. Growth of this enteric mixture on all of the prebiotics was significantly less (po0.05) compared with growth on glucose (Table 2). 3.2. Prebiotic activity score

2.5. Statistical analysis Prebiotic activity assays were repeated three times and the data were analyzed using the Statistical Analysis System (Version 9.1, copyright 2002–2003 by SAS Institute Inc., Cary, NC, USA). Statistical differences among the increase in cell density values for a given strain for each carbohydrate treatment were determined by the General Linear Model followed by the Least Significant Difference comparison of the means (po0.05). Statistical differences among prebiotic activity scores were determined by the

Prebiotic activity scores, shown in Fig. 1, were derived from the cell density values from Table 2. The highest prebiotic activity scores (po0.05) were for L. paracasei 1195 paired with Inulin-S, Raftiline HP, and Raftilose P95 (1.17, 1.10, and 0.99, respectively), followed by L. plantarum 4008, L. acidophilus 33200 and L. acidophilus NCFM grown on purified GOS, and L. acidophilus NCFM grown on Raftilose P95 (0.82, 0.70, 0.66, and 0.58, respectively). In contrast, the lowest scores (po0.05) were for B. bifidum NCI grown with purified GOS and Inulin-S

ARTICLE IN PRESS J. Huebner et al. / International Dairy Journal 17 (2007) 770–775

773

Table 2 Increase in cell density between time 0 and time 24 hb, reported as log10(cfu mL1)7standard deviation, for bacterial cultures grown with various carbohydrates Bacterial Culture

Glucose

L. paracasei 1195 L. plantarum 4008 L. plantarum 12006 L. acidophilus NCFM L. acidophilus 33200 B. breve 15698 B. infantis 17930 B. adolescentis 15706 B. longum 15708 B. bifidum NCI Enteric mixture a

NutraFlora P-95

1.6670.14 2.2770.03 1.8870.12 2.0470.14 1.6670.07 2.1370.08 2.2070.05 2.0870.11 2.1070.02 1.4870.04 1.9470.11

Raftilose P95

Inulin-S

a

1.8870.13 2.1170.11 1.0870.02a 1.9870.03 0.6970.06a 1.9170.04a 1.9370.05a 1.8370.03a 1.9370.03a 1.7270.04a 1.3370.07a

a

2.6370.31 1.5470.07a 0.4670.10a 0.8970.32a 0.4370.07a 1.5970.03a 1.6670.12a 1.6170.14a 1.6770.02a 1.1170.08a 0.8170.04a

2.4870.26 1.9970.07 0.9270.04a 2.2070.11 0.3370.12a 1.9270.06a 1.8470.06a 1.8170.10a 1.8370.02a 1.7070.07a 0.9870.06a

Raftiline HP

Purified GOS

a

0.9670.07a 2.9170.22a 1.5270.06a 2.2970.19 1.9870.07a 1.5570.02a 1.6770.08a 1.6470.02a 1.5270.08a 1.1470.21a 0.9070.04a

2.4870.27 1.8070.39a 0.4170.10a 0.9270.02a 0.3370.02a 1.5970.07a 1.5670.09a 1.5770.05a 1.6170.10a 0.3970.12a 0.7770.01a

Mean value (7standard deviation) for prebiotic-grown cells differ significantly (po0.05) from glucose-grown cells for the specific bacterial culture. Increase in cell density between time 0 and time 48 h reported for L. plantarum 12006 grown on GOS and B. bifidum NCI grown on Raftilose P95.

b

1.50 NutraFlora P-95 Raftilose P95 Inulin-S

1.00

Raftiline HP

0.50

-1.50

B. bifidum NCI

B. longum 15708

B. adolescentis 15706

B. infantis 17930

B. breve 15698

L. acidophilus 33200

L. acidophilus NCFM

-1.00

L. plantarum 12006

-0.50

L. plantarum 4008

0.00 L. paracasei 1195

Prebiotic activity score

Purified GOS

Bacteria

Fig. 1. Prebiotic activity scores of various bacteria grown on commercial prebiotics.

