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Nutrient Metabolism

Substitution of Casein by ß-Casein or of Whey Protein Isolate by -Lactalbumin Does Not Affect Mineral Balance in Growing Rats¹

Nestlé Research Centre, Vers-chez-les-Blanc, CH-1000 Lausanne, Switzerland

²To whom correspondence should be addressed. E-mail: denis.barclay{at}rdls.nestle.com.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Bovine milk protein fractions that enable modification of the protein composition and amino acid profile of infant formulas to mimic those of human milk have recently become available. To determine the effects on protein quality and mineral bioavailability of replacing casein by ß-casein and of whey protein isolate by -lactalbumin, 4 groups of growing rats were fed for 3 wk diets containing 10% protein as 1) casein (control); 2) ß-casein; 3) casein:whey (40:60); or 4) ß-casein:-lactalbumin (40:60). Protein quality, determined as protein efficiency ratio (PER), net protein utilization (NPU), biological value (BV) and protein digestibility (PD), as well as body weight gain, were higher (P < 0.05) with consumption of the whey-adapted diets [casein:whey (40:60); ß-casein:-lactalbumin (40:60)] compared with the casein diets (casein; ß-casein); however, there were no differences between the 2 casein diets or between the 2 whey-adapted diets. Apparent absorption of minerals (Ca, P, Fe, Zn) from the whey-adapted diets was higher than that from the casein diets (P < 0.05); but again, no differences were observed when casein or whey protein isolate were replaced by ß-casein or -lactalbumin, respectively. Thus, substitution of casein by ß-casein or of whey protein isolate by -lactalbumin does not affect protein quality or mineral bioavailability as determined in growing rats.

KEY WORDS: • protein quality • mineral balance • ß-casein • -lactalbumin

Mammalian milks contain 2 major protein fractions, i.e., caseins and whey proteins, independently of the species (1). Caseins are phosphate-containing proteins that occur as micelles and precipitate at pH 4.6 (2). Four major caseins, designated _S1-, _S2-, ß- and -caseins, have been identified. Whey proteins remain in solution at pH 4.6 and consist of a rather diverse group of proteins whose major components are -lactalbumin, ß-lactoglobulin, serum albumin, immunoglobulins, and lactoferrin (2). The total protein level, the ratio between casein and whey protein, and the levels of individual proteins differ markedly among species (1,3). For human nutrition, the differences between human and bovine milk are of most interest. The total protein level of human milk (10–12 g/L) is only one third that of bovine milk (32 g/L) (4). In addition, the protein composition also differs considerably between human and bovine milk, i.e., caseins make up 30–40% of total protein in human milk compared with 80% in bovine milk (4). ß-Casein and -lactalbumin are the major proteins in human milk, whereas _S1-casein, ß-casein, and ß-lactoglobulin are most abundant in bovine milk (4). These differences in the protein profiles of human and bovine milks reflect the differences in the nutritional and functional requirements of human and bovine neonates, respectively.

During the first months of life, the human neonate depends entirely for its nutrition on human milk or alternatively on infant formulas, which are based mainly on bovine milk. Although breast-feeding remains the reference for infant nutrition, considerable efforts have been made during recent decades to adapt the nutritional and functional properties of infant formulas to ensure adequate coverage of the requirements of formula-fed infants (5,6). With respect to protein nutrition, efforts have focused mainly on reducing the protein level and the casein:whey ratio of bovine milk-based formulas to be closer to those of human milk. So-called whey-adapted infant formulas were developed by reducing the proportion of casein from 80% as in bovine milk to 30–40% as in human milk. Due to the lower protein nutritional quality of bovine milk-based formulas, a higher protein level is required for growth compared with human milk (7). The protein content of starter infant formulas is typically 2.2–2.5 g/100 kcal (15–17 g/L),³ almost double the 1.3–1.8 g/100 kcal (8–12 g/L) in mature human milk (8). Recently, a whey-adapted formula with a reduced protein content at 1.8 g/100 kcal (12 g/L) was shown to sustain adequate growth of infants (8).

Further potential formula improvements with respect to protein nutrition could be obtained by adapting the protein composition to that found in human milk, in particular focusing on protein-related functional properties (5). This modification has become possible in recent years due to the commercial availability of specific bovine milk protein isolates, such as ß-casein, -lactalbumin, immunoglobulins, or lactoferrin (9,10). Structural and functional properties related to ß-casein, such as its surface-active properties and its phosphorylation profile, as well as the opioid, immunomodulatory, and anti-hypertensive activity of several peptides of ß-casein, should be evaluated before implementing the inclusion of ß-casein in infant formulas (11–14). Several caseino-phosphopeptides (CPP)⁴ formed during casein digestion were reported to influence mineral absorption, in particular calcium and zinc absorption (15). The effect of CPP on mineral absorption is most likely due to the formation of soluble mineral-phosphate complexes preventing mineral precipitation as insoluble, nonavailable compounds (16–19).

