ORIGINAL ARTICLE
Colonic Fermentation Capacity in 6itro: Development during Weaning in Formula-Fed Infants Alison M. Parrett, Ella Lokerse and Christine A. Edwards From the Department of Human Nutrition, Glasgow University, Glasgow, UK Correspondence to: Alison Parrett, Department of Human Nutrition, University of Glasgow, Yorkhill Hospitals, Glasgow G3 8SJ, UK. Tel.: + 141-201-0708; Fax: + 141-201-9275; E-mail:
[email protected]
Microbial Ecology in Health and Disease 2003; 5: 10–14 The objective of this study was to determine the effect of weaning on the ability of formula-fed infants to ferment complex carbohydrates. A cross-sectional study of 46 infants was performed. Fresh faeces were collected from normal exclusively formula-fed infants at pre(n= 15), early (n =15) and late weaning (n= 16). Faecal bacteria were incubated with simple and complex carbohydrates (glucose, lactose, raftilose and soyabean polysaccharide) in 6itro. In addition, a control culture with no added carbohydrate was also incubated. Short-chain fatty acids (SCFA) and lactate were measured in culture supernatant after anaerobic incubation for 24 hours. There were no significant differences between any stages of weaning for SCFA production for any substrate. However, there was a consistent trend for the percentage of butyric acid production to increase with age. The greater similarity of the colonic flora of the formula-fed infants to that of the adult results in less impact of weaning on the development of fermentation capacity for complex carbohydrates than that previously reported for the simpler flora of the breast-fed infant. Key words: formula-fed, infant, fermentation, carbohydrate, weaning.
INTRODUCTION Carbohydrates that escape digestion and absorption in the small intestine pass to the colon where they are metabolized anaerobically by the colonic bacteria to produce short-chain fatty acids (SCFA), mainly acetate, propionate and n-butyrate (1). SCFA have many potential beneficial roles. They act as a source of energy for the colonic mucosa and other tissues, providing a form of ‘colonic salvage’ and their absorption may also prevent osmotic water loss (2). Carbohydrate fermentation in adults has been well investigated, but knowledge of the colonic flora of neonates, and how fermentation develops in response to new foods during weaning, is limited. Early events in the bacterial colonization of the gut, at birth and at weaning, may be critical in determining the health of the infant as well as establishing the colonic flora of the adult. It is not known how quickly colonic bacterial colonization occurs in man or what dietary influences are important. However, inefficient fermentation of malabsorbed carbohydrate may result in a greater tendency to diarrhoea (3). Previous studies, using both traditional and new molecular techniques, have shown that before weaning exclusively formula-fed infants have a colonic microflora dominated by enterobacteriaceae and anaerobic bacteria in faeces in contrast to breast-fed infants who have a high proportion © Taylor & Francis 2003. ISSN 0891-060X
of bifidobacteria/lactobacilli (4 – 7). This difference in flora has also been established in the same population in Glasgow from which infants in the present study were recruited ((7), unpublished data, J Dore, G Gramet, F Martin, SAH Savage, CA Edwards, AM Parrett). The colonic microflora of formula-fed infants resembles that of adults more closely than breast-fed infants. This difference in flora is reflected in the SCFA produced, formula-fed infants produce mostly acetate and propionate with a small amount of butyrate before weaning, whereas breast-fed infants produce predominantly acetate and lactate (1). However, faecal SCFA may not reflect true bacterial activity, as they are the net result of both production and absorption and may not represent events in the proximal colon. They also give very little information on the ability of an infant to ferment carbohydrate. An alternative approach is to study changes in bacterial metabolism and fermentation capacity. It is difficult to study colonic fermentation in 6i6o in infants, but in 6itro studies using faecal bacteria have proved useful models of fermentation in adults (8). Previously in a cross-sectional study, breast-fed infants were shown to be able to ferment simple sugars equally well at all stages of weaning but their ability to ferment oligosaccharides did not develop until early weaning and the ability to ferment complex carbohydrates did not develop until late weaning (9). More recent work in our Microbial Ecology in Health and Disease DOI: 10.1080/08910600310015556
In 6itro carbohydrate fermentation during weaning
