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Nutritional and Physiological Criteria in the Assessment of Milk Protein Quality for Humans

Ce cile Bos, PhD, Claire Gaudichon, PhD, and Daniel Tome , PhD INRA, Nutrition humaine et physiologie intestinale, Institut National Agronomique Paris-Grignon, 16, rue Claude Bernard 75005 Paris, FRANCE Key words: dietary protein, quality, bioavailability, indispensable amino acids, requirements, peptides, bioactivity
Dietary protein quality is influenced by several factors and especially amino acid composition as well as the bioavailability of the protein. The method to assess the dietary protein quality recommended by the FAO/WHO (1985, 1990) is based on the ability of the protein to satisfy the indispensable amino acid requirements. The Protein Digestibility Corrected Amino Acid Score (PD-CAAS) has been proposed as a quality index and takes into account both the indispensable amino acid composition and the protein digestibility. This index can easily be used routinely, but some conceptual and methodological limits must be considered, such as the determination of both nitrogen and indispensable amino acid requirements, the bioavailability of dietary protein and the validation of the quality indexes. Another level in the evaluation of protein quality considers more specific activities related to specific protein-derived components. The compounds responsible for these activities include enzymes, immunoglobulins, mediator and hormone-like substances. These actions are linked to native proteins or to peptides cleaved from protein during digestion.

Key teaching points:


Cows milk represents a major source of dietary protein in young and adult humans. High milk protein quality arises both from its nutritional value and its physiological properties. The assessment of protein nutritional value raises methodological problems due to the difficulty of quantifying nitrogen and amino acid requirements and of defining the protein characteristics that have to be taken into account. The association of the amino acid content of a protein source and of its digestibility in the rat is the current index (PD-CAAS) recognized by the FAO (1990) for the assessment of protein nutritional quality. This index reduces the differences between high (such as milk) and poor quality protein sources. When considering the postprandial nitrogen retention as a criteria for nutritional value, milk proteins demonstrate a high metabolic utilization by the organism compared with other protein sources. These data obtained in vivo in humans could serve as reference for the validation of routine tools of protein quality evaluation. Several milk protein-derived compounds confer a functional quality to dairy products. These proteins and bioactive peptides are either found in the milk, released from protein digestion or from the processing of dairy products. Physiological properties of milk protein include acute regulatory effects on nutrient bioavailability or on immune mechanisms and longer term potential benefits for cardiovascular-system or tissue development. These effects have to be further confirmed.

Cows milk represents a major dietary source for young and adult humans, and cow milk proteins are considered to have a high nutritional quality. The nutritional value of dietary proteins is usually related to their ability to achieve N and amino acid requirements for tissue growth and maintenance [1 4]. This ability depends both on the protein content of indispensable amino acids and on the digestibility of the protein and subsequent metabolism of the absorbed amino acids. The bioavailability of each indispensable amino acid represents a major factor of nutritional quality

of the different dietary proteins. The general concept underlying this approach is of interest in implementing standards reflecting the relative nutritional values of different protein sources. The recommendations for dietary protein edited by the FAO/WHO (1990) [5] take into account both the protein composition of indispensable amino acids and its digestibility. In addition, milk proteins are also believe to play a potential functional role by acting on nutrient supply, as protective compounds against aggression or by a regulatory action on different physiological functions.

Address reprint requests to: Daniel Tome , PhD, INRA, Nutrition humaine et physiologie intestinale, Institut National Agronomique Paris-Grignon, 16, rue Claude Bernard 75005 Paris, FRANCE

Journal of the American College of Nutrition, Vol. 19, No. 2, 191S205S (2000) Published by the American College of Nutrition 191S

Assessment Criteria for Milk Protein Quality


These different aspects have also to be taken into account for the evaluation of milk protein quality, but further considerations are needed for the definition of reference nitrogen and amino acids requirements in humans, the determination of amino acid and protein bioavailability and the physiological role of milk protein-derived amino acids and peptides. intake, and studies investigating the metabolic response to variable protein intakes have addressed the broad ability of metabolic adaptation to a wide range of protein intake (0.75 to 2 g/kg/d). Indispensable Amino Acid Requirements. Nine amino acids are considered as indispensable in humans (lysine, threonine, valine, isoleucine, leucine, methionine, phenylalanine, tryptophan and histidine). Among these amino acids, lysine and threonine are strictly indispensable; that is, there is no precursor allowing for their synthesis after deamination, whereas the seven other amino acids can be obtained from their corresponding -ketoacid and are indispensable for their carbon skeleton. The remaining dietary amino acids are dispensable (arginine, glutamine, glutamic acid, tyrosine, cysteine, alanine, asparagine, proline, serine, glycine and aspartic acid). The quantification of an indispensable amino acid requirement has been subjected to numerous investigations [4,811]. The classical approach is based on a nitrogen balance method that comprises the measurements of the amount of a specific amino acid necessary to maintain a positive nitrogen balance, at a constant and well-balanced total nitrogen intake [12]. More recently, studies attempted to assess the indispensable amino acid requirement by performing a direct or indirect measurement of indispensable amino acid catabolism (Table 1) [13]. According to this approach, the oxidation rate of a specific amino acid, as a function of its level of ingestion, demonstrates an increase when the supply exceeds the requirements. In the direct oxidation method, the measurement of an amino acids oxidation is made through the infusion of this same 13C-labeled amino acid and the quantification of the expired 13CO2, at different intake levels. The indirect oxidation method allowed for the determination of the requirements for indispensable amino acids such as lysine, valine, methionine and tryptophan from the oxidation of another amino acid, generally 13C-leucine or 13C-threonine [14,15]. The data obtained by the nitrogen balance approach have long been considered accurate. In this respect, the international recommendations made by the FAO/WHO/UNU [7] were derived from Roses classical measurements. The resulting calculated requirements are relatively low, and most of the dietary regimen observed around the world permit their satisfaction, even those supplying a very poor quality protein source as in Third World countries. However, there are important limits to this method, and it appears that the estimated requirements arising from the method are less than required given the actual physiological needs [13,16]. The amount of indispensable