Human milk is richer than bovine milk-based infant formulas in the essential amino acid tryptophan (Trp) largely due to the presence of -lactalbumin, a Trp-rich protein. The addition of -lactalbumin to infant formulas enables the adaptation of Trp levels to those of human milk in addition to mimicking the human milk protein profile (10,20). However, any modification of the protein profile of infant formulas requires nutritional evaluation, in particular with respect to the protein nutritional quality and mineral utilization in comparison with existing formulas. Protein nutritional quality, expressed as protein efficiency ratio (PER), true protein digestibility (PD), biological value (BV), and net protein utilization (NPU), is generally assessed in rats using casein as the reference protein for biological quality of proteins in infant formulas (21). Rat models are also commonly used for the determination of mineral balances (16,18).

The present study was designed to determine the effect of substitution of casein by ß-casein, and of whey protein isolate by -lactalbumin in infant formula on protein nutritional quality and mineral balance for calcium (Ca), phosphorus (P), iron (Fe), and zinc (Zn) in growing rats.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Animals and diets. Male Sprague-Dawley rats (n = 40; Iffa-Credo), initial weight 60 g, were acclimated to standard housing conditions and fed a 10% casein diet for 4 d. The 10% casein diet (Table 1) is limiting for growth rate and allows differences in protein nutritional quality to be readily demonstrated (22). Rats were then randomized according to weight into 5 groups (8 rats/group). Four groups consumed ad libitum diets containing 10% protein as 1) casein (control); 2) ß-casein; 3) casein:whey (40:60); or 4) ß-casein:-lactalbumin (40:60) for 3 wk according to the study design shown in Figure 1. The 5th group was fed the 10% casein diet for 2 wk and then a 2.5% whey protein diet during wk 3. Apart from the protein content, nutrient levels of all diets were adequate for the growth of rats (23). The 2.5% whey protein diet, sufficient to maintain a constant body weight, was fed during wk 3 to allow determination of endogenous nitrogen losses (24). Tap water, freely available for wk 1 and 2, was replaced by deionized water during wk 3, i.e., the balance period. Body weight and food consumption were measured every 3–4 d during the 3 wk; food consumption was corrected for spillage. Rats were placed into metabolic cages for the balance period (wk 3), during which time food intake was determined, and feces and urine were collected for each rat. Fecal and urinary collections were made under clean-housing conditions to avoid mineral contamination. Samples were collected in acid-washed containers. The study was approved by the Veterinary Service of the Canton de Vaud (Lausanne, Switzerland, authorization N° 726.3).

View larger version (11K): [in this window] [in a new window]   FIGURE 1 Experimental design. During the adaptation days, the 10% casein diet was fed to all groups. After randomization, the rats were fed the experimental diets for 3 wk. Food consumption and body weight were measured every 2–3 d; wk 3 was spent in metabolic cages where urine and feces were collected separately.

    Protein nutritional quality. The PER was calculated using the body weight gain over 3 wk, standardized per unit of ingested protein during that period. The NPU, BV, and PD were calculated using data obtained during wk 3 when rats were housed in metabolic cages. Standard equations were applied to determine the protein nutritional quality markers (22):

where BWG is body weight gain, F is fecal nitrogen excretion, F₀ is endogenous fecal nitrogen excretion of the 2.5% whey protein group, I is ingested nitrogen, U is urinary nitrogen excretion, and U₀ is endogenous urinary nitrogen excretion of the 2.5% whey protein group.

    Apparent mineral absorption and retention. Apparent mineral absorption and retention were determined from total mineral intake and total fecal and urinary mineral excretions during the balance period (wk 3). Apparent mineral absorption was calculated as the difference between total mineral intake and fecal excretion. Apparent mineral retention was calculated as the difference between apparent mineral absorption and urinary excretion.

    Analytical methods. The nitrogen contents of diets, feces, and urine were determined by the classical Kjeldahl method (25,26). The Ca, Fe, and Zn contents in fecal digests were determined by inductively coupled plasma MS (ICP-MS) using an Elan 6000 (Perkin-Elmer). The instrument was calibrated using the ICP multi-element standard solution Merck VI. Isotopes selected for analysis were ⁴³Ca, ⁵⁷Fe, and ⁶⁷Zn. The internal standards ⁴⁵Sc and ¹⁰³Rh were added for ⁴³Ca and ⁵⁷Fe, and ⁶⁷Zn determination, respectively. The Ca and Fe contents of diet digests were determined by ICP-MS.

The Zn content of diet digests was determined by flame atomic absorption spectrophotometry (FAAS) using a SpectrAA 400 (Varian) against a calibration curve (0, 0.5, 1.0, 1.5 µg Zn/L), employing instrument conditions for Zn recommended by the manufacturer.