laboratory with breast-fed infants has confirmed this finding (see Discussion, unpublished data, CA Edwards, AM Parrett). This suggested a slow maturation of the colonic flora and its fermentation capacity. Other bacterial activities such as conversion of bilirubin to urobilinogen and mucin degradation ability have also been shown to develop slowly (10 – 12). Given the more diverse flora of the formula-fed infant these activities may mature more quickly than in breast-fed infants. In this cross-sectional study, we have used an in 6itro fermentation model to test the hypothesis that weaning has little impact on the development of colonic fermentation capacity for complex carbohydrates in formula-fed infants. The ability of faecal bacteria to ferment simple sugars (glucose, lactose), an oligosaccharide (raftilose) and complex carbohydrates (soyabean polysaccharide) was measured at different stages of weaning in an in 6itro model using faeces of exclusively formula-fed infants (pre-weaning), early weaning (4 weeks after introduction of first solid food) and late weaning (7 –12 months). MATERIALS AND METHODS Subjects Forty-six full-term, exclusively formula-fed healthy infants were recruited by personal contact. None of these infants or their lactating mothers had received any antibiotics at the time of recruitment or at any time before giving samples. Fresh faeces were obtained from 15 exclusively formula-fed infants (3 males, 12 females), 15 early weaning formula-fed infants (2 males, 13 females) and 16 late weaning formula-fed infants (6 males, 10 females). Early weaning was defined as 4 weeks after giving the first non-milk food, when liquidized/pure´ ed food was ingested and late weaning was defined as the period when chopped/solid food was ingested. The median (range) age of the pre-weaning group was 7 (4 –12) weeks, the early weaning group 12 (11 – 17) weeks and the late weaning group 9 (7–12) months. The mothers chose to wean at ages that were not influenced by the authors. Ethical appro6al This study was approved by the Ethics Committee of Yorkhill NHS Trust, Glasgow. The mothers of all infants gave written informed consent. In 6itro fermentation model Fresh faecal samples were obtained from infants at their homes as soon as possible after defecation, and were processed within 1 hour. After weighing the sample, a faecal slurry (32% w/v) was prepared with pre-reduced phosphate buffer, pH 7. One ml of this preparation was used to inoculate 9 ml of pre-reduced simple micronutrient and amino acid medium (8), containing 100 mg of one carbohydrate substrate: glucose (BDH, Loughborough,
11
UK) or lactose (Sigma Chemical Company, Poole, UK), for estimation of fermentation capacity for sugars; raftilose (Raffiniere tirlemontoise, SA), as a fructooligosaccharide and soyabean polysaccharide (Scientific Hospital Supplies, Liverpool, UK) as an example of complex carbohydrates. Cultures contained a total volume of 10 ml but if less faecal material was available all volumes were halved to give a total culture volume of 5 ml. Cultures were incubated in an anaerobic jar for 24 hours at 37°C. A control culture containing no carbohydrate was also incubated to allow for endogenous carbohydrate and production of SCFA. For total net SCFA the no-carbohydrate control values were subtracted from the test culture values. Measurements After 24 hours, the pH of fermentation fluid was measured before storage at − 20°C for later analysis of SCFA and lactic acid. SCFA were measured in acidified ether extracts of the faecal fermentation fluid by gas liquid chromatography (GLC) using b-methyl valeric acid as an internal standard (13). SFCAs were measured using a Pye Unicam 304 series GLC (Phillips Analytical Ltd, Cambridge, UK) with a packed glass column 4 feet, e.d. 14¦, i.d. 4 mm; 10% SP1000+ H3PO4 on Chromosorb WAW 80-100 mesh (Phase Separations, Deeside, Wales). Lactic acid was measured in the same way after methylation with methanol and sulphuric acid and with succinic acid as a standard (14). Lactic acid was measured on a similar column in a Pye Unicam 4550 series GLC (Phillips Analytical Ltd). Statistics The results obtained in this study were not normally distributed. Therefore the results of the three feeding periods were compared by Kruskal-Wallis and Mann-Whitney U tests after subtraction of the no-carbohydrate blank. Molar proportions of SCFA and lactic acid of total SCFA produced were compared by Kruskal-Wallis and MannWhitney U tests. RESULTS Total SCFA concentration Formula-fed infants were able to ferment sugars easily at all stages. Soyabean polysaccharide fermentation produced much less SCFA. There were substantial variations in SCFA production between individual infants. The total SCFA and lactate concentration (mmol/ml) in each culture was statistically compared after subtraction of values from the no-carbohydrate blank, to control for any exogenous SCFA or carbohydrate in the faeces (Table I). The trend was for total SCFA and lactate concentration to increase with all substrates from pre-weaning to late weaning.