(1.24 and 1.17, respectively), followed by L. acidophilus 33200 grown on Raftilose P95 and B. bifidum NCI grown on Raftiline HP (0.70 and 0.67, respectively). Also, L. plantarum 12006 and L. acidophilus 33200 had prebiotic activity scores below zero when grown on all the prebiotics, except purified GOS. A low or negative prebiotic activity score was obtained if the test strain grew less well (based on cell densities) on the prebiotic compared with that on glucose and/or had less growth on the prebiotic than the growth of the enteric mixture on the prebiotic carbohydrate. Strains within the same species had significantly different prebiotic activity scores for the same prebiotic. For example, L. plantarum 4008 had significantly higher scores (po0.05) compared with L. plantarum 12006 for all of the prebiotics tested. In addition, L. acidophilus 33200 had significantly lower scores (po0.05) compared with L.

acidophilus NCFM for all of the fructooligosaccharides and inulin type prebiotics, but a similar score for purified GOS.

4. Discussion Prebiotic carbohydrates are, by definition, metabolized only by selected members of the gastrointestinal tract. Accordingly, these sugars have the ability to influence the population of the gastrointestinal tract due to their selective utilization. Organisms that rapidly ferment prebiotic sugars are enriched, presumably at the expense of those that do not. The effectiveness of a prebiotic depends, therefore on its ability to be selectively fermented by and to support growth of specific targeted organisms. The goal of this study, therefore, was to quantify the extent to which

ARTICLE IN PRESS 774

J. Huebner et al. / International Dairy Journal 17 (2007) 770–775

prebiotic sugars express this activity using selected strains of Lactobacillus and Bifidobacterium. In vitro studies by Olano-Martin et al. (2002), Palframan et al. (2003), Vulevic et al. (2004), Sanz, Gibson et al. (2005), and Sanz, Polemis et al. (2005) have also developed equations to quantitatively evaluate the ability of carbohydrates to have prebiotic effects. In these studies, prebiotic indices were based on changes in bacterial populations (of specific genera), substrate assimilation, growth rates, and/ or short chain fatty acid production, with each characteristic assigned a positive or negative effect on the calculated index. Unlike previous studies, our method to assess prebiotic activity evaluates the combination of a prebiotic with specific strains of putative probiotic bacteria. Within the equation, the change in growth of the test strain on the prebiotic is compared with the growth on glucose and to the growth of a mixture of commensal bacteria on the prebiotic and glucose. This method is comparably more simple than previous methods because fecal samples are not required. In addition, it is a relatively quick way to evaluate a prebiotic’s ability to be utilized by specific strains of bacteria. As might be expected, given the known metabolic diversity of the lactobacilli, there was considerable variation in prebiotic activity scores for the different prebiotics utilized by a single probiotic strain. For example, L. paracasei 1195 had significantly higher scores (po0.05) for the inulin type prebiotics and Raftilose P95 compared with purified GOS. In contrast, L. plantarum 12006 and L. acidophilus 33200 had significantly higher scores (po0.05) for GOS compared with the other commercial prebiotics. In fact, it was observed that even strains within a single species (e.g., L. plantarum strains 4008 and 12006) had significantly different prebiotic activity scores, indicating that differences in the metabolic capacity of related strains apparently exist. Utilization of prebiotics by lactic acid and related bacteria requires the presence of specific hydrolysis and transport systems for the particular prebiotic (Barrangou, Altermann, Hutkins, Cano, & Klaenhammer, 2003; Gopal, Sullivan, & Smart, 2001; Imamura, Hisamitsu, & Kobashi, 1994; Kaplan & Hutkins, 2003; Muramatsu, Onodera, Kikuchi, & Shiomi, 1992, 1994; Perrin, Warchol, Grill, & Schneider, 2001; Rabiu, Jay, Gibson, & Rastall, 2001). Therefore, genes coding for these metabolic systems may be present or absent in the different strains, resulting in varied prebiotic activity scores. In contrast to the metabolic diversity observed for the lactobacilli, the prebiotic activity scores for the bifidobacteria on the different prebiotics varied relatively little (except for B. bifidum), with most scores ranging from 0.2 to 0.4. In previous studies using mixed flora samples (Palframan et al., 2003), the highest prebiotic index scores were generally obtained for GOS (as well as pectic, isomaltooligosaccharides and soybean oligosaccharides), compared with fructooligosaccharides and inulin. The prebiotic activity scores reported in this study reflect the extent to which a given carbohydrate would promote