The Ca, Fe, and Zn analyses were validated against standard reference materials National Institute of Standards and Technology (NIST) 1548 Total Diet, NIST 1577b Bovine Liver, and NIST 1549 Nonfat Milk Powder. Rat fecal material that had been analyzed for internal quality control was included in each batch of samples.

The urinary Ca level was determined by FAAS; 200 µL of urine was diluted to 10 mL after the addition of 2 mL of a 5% lanthanum solution in HCl. The Ca levels were determined against a calibration curve (0, 3, 6, and 9 mg Ca/L) according to manufacturer’s conditions. Dade Urine Chemistry Control Level 1 (Dade Diagnostics) was used for quality control.

The P contents of diets and fecal and urine samples were determined by spectrophotometry using malachite green on an Uvikon 943 apparatus (Bio-Tek Instruments). Digest or urine sample (20 µL) was mixed with the color reagent (malachite green:ammonium molybdate:H₂SO₄:Tween 20) and measured after 10 min at a wavelength of 630 nm against water. Calibration standards of 0, 40, 80, 120, and 160 mg P/L were prepared from a 1000 mg/L PO₄^– standard solution (Merck). Accuracy was assessed against NIST 1548 Total Diet, NIST1577b Bovine Liver, NIST 1549 Nonfat Milk Powder, and Dade Urine Chemistry Control Level 1. Rat fecal material that had been analyzed for internal quality control was included in each batch of samples.

    Statistical analysis. Statistical analysis was performed using the package SYSTAT (SYSTAT 7.0.1 for Windows, SPSS). Values are presented as means ± SEM. Diet effect was assessed by one-way ANOVA, followed by post-hoc pairwise mean difference probabilities according to Tukey. When relevant, pooled results for diets were compared by t test for independent means. Differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Protein nutritional quality. Weight gain did not differ for the casein and ß-casein diet groups or for the casein:whey and ß-casein:-lactalbumin groups (Table 2). Weight gain for the casein:whey group was greater than for the casein and ß-casein groups. The weight of the group fed the 2.5% whey-protein diet did not change. When the casein and ß-casein groups were pooled and compared with the pooled casein:whey and ß-casein:-lactalbumin diet groups, body weight differed (P < 0.05) on d 8, i.e., 1 wk after feeding the test diets.

    Mineral absorption and retention. The mineral contents of the diets and the total dietary mineral intakes of the 4 dietary groups during the balance period did not differ (Table 3). Apparent Ca absorption was high in all groups, but differed significantly between the casein and whey-adapted diet groups. Apparent Ca retention differed between the groups; it was highest for the casein:whey 40:60 diet group and lowest for the ß-casein diet group. Apparent P absorption was high in all groups and also differed significantly between the casein and whey-adapted diet groups. Apparent P retention was significantly higher for the whey-adapted diet groups compared with the casein diet groups. The highest apparent P retention occurred with consumption of the ß-casein:-lactalbumin 40:60 diet and the lowest value with the casein diet. Apparent Fe absorption was high for all 4 diet groups, with no significant differences among groups. The apparent Zn absorption from the ß-casein diet was significantly lower than that from the casein:whey diet.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The whey-adapted diets were superior to the casein diets for body weight accretion, a result that was expected on the basis of the less balanced amino acid profile of casein compared with whey proteins. Casein indeed contains less total sulfur amino acids (methionine, cysteine), lysine, threonine, and Trp (2), and the limited supplies of these essential amino acids are likely to limit the growth of rats (7).

The higher PER of whey-adapted diets demonstrated their superior protein quality compared with the casein diets. Comparison of protein intake and weight gain suggested that the higher PER of the whey-adapted diets was due to a more efficient body weight gain rather than to differences in protein intake. PER was similar for the 2 casein diets and for the 2 whey-adapted diets, indicating that the protein quality of ß-casein and -lactalbumin was similar to that of casein and whey protein isolate, respectively. Using the PER rat bioassay reference method for the assessment of protein nutritional quality of infant formulas, our data indicated that whey-adapted formulas have superior protein quality, which is unaffected by inclusion of ß-casein or -lactalbumin. However, because PER does not accurately represent protein digestion and utilization by humans, interpretation of these data warrants necessary caution (7).

In addition to PER, PD is an important variable in considering the nutritional adequacy of a protein source. This study indicated that the proteins of all 4 diets were well digested despite a slightly, albeit significantly, higher digestibility for whey-adapted diets. With PD close to 100% for all diets, the differences in PER described above cannot be explained by differences in digestibility. NPU, calculated on a complete nitrogen balance and corrected for endogenous nitrogen, was higher for whey-adapted diets compared with casein diets. Similarly, results for BV demonstrated superior protein nutritional quality for whey-adapted diets. No difference was observed for NPU or BV between the 2 casein diets or between the 2 whey-adapted diets. All markers of protein nutritional quality indicated that substitution of casein by whey proteins improved the protein quality. However, substitution of casein by ß-casein or whey protein isolate by -lactalbumin did not affect protein quality.

