Synergism of GOS and polyfructose

Abstract

The present invention relates to the field of prebiotics. Provided are uses for compositions comprising synergistically effective amounts of polyfructose and galactooligosaccharides (GOS).

Claims

1. Use of galacto-oligosaccharides and polyfructose for the manufacture of a composition for the treatment or prevention of abdominal bloating, gas formation, abdominal pain and/or flatulence.

2. Use of galacto-oligosaccharides and polyfructose for the manufacture of a composition for the treatment or prevention of allergy, eczema or atopic diseases

3. Use of galacto-oligosaccharides and polyfructose for the manufacture of a composition for the relaxation of contractions of the colon, preferably the tonic and/or phasic contractions.

4. Use of galacto-oligosaccharides and polyfructose for the manufacture of a composition for the treatment or prevention of irritable bowel syndrome or inflammatory bowel disease.

5. Use of galacto-oligosaccharides and polyfructose for the manufacture of a composition for the increase of intestinal barrier functioning and/or mucus production of the large intestine.

6. Use according to any one of the preceding claims, wherein intestinal and/or faecal short chain fatty acid levels are increased, intestinal and/or faecal acetate levels relative to total short chain fatty acids are increased, intestinal lactate levels are increased, intestinal and/or faecal pH is decreased, the sum of intestinal and/or faecal butyrate, isobutyrate, valerate and/or isovalerate relative to total short chain fatty acids is decreased and/or intestinal gas production is decreased, without having a significant effect on the total and/or relative number of intestinal bifidobacteria.

7. Use according to any one of the preceding claims, wherein the amount of galacto-oligosaccharides:polyfructose has a ratio of 3:97 to 97:3, preferably 5:95 to 95:5, more preferably 90:10 to 45:55.

8. Use according to any one of the preceding claims, wherein the galacto-oligosaccharide is transgalacto-oligosaccharide and/or the polyfructose is inulin.

9. Use according to any one of the preceding claims, wherein the polyfructose is inulin with an average degree of polymerisation of 20 or above.

10. Use according any one of the preceding claims, wherein said composition is suitable for decreasing the faecal and/or intestinal pH to below pH 6, preferably to pH 5.75 or less, and wherein said composition is suitable for oral administration to an infant.

11. Use according to any one of the preceding claims, wherein said composition is suitable for increasing the intestinal and/or faecal lactate to above about 10 mmol/kg faeces, and wherein said composition is suitable for oral administration to an infant.

12. Use according to any of the preceding claims, wherein said composition is suitable for increasing the intestinal and/or faecal acetate to above about 85% of the total SCFA.

13. Use according to any of the preceding claims, wherein said composition is suitable for decreasing the sum of intestinal and/or faecal butyrate, isobutyrate, valerate and isovalerate to below 7% of total SCFA.

14. Use according to any of the preceding claims for promoting the production of short chain fatty acids at the beginning, middle and end of the colon.

15. Use according to any one of the preceding claims, wherein the composition is a food composition or food supplement composition, preferably an infant nutrition, more preferably an infant or follow-on formula.

Description

FIGURE LEGENDS

[0096] FIG. 1

[0097] Formation of acetate, propionate and butyrate after 48 h in vitro fermentation of TOS, Inulin, a mixture of TOS/Inulin 9/1 and a mixture of oligofructose and inulin 1/1 by fresh faeces obtained from babies.

[0098] FIG. 2

[0099] Relative amounts of acetate, propionate and butyrate formed after 48 h in vitro fermentation of TOS, Inulin, a mixture of TOS/Inulin 9/1 and a mixture of oligofructose and inulin 1/1 by fresh faeces obtained from babies. The sum of acetate plus propionate plus butyrate formed was set at 100% for each of the fibre tested.

[0100] FIG. 3

[0101] Formation of gas after 48 h in vitro fermentation of TOS, Inulin, a mixture of TOS/Inulin 9/1 and a mixture of oligofructose and inulin 1/1 by fresh faeces obtained from babies. The amount of gas formed (ml was related to the amount of total SCFA formed (in mmol per g fibre)

[0102] FIG. 4

[0103] Effects of mixtures of SCFA (acteate/propionate/butyrate in a ratio of 90/5/5 on a molar basis) and of L-lactate.

