Low level of galacto-oligosaccharide in infant formula stimulates growth of intestinal Bifidobacteria and Lactobacilli

Abstract

AIM: To investigate the effect of a new infant formula supplemented with a low level (0.24 g/100 mL) of galacto-oligosaccharide (GOS) on intestinal micro-flora (Bifidobacteria, Lactobacilli and E. coli) and fermentation characteristics in term infants, compared with human milk and a standard infant formula without GOS.

METHODS: Term infants (n = 371) were approached in this study in three hospitals of China. All infants started breast-feeding. Those who changed to formula-feeding within 4 wk after birth were randomly assigned to one of the two formula groups. Growth and stool characteristics, and side effects that occurred in recruited infants were recorded in a 3-mo follow-up period. Fecal samples were collected from a subpopulation of recruited infants for analysis of intestinal bacteria (culture technique), acetic acid (gas chromatography) and pH (indicator strip).

RESULTS: After 3 mo, the intestinal Bifidobacteria, Lactobacilli, acetic acid and stool frequency were significantly increased, and fecal pH was decreased in infants fed with the GOS-formula or human milk, compared with those fed with the formula without GOS. No significant differences were observed between the GOS formula and human milk groups. Supplementation with GOS did not influence the incidence of crying, regurgitation and vomiting.

CONCLUSION: A low level of GOS (0.24 g/100 mL) in infant formula can improve stool frequency, decrease fecal pH, and stimulate intestinal Bifidobacteria and Lactobacilli as in those fed with human milk.

Key Words: Human milk, Prebiotic, Probiotic, Safety, Chinese

INTRODUCTION

Table 1 Clinical data of the infants enrolled in the study.

	GOS formula	GOS formula & human milk	Human milk	Control formula	F/P value
Babies recruited in 3 mo follow-up	n = 37	n = 58	n = 24	n = 45
Male/Female	20/17	30/28	13/11	24/21
Gestational age (wk)	38.7 ± 0.6	39.1 ± 0.8	39.4 ± 0.7	38.8 ± 0.8	1.40/0.24
Weight at birth (kg)	3.30 ± 0.42	3.20 ± 0.43	3.34 ± 0.43	3.35 ± 0.45	1.28/0.28
Length at birth (cm)	49.50 ± 0.96	49.47 ± 1.15	49.61 ± 1.23	49.57 ± 1.03	0.13/0.94
Head circumference at birth (cm)	34.15 ± 0.55	33.98 ± 0.72	34.07 ± 0.81	34.25 ± 0.77	1.35/0.26
Feeding volume (mL/kg-BW/d)	162 ± 27			157 ± 34
Weight gain during study period (g/d)	41.26 ± 5.22	43.35 ± 4.87	40.97± 5.06	40.59 ± 3.95	1.54/0.21
Length gain during study period (cm/wk)	0.95 ± 0.11	1.01 ± 0.11	0.93 ± 0.10	0.96 ± 0.11	1.94/0.13

Data are expressed as mean ± SD. GOS formula: Galactooligosaccharides in an amount of 0.24 g/dL. Control formula does not contain added GOS.

Table 2 Levels of intestinal bacteria at the end of a 3-mo feeding period as measured in fresh feces.

	GOS formula (n = 20)	GOS formula & human milk (n = 29)	Human milk (n = 15)	Control formula (n = 18)	F/P value
Bifidobacteria	9.01 ± 1.18	8.97 ± 0.85	9.25 ± 0.93	8.16 ± 0.99	4.08/0.01
Lactobacilli	5.91 ± 1.61	5.99 ± 2.12	5.45 ± 2.16	4.27 ± 2.02	3.17/0.03
E. coli	6.35 ± 1.59	5.90 ± 1.84	5.74 ± 1.68	5.68 ± 2.11	0.52/0.67

Data are presented as mean ± SD Log₁₀ cfu/g wet faeces. Control formula does not contain added GOS.

Table 3 Fecal concentration of acetic acid and pH values at the end of a 3-mo feeding period.

