Biomedical subjects

M Yvon

Publications and source records attributed to M Yvon.

At least 19 recordsLinked to original sources

PubMed2006

Addition of oxidizing or reducing agents to the reaction medium influences amino acid conversion to aroma compounds by Lactococcus lactis.

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 E(h) 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 alpha-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: E(h) 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.

Amino Acids↗

PubMed2001

Sulfur compound production by Geotrichum candidum from L-methionine: importance of the transamination step.

L-methionine degradation products and catabolic enzymatic activities involved in methanethiol generation were investigated in Geotrichum candidum GcG. L-methionine was easily degraded by G. candidum and the transamination product, 4-methylthio-2-oxobutyric acid (KMBA), was found to transiently accumulate. In parallel, considerable L-methionine aminotransferase activity was found in this microorganism. L-methionine and KMBA demethiolating activities were also detected. The degradation of KMBA corresponded to an overall increase in the production of volatile sulfur compounds. These results show that the transamination pathway is of major importance in the initial breakdown of L-methionine by this cheese-ripening microorganism.

Butyrates↗

PubMed2001

L-methionine degradation potentialities of cheese-ripening microorganisms.

Volatile sulphur compounds are major flavouring compounds in many traditional fermented foods including cheeses. These compounds are products of the catabolism of L-methionine by cheese-ripening microorganisms. The diversity of L-methionine degradation by such microorganisms, however, remains to be characterized. The objective of this work was to compare the capacities to produce volatile sulphur compounds by five yeasts, Geotrichum candidum, Yarrowia lipolytica, Kluyveromyces lactis, Debaryomyces hansenii, Saccharomyces cerevisiae and five bacteria, Brevibacterium linens, Corynebacterium glutamicum, Arthrobacter sp., Micrococcus lutens and Staphylococcus equorum of technological interest for cheese-ripening. The ability of whole cells of these microorganisms to generate volatile sulphur compounds from L-methionine was compared. The microorganisms produced a wide spectrum of sulphur compounds including methanethiol, dimethylsulfide, dimethyldisulfide, dimethyltrisulfide and also S-methylthioesters, which varied in amount and type according to strain. Most of the yeasts produced methanethiol, dimethylsulfide, dimethyldisulfide and dimethyltrisulfide but did not produce S-methylthioesters, apart from G. candidum that produced S-methyl thioacetate. Bacteria, especially Arth. sp. and Brevi. linens, produced the highest amounts and the greatest variety of volatile sulphur compounds includling methanethiol, sulfides and S-methylthioesters, e.g. S-methyl thioacetate, S-methyl thiobutyrate, S-methyl thiopropionate and S-methyl thioisovalerate. Cell-free extracts of all the yeasts and bacteria were examined for the activity of enzymes possibly involved in L-methionine catabolism, i.e. L-methionine demethiolase, L-methionine aminotransferase and L-methionine deaminase. They all possessed L-methionine demethiolase activity, while some (K. lactis, Deb. hansenii, Arth. sp., Staph. equorum) were deficient in L-methionine aminotransferase, and none produced L-methionine deaminase. The catabolism of L-methionine in these microorganisms is discussed.

Arthrobacter↗

PubMed2000

Characterization and role of the branched-chain aminotransferase (BcaT) isolated from Lactococcus lactis subsp. cremoris NCDO 763.

In Lactococcus lactis, which is widely used as a starter in the cheese industry, the first step of aromatic and branched-chain amino acid degradation is a transamination which is catalyzed by two major aminotransferases. We have previously purified and characterized biochemically and genetically the aromatic aminotransferase, AraT. In the present study, we purified and studied the second enzyme, the branched-chain aminotransferase, BcaT. We cloned and sequenced the corresponding gene and used a mutant, along with the luciferase gene as the reporter, to study the role of the enzyme in amino acid metabolism and to reveal the regulation of gene transcription. BcaT catalyzes transamination of the three branched-chain amino acids and methionine and belongs to class IV of the pyridoxal 5'-phosphate-dependent aminotransferases. In contrast to most of the previously described bacterial BcaTs, which are hexameric, this enzyme is homodimeric. It is responsible for 90% of the total isoleucine and valine aminotransferase activity of the cell and for 50 and 40% of the activity towards leucine and methionine, respectively. The original role of BcaT was probably biosynthetic since expression of its gene was repressed by free amino acids and especially by isoleucine. However, in dairy strains, which are auxotrophic for branched-chain amino acids, BcaT functions only as a catabolic enzyme that initiates the conversion of major aroma precursors. Since this enzyme is still active under cheese-ripening conditions, it certainly plays a major role in cheese flavor development.