[0104] FIG. 4A: Effect of 0, 0.1, 0.5, 1 and 4 mM of the SCFA mixture or L-lactate on muc-2 expression in a CCD18/T84 co-culture (n=6). Error bars show the SEM. The increase at 1 and 4 mM SCFA mixture and 0.5, 1 and 4 mM L-lactate is statistically significant.

[0105] FIG. 4B: Effect of 0, 50, 100, 250 and 500 μM SCFA mixture or L-lactate on the spontaneous PGE1 and PGE2 response in CCD18 cells (n=7). Error bars show the SEM. The increase of PGE1 at 100, 250 and 500 μM SCFA mixture and 100 μM L-lactate is statistically significant. The increase of PGE2 at 100, 250 and 500 μM SCFA mixture and 250 μM L-lactate is statistically significant (P<0.05)

[0106] FIG. 5

[0107] Effects of sodium acetate and sodium L-lactate on the spontaneous contraction in the distal and proximal part of the colon. The blanc (set to a tension of 1 g) is 0%. The tension after addition of 40 mM KCl was set to 100%.

EXAMPLES

Example 1

In Vitro Fermentation Studies Show Synergistic Effects on Fermentation Patterns

1.1 Materials and Methods

Microorganisms

[0108] Microorganisms were obtained from fresh faeces from bottle fed babies. Fresh faecal material from babies ranging 1 to 4 month of age was pooled and put into preservative medium within 2 h.

Compositions/Substrate

[0109] As substrate either prebiotics (TOS; TOS (from VivinalGOS, Borculo Domo Ingredients, The Netherlands) and inulin (raftilinHP from Orafti, Belgium) mixture in a 9/1 (w/w) ratio; inulin; oligofructose and inulin mixture in a 1/1 (w/w) ratio, or none (blanc) was used.

Media

[0110] McBain & MacFarlane medium: Buffered peptone water 3.0 g/l, yeast extract 2.5 g/l. mucin (brush borders) 0.8 g/l, tryptone 3.0 g/l, L-Cysteine-HCl 0.4 g/l, bile salts 0.05 g/l, K.sub.2HPO.sub.4.3H.sub.2O 2.6 g/l, NaHCO.sub.3 0.2 g/l, NaCl 4.5 g/l, MgSO.sub.4.7H.sub.2O 0.5 g/l, CaCl.sub.2) 0.228 g/l, FeSO.sub.4.7H.sub.2O 0.005 g/l. 500 ml Scott bottles were filled with the medium and sterilised for 15 minutes at 121° C.

[0111] Buffered medium: K.sub.2HPO.sub.4.3H.sub.2O 2.6 g/l, NaHCO.sub.3 0.2 g/l, NaCl 4.5 g/l, MgSO.sub.4.7H.sub.2O, 0.5 g/l, CaCl.sub.2) 0.228 g/l, FeSO.sub.4.7H.sub.2O 0.005 g/l. pH was adjusted to 6.3±0.1 with K.sub.2HPO.sub.4 or NaHCO.sub.3. 500 ml Scott bottles were filled with the medium and sterilised for 15 minutes at 121° C.

[0112] Preservative medium: Buffered peptone 20.0 g/l, L-Cysteine-HCl 0.5 g/l, Sodium thioglycollate 0.5 g/l, resazurine tablet 1 per litre. pH was adjusted to 6.7±0.1 with 1 M NaOH or HCl. The medium was boiled in microwave. 30 ml serum bottles were filled with 25 ml medium and sterilised for 15 minutes at 121° C.

[0113] The fresh faeces were mixed with the preservative medium. Fresh faeces can be preserved in this form for several hours at 4° C.

[0114] Faecal suspension: The preserved solution of faeces was centrifuged at 13,000 rpm for 15 minutes. The supernatant was removed and the faeces was mixed with the McBain & Mac Farlane medium in a weight ratio of 1:5.

Fermentation

[0115] 3.0 ml of the faecal suspension were combined with 85 mg glucose or prebiotic or with no addition (blanc) in a bottle and mix thoroughly. A t=0 sample was withdrawn (0.5 ml). 2.5 ml of the resulting suspension was brought in a dialysis tube in a 60 ml bottle filled with 60 ml of the buffered medium. The bottle was closed well and incubated at 37° C. Samples were taken from the dialysis tube (0.2 ml) or from the dialysis buffer (1.0 ml) with a hypodermic syringe after 3, 24, and 48 hours and immediately put it on ice to stop fermentation.