12
A. M. Parrett et al.
Table I Production of total SCFA and lactic acid (vmol/ml) in in 6itro cultures of faecal bacteria from formula-fed infants containing a range of carbohydrate substrates a Stage
Glucose
Lactose
Raftilose
Soyabean polysaccharide
Pre-weaning n=15 Early weaning n=15 Late weaning n=16
55.1 (8.0–107.5) 55.5 (23.6–142.1) 68.1 (10.9–142.0)
63.6 (21.9–124.7) 67.5 (28.4–164.1) 76.6 (11.9–168.1)
46.2 (10.0–132.9) 63.9 (19.5–152.3) 64.5 (13.8–135.9)
14.0 (8.0–42.7) 20.0 (3.0–100.8) 26.2 (2.2–69.4)
a
Results are expressed after subtraction of short-chain fatty acids (SFCA) produced in control cultures containing no added carbohydrate and given as median (range).
However, there were no statistically significant differences between the stages of weaning. At late weaning the amount of SCFA and lactate in cultures with soyabean polysaccharide was just over half that found in similar cultures of adult faeces (1). Indi6idual SCFA concentration The cultures of formula-fed infant faeces produced mainly acetic and propionic acid with lower amounts of butyric acid. When individual SCFAs were compared at different stages there were no statistically significant differences between any stage of weaning with any substrate. Lactic acid Very little lactic acid was produced in cultures of faeces from most infants. No statistically significant differences were seen in lactate production in formula-fed infant cultures at any stage of weaning with any substrate. Proportions of SCFA The proportions of acetate, propionate and n-butyrate did not vary significantly as weaning progressed (Table II). There was an increase in the percentage of n-butyrate in the control cultures from pre-weaning to early and late weaning (pB0.01 and pB 0.05, respectively). In cultures containing the glucose and complex carbohydrate soyabean polysaccharide there was a decrease in the percentage of propionate at early weaning compared with pre-weaning (pB0.01 and pB0.05, respectively). In all other cultures there was an increase in the proportion of n-butyrate as weaning progressed but this was not statistically significant. This was associated with a decrease in the proportion of acetate in control and glucose cultures and a decrease in the proportion of lactate in lactose cultures, but none of these trends achieved statistical significance. DISCUSSION Several studies have investigated the flora of infants in the first weeks of life (4, 5, 15), but very little is known about how the flora changes during the weaning process. The response of the infant microflora to weaning may depend on previous feeding practice. It is important to know how changes in microflora allow infants to cope with new
substrates they ingest at weaning. If fermentation capacity is sufficiently developed, the SCFA produced will provide 2 Kcal/g and may contribute to daily energy needs (16). However, if infants are presented with a substrate that they are unable to ferment, it will pass through unmetabolized, pulling water with it and causing an increase in stool output. This may result in increased faecal energy loss. We have previously shown in a study that used identical techniques to those described here that the ability of exclusively breast-fed infants to ferment oligosaccharides developed in early weaning but the ability to ferment complex carbohydrates did not increase until late weaning (9). More recent data from cultures using faecal samples from eight breast-fed