PROTEIN COMPOSITION AND AMINO ACID REQUIREMENT


The first level in the evaluation of protein quality is related to the amino acid composition in conjunction with nitrogen and amino acid requirements in humans. This approach needs to quantify these requirements precisely and to further evaluate the ability of each dietary protein to satisfy these requirements.

The Determination of Amino Acid Requirements


The concept of protein requirements includes both total nitrogen and indispensable amino acids requirements. From the beginning of the 70s, the FAOs priority was to define and reference indispensable amino acid requirement profiles which serve as a determining factor in the establishment of protein nutritional value. Nitrogen Requirement. Nitrogen requirements represent the amount of dietary nitrogen that is necessary for fulfilling nitrogen and amino acid losses without affecting protein and energy metabolism. The nitrogen balance method uses the measurement of the difference between nitrogen intake and nitrogen losses for various protein intakes. Minimal nitrogen losses (Obligatory Nitrogen Loss, ONL) are measured in subjects fed a protein-free diet for a week. Under these conditions, nitrogen losses include urinary (36 mg/kg/d), fecal (12 mg/kg/d) and miscellaneous (sweat, sebum, desquamations, nails, hair, 5 mg/kg/d) losses [6]. From these estimations, the total nitrogen losses represent 54 mg/kg/d and correspond to a protein requirement level of 0.34 g/kg/d [7]. As the dietary protein utilization does not achieve 100% efficiency, an intake of 0.6 g/kg/d of well-balanced protein has been stated to achieve body nitrogen balance. The adequacy of this amount has been reported in studies conducted for two or three months [7]. A security coefficient is added to the previous finding, and thus the recommendations for dietary protein are 0.75 g/kg/d. In children, the requirements for growth have to be added to maintenance requirements. It is of importance to emphasize that these values represent a minimal recommended protein

Table 1. Indispensable Amino Acid Requirements (mg/kg/j) Assessed in Adults with Nitrogen Balance (1) or Amino Acid Oxidation (2) Method
His (1) (2) 12 12 Ile 10 23 Leu 14 40 Val 10 20 Lys 12 30 Met Cys 13 16 Phe Tyr 14 39 Thr 7 15 Trp 3, 5 6

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amino acids derived from the direct oxidation measurements is nearly two to three times greater than that calculated from the nitrogen balance studies. However, when calculating the requirements for indispensable amino acids necessary to balance the obligatory nitrogen losses (54 mg/kg/d) considering a nitrogen fixation rate of 70%, the following amino acid pattern is very near that derived from the oxidation method. Under these conditions, the amino acid requirements have been subsequently reevaluated and a new pattern has been proposed [5]. The nutritional significance of these new recommendations is discussed on conceptual and technical grounds [10,11,1725]. Several aspects arise from this discussion including (i) the validity of the short period measurements in both the fasted and fed protocols for predicting a 24-hour oxidation rate, (ii) the underestimation of the actual oxidation rate due to its evaluation from the plasma enrichment and not from the intracellular enrichment, as well as that of the labeled carbon retention in the body, (iii) the sparing effect of non-indispensable amino acids on the utilization of the indispensable amino acids. The indirect oxidation method leads to higher lysine requirements than the direct method, with values ranging from 35 to 45 mg/kg/d [15,26]. notably feasible to obtain intrinsically labeled animal or vegetable protein intended for human consumption with stable isotopes (15N or 13C) [29,30]. These methods present the advantage of direct assessment of true digestibility, but their drawbacks are the heaviness and cost of implementation. True digestibility can be calculated from (Ningested (Nfeces Nfecal endogenous)/Ningested). After their transit through the distal ileum, the unabsorbed amino acids are mostly metabolized by colonic bacteria. Thus, the ileal rather than the fecal digestibility must be considered as a critical biological parameter for protein and amino acid digestibility. Different methods have been proposed to determine ileal flux and both endogenous and exogenous amino acid absorption rates [31,32]. Studies were conducted in animal [3338] and in humans [32,3942]. Analysis of milk protein digestion indicates a high true ileal digestibility of 95% in humans [38,4346]. For comparison, the true ileal digestibilities for soy and pea proteins were 91% and 89%, respectively. It must by now be clarified whether the nitrogen digestibility is a reliable reflection of the individual digestibility of each amino acid, particularly concerning the indispensable amino acids, since their metabolic availability is a limiting step in protein quality. It was demonstrated that casein and whey protein present different kinetics in the intestinal lumen since whey proteins are rapidly emptied from the stomach mainly in an intact form whereas casein slowly empties mainly in the form of degraded products [40,45]. These processes can be modified according to the treatment of milk since heat treatment or fermentation reduces the transit kinetic difference between casein and whey protein [43]. The Distal Metabolism of Nitrogen and Amino Acids. The nitrogen entering the colon is constituted by luminal dietary products and endogenous nitrogen secretion that were not absorbed in the small intestine and by urea arising from hepatic recycling. The evaluation of the recycling rates of urea have revealed the quantitative importance of nitrogen flux in the colon. This amount can be estimated as 15 g/d in adult, equivalent to the third of the daily total body nitrogen flux and near the dietary nitrogen supply [16,47]. It has been assumed that nitrogenous compounds might be hydrolyzed in the colon and reincorporated into bacterial protein [10,4749]. In pigs, bacterial nitrogen represents 25% to 30% of whole nitrogen in the cecum-colonic [50] and 62% to 76% at the fecal level. Studies in humans and animals generally underline a large degradation of most amino acids reaching the colon, while for some specific amino acids, for example methionine and lysine, a net synthesis occurs [37]. Some indispensable and dispensable amino acids are synthesized by small intestine bacteria; however, this amount has not yet been determined [47]. There are two categories of amino acids with regard to colonic metabolism. The first group comprises aspartic acid, threonine, proline, glycine and tryptophan, which are subject to an important deamination and oxidation in the colon. The second

