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Scientific
Publications - Work Done by Microbiology Reader
Journal of Applied Microbiology, 2002;93(3):505-511 Comparison of the in vitro bifidogenic properties of pectins and pectic-oligosaccharidesE. Olano-Martin, G.R. Gibson and R.A. Rastall
ABSTRACT Aims: To compare the in vitro fermentation properties of
pectins and oligosaccharides derived from them in pure and mixed faecal
cultures.
INTRODUCTION The human gut microflora is affected by many factors such as age, drug therapy, diet, host physiology, peristalsis, local immunity and in situ bacterial metabolism(Berg 1996). However, diet is probably the most significant factor determining the type of gut flora that develops since foodstuffs provide the main nutrient sources for colonic bacteria. This has led to the concept of prebiotics (Gibson and Roberfroid 1995) where selectively fermented carbohydrates result in elevated levels of bifdobacteria and lactobacilli in the colon. These genera are viewed as being positive for host health and welfare. All current prebiotics are nondigestible oligosaccharides (NDO). NDO are resistant to the digestive process but, to be considered as prebiotics, they have to exert a specific fermentation in the gut. Examples of prebiotic NDO include various fructans (i.e. oligofructose, neosugar), galacto-oligosaccharides and lactulose all of which are bifidogenic (Wang and Gibson 1993; Delzenne and Roberfroid 1994; Gibson and Wang 1994; Buddington et al. 1996; Kleessen et al. 1997). Crittenden and Playne (1996) have reviewed the production, properties and applications of food-grade oligosaccharides. Pectin is considered a soluble dietary fibre and exerts physiological effects on the gastrointestinal tract such as delayed gastric emptying (Schwartz et al. 1982; Flourine et al. 1985), reduced transit time (Spiller et al. 1980) and reduced glucose absorption (Jenkins et al. 1977). These effects are mainly due to its gel forming and water holding capacity (Roberfroid 1993). Isolated citrus pectin is also known to have hypocholesterolemic effects in humans in short-term trials (Kay and Truswell 1977). Other actions that have been attributed to the ingestion of pectin are interaction with medicines and interaction with the intestinal metabolism of ions (Seyrig et al. 1983). Like other dietary fibres, pectin reaches intact the large intestine (Englyst and Cummings 1987) where it is extensively fermented by the gut microflora, although it has never been reported to be prebiotic. Highly esterified pectins are more slowly degraded than less esterified forms, with fractions of pectin with a degree of esterification (DE) of 5% still being present after 24 h incubation with faecal bacteria (Dongowski and Lorenz 1998). In terms of the rapidly growing functional food industry the use of pectic-oligosaccharides as prebiotics has not yet been investigated. In vitro fermentations of other plant polysaccharides such as xyloglucan have shown that some intestinal bacteria, which were not able to grow on the polysaccharide, were able to completely ferment the oligosaccharides (Hartemink et al. 1996). This difference in fermentation pattern between some polysaccharides and oligosaccharides has been also reported for dextran and oligodextrans (Olano-Martin et al. 2000) and various glucose oligosaccharides of different degrees of polymerization (Rycroft et al. 2001). The aim of this paper was therefore to study the bifidogenic properties of pectins and pectic-oligosaccharides via their in vitro fermentation by human gut bacteria (pure and mixed cultures).