infants recruited from a similar area and using the same in 6itro fermentation techniques confirmed these results. Cultures containing raftilose produced increasing amounts of total SCFA as weaning progressed; 46.8 (13.7 –74.9), 63.6 (40.0 –101.1) and 84.8 (52.4 –145.7) mmol/ml; median (range) at pre-, early and late weaning respectively. However, with the complex carbohydrate soyabean polysaccharide much less total SCFA was produced throughout weaning and the rate of increase was much slower; 5.3 (0.0 –28.6), 22.8 (10.2 –33.3) and 34.3 (14.1 – 46.0) mmol/ml, median (range) for pre-, early and late weaning, respectively. The present study aimed to determine whether weaning had less impact on the fermentation capacity of formula-fed infants than on breast-fed infants given the more diverse microflora present in the formulafed infant gut. In this study there were no significant differences in total SCFA and lactate production in 6itro at any stage of weaning for any substrate in formula-fed infants. There was a trend towards an increase as weaning progressed but this did not reach statistical significance. As with breastfed infants there was a marked increase between pre- and early weaning, with cultures containing raftilose, and between pre- and late weaning in cultures containing soyabean polysaccharide, but in neither case was this statistically significant in cultures of faeces from formulafed infants. In these cultures the dominant SCFA produced were acetate and propionate. There was a trend for an increase in butyrate but even at late weaning the amount of butyrate produced with any substrate was very
In 6itro carbohydrate fermentation during weaning
13
Table II Proportions of SCFA produced in cultures of faeces from formula-fed infants at different stages of weaning containing a range of carbohydrate substrates a Carbohydrate
Proportion of individual SCFA of total SCFA (%) Acetate
Propionate
n-Butyrate
Branched chain
Lactate
Control Pre-weaning Early weaning Late weaning
59.0 (44.7–71.9) 58.0 (46.5–80.5) 59.4 (43.7–73.5)
18.1 (6.8–33.6) 11.2 (1.6–23.9) 14.1 (4.4–30.2)
4.9**† (1.5–12.2) 11.0 (5.1–16.2) 12.2 (0.0–18.1)
18.4 (4.9–24.1) 16.0 (4.5–34.8) 15.2 (4.6–22.7)
Glucose Pre-weaning Early weaning Late weaning
55.6 (13.7–79.0) 65.0 (3.2–78.7) 59.4 (30.5–86.2)
15.6** (6.1–42.0) 7.5 (0.0–24.3) 11.6 (0.7–43.6)
2.4 (0.1–13.3) 2.8 (0.4–19.0) 7.6 (0.0–32.7)
0.4 (0.0–2.8) 0.4 (0.0–15.0) 0.4 (0.0–12.1)
13.3 (0.0–75.4) 18.9 (0.0–96.4) 5.1 (0.0–64.9)
Lactose Pre-weaning Early weaning Late weaning
57.1 (12.3–71.4) 63.4 (25.7–77.2) 60.9 (39.4–80.3)
15.3 (3.3–51.7) 8.7 (1.0–25.5) 11.6 (0.8–44.0)
2.1 (0.0–13.3) 2.2 (0.2–20.5) 5.8 (0.0–27.1)
0.2 (0.0–17.9) 0.3 (0.0–1.0) 0.4 (0.0–24.1)
22.1 (0.0–70.2) 19.5 (0.0–72.4) 0 (0.0–54.8)
Raftilose Pre-weaning Early weaning Late weaning
55.1 (7.4–84.7) 65.5 (14.5–76.5) 63.2 (18.9–76.0)
15.3 (4.4–59.5) 12.7 (1.0–34.6) 13.1 (0.4–38.9)
4.2 (0.4–34.1) 8.0 (1.0–31.4) 6.9 (0.0–31.2)
0.5 (0.0–3.1) 0.4 (0.0–5.1) 0.7 (0.0–26.5)
4.2 (0.0–86.7) 3.2 (0.0–82.7) 0 (0.0–72.9)
Soy polysaccharide Pre-weaning Early weaning Late weaning
56.7 (46.6–72.6) 63.0 (12.5–75.0) 64.2 (56.4–80.2)
29.5* (8.5–45.7) 16.0 (2.5–31.7) 17.9 (8.0–29.1)
4.4 (0.6–16.8) 7.6 (0.9–30.7) 9.1 (0.0–18.6)
8.5 (4.3–11.8) 6.8 (0.5–84.1) 8.3 (2.4–17.4)
0 (0.0–22.4) 0 (0.0–2.3) 0 (0.0–4.5)
0 (0.0–0.0) 0 (0.0–2.3) 0 (0.0–0.0)
a
Median (range). *pB0.05, **pB0.01 compared with early weaning; †pB0.05 compared with late weaning.
