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Two wild-type Lactococcus lactis strains isolated from naturally Tunisian fermented milk (Leben), and one laboratory strain, were used to investigate the ability of L. lactis to transform amino acids into aroma compounds during milk fermentation. The α-ketoacid acceptor used for leucine transamination, the first step of catabolism, was identified by gas chromatography/mass spectrometry analysis of the 15 N-labelled amino acids that formed from 15 N-labelled leucine in fermented milk. Furthermore, the amino acids produced or catabolized by the laboratory strain via transamination were identified by comparing the free amino acids in milk fermented with the wild-type strain and the double mutant for aromatic and branched-chain aminotransferases, which cannot transaminate amino acids. The three L. lactis strains strongly catabolized leucine and valine during milk fermentation. The principal amino acid formed via leucine and valine transamination was glutamate indicating that α-ketoglutarate was the principal α-ketoacid acceptor and was generated during milk fermentation.

The autolysis of Lactococcus lactis starters accelerates the formation of free amino acids during cheese ripening. The purpose of this study was to evaluate the impact of L. lactis autolysis on the conversion of free amino acids to aroma compounds. The study was conducted with two L. lactis isogenic strains differing by their autolytic behaviour in cheese: the highly autolytic strain AM2 and a non-autolytic derivative. The effect of bacterial lysis on phenylalanine catabolism was first evaluated in buffer solution. L. lactis cells were also included in a cheese paste model with or without α-ketoglutarate, which stimulates the first step of amino acid degradation by lactococci. Amino acid catabolism and volatile aroma compound formation were analysed after 28 days of ripening. When α-ketoglutarate concentration was not limiting, autolysis of L. lactis cells stimulated the catabolism of aromatic amino acids and methionine and enhanced the formation of sulphur aroma compounds and benzaldehyde, which are desirable aroma compounds in cheese.

Recently, we demonstrated that mesophilic lactobacilli exhibiting glutamate dehydrogenase (GDH) activity were capable of degrading aromatic amino acids (ArAAs) and branched-chain amino acids (BcAAs) in vitro to precursors of aroma compounds and to aroma compounds, when they were combined with Lactococcus lactis. Indeed, they produced via their GDH activity the α-ketoglutarate required for the amino acid transamination. In the present study, we compared the ability of two Lactobacillus strains, with and without GDH activity, to produce aroma compounds from amino acids in a cheese model. The GDH-positive Lactobacillus INF15D produced mainly diacetyl and acetoin from catabolism of aspartate (Asp). However, there were no aroma compounds produced from BcAAs, even when Lactobacillus was combined with L. lactis. In fact, Lactobacillus INF15D exhibited 5–10-fold higher aminotransferase (AT) activity towards Asp than towards BcAAs. We concluded that due to competition of ATs for the α-ketoglutarate produced by GDH, the aroma compounds produced in cheese depends on the relative AT activities towards Asp, BcAAs and ArAAs of the GDH-positive strain.

In Gouda and Cheddar type cheeses the amino acid conversion to aroma compounds, which is a major process for aroma formation, is essentially due to lactic acid bacteria (LAB). In order to evaluate the respective role of starter and nonstarter LAB and their interactions in cheese flavor formation, we compared the catabolism of phenylalanine, leucine, and methionine by single strains and strain mixtures of Lactococcus lactis subsp. cremoris NCDO763 and three mesophilic lactobacilli. Amino acid catabolism was studied in vitro at pH 5.5, by using radiolabeled amino acids as tracers. In the presence of α-ketoglutarate, which is essential for amino acid transamination, the lactobacillus strains degraded less amino acids than L. lactis subsp. cremoris NCDO763 , and produced mainly nonaromatic metabolites. L. lactis subsp. cremoris NCDO763 produced mainly the carboxylic acids, which are important compounds for cheese aroma. However, in the reaction mixture containing glutamate, only two lactobacillus strains degraded amino acids significantly. This was due to their glutamate dehydrogenase (GDH) activity, which produced α-ketoglutarate from glutamate. The combination of each of the GDH-positive lactobacilli with L. lactis subsp. cremoris NCDO763 had a beneficial effect on the aroma formation. Lactobacilli initiated the conversion of amino acids by transforming them mainly to keto and hydroxy acids, which subsequently were converted to carboxylic acids by the Lactococcus strain. Therefore, we think that such cooperation between starter L. lactis and GDH-positive lactobacilli can stimulate flavor development in cheese.