MATERIALS AND METHODS General reagents All chemicals were purchased from Sigma (Poole, Dorset, UK). Microbiological culture media and media additions were obtained from Oxoid (Basingstoke, Hampshire, UK). High methylated citrus pectin (HMP) with a degree of methylation of 66% and low methylated apple pectin (LMP) with a degree of methylation of 8% were obtained from Fluka (Poole, Dorset, UK). Pectic oligosaccharides Pectic-oligosaccharides were manufactured according to Olano-Martin et al. (2001). Two different pectic-oligosaccharides were used in the experiments: POS I, obtained from the controlled hydrolysis of high methylated pectin, and POS II, obtained from the controlled hydrolysis of low methylated pectin. Pure culture growth experiments Organisms. The following bacteria were isolated from faeces and further characterized by sequencing specific regions of 16S-rRNA (McCartney and Gibson 1998): Bifidobacterium angulatum, Bif. lactis, Bif. infantis, Bif. adolescentis, Bif. pseudolongum, Lactobacillus casei shirota, L.pentosus, L. acidophilus, Bacteroides fragilis, Bact. ovatus, Bact. distasonis, Bact. thetaiotaomicron, Clostridium perfringens, C. inocuum and C. ramosum. L. lactis subsp cremoris LC5, L. plantarum 0207 and Bif. bifidum Bb12 are commercial probiotics obtained from Rhodia Texel (Woodley, Cheshire, UK) Enterococcus faecalis and Escherichia coli were obtained from the culture collection of the School of Food Biosciences, University of Reading (UK). Growth experiments.The basal culture medium consisted of (g l-1): peptone water, 2·0; yeast extract, 2·0; NaCl, 0·1; K2HPO4, 0·04; KH2PO4, 0·04; MgSO4·7H2O, 0·01; CaCl2· 2H2O, 0·01; NaHCO3, 2·0; cysteine HCl, 0·5; bile salts, 0·5. The following liquid additions were made (ml l-1): Tween 80, 2; vitamin K1, 0·01; haemin solution, 1. The organisms were grown in serum tubes containing 10 ml of prereduced basal medium with 1% (w/v) glucose, at 37°C and anaerobic conditions. Subculture was done every 5 days and the purity checked by plating on Wilkins-Chalgren agar and Gram staining. Pre-reduced basal media (0·9 ml) containing 1% (w/v) of carbohydrate (HMP, LMP, POS I, POS II) were inoculated with 100 µl of the above bacterial suspension. A multiwell plate was then inoculated with 300 µl of this culture and placed in an automatic spectrophotometer (Bioscreen A system, Labsystem, Cambridge, UK). Growth was measured turbidometrically every 20 min at 540 nm up to 48 h. Temperature was maintained at 37°C. An anaerobic environment was achieved by carrying out all the manipulations inside an anaerobic cabinet (10 : 10 : 80; H2:CO2:N2; Don Whitley Scientific, West Yorkshire, UK) and by airtight sealing of the multiwell plates before transfer to the Bioscreen. Growth rates.Growth of each microorganism was calibrated in order to relate absorbance with cell numbers. The specific growth rate was then calculated using the following equation: µ = (lnXt2 - lnXt1)/(t2 - t1), where lnXt1 and lnXt2 are cell number at time 1 (t1) and time 2 (t2), respectively, during the exponential growth phase and µ is the specific growth rate (µh -1). PH-controlled faecal bacterial batch culture fermentations Water jacketed fermenters (150 ml) were filled with a prereduced culture medium and inoculated with a faecal slurry to give a final concentration of 10% (w/v). Carbohydrates (HMP, LMP, POS I, POS II) were added separately before inoculation, to give a final concentration of 1% (w/v). Faecal slurries were prepared by homogenizing freshly voided faeces in anoxic sodium phosphate buffer (0·1 mmol/l), pH 7·0 (Wang and Gibson 1993). Each vessel was magnetically stirred and maintained under anaerobic conditions by continuous sparging with oxygen-free nitrogen. Temperature (37°C) and pH (6·8) were controlled automatically. Liquid samples (5 ml) were removed from each fermenter at 0, 8, 24 and 48 h. Six experiments were carried out for each carbon source. Four healthy volunteers (age 24-30 years) with a Western diet who had not been prescribed antibiotics for at least 3 months prior to the study were used as donors. Enumeration of bacteria in mixed cultures Bacteria were counted using fluorescent in situ hybridization (FISH) as described by Rycroft et al. (2001). Samples were fixed overnight at 4°C with 4% (w/v) filtered paraformaldehyde (pH 7·2) in a ratio of 1 : 3 (v/v). Samples were washed twice with filtered phosphate buffered saline (pH 7·0, 0·1 m), and stored at - 20°C in PBS/ethanol until further processing. When required for counting, the fixed cells were hybridized with the appropriate genus-specific probes for differential bacterial counts, or 4',6-diamidino-2-phenylindole (DAPI) for total cell counts. The probes used for each bacterial group have been previously designed and validated by various authors: Bifidobacterium, Langendijk et al. (1995); Bacteroides, Manz et al. (1996); Lactobacillus/Enterococcus, Harmsen et al. (1996) and Clostridium subgrp. histolyticum, Franks et al. (1998). Statistics Anova single factor analysis tool (Windows Excel 2000, Microsoft Corporation) was used to perform a simple analysis of variance to test the hypothesis that means from two or more treatments are equal. When differences in the treatments were found, a two-sample student's t-test was carried out between the samples. The differences were considered significant when P< 0·05.