	GOS formula	GOS formula & human milk	Human milk	Control formula	F/P value
Acetic acid (n)	25.93 ± 6.84 (21)	25.09 ± 5.49 (34)	23.76 ± 5.65 (17)	19.42 ± 5.35 (24)	6.03/< 0.01
pH (n)	5.22 ± 0.25 (25)	5.27 ± 0.25 (41)	5.32 ± 0.24 (19)	5.56 ± 0.51 (27)	5.57/< 0.01

Data of acetic acid are presented as mean ± SD mmol/g wet faeces. Control formula does not contain added GOS.

Table 4 Scores of stool characteristics and intensity of digestive symptoms in the infants enrolled in the study (mean ± SD).

	GOS formula	GOS formula & human milk	Human milk	1Control formula	F/P value
Babies recruited in 3 mo follow-up	n = 37	n = 58	n = 24	n = 45
Stool consistency	2.46 ± 0.62	2.55 ± 0.66	2.37 ± 0.83	3.11 ± 0.34	3.27/0.02
Crying	1.06 ± 0.03	1.04 ± 0.02	1.08 ± 0.05	1.05 ± 0.03	1.29/0.27
Regurgitation	1.34 ± 0.55	1.28 ± 0.63	1.41 ± 0.58	1.35 ± 0.67	1.18/0.34
Vomiting	1.22 ± 0.43	1.18 ± 0.34	1.14 ± 0.46	1.25 ± 0.38	1.24/0.30

¹Control formula does not contain added GOS.

Breastfeeding is the primary choice for newborns. However, for some reason, many infants are formula fed. Breast milk is superior over artificial formula in many aspects, including its effect on the development of intestinal microflora. Breast-fed infants have a higher level of intestinal Bifidobacteria and Lactobacilli[1,2], both of which are known to be potentially beneficial to the health of their hosts[3,4]. It is likely that oligosaccharides in human milk are more beneficial to intestinal flora[5-7].

The amount of oligosaccharides in mature human milk is in range of 12-15 g/L. Most of oligosaccharides are fermented in the large intestine[8,9]. Galacto-oligosaccharide (GOS) contained in oligosaccharides, is a short-chain galactose with a terminal glucose molecule[10], which is produced from lactose and commercially available. Studies have shown that GOS can selectively stimulate development of Bifidobacteria[11]. However, only a few studies are available on oligosaccharides in infant formula[12,13], and there is no study using only GOS in infant formula.

Recently, Moro et al[13] showed that the number of Bifidobacteria and Lactobacilli increases significantly compared with a control formula with maltodextrin instead of the oligosaccharides in term infants fed with formula at the dose of 0.4 g and 0.8 g of oligosaccharide per 100 mL (90% GOS and 10% low-molecular weight fructo-oligosaccharides). There was no difference between the two supplemented groups. These values have been adopted by the Scientific Committee on Food (SCF: April, 2003) of the European Commission and probably will be implemented in European regulations. In preterm infants, the same mixture of oligosaccharide (1.0 g/100 mL) could stimulate growth of Bifidobacteria and result in stool characteristics as seen in human milk-fed infants[12]. Recently, however, a rat study[14] showed that the intestinal cell wall is irritated and the risk of bacterial translocation increases (following orally Salmonella enteritidis infection) when fructooligosaccharides are provided at a high (3% and 6% of dry matter), but not an unrealistic amount (maximum level adopted by the SCF is about 6% of dry matter). Therefore, positive effects of oligosaccharides on intestinal bacteria may not always justify the levels tested.

Our hypothesis is that a prebiotic effect of the human milk-like GOS is already present at a much lower level than that currently adopted by the SCF. This study was to investigate the effect of 0.24 g GOS per 100 mL on intestinal microflora colonization and fermentation in formula-fed term infants compared with breast-fed and control formula-fed counterparts, and to detect the lowest and safe effective level of GOS.

MATERIALS AND METHODS

Subjects

Three hundred and seventy-one healthy term infants, appropriate for gestational age, were approached in the study by Nanjing Children’s Hospital of Nanjing Medical University (n = 181), Nanjing Maternal Hospital of Nanjing Medical University (n = 90) and Affiliated Hospital of South East University (n = 100). Finally, 164 infants were recruited for the 3-mo follow-up (Table 1). Those excluded from the study were due to refusal of their parents and failure in taking the fresh fecal samples. The Ethics Committees of the three hospitals approved the study and all the parents gave their informed consent before enrolment in the study.