selective growth of specific organisms in the presence of competitors unable to utilize that particular carbohydrate. Thus, these scores provide a rational basis for identifying synbiotics for incorporation into dairy and other foods (Chow, 2002; Fooks & Gibson, 2002; Rastall & Maitin, 2002). However, it is important to recognize two important factors. First, in the gastrointestinal tract, commensal organisms likely exist that, unlike the E. coli strains used in this study, will have some ability to utilize prebiotic carbohydrates (Hartemink, Van Laere, & Rombouts, 1997). Indeed, despite the definition of prebiotics as being fermented by a ‘‘limited number’’ of colonic bacteria, it now appears that Bacteroides and other resident members of intestinal microflora have the metabolic capacity to metabolize these substrates (Van der Meulen, Makras, Verbrugghe, Adriany, & De Vuyst, 2006). In addition, the specific means by which metabolism of prebiotic carbohydrates occurs is also relevant with respect to their prebiotic activity. If, for example, a particular organism initiates metabolism of an oligosaccharide via extracellular hydrolysis, the products (mono- or disaccharides) that are released may then ‘‘cross-feed’’ other organisms. In pure culture, the sugar would appear to be prebiotic and would have a prebiotic activity score, but in a mixed culture environment, even ‘‘nonfermentors’’ would still have access to fermentable hydrolysis products. 5. Conclusions Quantitative prebiotic activity scores that describe the extent to which five prebiotics support selective growth of five lactobacilli and five bifidobacteria were determined. The highest score was obtained for L. paracasei 1195 grown on inulin, while the lowest score was for B. bifidum NCI grown on galactooligosaccharides. This study provides a basis for the evaluation of combinations of probiotic and prebiotic ingredients for applications as synbiotics in dairy and other foods. Acknowledgements This work was supported by a grant through the United States Department of Agriculture Cooperative State Research, Education, and Extension Service (# 200435503-14118). The authors would like to thank GTC Nutrition, Orafti Group, and Yakult for their generous donation of commercial prebiotics. References Atlas, R. M. (1993). Handbook of microbiological media. Boca Raton, CA, USA: CRC Press. Barrangou, R., Altermann, E., Hutkins, R., Cano, R., & Klaenhammer, T. R. (2003). Functional and comparative genomic analyses of an operon involved in fructooligosaccharide utilization by Lactobacillus acidophilus. Proceedings of the National Academy of Sciences, 100, 8957–8962.