    Mineral absorption and retention. The protein composition of the milk-based diets considerably influenced mineral utilization, the effect depending on the specific nutrient as well as on the dietary protein composition. Mineral balances did not differ between casein diets or between whey-adapted diets.

Apparent absorption of Ca and P was high from all diets and was within the range of earlier reported Ca absorption data from milk-based formulas in growing rats (28). Although Ca and P were well absorbed from all diets, apparent absorption and retention of Ca and P were 15–20% higher from whey-adapted diets than from the casein diets. This observation suggests that either dietary casein reduces Ca and P absorption and retention, or alternatively, whey-adapted diets enhance Ca and P absorption and retention. Substitution of casein by ß-casein and of whey protein isolate by -lactalbumin did not affect Ca and P absorption or retention. The effect of casein on Ca absorption is thought to be associated with the presence of CPP formed by proteolytic digestion of casein in the gastrointestinal tract (4,29–31). However, data on the effect of CPP on Ca absorption are not unequivocal. The results of the present study are in agreement with earlier findings, suggesting an inhibitory effect of dietary casein on Ca absorption (32,33). In contrast, CPP had an enhancing effect on Ca absorption explained by the formation of soluble and thus available Ca-CPP complexes in the gut (28). The differences in the influence of casein on Ca absorption may also be related to variations in the design of the reported studies. Indeed, some studies compared the effect of dietary Ca-CPP directly, whereas others, such as the present study, evaluated the effect of dietary casein on Ca absorption. Hence a possible explanation might be related to the fact that CPP complexes Ca immediately and more effectively compared with peptides formed upon digestion of dietary casein and ß-casein, thus enabling a higher proportion of dietary Ca to be available for absorption. The higher proportion of dietary Ca retained from whey-adapted diets implies that a higher Ca amount is available for bone mineralization. However, the incorporation of retained Ca into bone is not only dependent on the amount of dietary Ca retained, but also on other dietary factors such as dietary protein, vitamin D, and P (34). In this context, the high P retention from both the casein and whey-adapted diets is a positive factor for bone mineralization. Our results, showing a higher Ca retention from the whey-adapted diets, may imply better bone mineralization as well, which would support the findings of Bonjour et al. (35), who reported an improved bone mineral density in prepubertal girls after supplementation with a Ca-rich whey extract.

Fe absorption by growing rats was high from all tested diets and was considerably higher than the absorption rates of 5–15% commonly seen in human infants (36). Contrary to previous findings reporting a positive effect of ß-casein–derived phosphopeptides on intestinal Fe absorption (37), apparent Fe absorption from the ß-casein diet was slightly, but not significantly lower than for the other diets. Moreover, upon pooling data according to the presence or absence of whey protein, Fe absorption was found to be slightly higher from whey-adapted diets, in agreement with the results of studies in humans (36). Although Zn absorption was somewhat lower from the ß-casein diet, the 4 groups did not differ, and absorption rates were similar to those previously reported for milk-based formulas in rats (38,39) and for a cow’s milk-based formula in infants (40).

In summary, protein nutritional quality was not affected by substitution of casein by ß-casein or of whey protein isolate by -lactalbumin. However, protein nutritional quality was significantly higher for the whey-adapted diets compared with the casein diets. Mineral balance did not differ after the replacement of casein or whey protein isolate by ß-casein or -lactalbumin, respectively. Apparent mineral absorption was higher from whey-adapted diets, indicating a small, but significant inhibitory effect of casein on mineral absorption. Therefore, substitution of casein by ß-casein or of whey protein isolate by -lactalbumin did not affect body weight gain, protein nutritional quality, or mineral balance in growing rats.

	ACKNOWLEDGMENTS

 
The authors are very thankful to D. Genoud for his excellent technical assistance and to Ms. D. Cavin and M. Ribeiro of the Nestlé Research Center animal house for their expert assistance in conducting the animal experiment. O. Ballèvre and P.-A. Finot are acknowledged for their helpful comments and suggestions.

	FOOTNOTES

 
¹ Supported by Nestlé Research Center.

³ 100 kcal = 418.4 kJ.

⁴ Abbreviations used: BV, biological value; CPP, caseino-phosphopeptides; FAAS, flame atomic absorption spectrophotometry; ICP-MS, inductively coupled plasma-MS; NIST, National Institute of Standards and Technology; NPU, net protein utilization; PD, protein digestibility; PER, protein efficiency ratio.

Manuscript received 16 December 2004. Initial review completed 27 January 2005. Revision accepted 18 March 2005.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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