Determination of the Short Chain Fatty Acids and Lactate

[0116] See Example 2. Values were corrected for blanc.

Gas Determination

[0117] At t=3, t=24 and t=48 the gas pressure in the head space of the 60 ml bottle was measured by a gas pressure meter (Druckmessumformer, Econtronic, Germany) by stinging a hypodermic 6 ml syringe through the rubber cap of the bottle and withdrawal of gas from the headspace by this syringe until the gas pressure was 0 bar. The volume in the syringe was the volume of gas formed. Values were corrected for blanc.

1.2 Results

[0118] In vitro fermentation was carried out using the following samples:

1.) 85 mg TOS (from VivinalGOS, Borculo Domo Ingredients, The Netherlands)
2.) 85 mg inulin (RaftilinHP, from Orafti, Belgium)
3.) 85 mg TOS/inulin with a ratio of TOS/inulin of 9/1 (w/w) and
4.) 85 mg OF (raftiloseP95, from Orafti, Belgium)/inulin (raftilinHP) in a ratio of OF/inulin of 1/1 (w/w).

Total Amount of SCFA Produced

[0119] Results are shown in FIG. 1. FIG. 1 shows that the mixture of TOS/Inulin resulted in a significantly higher amount of SCFA per g fibre than the single components, but also higher than the mixture of oligofructose (OF) and inulin. Also tested were 85 mg TOS/inuline in a ratio of 1/1 (data not shown), which also provided a synergistic effect.

L- and D-Lactate Production

[0120] L- and D-lactate could only be determined at t=3. Table 1 shows the metabolic end products formed at that time point.

TABLE-US-00001 TABLE 1 metabolic end-products (mmol/g fibre) formed after 3 hours in vitro fermentation Acetate Propionate Butyrate L-lactate D-lactate TOS 0.23 0 0 0.14 0.03 Inulin 0 0 0 0.00 0.00 TOS/Inulin 0.40 0 0 0.17 0.04 OF/Inulin 0.30 0 0 0.04 0.02

[0121] Again a synergistic higher formation of lactate is observed for the mixture TOS/Inulin compared to the single components TOS and Inulin. Compared to the mixture with OF and Inulin the percentage lactate (based on total acids) and ratio L-/D-lactate is higher in the TOS/Inulin mixture.

Relative Amounts of SCFA

[0122] FIG. 2 shows the pattern of fermentation products after 48 h. The mixture of TOS/inulin shows a significantly higher percentage of acetate than the single components, which is also higher than for the mixture of oligofructose (OF) and Inulin. Faeces of breast fed babies show a high percentage of acetate, so the mixture of TOS/Inulin results in a pattern of fermentation products which most resembles that of breast fed babies.

Gas Formation

[0123] Results are shown in FIG. 3. Regarding the formation of gas, TOS and the mixture TOS/Inulin form the lowest amount of gas per mmol SCFA formed. Inulin and the mixture of OF/inulin show a much higher amount of gas formation. Per mmol SCFA formed the amount of gas is lowest in the TOS/inulin mixture.

Kinetics of SCFA Formation

[0124] Table 2 shows the kinetics of SCFA formation. The combination of TOS/inulin still shows a high SCFA formation between 24 and 48 h, indicating that in the distal part of the colon still SCFA is formed and having a beneficial effect on the colon permeability, mucus formation and anti-pathogenic effects etc. Also in the first 3 h the highest amount of SCFA is formed, as is the case with human milk oligosaccharides (data not shown). A fast fermentation at the beginning of the colon is of importance because of the antipathogenic effects.

TABLE-US-00002 TABLE 2 kinetics of SCFA formation (mmol) SCFA (blanc corrected) Time interval (hours) Prebiotics 0-3 hrs 3-24 hrs 24-48 hrs TOS 0.23 3.85 0.13 TOS/inulin HP 0.40 4.49 0.24 Inulin HP 0.00 3.05 0.05 OF/Inulin HP 0.11 4.26 0.00 OF = oligofructose

Example 2

[0125] Clinical Study with TOS/Inulin: A Relative Increase of Acetate and Relative Decrease of Butyrate is not Correlated with an Increase of Bifidobacteria. TOS and Inulin have a Synergistic Effect.