The Determination of Protein Digestibility


A major concern in the assessment of the protein nutritional value is the determination of amino acid bioavailability. The evaluation of digestibility aims at predicting the profile of absorbed amino acids following protein consumption. Though several methods requiring enzymatic hydrolysis were proposed [27], the classical approach uses in vivo digestibility in an animal model. In vivo Digestibility of Nitrogen. The classical in vivo procedure for the measurement of protein and nitrogen digestibility is based on feces collection and determination of the nitrogen output for several days. The apparent fecal digestibility is calculated from Ningested Nfeces/Ningested. The two main difficulties in the measurement of true nitrogen digestibility is the presence of endogenous nitrogen secretion and that colonic metabolism is not taken into account in the fecal digestibility. The presence of endogenous nitrogen secretions implies the need for discriminating between exogenous nitrogen (food) and endogenous nitrogen (secretions, desquamations and the like) if the true (or real) digestibility has to be determined. To account for these endogenous secretions in nitrogen fecal or ileal fractions, it is necessary to determine them, either by direct or indirect methods. This may be achieved with a protein-free diet. In that case, it involves making the assumption that intestinal endogenous nitrogen secretions are not affected by the quality and quantity of food ingested. But it appears in contrast that food composition represents a major factor for the changes in intestinal enzymatic secretions [28]. However, some techniques based on stable isotope use make it possible to differentiate between dietary and endogenous nitrogen. It is

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group includes methionine, leucine, isoleucine, valine and lysine which are synthesized in the colon [51]. A study in humans has found higher fecal than ileal digestibilities for Arg, Asp, Gly, Phe, Pro, Ser, Thr and Trp, while the opposite result was found for Met. Fecal concentrations for Lys, Ala and Ile are also higher than the ileal concentrations, but without statistical significance [37]. These considerations emphasize the necessity to be able to measure ileal rather than fecal digestibility. It appears that the colon has not the ability to significantly assimilate amino acids. These findings are confirmed in pigs [5254], although the interpretation is difficult in this species due to the presence of a small intestinal flora [47]. The use of non-protein nitrogen (NH4Cl, urea) for lysine synthesis by the intestinal microflora has been documented [5558]. The contribution of this lysine production to the plasmatic flux might approximate 11 to 20 mg/kg/d in humans [59,60]. It was argued that this production originated from small intestine bacteria. This question probably represents a critical parameter for the assessment of lysine requirements, an amino acid frequently deficient in different protein sources. serious limitations and significantly underestimate actual physiological needs [13,16]. The requirements for all the amino acids tested with the direct amino acid oxidation technique have turned out to be approximately two to three times higher than the values recommended by FAO/WHO/UNU [7]. Accordingly the international requirement values have been reevaluated [5]. The observation that the amino acid requirements obtained from oxidation tracer balance studies in adult were closed to those previously proposed for pre-school children has led to two patterns of amino acid requirement, the first for the infants corresponding to the composition of human milk and the second for all the other categories based on the scoring pattern initially proposed for the pre-school child (Table 2). Chemical Score and Protein Digestibility Corrected Amino Acid Score (PD-CAAS). The reference amino acid profiles are of great importance since they are used in the determination of chemical score. In the chemical score method, the product content of each indispensable amino acid is expressed as a fraction of the same amino acid content in a reference protein. The chemical score of the test product is then calculated from the lowest percentage. This value originates from the apparently limiting amino acid. The chemical score is leveled off at 100% given that beyond the satisfaction of the requirements, the surplus is not used. The chemical score method is a helpful tool for underlining the limiting amino acid(s) and for assessing the protein quality in a specific food ration. This method has also the advantage of being fast (an amino acid analysis takes only few hours) and less expensive than in vivo measurements. It assumes moreover that the protein value is directly and only proportional to the limiting amino acid amount. However it is likely that, at least in some cases, the levels of the other amino acids, sometimes higher than the requirements, might have an effect on the limiting amino acid metabolic utilization. In other respects, the method does not consider protein digestibility and amino acid bioavailability, although it is obvious that certain protein sources Table 2. Ideal Composition of Protein to Satisfy Amino Acid Requirements in Adults and Children
FAO/WHO/UNU1985 Children Preschoolers (1012 (mg/g of proteins) Infants (25 years) years) Adults FAO/WHO 1990 Infants Other Ages Histidine 26 19 19 16 Isoleucine 46 28 28 13 Leucine 93 66 44 19 Lysine 66 58 44 16 Methionine Cystine 42 25 22 17 Phenylalanine Tyrosine 72 63 22 19 Threonine 43 34 28 9 Tryptophane 17 11 9 5 Valine 55 35 25 13
From the Report of a Joint FAO/WHO Expert Consultation held in Bethesda, Maryland, 4 8 December 1989.

