Mireille Yvon

Cheese flavour formation by amino acid catabolism

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.

Adding α-Ketoglutarate to Semi-hard Cheese Curd Highly Enhances the Conversion of Amino acids to Aroma Compounds

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.

Characterization and Role of the Branched-Chain Aminotransferase (BcaT) Isolated from Lactococcus lactis subsp. cremoris NCDO 763

ABSTRACT 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.

Cooperation between Lactococcus lactis and Nonstarter Lactobacilli in the Formation of Cheese Aroma from Amino Acids

ABSTRACT 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.

Glutamate dehydrogenase activity: a major criterion for the selection of flavour-producing lactic acid bacteria strains

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.

Ability of Thermophilic Lactic Acid Bacteria To Produce Aroma Compounds from Amino Acids

ABSTRACT 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.

Enhancement of amino acid catabolism in Cheddar cheese using α-ketoglutarate : amino acid degradation in relation to volatile compounds and aroma character

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.

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, 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.

Conversion of l-Leucine to Isovaleric Acid by Propionibacterium freudenreichii TL 34 and ITGP23

ABSTRACT Several branched-chain volatile compounds are involved in the flavor of Swiss cheese. These compounds are probably produced by enzymatic conversion of branched-chain amino acids, but the flora and the pathways involved remain hypothetical. Our aim was to determine the ability of Propionibacterium freudenreichii, which is one of the main components of the secondary flora of Swiss cheese, to produce flavor compounds during leucine catabolism. Cell extracts and resting cells of two strains were incubated in the presence of l-leucine, α-ketoglutaric acid, and cofactors, and the metabolites produced were determined by high-performance liquid chromatography and gas chromatography. The first step of leucine catabolism was a transamination that produced α-ketoisocaproic acid, which was enzymatically converted to isovaleric acid. Both reactions were faster at pH 8.0 than at acidic pHs. Cell extracts catalyzed only the transamination step under our experimental conditions. Small amounts of 3-methylbutanol were also produced by resting cells, but neither 3-methylbutanal norα-hydroxyisocaproic acid was detected. l-Isoleucine and l-valine were also converted to the corresponding acids and alcohols. Isovaleric acid was produced by both strains during growth in a complex medium, even under conditions simulating Swiss cheese conditions (2.1% NaCl, pH 5.4, 24°C). Our results show that P. frendenreichii could play a significant role in the formation of isovaleric acid during ripening.

Expression of a Heterologous Glutamate Dehydrogenase Gene in Lactococcus lactis Highly Improves the Conversion of Amino Acids to Aroma Compounds

ABSTRACT 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.