Randomized Double-Blind Study of the Nutritional Efficacy and Bifidogenicity of a New Infant Formula Containing Partially Hydrolyzed Protein, a High β-Palmitic Acid Level, and Nondigestible Oligosaccharides

Abstract

Objectives 

The aim of this study was to evaluate the nutritional efficacy and bifidogenic characteristics of a new infant formula containing partially hydrolyzed whey protein, modified vegetable oil with a high β-palmitic acid content, prebiotic oligosaccharides, and starch.

Methods 

In a double-blind study, healthy formula-fed term infants aged younger than 2 weeks were randomized to receive either the new infant formula (NF) or a standard formula (SF) until the age of 12 weeks. Anthropometric measurements were taken at enrollment, 6 weeks, and 12 weeks. In a subsample of infants, blood samples were taken at 6 weeks and stool samples were taken at enrollment and 6 weeks. Blood samples were analyzed for biochemical measures of protein status and amino acids, and stools were analyzed for total bacteria and bifidobacteria. Mothers completed a feeding diary and questionnaire at 6 and 10 weeks.

Results 

One hundred fifty-four infants were enrolled in the study; 102 completed the trial. The growth of infants in both formula groups was in line with published growth curves. During the first 6 weeks, NF girls gained more weight and head circumference than the SF girls. These velocity differences were not maintained throughout the 12-week study period. The NF stools had a higher proportion of bifidobacteria at 6 weeks compared with the SF stools, and they were softer. There were no clinically significant differences in the blood biochemical and amino acid values between groups. Both formulas were well tolerated by the infants.

Conclusions 

When compared with a standard infant formula, the new formula supported satisfactory growth, led to higher counts of bifidobacteria in the feces, produced blood bio-chemical values typical of formula-fed infants, and was well tolerated.

The importance of evaluating the nutritional efficacy of new infant formulas has been the subject of two recent reports by expert committees (^1,2). Both of these reports recommended that formulas that include new ingredients or new combinations of ingredients should be assessed nutritionally and that the results of such evaluations should be made publicly available. This report concerns the double-blind randomized nutritional evaluation of a new infant formula (NF; Omneo/Comformil, Numico, Zoetermeer, The Netherlands) that contains a mixture of prebiotic oligosaccharides comprising galactooligosaccharides (GOS) and fructooligosaccharides (FOS), fat with 41% of the palmitic acid in the β position, partially hydrolyzed whey protein, and starch.

The rationale for adding oligosaccharides to this formula is to provide some of the protective functions associated with human milk. Human milk contains approximately 10 g/L oligosaccharides, which are resistant to digestion in the upper gastrointestinal tract (^3,4). These oligosaccharides are thought to play an important role by promoting the bifidogenic-dominant colonic microflora observed in breast-fed infants (⁵). Bifidobacteria are desirable because they are nonpathogenic and are thought to protect infants from pathogenic intestinal microorganisms during a phase of insufficient immune response (⁶). Also, it has been observed that increased levels of bifidobacteria in the gut microflora of infants with a high risk for atopic disease at age 3 weeks are associated with reduced atopic sensitization at age 12 months (⁷). Normal infant formulas based on cows' milk do not contain prebiotic oligosaccharides; therefore, these have been added to NF as a mixture comprising 90% GOS and 10% FOS. The GOS is prepared enzymically from lactose and has a chain length mainly between 2 and 6 monomers, which are joined by β(1–4) and β(1–6) glycosidic linkages. The FOS is extracted from chicory roots and comprises linear chains of β(1–2) glycosidic-linked fructose. The FOS component used in NF is especially selected to contain a reduced proportion of molecules with a chain length of less than 10 monomer units. The GOS–FOS oligosaccharide mixture is similar to the oligosaccharides in human milk with respect to the molecular weight profile and high galactose content. However, this mixture differs in other respects from the oligosaccharides in human milk, the latter comprising a complex mixture of more than 100 different oligosaccharide structures.

Previous studies have shown that some infant formulas based on hydrolyzed protein were not nutritionally optimal. In one study involving formulas containing several different types of hydrolyzed protein, Rigo et al. (⁸) found lower weight gains and reduced nitrogen retention with some hydrolyzed protein-based formulas compared with human milk or a conventional formula based on whole cow milk protein.