low. As expected the sugars and oligosaccharide were fermented to greater amounts of SCFA than the soyabean polysaccharide, which even at late weaning only produced 57% of SCFA produced in a similar model using adult faecal bacteria (9). There is substantial evidence that the flora of the breastfed infant differs from that of the formula-fed infant in both composition and function in the first weeks of life. This would be likely to affect the impact of weaning. Other studies have reported differences in the development rate of different bacterial functions. The faecal SCFA profile of infants who had received formula milk tended to be more varied and adult-like, in a Swedish study, than infants who were exclusively breast-fed (11). Mucin degradation was initiated later in exclusively breast-fed infants (by at least 4 months) compared with infants who had received formula (either with or without breast milk) at 1 month of age (12). The establishment of conversion of cholesterol to coprostanol was also delayed by breast-feeding, only one child was shown to have coprostanol in their faeces while still being breast-fed (17). However, once conversion was initiated previous breast-feeding resulted in a stable, high rate of conversion of cholesterol. Conversion of bilirubin to urobilinogen was higher in formula-fed infants at younger ages than in breast-fed infants and at 6 months
faecal tryptic activity was higher in infants receiving formula milk (10). Breast-feeding was not shown to have any consistent effect on urease, b-glucosidase or b-glucuronidase activity in one study (18), but Gro¨ nlund et al. (19) reported that formula feeding (before 2 months of age) resulted in an increase in the activity of these enzymes. These studies were all carried out in Scandinavian countries where the rate of breast-feeding is higher than in Scotland (20, 21). As such, none of the infants studied were exclusively formula-fed. If infants had been exclusively formula-fed differences in metabolic activities may have been more marked between breast-fed and formula-fed infants. In conclusion, the flora of the formula-fed infant is different to that of the breast-fed infant in terms of its composition, which is more like that of the adult microflora, and its metabolic activity, which is more developed due to the greater diversity of organisms present. Weaning had less impact on the in 6itro fermentation capacity of the faecal flora of the formula-fed infant compared with the breast-fed infant. However, the amount of SCFA produced during fermentation for complex carbohydrates had not reached adult levels by late weaning. More research is needed to determine the factors influencing the maturation of colonic bacterial function after
14
A. M. Parrett et al.
weaning in infants and young children as this may have important implications for dietary advice and food product formulation at this critical stage of development.