The Chemical Score and the Protein Digestibility Corrected Amino Acid Score
The initial method of the chemical score [7] is based on the complete analysis of food amino acid content and its comparison to amino acid pattern of a reference protein. The PDCAAS (Protein Digestibility Corrected Amino Acid Score) more recently adopted by the FAO/WHO [5] includes both the amino acid composition and the digestibility of a protein. The accuracy of amino acid analysis constitutes a first limitation for the calculation of the amino acid score. Another important limitation is the calculation of reference profiles for indispensable amino acids based on the determination of the requirements. Reference Profiles for Indispensable Amino Acids. Since 1985, FAO edited an amino acid profile reflecting a virtual ideal protein covering the human amino acid requirements. The relevance of the level of the amino acid requirements is then of major concern. Until relatively recently, the international FAO/ WHO/UNU [7] requirement values were based on the classical N balance studies of Rose and others [12]. Four amino acid reference patterns were first established for infant, pre-school children, school children and adult. The pattern for infants was derived from the human milk protein composition, whereas the other were derived from nitrogen balance studies. In this last case, reference profiles were calculated from an intake of 0.7 g/kg/d of protein and a metabolic efficiency of 70% for dietary nitrogen. The N-balance derived estimates are relatively low, and they would be easily met by nearly every human diet around the world, including diets of relatively poor quality that are consumed in many Third World countries [61]. The N-balance derived requirement estimates have however

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present poor digestibility. In order to take into account the differences in protein digestibility, a new index weighting the chemical score with the digestibility factor was proposed (PDCAAS: protein digestibility corrected amino acid score). The PD-CAAS is calculated from the product of the amino acids chemical score and the protein digestibility. The digestibility is determined in the rat over five days; the contribution of endogenous nitrogen is then derived from a group of rats receiving a protein-free diet [62]. The experimental protocol is detailed in the FAO report [5]. Growth in the Rat. Rat growth assays permit the calculation of the Protein Efficiency Ratio (PER). This method is the one most commonly used and is standardized by the A.O.A.C. (art. 43.253 to 43.257). The principle lies in the calculation of the ratio between the average animal (rat) weight gain and the amount of ingested protein over 28 days. The experiment includes a group of rats fed with a reference protein (casein). This method cannot be applied to the food with a nitrogen content less than 1.8%. The protein in the diet is maintained at a low level (around 10%) so that the supply is under the requirements. Under these conditions, growth is slow and protein is efficiently utilized, rather for synthesis processes than for energetic metabolic pathways, thus improving the test sensitivity. The PER method is simple but presents several shortcomings and inaccuracies. The coefficients of variation between laboratories range from 12.3 to 29.4 depending on protein sources. Casein values are between 2.15 and 3.31, with a mean value of 2.79. In addition to the duration of the test (28 days), the method can be criticized under several aspects. First, the results are dependent on the actual amount of ingested protein, a factor that is not easily measured with accuracy. The taste and smell of food influence the consumption and, as such, are source of variation. Secondly, PER values do not account for the protein utilization for tissue maintenance and consequently are not proportional: a protein source with a PER of 2 cannot be considered to be twice as good as another with a PER of 1. Moreover, the values depend upon the protein concentration in the diet, and the arbitrary choice of the concentration penalizes the high quality protein sources as well as those of poor quality. However, it is possible to discriminate between the capacity of a protein to meet the requirements of an organism for both growth and maintenance by considering the weight loss occurring in a group of rats receiving a protein-free diet. The measurements last for two weeks. The resulting value is the Net Protein Efficiency Ratio (NPER) or Net Protein Ratio (NPR): NPER (weight gain weight loss in the protein deprived group)/ingested protein. Rat growth assays have been improved by using regimens with different protein levels in the rat for 21 days. Two techniques have been developed [63]. The first method is based on diets containing three different protein contents and a proteinfree diet. Weight variations are plotted as a function of protein intake, and the regression line is compared to that of a reference protein (-lactalbumin, casein). The result is the Net Relative Protein Efficiency Ratio (NRPER) or the Relative Nutritive Value (RNV). The second technique uses the same principle but does not include a protein-free diet and establishes a regression line in the linearity zone between the weight change and the protein intake. Results are always expressed as percentages in comparison with the reference protein value and are the Relative Protein Value (RPV). Shortcomings have been underlined for these different methods since they are based on the growth requirements in

AMINO ACID AND PROTEIN METABOLISM


A second level in the evaluation of dietary protein value takes into account the overall metabolism of the ingested protein in relation to body nitrogen and amino acid metabolism.