2.1 Materials and Methods

[0126] 63 pregnant woman who had decided to breast-feed and 57 who chose not to, were recruited during their last trimester of pregnancy. Infants with normal birth weight, no congenital abnormality, congenital disease or gastrointestinal disease were enrolled within 3 days after delivery. The study was approved by the ethical committee of the Medical Center, St. Radboud, Nijmegen, The Netherlands. Written informed consent was obtained from the parents before enrolment in the study.

[0127] Infants of mothers who had decided not to breast-feed, were randomly and double blindly allocated to one of two formula groups (OSF, SF). The standard formula group (SF; n=19 received a regular, non-supplemented infant formula (Nutrilon I, Nutricia, The Netherlands). The main compositional data of the standard formula at standard dilution of 131 g/l are given in Table 3. The prebiotic formula group (OSF; n=19) received the same standard infant formula supplemented with a mixture of 6 g/l transgalacto-oligosaccharides (TOS; Vivinal GOS, Borculo Domo Ingredients, Zwolle, The Netherlands) and inulin (PF; RaftilineHP, Orafti active food ingredient, Tienen, Belgium). The mixture comprised 90% TOS and 10% inulin (polyfructose). The study formulas were fed ad libitum during the study period. Mothers who decided to breast feed were stimulated to continue breast feeding during the course of the study and were supported by a lactation consultant when needed. At termination of breast feeding their infants received one of two formulae. Compliance was assessed by counting the number of unused formula tins during each visit and comparing the amount of consumed formula with the recorded food intake.

TABLE-US-00003 TABLE 3 Composition of the standard formula per litre Energy kcal 670 Protein g 14 Casein/whey ratio 40/60 Fat g 35 Total carbohydrates g 75 Lactose g 75 Minerals Calcium mg 540 Phosphorus mg 270 Magnesium mg 50 Sodium mg 190 Potassium mg 680 Chloride mg 430 Iron mg 5 Zinc mg 5

Questionnaires

[0128] Demographic, clinical and anthropometrical data of the mother are collected prior to delivery. Information on delivery was obtained from the mothers at day 5 after delivery. Information of the infants' food intake, formula tolerance, stool characteristics, health and anthropometrics was obtained from questionnaires at postnatal day 5, 10, 28 and once every 4 weeks thereafter until the end of the study.

Faecal Samples

[0129] Parents were asked to take faecal samples from their infants, at postnatal day 5, 10, 28 and once every 4 weeks thereafter. The samples were taken from the diaper, as soon as possible after defecation, collected in faeces containers (Greiner Labortechnik, the Netherlands) and stored immediately at −20° C. by the parent and transported in a portable freezer to the laboratory by the investigators.

Preparation of Faecal Samples:

[0130] For the determination of SCFA, 1 gram of the samples was thawed in ice water diluted 10× in MilliQ and homogenised for 10 minutes using a stomacher (IUL Instruments, Barcelona, Spain). 350 μl homogenised faeces was mixed with 200 μl 5% (v/v) formic acid, 100 μl 1.25 g/l 2-ethylbutyric acid (Sigma-Aldrich, Zwijndrecht, The Netherlands) and 350 μl MilliQ. The samples were centrifuged for 5 minutes at 14,000 rpm to remove large particles and the supernatant was stored at −20° C. For the FISH analysis and lactic acid measurements, the samples were thawed in ice water, diluted 10× (w/v) in phosphate buffered saline, pH 7.4 (PBS) and homogenized for 10 minutes using a stomacher. The homogenised faeces were stored at −20° C.