In view of these results, an ESPACI/ESPGHAN joint expert committee recommended that all formulas based on hydrolyzed protein should be carefully evaluated (⁹). Previously, the ESPGHAN Committee on Nutrition recommended that these formulas should be evaluated in growth studies lasting at least 3 months and that information should be provided on the plasma levels of albumin, proteins with short half-lives (e.g., prealbumin), and amino acids (¹⁰).

The aim of this study was to investigate the nutritional adequacy of NF, as recommended by ESPGHAN, and to determine whether the proportion of bifidobacteria in the colonic microflora was changed compared with the microflora of infants who received a standard infant formula (SF; Pre-Aptamil with Milupan, Milupa, Friedrichsdorf, Germany).

SUBJECTS AND METHODS

Subjects

In this double-blind randomized study, healthy term neonates whose mothers could not or chose not to give breast milk for medical, social, or personal reasons were recruited from four centers in Germany. The inclusion criteria were gestational age between 37 weeks and 42 weeks, birth weight between the tenth and ninetieth percentiles, and exclusive formula feeding by age 14 days. Infants with any of the following characteristics were excluded from the study: multiple births, mothers with significant illness or disability, adopted or fostered, major congenital abnormality or chromosomal disorder, disease requiring mechanical ventilation or antibiotic treatment, or born with an umbilical artery pH <7.0 or with a 5- or 10-minute Apgar score <7.

The newborns were randomized to receive NF or SF using sealed envelopes. The randomization was constructed using a random numbers table with a block design for groups of four subjects and stratified by sex and study center. The study formulas were fed ad libitum from or before age 2 weeks until aged 12 weeks. The composition of the study formulas is given in Table 1. The primary outcome measure used for assessing nutritional adequacy was weight gain, and the secondary measures were gains in length, head circumference, and skinfold thickness, and blood biochemical measurements of protein status. At the initial interview, information about delivery, age, and occupation of the parents was recorded. Weight, height, head circumference, and skinfold thickness were measured at enrollment, and these measurements were repeated at ages 6 and 12 weeks. The infants were observed until the age of 12 weeks.

TABLE 1A: Composition of the study formulas ^†

TABLE 1B: Composition of the study formulas ^†

Anthropometry

Weight and Length

Weight of the naked infants was measured to the nearest 10 g using either an electronic or standard beam balance. Accuracy was confirmed using calibrated weights of known mass. Recumbent length was measured in triplicate to the nearest 0.5 cm using a measuring board fitted with a head and footboard. Head circumference was measured in triplicate to the nearest 0.1 cm using a metal tape measure.

Skinfold Measurements

Triceps, biceps, and suprailiac and subscapular skinfold thickness were measured in triplicate under standard conditions on the left side of the body using a standard Holtain skinfold caliper (Holtain Ltd., Crymych, UK) as previously described (¹¹).

A single observer at each participating center obtained the majority of the measurements. A well-trained medical technical assistant provided holiday coverage. To standardize the measurement techniques used at all centers, an anthropometrics workshop was held before the study started, at which the detailed procedures of each measurement were agreed on and practiced by each person responsible for this aspect of the study.

Fecal Bacteriology

In a subsample of 54 infants who had either been bottle-fed from birth or breast-fed for less than 7 days, one 5-g stool sample was collected on each of two consecutive days: immediately after study enrollment and at age 6 weeks (four samples per subject). Stools samples were collected as freshly as possible and immediately frozen at −20°C. The stools were analyzed for total bacterial count by staining the bacterial cells with 4´,6-diamidino-2-phenylindole (DAPI) and for the total number of bifidobacteria using fluorescent in situ hybridization (FISH), using probe Bif164 as described (¹²). At least 25 microscopic fields per sample were automatically examined using an Olympus AX70 epifluorescence microscope and appropriate image analysis software.

Biochemistry

A preprandial venous blood sample (3–4 hours after the last feeding) was taken at age 6 weeks from 40 children whose parents volunteered to participate in this part of the study. The blood was centrifuged, and the serum samples were stored at −20°C until they were analyzed for protein, urea nitrogen, albumin, prealbumin, and amino acids using standard laboratory procedures.

Diet

At 6 weeks and 10 weeks, postpartum mothers were asked to complete a diary and questionnaire in which the amounts of formula consumed and the quality and frequency of stools were recorded for 5 days. Stool consistency was classified as watery, runny, mushy/soft, formed/soft, or hard. The diary and questionnaire were carefully explained to the mothers before they were issued. The mothers were also asked to report on the ease of preparation of the formula and its acceptance and tolerance characteristics. A 24-hour dietary record was made at age 12 weeks.