Growth and Other Functional Responses in vivo


A first approach for evaluating the efficiency of protein utilization considers the interaction with a physiological process such as growth. These studies are hardly feasible in humans. In contrast, they are easily performed in the rat or using microorganisms. The difficulty lies in the significance of extrapolation to humans. Microbiological Responses. Protein quality can be estimated by using microbiological tests. The bioavailability of amino acids is assessed as a function of microorganisms growth. Several microorganisms have been used: Streptococcus faecalis, Leuconostoc mesenteroides, Streptococcus zymogenes, Tetrahymena pyriformis or Tetrahymena thermophyles [63,64]. A protozoan, Tetra pyriformis, is used for the determination of lysine and methionine availability. This microorganism has an indispensable amino acid requirement near that of humans and rats and is endowed with its own proteolytic material so that it can digest particles. A combination of a predigestion of food by means of proteolytic enzymes and a growth measurement with Tetra pyriformis has led to good correlation growth results in the rat for the same protein. The experiment duration can be reduced by replacement of Tetra pyriformis by Tetrahymena thermophyla WH14, the growth being determined with a coulter counter. The growth of WH14 is a direct function of the nitrogen content of the tested sample and is well correlated with rat growth values [65]. Streptococcus zymogenes has also been used insofar as it requires for its growth the same indispensable amino acids as the higher animals, except for lysine. Some interlaboratory studies have shown that the in vitro assays with Streptococcus zymogenes allow for the determination of methionine bioavailability, particularly in fish and meat flour. However, the sensitivity of microorganisms to certain dietary components, additives and spices is a limiting factor of these methods.

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rats, which do not accurately reflect human needs for maintenance. However, they offer the advantage of enabling a fast comparison of different protein sources and of establishing relative scales. the utilization of dietary amino acids [32,39,42]. The measurement of protein postprandial utilization could represent a reliable method for the assessment of protein nutritional value, since this parameter influences body protein turnover. Studies conducted in humans provide, for instance, values for the net postprandial protein utilization (NPPU) of 80%, 72% and 64% for milk, soy and pea proteins, respectively [32,39,42]. These data can be used as reference values in vivo for the validation of calculated indices. The biological value reflects the balance of indispensable amino acids in the absorbed protein digesta. Nitrogen balance techniques raise some methodological problems. It is effectively difficult to assess precisely the whole nitrogen loss. Moreover, a cycling variation of nitrogen balance has been shown [66], and this may lead to different results according to the time interval of measurement. It has also been recognized that the nitrogen metabolism is a dynamic process that is influenced by the energy and substrates consumed [67]. Thus, it is necessary to take these limits for the interpretation of nitrogen balance studies in the protein quality evaluation. Several techniques have been developed to investigate more precisely the protein metabolism. For instance, the urinary 3-methylhistidine excretion rate permits an evaluation of protein degradation. The use of tracers such as 13C, 15N or 2H has allowed for a better knowledge of the nature and quantity of nitrogen losses, protein synthesis, degradation and oxidation than the information provided by nitrogen balance studies.

Nitrogen Retention
Another approach of the dietary protein bioavailability is based on the determination of the extent to which the nitrogen absorbed in the intestine is retained and metabolized by the organism. This implies the measurement of fecal and urinary nitrogen losses. On this basis, the dietary nitrogen retention can be calculated. This method allows for a very accurate approach of the protein nutritional value. Such methods may act as reference methods for indices validation. The nitrogen balance indicates the amount of nitrogen that is retained in the body according to the following formula: Nitrogen balance Ingested nitrogen Excreted nitrogen (urine and feces) The Net Protein Utilization (NPU) permits the evaluation of the part of the N ingested that is retained: NPU N retained/N ingested The Biological Value (BV) gives the percentage of absorbed nitrogen that is retained: BV N retained/N absorbed In the same manner as with the digestibility, these values are true or apparent depending on whether the endogenous nitrogen is taken into account. Endogenous losses may be estimated either from a situation of protein-free diet or by using stable isotope tracer methodologies. Thus, it follows that:

NPU [Ningested (Nfecal Nendogenous) (Nurinary Nendogenous urinary)]/Ningested


and

PHYSIOLOGICAL PROPERTIES OF MILK PROTEIN COMPONENTS


The third level in the evaluation of protein quality considers more specific activities related to specific protein-derived components. The compounds responsible for these activities include enzymes, immunoglobulins, mediators and hormone-like substances. These actions are linked to native proteins or to peptides cleaved from protein during digestion. An important question is to understand whether the protein components of cow milk and milk products are able to have any regulatory beneficial effect in humans. Another aspect is the production of functional products in which a specific activity is favored.

NPU BV digestibility
This value includes the true digestibility of the protein. When the measurements involve small animals, it is possible to determine directly the nitrogen retained by analysis of the total nitrogen content from the carcass. If the experiments are made in conditions such as a single amino acid in its limiting amount, the result provides a valuable insight into the bioavailability or the digestibility of this amino acid. The classical approach for the measurement of net nitrogen retention usually originates from nitrogen balance data measured in subjects after adaptation to different protein levels for several days [1,4]. One of the major limitation to this method is the existence of a diurnal cycling for the transition between the fasted and fed states, leading alternately to nitrogen postprandial accretion and postabsorptive loss phases. From these considerations, the retention calculated on a daily basis is lower than that derived from the postprandial phase [66]. Another point of view concerns the deposition of dietary nitrogen during the postprandial phase, which is likely to be the critical step for

BIOACTIVE PEPTIDES AND PROTEINS IN MILK AND DAIRY PRODUCTS


Cow milk and dairy products are an important dietary source in humans. Fermented milk products were often reported for their health benefit for consumers due to the presence of lactic bacterial strains. Another aspect is the presence of active components that originate from milk protein fraction or produced during milk protein fermentation.