Fluorescence In Situ Hybridisation

[0131] FISH analysis was performed as described (Langendijk et al, 1995, Appl. Environ. Microbiol. 61:3069-3075.) with some slight modifications. Paraformaldehyde fixed samples were applied to gelatin coated glass slides (PTFE coated 8-wells [1 cm2/well] object slides, CBN labsuppliers, Drachten, The Netherlands) and air-dried. The dried samples were dehydrated in 96% ethanol for 10 minutes. Hybridisation buffer (20 mm Tris-HCl, 0.9 M NaCl, 0.1% SDS [pH 7.1[) with 10 ng/l Cy3 Labeled Bifidobacterium specific probe Bif164mod (5′-CAT CCG GYA TTA CCA CCC), was preheated and added to the dried samples. Bif 164 mod is modified version of probe S-G-Bif-a-0164-a-A-18 ((Langendijk et al, 1995, Appl. Environ. Microbiol. 61:3069-3075.). The slides were incubated overnight in a dark moist chamber at 50° C. After hybridisation the slides were washed for 30 minutes in 50 ml preheated washing buffer (20 mM Tris-HCl, 0.9 M NaCl [pH 7.2]) and briefly rinsed in MilliQ. For staining all bacteria, the samples were incubated with 0.25 ng/l 4′,6-diamidino-2-phenylindole (DAPI) in PBS for 5 minutes at room temperature. After DAPI staining the slides were briefly rinsed in MilliQ, dried, mounted with Vectashield (Vector Laboratories, Burlingame, Calif., U.S.A.) and covered with a coverslip. The slides were automatically analysed using an Olympus AX70 epifluorescence microscope with automated image analysis software (Analysis 3.2, Soft Imaging Systems GmbH, Münster, Germany). The percentage of bifidobacteria per sample was determined by analysing 25 randomly chosen microscopic positions. At each position the percentage of bifidobacteria was determined by counting all cells with a DAPI filter set (SP 100, Chroma Technology Corp., Brattleboro, U.S.A.) and counting all bifidobacteria using a Cy3 filter set (41007, Chroma Technology, Brattleboro, U.S.A.).

Short Chain Fatty Acids Analysis

[0132] The short chain fatty acids (SCFA) acetic, propionic, n-butyric, iso-butyric and n-valeric acids were quantitatively determined by a Varian 3800 gas chromatograph (GC) (Varian Inc., Walnut Creek, U.S.A.) equipped with a flame ionisation detector. 0.5 μl of the sample was injected at 80° C. in the column (Stabilwax, 15×0.53 mm, film thickness 1.00 μm, Restek Co., U.S.A.) using helium as a carrier gas (3.0 psi). After injection of the sample, the oven was heated to 160° C. at a speed of 16° C./min, followed by heating to 220° C. at a speed of 20° C./min and finally maintained at a temperature of 220° C. for 1.5 minutes. The temperature of the injector and detector was 200° C. 2-ethylbytyric acid was used as an internal standard.

Lactate Analysis

[0133] Homogenised faeces was thawed on ice and centrifuged for 5 minutes ant 14,000 rpm 100 μl supernatant was heated for 10 minutes at 100° C. to inactivate all enzymes. Lactate was determined enzymatically, using a L-lactate acid detection kit with D- and L-lactate-dehydrogenase (Boehringer Mannheim, Mannheim, Germany). Lactate was only determined in those faecal samples which were large enough.

pH Analysis

[0134] After storage at −20° C., faecal samples were thawed and the pH was directly measured in the faeces at room temperature using a Handylab pH meter (Scott Glas, Mainz, Germany) equipped with an Inlab 423 pH electrode (Mettler-Toledo, Columbo, U.S.A.)

Data Analysis

[0135] Prior to the study, power calculations showed that to detect a difference in percentage of bifidobacteria between the intervention formula group and the standard formula group of 30% with a SD of 25%, 13 infants per group should be included. Because of an expected drop out of 30% in the formula groups, more infants than calculated were included in the study. Statistical package SPSS (version 11/0) was used for statistical analysis of the results. All values were checked for normality by visual inspection of the normal probability plots. Differences in percentage bifidobacteria, pH, relative amounts of SCFA and lactate between the groups were tested for significance using analysis of variance. In case of a significant difference (p<0.05), groups were compared by using the Bonferroni post hoc test. Because it is not possible to double blind assign breast and bottle-feeding and to ensure adequate randomisation, no statistical analysis were performed to compare the breast-feeding with any of the formula feeding groups. Data from the breast-fed group are only given when the infant was only fed breast milk at that time point.

2.2 Results

[0136] In total, 120 (-pro group infants were included. 57 infants started on formula feeding directly after birth and were equally divided among the formula groups. Of the 63 infants that were fed breast milk directly after birth, 24 switched to formula feeding before the age of 16 weeks and 5 infants dropped out. The characteristics of the study subjects are shown in Table 4. In the formula groups, 9 infants dropped out of the study within the first 16 weeks after birth (4 in the SF, 5 in the OSF group. Reasons for drop out included: colics, suspicion of cow's milk allergy, constipation and practical problems.