Sample Size and Statistics

The sample size required to detect a difference in weight gain of 3 g/d (approximately one half of a standard deviation) at a 5% significance level and with 80% power was calculated to be 64 infants per group. After allowing for a 20% dropout rate, the target group size became 80 infants. This group size is in line with the advice of The American Academy of Pediatrics, which recommends that growth studies conducted for the purpose of assessing infant formulas should have the capability to detect a difference of 3-g/d weight gain during the first 3 months of life (¹³).

The results are presented on a per protocol basis. The data were also analyzed on an intention-to-treat basis, and any important differences in the statistical interpretation of the two sets of data are referred to in the Discussion section. The results of the two formula groups were evaluated using t tests for parametric data and the Mann-Whitney test for nonparametric data. Binary and categorical data were assessed by χ² analysis. A significance level of P ≤ 0.05 was considered to be significant.

Ethics

Because the Children's Hospital of Greifswald was the coordinating center of the study, the protocol was submitted to and approved by the Ethical Committee of the University of Greifswald. This approval was accepted by the authorities of the other three participating centers in accordance with the rules for multicenter studies. All parents gave written informed consent.

RESULTS

Sixty-six girls and 88 boys were entered into the study between September 1999 and February 2001; 78 subjects were given SF, and 76 were given NF. The characteristics of the study infants are shown in Table 2. These data show that the infants in the two formula groups were appropriately matched and were not different in their baseline characteristics. There was also no difference between groups regarding the socioeconomic status of the parents (data not shown). Fifty-two infants left the study before completion for a variety of reasons (Fig. 1). The majority of dropouts occurred within the first 6 weeks of the study when 21 and 22 of the SF and NF infants, respectively, left the study. The reasons given for dropping out were mainly because of feeding problems, e.g., baby not satisfied, flatulence, stool quality, or vomiting/posseting. The number of dropouts and the reasons for dropping out were not statistically significantly different between the two formula groups.

TABLE 2: Characteristics of study subjects

FIG. 1.: Study course and dropout characteristics of subjects.

Anthropometry

The growth velocity data show that the NF girls gained more weight than the SF girls between study entry and 6 weeks (P < 0.05) and had larger gains in head circumference between entry and 12 weeks (P < 0.05;Table 3). The NF boys had greater gains in total skinfold thickness during the 12-week study period (P < 0.05). There were no differences in the length gain measurements. There were no differences in weight gain and length gain between entry and 12 weeks for girls and boys analyzed either separately (Table 3) or combined (data not shown).

TABLE 3: Gains in weight, length, head circumference, and skinfold thickness of study subjects ^†

Fecal Bifidobacteria

The number of bacteria and bifidobacteria in the feces is shown in Table 4 and Figure 2. The number of bifidobacteria in the NF group increased substantially from 3.87 × 10⁹ to 1.03 × 10¹⁰ per gram of wet feces (P < 0.005), whereas there was no change in the stools of the SF group (entry, 3.50 × 10⁹; 6 weeks, 5.60 × 10⁹). The data shown in Figure 2 demonstrate that the amount of bifidobacteria, as a percentage of the total number of bacteria, was significantly different from the initial percentage of bifidobacteria in the NF stools (P = 0.002) and from the stools of the SF infants taken at 6 weeks (P = 0.003).

TABLE 4: Fecal total bacteria and bifidobacteria ^†

FIG. 2.: Percentage of bifidobacteria found in the stools of infants at study entry and at 6 weeks (± SE). SF, standard formula; NF, new formula.

Biochemistry

At 6 weeks, the serum concentrations of total protein, albumin, and urea were similar in the two dietary groups (Table 5 and Fig. 3). The mean serum prealbumin level was slightly lower in the NF infants (P < 0.002). The NF infants had a lower serum concentration of tyrosine (P < 0.05) and higher serum concentration of threonine (P = 0.001), isoleucine (P < 0.05), and lysine (P = 0.007) than the SF infants (Fig. 3). The mean level of threonine in the NF group was above normal (mean ± 2 SD) for breast-fed infants (⁸).

TABLE 5: Serum biochemistry at 6 weeks of age ^†

FIG. 3.: Serum levels of amino acids in infants at age 6 weeks. SF, standard formula; NF, new formula. Breast-fed values from (⁸). NF different to SF * P < 0.05, ** P = 0.07, *** P = 0.001.