RESULTS Pure culture growth experiments Selected gut bacteria were grown on HMP, LMP, POS I and POS II and their specific growth rates are shown in Table 1. In general, low methylated sugars were a better substrate for bacterial growth than high methylated sugars. Bif. angulatum, Bif. infantis and Bif. adolescentis were not able to grow on HMP, but they grew on the corresponding oligosaccharide. B. thetaiotamicron and C. ramosum could grow on HMP but not on POS I. However, other beneficial bacteria often used as probiotics like B. lactis Bb12, L.plantarum and L. pentosus did not show any growth after 48 h incubation on POS I, but they did on HMP. C. inocuum failed to grow on POS II, although it had a very high growth rate on LMP (13·92 h -1). Also the growth rate of C. perfringens was lower on POS II than on LMP, whereas Bif. pseudolongum and Bif. adolescentis had higher growth rates on POS II than on LMP. Lactobacilli, however, displayed higher growth rates on LMP as a carbon source. pH-controlled mixed faecal bacteria batch culture fermentations Counts of selected bacterial genera from mixed culture are shown in Table 2. Bacterial groups studied in mixed culture experiments were chosen to represent the most predominant groups present in the human colon, hence Bacteroides, Bifidobacterium, clostridia and lactic acid bacteria were enumerated. Generally, high levels of total bacteria were maintained in the four batch systems. The largest changes in bacterial populations took place during the first eight hours of fermentation and significant differences between the sugars were also observed at this point. For the bifidobacteria, differences between HMP and POS II after eight hours were significant, as well as the differences between POS I and POS II. Differences in the number of lactic acid bacteria were observed between HMP and POS II. HMP and LMP varied in the number of bacteroides with POS I and POS II. Fermentation of pectins (HMP and LMP) proceeded more slowly and elevated levels of bifidobacteria and Bacteroides could still be observed at 48 h. The pectic-oligosaccharides were more rapidly fermented and were depleted by 24 h. Both of the pectins and the pectic-oligosaccharides significantly increased (P < 0·01) the number of bifidobacteria with increases of 0·56-0·68 log recorded. The number of lactobacilli increased only during the fermentation of pectic-oligosaccharides, although this increase was not statistically significant. In addition, pectic-oligosaccharides did not sustain growth of clostridia and they maintained numbers of Bacteroides at the inoculum level. LMP significantly increased (P < 0·01) the number of Bacteroides. To be able to compare the prebiotic efficiency of the different sugars, a prebiotic index (PI) was calculated at times 8, 24 and 48 h. The PI for a time point tx was obtained using the following equation: [ ] The results are shown in Table 3. The PI for previously published (Wang and Gibson 1993) pH controlled fermentation data in similar conditions (12 h, pH 7·0) was also calculated. The PI12 for pectin was 0·033, which agrees with the PI found in this study for LMP after 24 h. The PI12 for oligofructose, known to be a prebiotic (Wang and Gibson 1993; Gibson and Wang 1994; Gibson et al. 1995; Buddington et al. 1996; Kleessen et al. 1997; Kruse et al. 1999), was 0·118. Compared to this value none of the sugars tested here were found to be a particularly effective prebiotic, but in all cases oligosaccharides had a higher PI than their parent polysaccharides. As the fermentation progressed, the PI for the oligosaccharides improved up to 48 h. The PI for the low methylated pectin decreased with time from 0·066 to 0·012, and the PI for HMP was negative after 24 h. An effect of the degree of methylation on the fermentation properties of the carbohydrates can also be seen from the PI results. LMP had a higher PI than HMP and the PI for POS II was higher than for POS I, indicating greater fermentation selectivity with lower degrees of methylation.