TABLE-US-00004 TABLE 4 Characteristics of study objects Standard Prebiotic Breast Formula, SF formula, OSF milk, BF N = 19 N = 19 N = 63 Sex Male 5 12 33 Female 14 7 30 Place of At home 7 8 40 birth Hospital 12 11 23 Mode of Vaginal 14 16 59 delivery Caesarean 5 3 4 Birth weight 3600 ± 501 3318 ± 602 3651 ± 601

Faecal Bifidobacteria

[0137] The percentages of bifidobacteria in faeces at the age of 5 days, 10 days, 4, 8, 12, and 16 weeks of the feeding groups are shown in Table 5 and the amounts in Table 5. The OSF group tends to have a higher bifidobacteria % than the SF group from total bacterial count at all ages, but the differences were not statistically different. Unexpectedly, the percentage of Bifidobacteria in breast fed babies was also relatively low and were in line with the formula fed groups.

[0138] Preliminary data also show an increase in Lactobacilli in the BF and OSF group, but the amounts of Lactobacilli in the faecal flora are at least one order of magnitude lower than the Bifidobacteria, the overall pattern is changes very little.

pH Results

[0139] The pH values measured in the faeces of the formula-fed infants are shown in table 6. Lowest pH was found in infants fed on breast milk. Faecal pH of faeces of infants fed the OSF formula were lower than the SF group (p<0.045 at all ages except day 5).

SCFA Results

[0140] The total amount of SCFA in the faeces is shown in Table 5 below.

[0141] The percentage of the different SCFA from total SCFA are shown in Table 6. There are no statistically significant differences found in total SCFA concentration between the formula groups. Also the amount of SCFA is comparable to those of the other feeding groups. However, already after 10 days, differences in the SCFA profiles can be seen between infants fed on OSF or breast milk compared to infants fed on standard formula. Infants fed the formula containing GOS and polyfructose and fed breast milk, have higher percentages of acetate and lower percentages of propionate, butyrate, iC4-6 SCFA when compared to infants fed the standard formula.

Lactate Results

[0142] The concentrations lactate (mmol/kg faeces) of all groups are shown in Table 5. Already from 5 days of age, the OS formula (not sign.) and the groups of breast milk have higher amounts of lactate compared to the standard formula group. The relative amount of lactate (as a percentage of the sum of SCFA and lactate) is highest in breast fed babies and lowest in standard formula fed babies. Babies fed a formula containing TOS/inulin have an intermediate relative amount of acetate. The percentage of lactate in OSF fed babies at 16 weeks (relative to total acids) significantly differs from that of SF babies.

TABLE-US-00005 TABLE 5 Concentration of lactate and total SCFA (mmol/kg faeces) and Bifidobacteria (*1.10.sup.13/kg faeces) pH between birth and 16 weeks of age. Mean ± SEM. Except for pH, no statistically differences were found. Lactate SCFA pH Bifidobacteria 5 days.sup.  SF 13.5 ± 7.7  54.7 ± 12.6 5.93 ± 0.15 0.58 ± 0.49 OSF 10.7 ± 4.3 56.5 ± 7.7 5.49 ± 0.15 1.20 ± 2.24 BF 13.3 ± 2.8 48.7 ± 4.4 5.27 ± 0.07 0.47 ± 0.39 10 days .sup.  SF  4.6 ± 3.0 62.0 ± 7.9 6.88 ± 0.15 0.96 ± 0.83 OSF  9.7 ± 3.6 62.3 ± 7.2 5.95 ± 0.20 1.10 ± 0.99 BF 15.1 ± 3.2 54.7 ± 4.9 5.35 ± 0.07 0.48 ± 0.61  4 weeks SF  2.6 ± 1.4  68.3 ± 10.3 6.77 ± 0.21 0.97 ± 0.96 OSF  9.9 ± 3.4 83.1 ± 8.8  5.88 ± 0.18* 1.20 ± 0.55 BF 22.8 ± 4.4 59.8 ± 4.8 5.45 ± 0.12 0.56 ± 0.64  8 weeks SF  7.6 ± 6.8  76.5 ± 13.2 6.80 ± 0.20 0.89 ± 0.56 OSF 24.4 ± 5.3 76.0 ± 8.4  5.68 ± 0.18* 1.00 ± 0.52 BF 30.9 ± 5.3 62.8 ± 5.4 5.27 ± 0.15 0.58 ± 0.59 12 weeks SF 14.1 ± 9.4  73.9 ± 11.9 6.88 ± 0.20 0.91 ± 0.80 OSF 18.4 ± 7.0  76.1 ± 12.1  5.60 ± 0.18* 1.30 ± 0.99 BF 42.1 ± 7.1 60.4 ± 4.9 5.29 ± 0.17 1.40 ± 1.38 16 weeks SF  1.7 ± 1.2  68.6 ± 14.0 7.09 ± 0.15 1.00 ± 0.80 OSF 18.5 ± 5.7  67.7 ± 11.7  5.60 ± 0.20* 1.30 ± 0.76 BF 45.1 ± 9.0 59.2 ± 6.9 5.68 ± 0.24 0.89 ± 0.78