Formula Intake and Tolerance

The two trial formulas were equally well accepted by more than 90% of the infants who completed the study, and the mothers reported that the infants were satisfied by their feeds and had no feeding problems (Tables 6 and 7). About one third of the mothers had some occasional concern about posseting, but there was no difference between the two formulas in this respect. The NF infants consumed less formula (P < 0.001) and received less energy per kg body weight than the SF infants (Table 6). After allowing for a potential energy contribution of 1.9 kcal/g GOS/FOS from the short-chain fatty acids produced by bacterial fermentation of the prebiotic oligosaccharides in the colon, the difference in energy intake disappeared at 6 weeks but remained at 12 weeks (P < 0.03).

TABLE 6: Intake of formulas ^†

TABLE 7: Percentage distribution of stool consistencies

The infants in the two groups passed a similar number of stools, which were predominantly yellow or yellow–green. The NF infants passed a higher proportion of softer stools than the SF infants (P = 0.005;Table 7).

DISCUSSION

In this study, we have demonstrated that infants fed a formula containing partially hydrolyzed protein with high β-palmitic acid level and nondigestible oligosaccharides grow normally and have a stool microflora with higher numbers of bifidobacteria compared with infants fed a standard formula. Although both formulas were well accepted and tolerated by the infants, approximately one third of the subjects enrolled did not complete the study. This dropout rate is of concern because it may potentially introduce a bias into the study. However, this is unlikely because an analysis of the baseline anthropometric characteristics of the infants who dropped out showed them to be not different than the remaining infants. The most likely explanation is that many mothers of young babies find the additional appointments and the completion of detailed questionnaires, which are an inevitable part of feeding studies, to be too time consuming and an unacceptable burden. This explanation is supported by the fact that nearly all the dropouts occurred during the first part of the study period. In one half of the dropouts, the reasons for leaving the study could not be ascertained despite repeated attempts by the investigators to contact the parents.

The actual dropout rate was higher than the anticipated value of 20%, leading to a group size at 12 weeks of 53 and 49 infants for the SF and NF groups, respectively. This effectively enabled the study to detect a difference of approximately 0.55 of a standard deviation at a 5% significance level and with 80% power. With respect to weight gain, this amounts to a difference of 4.8 g/d, which is still clinically relevant, instead of 3 g/d as originally planned.

Some minor differences in growth rates between the groups were observed during the first 6 weeks, but these had largely disappeared by age 12 weeks. The higher weight gain observed in the NF girls between study entry and 6 weeks (but not at 12 weeks) was also seen in the intention to treat NF girls. The latter also had higher gains in head circumference between study entry and 6 weeks. The higher weight gains in the NF infants are reflected to some extent in the skinfold thickness data (Table 3). Thus, the NF boys had higher gains in total skinfold thickness than their SF counterparts between study entry and 12 weeks (P < 0.05). The same trend was apparent in the girls but did not reach statistical significance. Between study entry and age 12 weeks, the weight gains of the girls and boys, either individually or combined, were not significantly different for the two formula groups. Furthermore, the mean weight of the NF girls and boys combined at 12 weeks (6,270 g ± 730 g) is similar to the weight of infants fed standard formulas previously published by Kennedy et al. (¹⁴) (6,120 g ± 720 g) and Morris et al. (¹⁵) (6,090 g ± 750 g). Comparison of the anthropometric data presented in Table 3 with previously published growth curves confirms that the infants in both formula groups grew normally.

Although NF has higher energy content compared with SF, the average energy intake of the NF infants was lower than that of the SF infants at 6 and 12 weeks. In a previous study, Hauser et al. (¹⁶) also reported a lower intake of energy from a formula containing hydrolyzed protein compared with a formula containing whole protein (80 kcal/kg/d vs. 97 kcal/kg/d). These authors considered that subjective factors, such as taste, might have been partly responsible for this effect. However, as with the present study, they found no difference in weight gain between the two formula groups. Another possible explanation for the difference in energy intake between SF and NF is that the metabolizable energy content of NF is higher than 70 kcal/dL because the method used for calculating energy, which is prescribed by law, excludes any energy contributed by FOS and GOS (¹⁷). Because these oligosaccharides have been reported to have an available energy content of between 1.0 to 2.0 kcal/g (^18,19), it is possible that the true metabolizable energy content of NF is 71.5 kcal/dL and not 70 kcal/dL as stated in Table 1A. However, after adjusting for the potential energy contribution from oligosaccharides, the energy intake from NF at 12 weeks still remained lower than from SF (P < 0.03). The 12-week intake data, based on a 24-hour recall, may have been less reliable than the 6-week information because the latter was collected in a 5-day diary. Whether the colonic microflora of young infants is sufficiently developed to fully realize this additional energy is questionable because Parrett et al. (²⁰) found that breast-fed infants were less able to ferment oligosaccharides before weaning compared with weaned infants.