Amino acid catabolism plays a major role in cheese aroma development. Previously, we showed that the lactococcal aminotransferases AraT and BcaT initiate the conversion of aromatic amino acids, branched-chain amino acids and methionine to aroma compounds. In this study, we evaluated the importance of these two enzymes in the formation of aroma compounds in a cheese model by using single araT and bcaT mutants and a double araT/bcaT mutant. We confirmed that addition of ¿-ketoglutarate, a co-substrate of aminotransferases, stimulates the conversion of amino acids to aroma compounds in cheese. The results demonstrated that AraT and BcaT are essential for conversion of aromatic and branched-chain amino acids to aroma compounds by Lactococcus lactis in the cheese model and that they also play a major role in the formation of volatile sulphur compounds from methionine. However, another pathway or another aminotransferase appears also to be weakly involved in the formation of these sulphur compounds.

The effectiveness of the transaminase acceptor α-ketoglutarate in enhancing amino acid catabolism and manipulating the aroma profile of Cheddar cheese has been studied. Utilisation of α-ketoglutarate, catabolism of amino acids, volatiles production, and aroma profile of the cheese were monitored after 6, 12 and 24 weeks ripening. Glutamate and GABA were considerably enhanced on addition of the transaminase acceptor while levels of phenylalanine, leucine, isoleucine, alanine, valine, methionine and threonine were reduced. Addition of α-ketoglutarate increased volatile components originating from the catabolism of branched chain and aromatic amino acids. These compounds included acetic, propanoic, 2-methylpropanoic and 3-methylbutanoic acids, 3-methylbutanol, phenylacetaldehyde and benzaldehyde. Additionally enhanced production of 3-OH-2-butanone was evident. Addition of α-ketoglutarate increased aroma intensity, creamy and fruity aromas. Effects obtained must be verified by tasting cheeses made with food grade α-ketoglutarate, but results suggest potential benefits in accelerated maturation, low fat systems and manipulation of flavour profiles.

Amino acid catabolism is a major process for flavour formation in cheese. The ability of lactic acid bacteria (LAB) and other cheese micro-organisms to degrade amino acids to aroma compounds is highly strain dependent. Generally, amino acid catabolism proceeds by 2 different pathways. The first one, mainly observed for methionine, is initiated by elimination reaction and leads to major sulphur aroma compounds. The second pathway is generally initiated by a transamination reaction and is the main pathway for degradation of all amino acids by LAB. The resulting α-keto acids are then degraded to various aroma compounds via 1 or 2 additional steps. The lactococcal enzymes initiating both pathways have been well characterised, and their importance in the formation of aroma compounds has been demonstrated by using isogenic strains lacking each enzyme. From the new knowledge several applications have been successfully developed, especially for intensifying or diversifying cheese flavour by controlling amino acid transamination.

St Paulin type cheeses made with three different starters were supplemented with alpha;-ketoglutarate, the main alpha;-ketoacid acceptor for amino acid transamination. Amino acid catabolism was monitored during ripening by free amino acid analysis and by analysis of metabolites produced from radiolabelled amino acids introduced as tracer into cheese curd. Also, odour development in cheese was evaluated by sniffing. Amino acid degradation in control cheeses was low and did not lead to aroma compounds. In contrast, adding alpha;-ketoglutarate in cheeses highly enhanced the degradation of aromatic and branched-chain amino acids and methionine. The degradation intensity was related to the amount of alpha;-ketoglutarate added, and alpha;-ketoglutarate used for amino acid transamination was transformed to glutamate. This degradation led to the formation of potent aroma compounds such as isovalerate for leucine and benzaldehyde for phenylalanine. As a result, cheese odour was significantly intensified by alpha;-ketoglutarate addition.