TABLE-US-00006 TABLE 6 relative amounts of SCFA (% of total SCFA), lactate (% of total acids), % Bifidobacteria between birth and 16 weeks of age. Sum % Day/week Acetate Propionate Butyrate iC4-C5 Bifidobacteria lactate  5 d SF 84.3 ± 3.4 12.9 ± 3.2 1.7 ± 0.5 1.1 ± 0.4 45 ± 3.6 13.8 ± 18.5 OSF 85.8 ± 5.1 12.0 ± 4.7 0.5 ± 0.5 1.7 ± 0.7 50 ± 8.6  8.7 ± 13.3 BF 89.5 ± 1.8  7.0 ± 1.5 1.6 ± 0.4 2.0 ± 0.4 54 ± 4.1 12.5 ± 14.0 10 d  SF 70.9 ± 2.0 21.3 ± 2.6 4.6 ± 1.1 3.2 ± 0.5 65 ± 6.0  4.3 ± 10.8 OSF  .sup. 84 ± 2.4* 13.5 ± 2.3  1.4 ± 0.4* 1.1 ± 0.4 61 ± 6.3 8.2 ± 9.8 BF 89.3 ± 1.9  5.8 ± 1.3 2.3 ± 0.3 2.6 ± 0.4 42 ± 4.1 13.4 ± 13.8  4 w SF 71.8 ± 2.8 17.8 ± 3.3 5.0 ± 1.1 5.5 ± 2.6 52 ± 5.4  6.9 ± 16.4 OSF 77.7 ± 2.2 15.4 ± 2.0 5.8 ± 2.2 1.1 ± 0.3 71 ± 4.5  8.4 ± 10.7 BF 91.0 ± 1.8  4.3 ± 1.2 2.6 ± 0.6 2.1 ± 0.4 47 ± 5.4 19.6 ± 17.0  8 w SF 74.6 ± 2.9 16.4 ± 2.0 6.1 ± 1.2 2.9 ± 0.7 50 ± 6.3 3.5 ± 9.9 OSF 83.5 ± 2.7 11.4 ± 2.1 3.7 ± 1.2 1.4 ± 0.4 64 ± 4.1 17.7 ± 15.2 BF 91.2 ± 1.6  5.4 ± 1.4 1.9 ± 0.5 1.6 ± 0.3 41 ± 4.5 21.7 ± 13.4 12 w SF 73.9 ± 2.9 17.8 ± 3.3 5.0 ± 1.1 3.2 ± 0.5 56 ± 5.4  5.4 ± 11.9 OSF 86.5 ± 2.1 11.2 ± 1.8  1.2 ± 0.3* 1.0 ± 0.4 60 ± 5.0 13.6 ± 13.2 BF 86.1 ± 3.3  7.5 ± 2.2 3.0 ± 0.7 3.5 ± 0.8 59 ± 4.5 30.2 ± 16.3 16 w SF 69.9 ± 3.9 19.6 ± 2.7 5.6 ± 0.9 4.9 ± 0.8 52 ± 6.3 0.6 ± 0.9 OSF 82.2 ± 5.3 14.3 ± 4.9  2.1 ± 0.5*  1.5 ± 0.4* 69 ± 7.7  16.4 ± 12.0* BF 89.7 ± 2.7  6.4 ± 2.1 1.6 ± 0.4 2.2 ± 0.5 47 ± 6.3 35.6 ± 20.4 P < 0.05 compared to SF; SF = standard formula fed, OSF = SF supplemented with GOS + inulin; BF = breast fed.