Another explanation could be that the higher level of β-palmitic acid in NF could increase the amount of fat absorbed in the infant's gut and thus increase the energy available from NF. Carnielli et al. (²¹) showed that a level of β-palmitate similar to that in NF increased the total fat absorption from SF from 90.4% to 93.2%. Because NF has a fat content of 3.3%, the improved fat absorption would amount to an increase of available energy of only 0.8 kcal/100 mL. This effect is too small to explain the lower energy intake of NF. Because the NF infants grew satisfactorily, these results suggest that the energy content of NF may be more efficiently used than the energy of SF.

The increased proportion of bifidobacteria in the stools of the NF infants is in line with the results of three previous studies that investigated a GOS–FOS prebiotic mixture (^22–24). In these studies, a bifidogenic effect was demonstrated with preterm and term infants, and in the latter the effect was dose dependent at levels of 0.4 g/dL and 0.8 g/dL (²³). The prebiotic characteristics of FOS are well established in adults; however, a previous study in which FOS alone was added to a term formula failed to demonstrate a bifidogenic effect in infants (²⁵). Therefore, the GOS in NF appears to be important for the prebiotic effect of this formula. The bifidogenic property of the GOS–FOS mixture matches the effect of human milk oligosaccharides, although the GOS–FOS mixture and human milk oligosaccharides differ chemically and are similar only with respect to their molecular weight profile and galactose content. Therefore, these two compositional characteristics may be important with respect to their bifidogenic function. Further studies should be performed to investigate whether the observed bifidogenic effect leads to health benefits in formula-fed infants, e.g., a reduction of infections or atopic symptoms.

The blood biochemical data confirm that the protein status of the infants fed NF was normal (²⁶). Also, the values obtained for total serum protein and urea are comparable with those previously published for infants fed formulas containing hydrolyzed whey protein (⁸). Although the prealbumin level of the NF infants was slightly lower than the SF group, all the observed values for both formula groups were within the reference range of 0.107 g/L to 0.280 g/L for 1-month-old term infants published by Kanakoudi et al. (²⁷). Therefore, the difference is not considered to be clinically significant. The high serum threonine value seen in the NF infants is not unexpected because previous reports have confirmed this phenomenon (²⁸). This effect is attributed to the threonine-rich glycomacropeptide protein fraction that is present in the sweet whey commonly used for the manufacture of whey-dominant infant formulas (²⁸). The higher serum threonine level observed in infants fed whey-dominant infant formulas is not thought to be clinically significant.

The mothers' responses in the questionnaires indicated that the study formulas were well accepted by their infants and that there was a low incidence of feeding-related problems. No adverse effects were reported during the study. The softer stools produced by the NF infants were more like the stools of breast-fed infants, which have been described as 100% runny or watery (¹⁴). The softer texture is most likely caused by the presence of a relatively high proportion of β-palmitic acid in the fat content of NF, leading to better palmitic acid absorption. This effect, which has also been reported by Carnielli et al. (²¹), is attributed to a decrease in the level of calcium palmitate in the stools (²⁹). Previous studies have also shown that formulas containing GOS–FOS or hydrolyzed protein can produce softer stools; therefore, these components in NF may also have contributed to this effect (^22,23,30).

In conclusion, this study has demonstrated that the new infant formula was well accepted and tolerated by infants aged 0 months to 3 months and resulted in a satisfactory growth pattern and biochemical values of protein status typical of formula-fed infants. Infants fed this formula produced softer stools with a microflora having a higher proportion of bifidobacteria compared with infants given a standard infant formula. This latter difference may confer health advantages by providing some protection against gastrointestinal infections and allergy. These potential benefits should form the basis for further studies.

Acknowledgments:

The authors thank Dr. I. Kadow and Mrs. M. Fusch for helping to recruit subjects and for collecting data, and Dr. Susanne Unverzagt for providing statistical advice and carrying out the statistical analysis of the study results.