Aromatic and branched chain amino acids cataboIism in Lactococcus lactis. The enzymatic degradation of amino acids in cheeses is believed to generate aroma compounds. In lac- tococci, transamination is the first step in the degradation of aromatic and branched-chain amino acids. Several aminotransferase activities have been detected and, an aromatic-aminotranferase has been purified and characterised. Ketoacids resulting from transamination are mainly degra- ded to carboxylic acids or to hydroxyacids, depending on the reaction conditions. These degra- dations could be catalysed by lactate dehydrogenase and pyruvate dehydrogenase which are involved in pyruvate catabolism. However, we also suggest the existence of other dehydrogenases, more specifie for ketoacids from amino acids. Whereas amino acids catabolism need to be ela- rified, we can state that lactococci have the enzymatic equipment required for the transformation of aromatic and branched chain ami no acids to volatile aroma compounds. © Inra/Elsevier

Hydroxyacid dehydrogenases limit the conversion of α-keto acids into aroma compounds. Here we report that inactivation of the panE gene, encoding the α-hydroxyacid dehydrogenase activity in Lactococcus lactis, enhanced the formation of 3-methylbutanal and 3-methylbutanol. L. lactis IFPL953ΔpanE was an efficient strain producing volatile compounds related to cheese aroma., This work was supported by the Spanish Ministry of Science and Innovation (grants AGL2006-12100, AGL2009-13361-C02-02, RM2011-00003-00-00, and Consolider Ingenio 2010 FUN-C-FOOD-CSD2007-00063) and Comunidad de Madrid (grant ALIBIRD P2009/AGR-1469).

The efficiency of colostral protein digestion was studied in nine newborn lambs fed one meal of bovine colostrum 3 h after birth. The results were compared with those obtained in two unfed lambs and four lambs fed bovine milk. The protein and peptide composition [immunoglobulins G1 and (IgG1), beta-lactoglobulin, alpha-lactalbumin, caseins and peptides resulting from casein hydrolysis] of digesta, gastrointestinal tissues, blood and urine were determined in samples taken 0.75 or 4 h after feeding. The amounts of ingested proteins in lambs fed colostrum were much higher than in those fed the milk diet, and their abomasal emptying was faster. alpha-Lactalbumin was highly degraded by abomasal and intestinal proteases, whereas beta-lactoglobulin and in particular the immunoglobulins were less sensitive. The gastric emptying of caseins was delayed in and the kinetics of appearance of peptides originating from casein hydrolysis was comparable to that observed in lambs fed milk and in 1-mo-old preruminant calves. Thirty-five percent of dietary amino acids ingested as colostrum were available within 4 h for amino acid metabolism; this percentage was 54% in the milk-fed lambs. In the lambs fed colostrum, these amino acids were provided by beta-lactoglobulin, casein and IgG1 (0.52, 0.43 and 0.30 g/kg body wt, respectively), whereas in milk-fed animals casein and beta-lactoglobulin were the most important sources of these amino acids (0.40 and 0.20 g/kg, respectively).

The redox potential plays a major role in the microbial and sensorial quality of fermented dairy products. The redox potential of milk (around 400 mV) is mainly due to the presence of oxygen and many other oxidizing compounds. Lactococcus lactis has a strong ability to decrease the redox potential of milk to a negative value (−220 mV), but the molecular mechanisms of milk reduction have never been addressed. In this study, we investigated the impact of inactivation of genes encoding NADH oxidases ( noxE and ahpF ) and components of the electron transport chain (ETC) ( menC and noxAB ) on the ability of L. lactis to decrease the redox potential of ultrahigh-temperature (UHT) skim milk during growth under aerobic and anaerobic conditions. Our results revealed that elimination of oxygen is required for milk reduction and that NoxE is mainly responsible for the rapid removal of oxygen from milk before the exponential growth phase. The ETC also contributes slightly to oxygen consumption, especially during the stationary growth phase. We also demonstrated that the ETC is responsible for the decrease in the milk redox potential from 300 mV to −220 mV when the oxygen concentration reaches zero or under anaerobic conditions. This suggests that the ETC is responsible for the reduction of oxidizing compounds other than oxygen. Moreover, we found great diversity in the reducing activities of natural L. lactis strains originating from the dairy environment. This diversity allows selection of specific strains that can be used to modulate the redox potential of fermented dairy products to optimize their microbial and sensorial qualities.