Diets

When not breast-fed, the infants were randomly assigned to test formula group and control formula group (Table 1). The test formula (Frisolac Advanced, Friesland Nutrition, Netherlands) group was supplemented with GOS (0.24 g/100 mL), while the control formula (Frisolac H, Friesland Nutrition, Netherlands) group was not supplemented with GOS. The other infants were either breastfed or received a combination of breast milk and test formula.

Growth and stool characteristics of, and side effects in the infants were recorded during the 3-mo follow-up. The body weight of all infants was measured using a scale with an accuracy of ± 5 g. The crown-heel length and head circumference were measured using a special board for newborn infants with an accuracy of ± 1 mm. Stool consistency (score 1-4: 1 = watery, 2 = loose/mushy, 3 = soft formed, 4 = hard formed) and frequency were recorded based on an interview with the mother. Crying (score 1-3: 1 = practically not crying, 2 = crying in connection with feeding, 3 = crying independently from the meals), regurgitation (score 1-3: 1 = no regurgitation, 2 = 1-2 regurgitations, 3 = > 2 regurgitations per day), and vomiting (score 1-3: 1 = no vomiting, 2 = 1 episode of vomiting, 3 = > 1 episode of vomiting per day) were also recorded based on an interview with the mother.

Fecal samples were collected from a subpopulation (Tables 2 and 3) for analysis of intestinal bacteria (n = 82), short chain fatty acids (SCFAs) (n = 96) and pH (n = 112). For analysis of Bifidobacteria and Lactobacilli[1,12,13], one gram of fresh feces was homogenized and diluted in 10 mL of a pre-reduced brain and heart infusion broth in an anaerobic glove box within 1 h of collection. Ten μL of each dilution was spread on the surfaces of Rogosa SL agar (Difco, Detroit, USA) dishes and incubated anaerobically at 37°C. Colony forming units (cfu) of Lactobacilli were marked after 2 d, whereas cfu of Bifidobacteria were counted after 4 d. For detection of E. coli, 1 g of fresh feces was homogenized and diluted in 10 mL of a pre-reduced brain and heart infusion broth in a clean airflow bench. Ten μL of each dilution was spread on the surfaces of MacConkey agar (Difco, Detroit, USA) dishes and incubated aerobically at 37°C for 1 d. Cfu were expressed as per gram of feces.

Concentration of SCFA acetic acid was determined by gas chromatography as previously described[15]. Briefly, weighed fecal samples were diluted at approximately 1:10 in a 0.1 mol/L sodium phosphate broth (pH 6.5), and suspensions were used to determine the concentration of acetate using a Hewlett-Packard 5890A Series II gas chromatograph (Agilent, Wilmington, DE) and a glass column (180 cm × 4 mm i.d.) packed with 10% SP-1200/1% H₃PO₄ on 80/100 mesh Chromosorb WAW (Supelco, Bellefonte, PA). Nitrogen was the carrier gas with a flow rate of 75 mL/min. Temperature of the oven, detector and injector was 125°C, 175°C and 180°C, respectively. SCFA concentrations in the blank tube were used to correct non-substrate SCFA production. pH of the fresh stool sample was measured with a piece of multicolor indicator paper with an accuracy of 0.2 U (Spezialindicatorpapier Merck Eurolab GmbH, Darmstadt, Germany).

Statistical analysis

All data were given as mean ± SD. An overall group effect on a measured variable was evaluated by ANOVA with F value. When significant, this was followed by t test for single factor group comparisons. P < 0.05 was considered statistically significant. SPSS 12 (SPSS Institute Inc., Chicago, USA) was used in analysis of data.

RESULTS

At the end of a 3-mo feeding period, the number of intestinal Bifidobacteria and Lactobacilli was significantly increased both in GOS-supplemented formula-fed infants and in breast-fed infants, compared with those fed with the control formula. No difference was seen between the GOS formula-feeding and breast feeding groups. The number of cfu in E. coli did not differ between the 3 groups (Table 2).

Intestinal acetic acid production and stool frequency were significantly increased in infants fed with GOS formula or with breast milk, compared with those fed with standard formula. Fecal pH was significantly higher in those fed with the control formula. No difference in stool frequency, fecal pH and intestinal acetic acid production was found between GOS formula-fed and breast-fed infants, (Tables 3 and 4).