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Milk proteins are a complex mixture of various components with quantitative and qualitative differences according to mammalian species. The two major fractions include a micellar casein fraction and the soluble whey protein fraction. Bioactive components in the protein fraction of milk include enzymes, bactericides, hormones, mediators and growth factors. These activities have various profiles in relation with the different steps of lactation [68]. The first group include proteins and polypeptides active in their native form such as lactoferrin, enzymes (amylase, lipase), immunoglobulins and hormones, the concentration of which is higher than in blood, probably in relation to a biological role for the mother and the young [69]. Lactoferrin has been particularly studied for its iron-chelating activity that could be implicated in different activities including cations transport, anti-infectious action or regulation of immune function [70,71]. Another group of compounds is constituted by peptides cleaved from larger precursors. Casein, particularly considered, has exogenous prohormones since numerous active peptides are present in its sequences. Lactoferrin which is active in its native form is also a precursor of different active peptides. Opiate agonist and antagonist are an important family of peptides cleaved from milk protein including casein, lactoferrin, -lactoglobulin, -lactalbumin and serum albumin [72 82]. Another important peptide with different activities is the caseinomacropeptide cleaved from -casein: it was reported to inhibit the adhesion of actinomyces and streptococci to the erythrocytes and to the polystyrene, the binding of the cholera toxin to its receptor, the hemaglutination of the influenza virus and the proliferation of T-lymphocytes, to stimulate the growth of Bifidobacteria and to modulate intestinal motility and digestive hormones secretion. Other peptides are able to inhibit angiotensin converting enzymes and platelet aggregation [83,84]. The phosphopeptides from casein could play a role in cation transport [85,86]. Lactoferricin cleaved from lactoferrin has bactericidal activities. Lastly numerous peptides were demonstrated to act has growth factors in different cell types including immune cells. A first approach is the presence of active components in cow milk. The concentration of the different compounds is different in human and in cow milk. The concentration of protein is four time higher in cow milk than in human milk, i.e. 30 34 g/L vs. 9 14 g/L, in relation with a higher casein level, respectively. Four casein components are present in cow milk including S1-casein, S2-casein, -casein et k-casein. Human casein is mainly constituted by -casein and a small level of k-casein. Casein fraction is a source of phosphorus, calcium and other divalent cations. The concentration of whey protein is equivalent, but qualitative differences exist between cow milk and human milk. -lactoglobulin, the main protein in cow whey, is absent in human milk, whereas lactoferrin, the main component in human whey, is only present at a low level in cow whey. The immunoglobulin fraction is mainly constituted by secretory IgA in human milk and by IgG in cow milk. In addition, various modifications occur during the processing of dairy products: fermentation, heat treatment, separation. During these treatments, protein denaturation can produce the deactivation of different activities (enzymes, hormones, growth factors). It was observed that lactoferrin was active after five minutes at 90 100C whereas it was inactivated at 120. In contrast, peptides, including active peptides, can be produced during milk fermentation with different bacterial strains [87]. According to the bacterial strain, it is possible to favor the release of casomorphins [88], of antihypertensive peptides [89] or of antimutagen activities [90 91]. The quantity of active peptides present in milk and dairy products is low. The release of these peptides from casein and whey protein is estimated as 25130 mg/g protein [92]. However, in vivo effects were observed with dietary level [93,94]. Following milk ingestion, whey protein (-lactoglobulin, -lactalbumin, lactoferrin, immunoglobulins) were detected in the intestinal lumen [41,95], and only traces of casein could be detected, whereas casein derived-peptides could be identified [29,45,46,96,97]. These peptides include the caseinomacropeptide (CMP) and other casein fragments including -casomorphins, immunomodulator peptides and phosphopeptides [92,98]. Intact bioactive protein and polypeptide absorption is an important and limiting step for their action. Transcytosis was demonstrated for -lactoglobulin, -lactalbumin, prolactin and lactoferrin [99 104]. These proteins could be detected in the blood after their ingestion. Immunoreactive CMP was detected in the blood [105]. In contrast immunoreactive -casomorphin was detected in the young, but in adult their absorption seems limited by the action of membrane peptidases [106 107].

Acute Regulatory and Physiological Effect of Milk Protein Components


Nutrients Bioavailability. The digestion of fat and carbohydrate is neither fully developed in new-borns due to low endogenous pancreatic lipase and amylase activities [108]. Enzymes are present in human milk and could compensate for these digestive functions [109]. Human milk contains amylase which is identical to salivary amylase and a bile salt-dependant lipase, which is also present in other many species milk [108]. This enzyme contributes significantly to fat digestion in breastfed new-borns [110] by its ability to completely hydrolyze tri-glyceride and release LC-PUFA [111]. These enzymatic activities are low in milk formula, but there are no data on attempts to supplement milk formula with digestive enzymes. If such a supplementation were attempted, it would be necessary to prevent nutrient hydrolysis in the supplemented milk [109]. Milk and milk products represent an important source of calcium for humans. This is due to both a high content of calcium and a high bioavailability. Lactose is known to en-