Gastric digestion of three milk replacers containing either total milk proteins or a mirture (50:50) of total milk proteins and native or heated whey protein concentrate was studied in vivo with three preruminant calves and in vitro using rennet in an artificial stomach. Stomach effluents were collected during 6 h in vivo and 3 h in vitro and were analyzed for amino acid composition. Proteins and large peptides were characterized by SDS-PAGE. With the skim milk powder diet, casein coagulated almost immediately and was evacuated later as degraded products

The effects of technological treatments of milk on the in vivo immunoreactive ~-Iactoglobulin and lactoferrin gastric emptying were studied in calves given 3 different diets: raw skim milk (RSM), pasteurized skim milk (PSM) and pasteurized and acidified skim milk (Y). The emptying of these proteins was evaluated in collected effluents leaving the stomach over a 12-h period by a competitive enzyme Iinked immunoassay (ELISA). The sensitivity of developed ELISA was 1.5 l!g/ml for ~-Iactoglobulin and 50 ng/ml for lactoferrin. Immunoreactive ~-Iactoglobulin was detected throughout the digestion period and was emptied, as was the fresh matter with RSM and Y, whereas it was strongly retained in the stomach with PSM. Amounts equivalent to 60% of ingested ~-Iactoglobulin were emptied 7 h after the meal for RSM and Y and 40% for PSM. Immunoreactive lactoferrin was detectable only for 1.5 h after the meal and only in digesta from calves fed RSM. An amount equivalent to 22% of ingested lactoferrin was emptied in an immunoreactive form.

Aims: The aim of this research was to investigate the impact of extracellular redox potential (Eh) on amino acid conversion to aroma compounds by Lactococcus lactis that is commonly used as a starter in the cheese industry. Methods and Results: The study was realized in vitro by incubating resting cells of L. lactis in reaction media in which Eh was modified by the addition of oxidizing or reducing agents. Oxidative condition (+300 mV) favoured the production of aldehydes and volatile sulfur compounds responsible for malty, floral, fruity, almond and cabbage aroma. This production was mainly the result of a chemical oxidation of the α-keto acids produced by amino acid transamination. In contrast, reducing condition (−200 mV) stimulated the production of carboxylic acids such as phenylacetic, methylthiopropionic and isovaleric acids, which contribute to the very-ripened-cheese aroma as well as the production of hydroxy acids. Conclusions: Eh of the medium highly influences the nature of aroma compounds produced from amino acid catabolism by the resting cells of L. lactis. Significance and Impact of the Study: Eh is a parameter that is not controlled during cheese production. Its control throughout cheese making and ripening could permit control of aroma formation in cheese.

The production of greek small letter alpha-ketoglutarate, which is required for amino acid transamination, by lactic acid bacteria is often a limiting factor in the conversion of amino acids to aroma compounds in cheese. In theory, there are three main pathways for its formation. The direct pathway, involving a glutamate dehydrogenase (GDH), has already been demonstrated to be functional for certain Lactococcus lactis strains. In this work, we explored two other pathways for the formation of greek small letter alpha-ketoglutarate by L. lactis and evaluated their impact on amino acid catabolism. The pathway that requires citrate permease (CitP), citrate lyase (CitL) and aspartate aminotransferase was operative for L. lactis subsp. diacetylactis and stimulated the conversion of amino acids. However, the pathway requiring aconitase and isocitrate dehydrogenase activities was never operative; none of the 13 strains tested had detectable aconitase and only one had detectable isocitrate dehydrogenase. Therefore, only the GDH and CitP/CitL pathways can stimulate flavour development during cheese ripening.