ARTICLE IN PRESS J. Huebner et al. / International Dairy Journal 17 (2007) 770–775 Chow, J. (2002). Probiotics and prebiotics: A brief overview. Journal of Renal Nutrition, 12, 76–86. Fooks, L. J., & Gibson, G. R. (2002). Probiotics as modulators of the gut flora. British Journal of Nutrition, 88, S39–S49. Gibson, G. R., & Roberfroid, M. B. (1995). Dietary modulation of the human colonic microbiota: Introducing the concept of prebiotics. Journal of Nutrition, 125, 1401–1412. Gopal, P. K., Sullivan, P. A., & Smart, J. B. (2001). Utilisation of galactooligosaccharides as selective substrates for growth by lactic acid bacteria including Bifidobacterium lactis DR10 and Lactobacillus rhamnosis DR20. International Dairy Journal, 11, 19–25. Hartemink, R., Van Laere, K. M. J., & Rombouts, F. M. (1997). Growth of enterobacteria on fructo-oligosaccharides. Journal of Applied Microbiology, 83, 367–374. Imamura, L., Hisamitsu, K., & Kobashi, K. (1994). Purification and characterization of b-fructofuranosidase from Bifidobacterium infantis. Biological & Pharmaceutical Bulletin, 17, 596–602. Kaplan, H., & Hutkins, R. W. (2000). Fermentation of fructooligosaccharies by lactic acid bacteria and bifidobacteria. Applied and Environmental Microbiology, 66, 2682–2684. Kaplan, H., & Hutkins, R. W. (2003). Metabolism of fructooligosaccharides by Lactobacillus paracasei 1195. Applied and Environmental Microbiology, 69, 2217–2222. Muramatsu, K., Onodera, S., Kikuchi, M., & Shiomi, N. (1992). The production of b-fructofuranosidase from Bifidobacterium ssp. Bioscience, Biotechnology, and Biochemistry, 56, 1451–1454. Muramatsu, K., Onodera, S., Kikuchi, M., & Shiomi, N. (1994). Substrate specificity and subsite affinities of b-fructofuranosidase from Bifidobacterium adolescentis G1. Bioscience, Biotechnology, and Biochemistry, 58, 1642–1645. Olano-Martin, E., Gibson, G. R., & Rastall, R. A. (2002). Comparison of the in vitro bifidogenic properties of pectins and pectic-oligosacchrides. Journal of Applied Microbiology, 93, 505–511. Palframan, R., Gibson, G. R., & Rastall, R. A. (2003). Development of a quantitative tool for the comparison of the prebiotic effect of dietary oligosaccharides. Letters in Applied Microbiology, 37, 281–284.

775

Perrin, S., Warchol, M., Grill, J. P., & Schneider, F. (2001). Fermentations of fructo-oligosaccharides and their components by Bifidobacterium infantis ATCC 15697 on batch culture in semi-synthetic medium. Journal of Applied Microbiology, 90, 859–865. Rabiu, B. A., Jay, A. J., Gibson, G. R., & Rastall, R. A. (2001). Synthesis and fermentation properties of novel galacto-oligosaccharides by bgalactosidases from Bifidobacterium species. Applied and Environmental Microbiology, 67, 2526–2530. Rastall, R. A., & Maitin, V. (2002). Prebiotics and synbiotics: Towards the next generation. Current Opinion in Biotechnology, 13, 490–496. 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. Sanz, M. L., Polemis, N., Morales, V., Corzo, N., Drakoularakou, A., Gibson, G. R., et al. (2005). In vitro investigation into the potential prebiotic activity of honey oligosaccharides. Journal of Agricultural and Food Chemistry, 53, 2914–2921. Schrezenmeir, J., & de Vrese, M. (2001). Probiotics, prebiotics, and synbiotics-approaching a definition. American Journal of Clinical Nutrition, 73, 361S–364S. Tuohy, K. M., Probert, H. M., Smejkal, C. W., & Gibson, G. R. (2003). Using probiotics and prebiotics to improve gut health. Drug Discovery Today, 8, 692–700. Van der Meulen, R., Makras, L., Verbrugghe, K., Adriany, T., & De Vuyst, L. (2006). In vitro kinetic analysis of oligofructose consumption by Bacteroides and Bifidobacterium spp. indicates different degradation mechanisms. Applied and Environmental Microbiology, 72, 1006–1012. Vulevic, J., Rastall, R. A., & Gibson, G. R. (2004). Developing a quantitative approach for determining the in vitro prebiotic potential of dietary oligosaccharides. FEMS Microbiology Letters, 236, 153–159. Wang, X., & Gibson, G. R. (1993). Effects of the in vitro fermentation of oligofructose and inulin by bacteria growing in the human large intestine. Journal of Applied Bacteriology, 75, 373–380.