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hance intestinal permeability for calcium salts [112]. Extensive studies have also been conducted on the role of casein phosphopeptides, which are likely to enhance intestinal calcium absorption by preventing the formation of insoluble calcium phosphate [75,86,113116]. Other results have also indicated a role of phosphopeptides in iron absorption [117]. Other milk proteins like lactoferrin, vitamin B12 binding protein, folate binding protein, -lactoglobulin and -lactalbumin are assumed to interact with minerals and vitamins absorption. Particularly, lactoferrin is believed to play a role as an iron-binding protein that could be involved in iron transport [71,118,119]. Whey proteins were also suspected to play a role in mineral absorption [120122]. Protective Role against Bacterial and Other Aggression. Milk contains substances which provide passive protection against infection in the intestinal lumen. This protection involves both immunoglobulins and enzymes (lysozyme, lactoperoxydase), but also other proteins and peptides including lactoferrin, casocidin, isracidin or CMP. Immunoglobulins present in milk are involved in the passive protection of the young and have been shown to resist partially to degradation in the intestinal lumen [41]. Lactoferrin is widely considered to be an important component of the host defence against microbial infection. Its antibacterial property is effective against both bacterial strains (Escherichia coli, Staphylococcus albus, Staphylococcus aureus, Staphylococcus mutans, Vibrio cholerae) and yeast (Candida albicans) [123]. This effect could be linked to its iron-scavenging activity since lactoferrin binds the iron needed for bacterial growth [124]. Bactericide properties of lactoferrin are often associated with lyzozyme and lactoperoxydase [125]. Lactoferricin, a peptide cleaved from lactoferrin by pepsin, also presents an antimicrobial activity that originate from a direct interaction with bacterial surface [126]. Lactoferricin showed a marked growth-inhibiting effect on several bacterial strains including Escherichia coli, Klebsiella pneumonia, Salmonella enteritidis, Staphylococcus haemolyticus, Streptococcus thermophilus, Corynebacterium ammoniagenes, Bacillus subtilis or Bifidobacterium infantis [127]. Casocidin I, an S2casein fragment (position 165203) cleaved by chymosin, was demonstrated to inhibit the growth of Echerichia coli and Staphylococcus carnosus [128]. Isracidin, the N-terminal segment (123) of S1-casein B, was demonstrated to be effective in vivo against lethal infection by Staphylococcus aureus strain and protected mice against Candida albicans by stimulation of both phagocytosis and immune responses [129]. Different compounds of milk were demonstrated to stimulate the development of Bifidobacteria that play a protective role against pathogenic strains such as E. coli. This probiotic activity is associated to the presence of lactose and other oligosaccharides in milk, but also to the presence of caseinomacropeptide and of lactoferrin [130132]. Regulatory Effect on Digestion and Metabolism. Dietary proteins and especially milk proteins are suspected of stimulating gastrointestinal hormones (gastrin, cholecystokinin, secretin and gastrin inhibitory peptide) that control both gastric as well as intestinal motility and gastric and bilio-pancreatic secretions [133]. This effect is suspected to be due to the presence of specific bioactive peptides cleaved from dietary proteins during digestion and acting either at the luminal level or after absorption [134]. Mahe et al. [46] observed that large amounts of ingested casein stimulate endogenous nitrogen secretions more efficiently than whey protein, thus suggesting that caseinderived peptides are responsible for a part of this stimulation [45,96,134]. Indeed, it is observed that casein slowly empties from the stomach in the form of degraded products, including putative bioactive peptides such as caseinomacropeptides (CMP, GMP), opioid peptides (casomorphins) or peptidase inhibitors. These activities could include the hormone modulation effect of GMP through both antigastrin and CCK stimulating properties [96,135]. This effect could explain, at least in part, the specific effect of casein on gastric and pancreatic digestion. Numerous studies have focused on the presence of opiate peptides in the sequence of milk proteins including -casein, -casein, -lactoglobulin, -lactalbumin, lactoferrin and serum albumin [75]. Furthermore, lactoferroxin derived from lactoferrin and a fragment of S1-casein have been found in human milk as opioid antagonist [75]. Casoxin C has been recently described as an ileum contracting peptide [136]. Studies have shown an interaction of casomorphins with opiate receptors located on the serosal side of the intestinal epithelium with a subsequent increase on electrolyte transport which could be linked to an antisecretory activity [106,137139]. Studies have also shown an antisecretory activity after intragastric administration of casomorphin in dogs. Indeed, modulation of gastrointestinal motility, postprandial insulin, somatostatin and pancreatic polypeptide release have been observed [140142]. Furthermore, a recent study suggested a possible involvement of casomorphins on food intake regulation. Froetschel [143] measured a decreased reticular contraction frequency, duration and amplitude of the abomasum after casomorphin infusion, implying a reduction in gastric motility and emptying.