The GOS formula did not influence the incidence of side effects (crying, regurgitation, vomiting) (Table 4). Weight gain and body height increase were similar among the groups (Table 1).

DISCUSSION

This study showed that a 3-mo feeding period of a relatively low amount of GOS (0.24 g/dL) in infant formula stimulated the growth of Bifidobacteria and Lactobacilli as seen in breast-fed infants but not the growth of potentially harmful E. coli. Stool frequency, fecal pH and amount of produced acetic acid were also comparable in the breast-fed infants, indicating hat a low GOS formula has the same prebiotic effect as a high GOS formula, but a minimized risk of intestinal irritation.

Breast-fed infants have an intestinal microflora dominated by Bifidobacteria and Lactobacilli[1,2] and are quite different from those fed with a standard infant formula[16]. Both Bifidobacteria and Lactobacilli are beneficial to infants. As a result, manufacturers of formula try to mimic the gastrointestinal flora in breast-fed infants by adding probiotics and/or prebiotics such as GOS. Although the addition of probiotics is able to manipulate the infants’ microflora towards the breast-fed infants’ microflora[17,18], this concept may be regarded as unphysiological since breast milk itself does not contain bacteria. Prebiotics are, therefore, the first choice.

Prebiotics serve as food for Bifidobacteria and Lactobacilli and increase their number by their competitive edge over the pathogenic bacteria. During the fermentation of prebiotics, organic acids (lactic acid and SCFA) are produced that can inhibit the growth (increase colonization resistance) of acid-sensitive pathogens like salmonella[19]. SCFA, also the preferred source of energy for colonic epithelial cells, may stimulate colonocyte proliferation, and are suggested to enhance small intestinal glucose uptake[20]. On the other hand, the same organic acids may induce injury to the intestinal mucosa and impair its barrier function, as indicated by increased cytotoxicity of fecal water and fecal mucin excretion[14]. The fermentation rate might play an important role in this detrimental effect. Since slow fermentation as seen in case of resistant starch does not increase fecal mucin and luminal cytotoxicity, providing a lower amount of fast fermentable prebiotics to the intestine may also prohibit irritation[21].

Ten Bruggencate et al[14], in a rat study, showed that 6% of FOS on dry matter increases the number of Bifidobacteria, but 3% of FOS significantly (100-fold) increases the number of Enterobacteria, indicating that the selectivity of fibers can be questioned. The increased levels of Enterobacteria in combination with an impaired barrier function may increase the risk of bacterial translocation[14]. Moro et al[13] reported that the number of Bifidobacteria and Lactobacilli increases significantly in full term infants following oligosaccharide supplementation of 0.4 g/dL and 0.8 g/dL (3% and 6% on dry matter respectively). Although the authors reported no significant increase in the number of infants with positive culture of Enterobacteria, this statement does not say anything about the levels of these gram negative bacteria in those with a positive culture. Boehm et al[22] showed that 1.0 g of a mixture of GOS and FOS per 100 mL preterm formula has a bifidogenic effect but no significant effect on the number of Lactobacilli and Enterobacteria.

In the present study, the effect of only 0.24 g/dL of GOS on intestinal microflora and fermentation was observed in term infants. The results show that even such a low amount of GOS could stimulates the growth of Bifidobacteria and Lactobacilli as in breast-fed counterparts, decrease fecal pH, and increase the production of intestinal SCFA. The frequency of stools was shorter and the stools became softer, as seen in breast milk-fed infants. These changes in stool characteristics could not be explained by the increased osmolarity (about 1 mOsmol/L) of the formula because of the addition of GOS, and are, therefore, probably related to the changes in bacterial flora and fermentation. Studies in adults showed that a greater amount of dietary oligosaccharides may lead to adverse effects, flatulence in particular[23]. In the present study, no adverse side effects were reported.

This study certainly has its limitations. The number of infants involved in bacterial and SCFA analysis as very small mainly due to the refusal of parents and the failure in taking fresh fecal samples. However, despite such limitations, the difference between GOS-fortified and non-fortified groups was significant. Furthermore, files of infants are not complete because of the poor communication and traffic facility for the follow-up. Therefore, this study was a pilot study with promising results that need to be confirmed in a larger and more appropriate study.