TABLES Table 1 Specific growth rates of selected gut bacteria on 1% high methylated pectin (HMP), 1% low meth...
DISCUSSION The utilization of HMP, LMP, POS I and POS II as carbon sources by pure cultures of selected gut bacteria was investigated. The various bacteria were chosen to cover the main constituents of the gut microflora although particular attention was given to bifidobacteria, lactobacilli, clostridia and Bacteroides. There was a clear influence of the degree of esterification (DE) on the fermentations, with highly methylated carbon sources giving lower growth rates than the lower methylated ones. This is in agreement with Dongowski and Lorenz (1998) who reported that pectin with a DE of 95% was degraded more slowly than the low-esterified substrate with a DE of 35%. They found that highly esterified pectin and oligogalacturonic acid fractions were still present after 24 h of incubation with gut microflora whereas there was no trace of the low esterified samples. In the human colon, pectinolytic enzymes have been isolated from Bacteroides sp. (Jensen and Canale-Parola 1985; MacCarthy et al. 1985) and the Clostridium butyricum-Clostridium beijerinckii group (Matsuura 1991). The enzymes involved in the breakdown of pectin are mainly endo-type pectate lyases (EC 4·2.2·2), giving unsaturated pectic-oligosaccharide as the product. In this study, all of the Bacteroides and Clostridium species could grow on pectins, but some of them failed to do so on the derived oligosaccharides. On the other hand, most of the bifidobacteria grew better on the oligosaccharides than the pectins. This indicated that the oligosaccharides might have a prebiotic effect by failing to sustain the growth of potentially pathogenic bacteria and promoting the growth of bifidobacteria. In pure cultures, most microorganisms adapt to the utilization of their carbon sources (Roberfroid 2001), but these studies are of limited use unless the results are supported by the results of mixed cultures. The fermentation process in the gut is a sequence of different metabolic pathways carried out by bacteria growing in the colon. Each species has its own specialized ecological niche and frequently, the end products of one species are used as substrates by other. In this way, some microorganisms benefit from substrates they are not able to ferment. (Gibson and Roberfroid 1995). Therefore pH-controlled batch culture fermentations with mixed faecal bacteria were carried out, in order to evaluate the potential use of pectic-oligosaccharides as prebiotics. Pectins are metabolized by many species of the human gut microflora. Isolates reportedly obtained after pectin fermentation include Bacteroides distasonis, Bacteroides ovatus and Bifidobacterium infantis (Bayliss and Houston 1984). These authors observed no selectivity of pectin fermentation towards bifidobacteria: all of the bacteria analysed grew well with both high methylated and low methylated pectin. In addition, acidic oligogalacturonides in the form of a carrot soup, have been fed to animals (Guggenbichler et al. 1997; Jugl et al. 2000). In both cases a reduction in the incidence of diarrhoea and gut infections in the treated animals was observed. Guggenbichler et al. (2000) also performed autopsies on the animals to investigate their intestinal flora. A substantial decrease in the number of E.coli was found to occur in the animals fed with the acidic oligogalacturonides, although no other bacterial genera were enumerated. In the present study, when oligosaccharides were used as carbon sources the number of bifidobacteria increased but the numbers of Bacteroides and clostridia were maintained. This makes POS I and POS II better candidate prebiotics than the pectins, as reflected in the PI score for each carbon source. Importantly, in all cases a higher PI was seen for the oligosaccharide than for the parent pectin. This effect of size on prebiotic potential has been also seen in xylo-oligosaccharides (Hartemink et al. 1996), oligodextrans (Olano-Martin et al. 2000) and glucose-based oligosaccharide (Rycroftet al. 2001). As the fermentation progressed the PI for the pectins reduced, whereas it increased for the oligosaccharides. Also, higher PI values were obtained with low methylated sugars, since they are more easily fermented. The prebiotic effect of the oligosaccharides was, however, low compared to others reported in the literature (Wang and Gibson 1993; Gibson et al. 1995; Kruse et al. 1999).
ACKNOWLEDGEMENTS The authors would like to acknowledge Uniq Convenience Foods for their financial support of this research.
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