Amino Acids, Branched-Chain↗

PubMed2000

Expression of a heterologous glutamate dehydrogenase gene in Lactococcus lactis highly improves the conversion of amino acids to aroma compounds.

The first step of amino acid degradation in lactococci is a transamination, which requires an alpha-keto acid as the amino group acceptor. We have previously shown that the level of available alpha-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 alpha-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 alpha-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 alpha-ketoglutarate to the same extent that the wild-type strain degraded amino acids with added alpha-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 alpha-ketoglutarate to improve aroma development in cheese.

Amino Acids↗

PubMed1999

Genetic characterization of the major lactococcal aromatic aminotransferase and its involvement in conversion of amino acids to aroma compounds.

In lactococci, transamination is the first step of the enzymatic conversion of aromatic and branched-chain amino acids to aroma compounds. In previous work we purified and biochemically characterized the major aromatic aminotransferase (AraT) of a Lactococcus lactis subsp. cremoris strain. Here we characterized the corresponding gene and evaluated the role of AraT in the biosynthesis of amino acids and in the conversion of amino acids to aroma compounds. Amino acid sequence homologies with other aminotransferases showed that the enzyme belongs to a new subclass of the aminotransferase I subfamily gamma; AraT is the best-characterized representative of this new aromatic-amino-acid-specific subclass. We demonstrated that AraT plays a major role in the conversion of aromatic amino acids to aroma compounds, since gene inactivation almost completely prevented the degradation of these amino acids. It is also highly involved in methionine and leucine conversion. AraT also has a major physiological role in the biosynthesis of phenylalanine and tyrosine, since gene inactivation weakly slowed down growth on medium without phenylalanine and highly affected growth on every medium without tyrosine. However, another biosynthesis aromatic aminotransferase is induced in the absence of phenylalanine in the culture medium.

Amino Acid Sequence↗

PubMed1998

Specificity of the human IgE response to the different purified caseins in allergy to cow's milk proteins.

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, alpha(s1)-, alpha(s2)-, beta- and kappa-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 (alpha[s1]- and beta-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.

Animals↗

PubMed1998

Allergy to bovine beta-lactoglobulin: specificity of human IgE using cyanogen bromide-derived peptides.

BACKGROUND: Bovine beta-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.

Adolescent↗

PubMed1997

An aminotransferase from Lactococcus lactis initiates conversion of amino acids to cheese flavor compounds.

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.

Amino Acids↗

PubMed1994

Effects of caseinomacropeptide (CMP) on digestion regulation.

Caseinomacropeptide (CMP) is a 64-amino-acid-residue peptide which is released from kappa-casein by gastric proteinases. This review sums up the knowledge concerning its effects on the digestive function. Part 1 recalls the origin and heterogeneity of CMP. Here we underline that there are various forms of CMP which differ by their glycosylation level and genetic mutation. Consequently the forms used for studying biological activities need to be defined accurately. Part 2 summarizes the effects of CMP on digestive secretions. The major effect is an inhibitory effect on acid gastric secretions. Simultaneously, the blood concentration of regulatory digestive peptides is modified. In part 3 we try to clarify the mechanisms of action of CMP. A slightly glycosylated form of CMP, the A variant, appears to be responsible for the biological activity. Evidence suggests that CMP triggers stimuli from intestinal receptors without being absorbed. The signal would be then transmitted to organs through regulatory digestive peptides.

Amino Acid Sequence↗

PubMed1994

In vivo and in vitro gastric emptying of milk replacers containing soybean proteins.

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.

Animal Feed↗

PubMed1993

Colostrum protein digestion in newborn lambs.

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).

Abomasum↗

PubMed1992

Prediction of peptide retention time in reversed-phase high-performance liquid chromatography.

Peptide retention in reversed-phase chromatography depends mainly on the amino acid composition of peptides and can therefore be predicted by summing the relative hydrophobic contributions of each constitutive amino acid residue. The prediction is correct for small peptides but overestimates the retention times of peptides larger than 10-15 residues. A new prediction model is proposed in which the contribution to peptide retention of each amino acid residue is not a constant but a decreasing function of peptide length. From the retention times of 104 peptides, the parameters of decreasing functions were estimated by a non-linear multiple regression analysis. The contribution to peptide retention of charged, polar and non-polar residues appears to be differently affected by peptide length. The secondary structure of most peptides during reversed-phase high-performance liquid chromatography could be responsible for this. The high correlation between the predicted and observed retention times of peptides which were not used to establish the model indicates a good predictive accuracy of the new model.