Although a large number of key odorants of Swiss-type cheese result from amino acid catabolism, the amino acid catabolic pathways in the bacteria present in these cheeses are not well known. In this study, we compared the in vitro abilities of Lactobacillus delbrueckii subsp. lactis , Lactobacillus helveticus, and Streptococcus thermophilus to produce aroma compounds from three amino acids, leucine, phenylalanine, and methionine, under mid-pH conditions of cheese ripening (pH 5.5), and we investigated the catabolic pathways used by these bacteria. In the three lactic acid bacterial species, amino acid catabolism was initiated by a transamination step, which requires the presence of an α-keto acid such as α-ketoglutarate (α-KG) as the amino group acceptor, and produced α-keto acids. Only S. thermophilus exhibited glutamate dehydrogenase activity, which produces α-KG from glutamate, and consequently only S. thermophilus was capable of catabolizing amino acids in the reaction medium without α-KG addition. In the presence of α-KG, lactobacilli produced much more varied aroma compounds such as acids, aldehydes, and alcohols than S. thermophilus, which mainly produced α-keto acids and a small amount of hydroxy acids and acids. L. helveticus mainly produced acids from phenylalanine and leucine, while L. delbrueckii subsp. lactis produced larger amounts of alcohols and/or aldehydes. Formation of aldehydes, alcohols, and acids from α-keto acids by L. delbrueckii subsp. lactis mainly results from the action of an α-keto acid decarboxylase, which produces aldehydes that are then oxidized or reduced to acids or alcohols. In contrast, the enzyme involved in the α-keto acid conversion to acids in L. helveticus and S. thermophilus is an α-keto acid dehydrogenase that produces acyl coenzymes A.

Aminotransferases, which catalyze the last step of biosynthesis of most amino acids and the first step of their catabolism, may be involved in the growth of Lactococcus lactis in milk. Previously, we isolated two aminotransferases from L. lactis , AraT and BcaT, which are responsible for the transamination of aromatic amino acids, branched-chain amino acids, and methionine. In this study, we demonstrated that double inactivation of AraT and BcaT strongly reduced the growth of L. lactis in milk. Supplementation of milk with amino acids and keto acids that are substrates of both aminotransferases did not improve the growth of the double mutant. On the contrary, supplementation of milk with isoleucine or a dipeptide containing isoleucine almost totally inhibited the growth of the double mutant, while it did not affect or only slightly affected the growth of the wild-type strain. These results suggest that AraT and BcaT play a major role in the growth of L. lactis in milk by degrading the intracellular excess isoleucine, which is responsible for the growth inhibition. The growth inhibition by isoleucine is likely to be due to CodY repression of the proteolytic system, which is necessary for maximal growth of L. lactis in milk, since the growth of the CodY mutant was not affected by addition of isoleucine to milk. Moreover, we demonstrated that AraT and BcaT are part of the CodY regulon and therefore are regulated by nutritional factors, such as the carbohydrate and nitrogen sources.

Lactic acid bacteria (LAB) have the enzyme potential to transform amino acids into aroma compounds that contribute greatly to cheese flavour. Generally, amino acid conversion by LAB is limited by their low production of α-ketoglutarate since this α-ketoacid is essential for the first step of the conversion. Indeed, we have demonstrated that adding exogenous α-ketoglutarate to cheese curd, as well as using a genetically modified L. lactis strain capable of producing α-ketoglutarate from glutamate, greatly increased the conversion of amino acid to potent aroma compounds in cheese. Here we report the presence of glutamate dehydrogenase (GDH) activity required for the conversion of glutamate to α-ketoglutarate in several ‘natural’ LAB strains, commonly used in cheese manufacturing. Moreover, we show that the ability of LAB to produce aroma compounds from amino acids is closely related to their GDH activity. Therefore, GDH activity appears to be a major criterion for the selection of flavour-producing LAB strains, which could be used as a starter or as an adjunct to intensify flavour formation in some cheeses.

The first step of amino acid degradation in lactococci is a transamination, which requires an α-keto acid as the amino group acceptor. We have previously shown that the level of available α-keto acid in semihard cheese is the first limiting factor for conversion of amino acids to aroma compounds, since aroma formation is greatly enhanced by adding α-ketoglutarate to cheese curd. In this study we introduced a heterologous catabolic glutamate dehydrogenase (GDH) gene into Lactococcus lactis so that this organism could produce α-ketoglutarate from glutamate, which is present at high levels in cheese. Then we evaluated the impact of GDH activity on amino acid conversion in in vitro tests and in a cheese model by using radiolabeled amino acids as tracers. The GDH-producing lactococcal strain degraded amino acids without added α-ketoglutarate to the same extent that the wild-type strain degraded amino acids with added α-ketoglutarate. Interestingly, the GDH-producing lactococcal strain produced a higher proportion of carboxylic acids, which are major aroma compounds. Our results demonstrated that a GDH-producing lactococcal strain could be used instead of adding α-ketoglutarate to improve aroma development in cheese.