REFERENCES 1. Edwards CA, Parrett AM, Balmer SE, Wharton BA. Faecal short chain fatty acids in breast-fed and formula-fed babies. Acta Pediatr 1994; 83: 459 –62. 2. Kien CL, Liechty EA, Mullett MD. Contribution of lowmolecular weight compounds to the faecal excretion of carbohydrate energy in premature infants. Gastroenterology 1990; 99: 165 – 74. 3. Rao SSC, Edwards CA, Austen C, Read NW, Holdsworth CD. Impaired colonic fermentation of carbohydrates after ampicillin. Gastroenterology 1988; 94: 928 – 32. 4. Balmer SE, Wharton BA. Diet and faecal flora in the newborn: breast milk and infant formula. Arch Dis Child 1989; 64: 1672 – 7. 5. Stark PL, Lee A. The microbiology of the large bowel of breast-fed and formula-fed infants during the first year of life. J Med Microbiol 1982; 15: 189 –203. 6. Harmsen HJ, Wildeboer-Veloo AC, Raangs GC, Wagendorp AA, Klijn N, Bindels JG, Welling GW. Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods. J Pediatr Gastroenterol Nutr 2000; 30: 61 – 7. 7. Martin F, Savage SAH, Parret AM, Gramet G, Dore J, Edwards CA. Investigation of bacterial colonisation of the colon in breast-fed infants using novel techniques, Proc Nutr Soc 2000; 64. 8. Adiotomre J, Eastwood MA, Edwards CA, Brydon WG. Dietary fibre; in vitro methods that anticipate nutrition and metabolic activity in humans. Am J Clin Nutr 1990; 68: 741 – 51. 9. Parrett AM, Lokerse E, Edwards CA. Colonic fermentation in vitro: development during weaning in breast fed infants is slower for complex carbohydrates than for sugars. Am J Clin Nutr 1997; 65: 927 –33.
10. Midtvedt A-C, Carlstedt-Duke B, Norin KE, Saxerholt H, Midtvedt T. Development of five metabolic activities associated with the intestinal microflora of healthy infants. J Pediatr Gastroenterol Nutr 1988; 7: 559 – 67. 11. Midtvedt A-C, Midtvedt T. Production of short chain fatty acids by the intestinal microflora during the first two years of human life. J Pediatr Gastroenterol Nutr 1992; 15: 395 – 403. 12. Midtvedt A-C, Carlstedt-Duke B, Midtvedt T. Establishment of a mucin-degrading intestinal microflora during the first two years of human life. J Pediatr Gastroenterol Nutr 1994; 18: 321 – 6. 13. Spiller GA, Chernoff MC, Hill RA, Gates JE, Nassar JJ, Shipley EA. Effect of purified cellulose pectin and a low residue diet of faecal volatile fatty acids, transit time and faecal weight in humans. Am J Clin Nutr 1980; 33: 754 – 9. 14. Holdeman LV, Moore WEC, eds. In: Anaerobe Laboratory Manual, 2nd edn. Blacksburg, VA: Virginia Polytechnic Institute and State University, 1973. 15. Gothfors L. Effects of diet on intestinal flora. Acta Paediatr Scand Suppl 1989; 351: 118 – 21. 16. Livesey G. Energy values of unavailable carbohydrate and diets: an inquiry and analysis. Am J Clin Nutr 1990; 51: 617 – 37. 17. Midtvedt A-C, Midtvedt T. Conversion of cholesterol to coprostanol by the intestinal microflora during the first two years of human life. J Pediatr Gastroenterol Nutr 1993; 17: 161 – 8. 18. Mykka¨ nen H, Tikka J, Pitka¨ nen T, Ha¨ nninen O. Fecal bacterial enzyme activities in infants increase with age and adoption of adult-type diet. J Pediatr Gastroenterol Nutr 1997; 25: 312 – 6. 19. Gro¨ nlund M-M, Salminen S, Mykka¨ nen H, Kero P, Lehtonen O-P. Development of intestinal bacterial enzymes in infants — relationship to mode of delivery and type of feeding. APMIS 1999; 107: 655 – 60. 20. The Scottish Diet Report. Scottish Office. Home & Health Dept, Edinburgh: HMSO, 1993. 21. Foster K, Lader D, Cheesebrough S. Infant Feeding 1995. Office for National Statistics, London: The Stationery Office, 1997.