Amino Acid Sequence↗

PubMed1992

Characterization and kinetics of gastric emptying of peptides derived from milk proteins in the preruminant calf.

The gastric emptying kinetics of peptides derived from milk protein were studied in vivo in preruminant calves by collecting and characterizing the whole effluent leaving the stomach for 12 h after ingestion of crude skim milk. Peptides were isolated by reversed-phase HPLC and identified. Particular attention was paid to biologically active peptides and to peptides that could be precursors of biologically active sequences. A gastrin inhibitor, the caseinomacropeptide, was emptied from the stomach only during the first 0.5 h of digestion and rapidly hydrolysed. Precursors of immunostimulatory peptides from alpha s1- and beta-caseins were emptied throughout digestion in the gastric effluent. A precursor of beta-casomorphins (peptide 58-93 of beta-casein) was emptied from the stomach 3.5 h after the meal when it was taken on an empty stomach. From this precursor, peptides that may be resistant to hydrolysis by intestinal peptidase were obtained after in vitro hydrolysis by pancreatic enzymes. A phosphopeptide (fragment 110-142 of alpha s1-casein) was also found in digesta after a few hours of digestion. When the meal was not taken on an empty stomach, these peptides were emptied in the first digesta at a low concentration. The potential activity of these peptides is discussed. The results support the hypothesis that active sequences could still be present in the gut after the action of pancreatic enzymes.

Animals↗

PubMed1992

In vivo and in vitro gastric emptying of protein fractions of milk replacers containing whey proteins.

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.

Animals↗

PubMed1991

Tandem immunoaffinity and reversed-phase high-performance liquid chromatography for the identification of the specific binding sites of a hapten on a proteic carrier.

Immunoaffinity (IA) and reversed-phase (RP) high-performance liquid chromatography were combined for the identification of the specific binding sites of benzylpenicilloyl (BPO) groups on human serum albumin (HSA). Tryptic hydrolysates of BPO-HSA were loaded on the IA column. BPO-peptides were desorbed and concentrated directly on the RP column, coupled via a switching valve, then separated by using gradient elution and identified by the amino acid sequences. This single-step procedure permitted more than 95% recovery of the BPO-peptides present in minute amounts, with good specificity.

Amino Acid Sequence↗

PubMed1991

Immobilized Fe3+ affinity chromatographic isolation of phosphopeptides.

Immobilized Fe3+ affinity chromatography is suggested as a means of concentrating phosphopeptides that are present in too low a proportion in a complex mixture to be purified by a single-step method. A high-performance liquid chromatographic system and a chelating Superose HR 10/2 column were used. The chromatographic conditions were optimized using a tryptic hydrolysate of whole casein. The unbound fractions did not contain any phosphorylated peptide. All caseinophosphopeptides were retained. Only four other strongly basic peptides were also retained. The quantitative accuracy of the method was evaluated. This method allowed the isolation of phosphopeptides in gastric effluents of calves fed with milk.

Animals↗

PubMed1991

Enzyme immunoassay of benzyl penicilloyl (BPO) groups using acetylcholinesterase as label. Application to the study of the BPO-binding sites on albumin.

Benzyl penicilloyl groups (BPO) derive from penicillin G by cleavage of the beta lactam ring; they covalently bind to proteins to give conjugates which have lost all antibiotic properties but are considered as the major allergenic determinants in penicillin allergy. A solid-phase Enzyme Immuno Assay (EIA) of BPO groups in different biological fluids is described. It is a competitive immunoassay using acetylcholinesterase as label. In all biological fluids, very low non-specific binding values are observed. The sensitivity and the precision of the assay are good since ca. 0.5 ng/ml can be measured with a coefficient of variation less than 10%. Cross reactions between BPO and penicillin or penicillin derivatives are nil or very low. This assay is more sensitive, much more rapid and easier to handle than the other methods available and is thus suitable for routine determinations. In association with reversed-phase high performance liquid chromatography this EIA has allowed an initial investigation of the location of BPO-binding sites on micro quantities of serum albumin (ca. 1 mg) from penicillin treated patients.

Acetylcholinesterase↗