The enzymatic conversion of aromatic amino acids to aroma compounds plays a role in the formation of an undesirable floral aroma in Cheddar-like cheeses. In lactococci, the first step of aromatic amino acid degradation is a transamination, catalysed by an aromatic aminotransferase (AraT). We observed previously that in vitro, araT inactivation prevented degradation of aromatic amino acids and decreased degradation of Met and Leu. In this study we evaluated the effect of araT inactivation in Lactococcus lactis on flavour development in St. Paulin-type cheese. The degradation of amino acids was monitored by using radiolabelled amino acids and the volatile compounds formed were analysed by GC-MS. The development of cheese odour was also evaluated by sniffing. We confirmed that the availability of an α-ketoacid acceptor for transamination is the first limiting factor for amino acid conversion to aroma compounds in cheese. In the presence of α-ketoglutarate, araT inactivation greatly prevented formation of floral aroma compounds from aromatic amino acids while it did not affect the formation of volatile aroma compounds from branched-chain amino acids and methionine. However, the sensory analysis by sniffing did not reveal any significant effect of the gene inactivation although the odour of cheese made with the mutant tended to be less floral than that of cheese made with the wild type strain.

Background: Bovine β–Lactoglobulin (Blg) is a major allergen involved in allergy to cow's milk proteins. Hydrolyzing Blg did not totally suppress its allergenicity; moreover its immunoreactivity may be increased. The aim of this work was to evaluate the specificity of serum IgE to different fragments of Blg in a group of 19 individuals allergic to cow's milk. Methods: This study was performed using both direct and competitive inhibition ELISA involving immobilized native protein or peptides derived from Blg cyanogen bromide cleavage. Results: Analyses of responses to each peptide revealed a large number of epitopes recognized by specific IgE of human allergic sera. However, there were differences in the specific determinants recognized, depending on the serum. Generally, peptides (25–107) and (108–145) retained substantial proportions of the immunoreactivity of the whole protein. Two other peptides, i.e. (8–24) and (146–162), were less recognized but were not inert. Conclusion: The main conclusion is that many epitopes were identified all along the Blg sequence by specific anti–Blg IgE from allergic humans.

Background: Cow’s milk is one of the most frequent food allergens. Whole casein appears to be highly allergenic. It corresponds to an association of four different proteins, αs1-, αs2-, β- and *-caseins in approximate proportions of 40, 10, 40, and 10%, respectively. Methods: These different components were thus purified and used as immobilized antigens in an original enzyme immunoassay to measure specific serum IgE response in a population of 58 children (median age 11 months) allergic to cow’s milk who were sensitive to whole casein. Results: A great variability was observed in the affinity and specificity of specific IgE responses in milk-allergic patients’ sera. 85% of the patients presented IgE against each of the four caseins. Statistically higher amounts of specific IgE were found to be directed against the most abundant fractions (αs1- and β-casein). Co- and/or cross-sensitization to the different caseins were seen in most of the patients sensitive to whole casein. Conclusion: These results suggest that both distinct and common epitopes may occur on these different caseins. The major site of phosphorylation which is the most conserved domain in three caseins could be involved in the IgE response to casein and in immunocross-reactivity between these proteins.

The enzymatic degradation of amino acids in cheese is believed to generate aroma compounds and therefore to be involved in the complex process of cheese flavor development. In lactococci, transamination is the first step in the degradation of aromatic and branched-chain amino acids which are precursors of aroma compounds. Here, the major aromatic amino acid aminotransferase of a Lactococcus lactis subsp. cremoris strain was purified and characterized. The enzyme transaminates the aromatic amino acids, leucine, and methionine. It uses the ketoacids corresponding to these amino acids and alpha-ketoglutarate as amino group acceptors. In contrast to most bacterial aromatic aminotransferases, it does not act on aspartate and does not use oxaloacetate as second substrate. It is essential for the transformation of aromatic amino acids to flavor compounds. It is a pyridoxal 5'-phosphate-dependent enzyme and is composed of two identical subunits of 43.5 kDa. The activity of the enzyme is optimal between pH 6.5 and 8 and between 35 and 45 degrees C, but it is still active under cheese-ripening conditions.