Effect of the Regular Ingestion of Milk Protein Components


Regulatory Effect on Cardiovascular System. Peptides cleaved from casein called casokinins are angiotensin-converting enzyme (ACE) inhibitors [89,144149]. For example, highly active casokinin are cleaved from - and S1-casein, respectively. Recently, opioid sequences, corresponding to -casomorphin 7 or -lactorphin, have also been identified as ACE inhibitory peptides with low activities [75]. These peptides could act on ACE at the surface of the vascular endothelium that could result in anti-hypertensive action [150]. This

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antihypertensive effect has been demonstrated in rats after oral administration of inhibitors containing milk products (Val-ProPro and Ile-Pro-Pro) [89,94,149,151]. A similar effect was also suspected in humans [93]. Two peptides isolated from bovine -casein [83] and human lactoferrin [84] were shown to inhibit platelet function and could have anti-thrombus properties. The main anti-thrombus peptides are the sequence 106116, 106112, 112116 and 113116 of -casein and the residue 3942 of human lactotransferrin. They inhibited both aggregation of ADP-treated platelets and binding of 125I-fibrinogen to ADP-treated platelets [152]. Recently, two antithrombus peptides derived from the corresponding -casein were detected in physiologically active concentrations in the plasma of five-day-old infants after ingestion of cow-milk-based formula or human milk respectively. It suggests that these bioactive peptides are released from milk proteins during digestion and may exert anti-thrombus activity [153]. Immunomodulatory Effect. Lactoferrin, prolactin or other factors in milk contribute to the active immunity of the neonate by modulating the ontogeny of immune function and surface antigen expression of specific sets of cells [154156]. Prolactin was demonstrated as acting as a immunoregulatory agent on T cells and B cells [101,157,163]. IGF-1 stimulates DNA synthesis and proliferation in many cell types such as endothelial cells, fibroblast cells, muscle cells and lymphohemopoietic cells [162,164]. Lactoferrin promotes in vitro lymphocyte proliferation [165,166]. Specific receptor-mediated binding of lactoferrin has been reported for intestinal epithelial cells, hepatocytes, B lymphocytes and macrophages/monocytes [85,167170]. Furthermore, lactoferrin can stimulate the humoral immune response to sheep red blood cells (SRBC) in mice and induce in vitro CD4-CD8- thymocytes to mature into the T CD4 helper phenotype [171]. Oral administration of bovine lactoferrin induced both intestinal and peripheral specific antibody responses in mice [172]. Other studies have reported that lactoferrin can modulate production of different lymphokines including IL-1, IL-2, IL-6 and TNF- [173177]. Lactoferrin administered orally to mice increased the antigen-specific proliferation of Peyers patch and spleen cells [172]. In mice fed lactoferrin, CD4-positive cells were enhanced in both Peyers patch and spleen cells type, intracellular IL-2 and IL-4 production was increased in Peyers patches, and IL-4 production was increased in spleen cells. Bovine milk -casein and its glyco-macropeptide (residues 106169) inhibited in vitro mouse mitogen-induced proliferation of mouse spleen B lymphocytes and rabbit Peyers patch cells and antibody responses to SRBC in mouse splenocyte cultures [178,179]. Moreover, enzymatic digests of S1-casein, -casein and -casein inhibited in vitro proliferation of mouse spleen lymphocytes and rabbit Peyers patch cells induced by B-mitogens (LPS, pokeweed mitogen) or T-mitogens (PHA, ConA) [180182]. Another group of peptides was shown to act on immunocompetent cells, i.e. macrophage function, B lymphocytes or T lymphocytes [183186]; these could play a role in the stimulation of the immune system. Tissue Development and Antimutagenic Activity. A regular ingestion of milk protein components could also act on the development of tissues and organs [69]. Lactoferrin has been identified as a growth factor for various cell types [187191]. Peptides cleaved from -casein were shown to stimulate mitosis of different cell types [192194], whereas other peptides inhibited protein synthesis, proteolysis and enhanced ureogenesis [195]. -lactalbumin and derived peptides could also modulate intestinal cells proliferation and differentiation [196]. In addition antimutagnic activity was demonstrated in lactic bacteria whey protein hydrolysate [9091].

CONCLUSION
Among the available methods for the evaluation of protein quality in food, those based on indices calculations progressively stand out due to their scientific logic and their ease in implementation. However they present limits that have to be taken into account. For instance, some questions are raised by the use of the PD-CAAS, despite its interest. Both the criteria and the methods relative to the assessment of reference requirements for indispensable amino acids have to be further examined. The initial requirement values derived from nitrogen balance studies are generally taken as too low with regard to the new data obtained from the oxidation methods. These latter present as well some shortcomings, and there is not always agreement between results calculated by either direct or indirect methods. A subsequent question concerns nitrogen metabolism in the colon. The possibility of amino acid synthesis available for the organism has to be documented. A second problem lies in the assessment of amino acid digestibility and bioavailability. Ileal digestibility for individual amino acids should probably be considered, but this methodology is difficult. Moreover, this implies the establishment of correlations between methods with a view to defining and validating a fast measurement method reliable in comparison to data collected in humans. It is thus necessary to validate the PD-CAAS values against bioavailability measurements. The limitation of this index at 100% should not only be considered from a theoretical point of view but also in the aim of obtaining the best correlation between calculated and in vivo experimental data. Another level of milk protein evaluation is their potential role as bioactive components. Research conducted during the last 30 years strongly suggested that milk and other dietary proteinderived peptides could play a functional role as specific metabolic regulators of various physiological functions. A clear confirmation of this concept is highly relevant for progression of knowledge in the fields of physiology, nutrition and medical sciences and for an understanding of the complex relationships of the organism with foods. It is also of great innovative interest

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for the identification of new bioactive molecules and their application in the development of new dietary protein-derived products with specific nutritional and physiological applications. Such innovative developments could contribute an economical added value for dietary protein production and food industry.
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