In summary, supplementation of GOS stimulates the growth of Bifidobacteria and Lactobacilli. Both bacteria are beneficial to infants. However, an increase in Enterobacteria cannot be excluded, although it may be dose-dependent. A small amount of GOS can stimulate the growth of Bifidobacteria and Lactobacilli, but not the growth of Enterobacteria in breast-fed infants.

COMMENTS

Background

Breast milk is superior over artificial formula in terms of newborn nutrition. Breast-fed infants have a higher level of intestinal Bifidobacteria and Lactobacilli, both of which are potentially beneficial to the health of their hosts. Oligosaccharides in human milk are more beneficial to intestinal flora. The amount of oligosaccharides in mature human milk ranges 12-15 g/L. Galacto-oligosaccharide (GOS) is a short chain galactose with a terminal glucose molecule. Studies have shown that GOS can selectively stimulate the development of Bifidobacteria. However, a large amount GOS (3%-6% of dry matter) supplementation to the artificial formula showed irritation of the intestinal cell wall and increased risk of bacterial translocation. This study investigated the effects of artificial formula supplemented with 0.24 g GOS per 100 mL (1.8% of dry matter) on intestinal microflora colonization and fermentation in infants, and detected the lowest and safe effective level of GOS.

Research frontiers

Moro et al[13] showed that in term infants fed with formula at the doses of 0.4 and 0.8 g of oligosaccharides per 100 mL (90% GOS and 10% low-molecular weight fructo-oligosaccharides), the number of Bifidobacteria and Lactobacilli increased significantly compared with a control formula with maltodextrin instead of oligosaccharides. These values have been adopted by the Scientific Committee on Food of the European Commission and probably will be implemented in European regulations. In preterm infants, the same mixture of oligosaccharides (1.0 g/100 mL) stimulated the growth of Bifidobacteria and resulted in stool characteristics as seen in human milk-fed infants. However, a recent rat study showed irritation of the intestinal cell wall and increased risk of bacterial translocation (following orally Salmonella enteritidis infection) when a large amount of fructooligosaccharide (3% and 6% of dry matter), but not an unrealistic amount (maximum level adopted by the SCF is about 6% of dry matter) was provided. Therefore, positive effects of oligosaccharides on intestinal bacteria may not always justify the levels tested.

Innovations and breakthroughs

This study showed that a 3 mo feeding period of a relatively small amount of GOS (0.24 g/dL) in infant formula could stimulate the growth of Bifidobacteria and Lactobacilli, but not the growth of potentially harmful E. coli in breast-fed infants. Stool frequency, fecal pH and the amount of produced acetic acid were also comparable, indicating that low GOS formula may have the same prebiotic effect as high GOS formula, but a minimized risk of intestinal irritation.

Applications

The present study was designed to investigate the effect of only 0.24 g/dL of GOS on intestinal microflora and fermentation in term infants. The data show that even such a small amount of GOS could stimulate the growth of Bifidobacteria and Lactobacilli, decrease fecal pH, and increase the production of intestinal SCFA. Stools came more frequently and became softer in breast milk-fed infants, indicating that a mall amount of GOS (0.24 g/dL) supplementation can stimulate the growth of Bifidobacteria and Lactobacilli, but not the growth of Enterobacteria in breast-fed infants. It is, therefore, safe and effective when used in artificial infant formula.

Terminology

Prebiotics are "selectively fermented ingredients that allow specific changes both in composition and/or activity of gastrointestinal microflora that confers benefits to host well being and health". Probiotics are defined viable microorganisms, a sufficient amount of which can reach the intestine in an active state and thus exerting positive health effects.Synergistic combinations of pro- and prebiotics are called synbiotics. Today, only bifidogenic, non-digestible oligosaccharides (particularly inulin, its hydrolysis product oligofructose, and galactooligosaccharides), fulfill all the criteria for prebiotic classification.

Peer review

This study determined the effect of a lower-than-normal dose of a prebiotics on the gastrointestinal tract of infants. Its results show that a mall amount of GOS (0.24 g/dL) in artificial formula could stimulate the growth of intestinal Bifidobacteria and Lactobacilli but not E. coli in term infants, indicating that it is safe and effective when used in artificial infant formula.