Gastric digestion of three milk replacers for which protein was provided either exclusively by milk powder or partially (50%) by heated soybean flour or soybean protein concentrate was studied in vivo and in vitro. In vivo gastric emptying of protein fractions of the diets was measured in six preruminant calves fitted with reentrant duodenal cannulas and used in a double 3 x 3 Latin square design. In vitro gastric emptying was studied after hydrochloric acid and rennet digestion in an artificial stomach. In vivo and in vitro flow rates of 12% TCA-insoluble N and total N were higher for the soybean diets than for the milk protein diet, indicating that the incorporation of soybean protein prevented casein from clotting. Because of this faster gastric emptying, proteolysis in the stomach was reduced. However, gastric emptying of NPN (12% TCA-soluble N) was significantly decreased only in vitro. No difference existed between the two milk replacers containing either soybean flour or soybean protein concentrate. In vivo and in vitro results were correlated, suggesting that the in vitro method could be used to predict gastric digestion of protein fractions in vivo.

In vivo and in vitro gastric emptying of protein fractions of three milk replacers containing either milk protein (control), a mixture (50:50 on a CP basis) of milk protein and native whey protein concentrate, or a mixture (50:50 on a CP basis) of milk protein and heated whey protein concentrate was studied. In vivo gastric emptying was measured in three preruminant calves fitted with reentrant duodenal cannulas and used in a 3 x 3 Latin square design. In vitro gastric emptying was determined after enzymatic digestion in an artificial stomach. In vivo and in vitro flow rates of protein N (12% TCA-insoluble N) and total N were higher for milk replacers containing whey proteins than for control. Gastric emptying of NPN (12% TCA-soluble N) was slightly higher for diets containing whey proteins than for that containing milk proteins. Gastric emptying of all protein fractions was similar for the two milk replacers containing whey proteins. In vivo and in vitro results were significantly correlated, suggesting that the in vitro method reproduced conditions for proteolysis and could be used to predict gastric digestion of protein fractions.

Gastric digestion of milk proteins has been studied in the preruminant calf by analysing gastric effluents in the duodenum after the ingestion of 5 different diets: whole milk, skim milk, casein solution in water, casein solution with minerals, whey. The amino acid composition of the gastric effluents and HPLC analysis of the latter show that the major part of the whey proteins were evacuated rapidly without any proteolysis and after milk ingestion. On the contrary, all the caseins were largely proteolysed and arrived slowly in the gut. The major part of the peptides obtained came from alpha s1 casein. Only a few peptides, principally CMP, were quickly emptied from the stomach.

Calves were fed five different test meals: whole milk, skim milk, 3% whole casein solution, 3% whole casein in simulated milk ultrafiltrate, and whey. The digesta leaving the abomasum before feeding and during the first 7 postprandial hours were collected by fractions. After precipitation with 12% TCA, the amino acid compositions of the sediments and the supernatants were determined and compared by multivariate analysis. The composition of prefeeding digesta was similar to that of gastric juice. When the calves were fed the two casein diets, the amino acid composition of the sediments changed little with time. In contrast, the changes observed in the composition of the supernatants suggested rapid abomasal emptying of caseino-macro peptide. With the whey diet, it was not possible to evidence more rapid hydrolysis or abomasal emptying of any particular whey protein. During the first 10 min following the ingestion of whole or skim milk, the amino acid composition of the sediment was close to that of milk protein. Immediately afterwards, the composition of the sediment was similar to that of whey protein. Thereafter, the composition of the sediment became more like that of casein and almost reached that of casein during the 7th hour. The amino acid composition of the supernatant was similar to that obtained with the casein diets; this fact suggests that the small peptides produced by proteolysis in the abomasum originated more from casein than from whey proteins.