[0143] The above demonstrates that in faeces of infants fed with this combination of GOS and polyfructose the pH is lowered, that more lactate is formed, and that an acid (acetate and lactate) pattern is formed which is more similar to that of breast fed babies and that this effect cannot be attributed to the quantitative increase of Bifidobacteria.

Example 3—Beneficial Effects of Lactate and SCFA Mixture on Muc-2, PGE1 and PGE2 Expression

3.1 Material and Methods

[0144] The effect of lactate and a SCFA mixture was analysed as described in Willemsen, LEM, Koetsier Mass., van Deventer S J H, van Tol E A F (2003), Gut 52:1442-1447, with the following modifications: Lactate and a mixture of acetate/propionate/butyrate 90/5/5 was tested. For the mucus production experiments a co-culture of CCD18 and T84 cells was used, whereas for the PGE1 and PGE2 production experiments a monoculture of CCD18 cells was used.

3.2 Results

[0145] SCFA, in a mixture of 90/5/5 (acetate/propionate/butyrate), and L-lactate dose dependently induces MUC-2 expression in a co-culture of CCD18 and T84 cells as can be seen in FIG. 4A. Also the concentration of PGE1 and PGE2 increases in CCD18 cells as can be seen in FIG. 4B. At higher concentrations of the added organic acids the increases reaches statistically significance.

Example 4: Effect of Lactate on Colon Contraction

4.1 Material and Methods

[0146] Male Wistar rats (CKP/Harlan, Wageningen/Horst, Netherlands) were housed under conditions of controlled temperature and light cycle and were provided free access to food pellets and water. Animals were anaesthetised by a mixture of N.sub.2O, O.sub.2 and isofluran, the abdomen was opened and the colon was removed immediately. The tissue was placed in Krebs-Henseleit buffer pH 7.4 (composition in mM: 118.0 NaCl, 4.75 KCl, 1.18 MgSO.sub.4, 2.5 CaCl.sub.2, 10 glucose, 1.17 KH.sub.2PO.sub.4 and 24.9 NaHCO.sub.3).

[0147] The colon was cut into a distal and a proximal portion and was rinsed with Krebs-Henseleit buffer while gently squeezing out faecal content. To approximate in vivo conditions as closely as possible, 1 cm fully intact segments were attached longitudinally to an isometric force transducer (F30 type 372, HSE, Germany) in 20 ml water-jacketed (37° C.) organ baths (Schuler, HSE, Germany) containing Krebs-Henseleit buffer gassed continuously with 95% O.sub.2-5% CO.sub.2. The segments were gradually stretched to a resting tension of 1 g and allowed to equilibrate for 45 minutes with intermittent washings. The tensions of the segments in rest and in response to different stimuli were amplified by a transducer amplifier module (HSE, Germany) and recorded on a multi-pen recorder (Rikadenki, HSE, Germany).

[0148] The segments were incubated with 40 mM KCl for 5 minutes and the contractile responses were measured. KCl was washed out by three consecutive washes at 5 minutes intervals. The segments were then incubated with increasing concentrations up to 100 mM of acetate or sodium-L-lactate. The acid solutions were prepared freshly in distilled water. NaOH was added to acetate to obtain a neutral pH. At the end of the incubation with a fatty acid, 40 mM KCl was added to determine whether the contractile response to KCl was influenced by the fatty acid. Before a new incubation the segments were allowed to equilibrate for 45 minutes in fresh Krebs-Henseleit buffer with intermittent washings.

[0149] The experimental protocol consisted of two proximal and two distal sections of the colon. For data analysis (n=3), the contraction level induced by the stimuli was defined as the tension in g after 5 minutes incubation. Data obtained from identical segments (proximal or distal) were used to calculate a mean value and each segment served as its own control sample.

4.2 Results

[0150] As can be seen from FIG. 5, sodium acetate and especially sodium-L-lactate decrease the tension of tonic contractions. The relaxation effects are higher in the distal part of the colon than in the proximal part of the colon.

[0151] Also the number of spontaneous contractions, the phasic contractions, decrease in the proximal part of the colon upon addition of the sodium acetate and sodium-L-lactate, whereas no effects are observed in the distal part of the colon.

[0152] In the proximal part of the colon the tonic contractions as a response to KCl, however, are comparable in the presence or absence of 25 mM sodium actetate or sodium L-lactate. At higher concentrations a significant relaxation is observed even after addition of KCl.