Lactococcal aminotransferases AraT and BcaT are key enzymes for the formation of aroma compounds from amino acids 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 α-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.     

Contribution of lactic acid bacteria to flavour compound formation in dairy products

Cheese flavour cannot be produced without starter bacteria. Lactic acid bacteria convert lactose to lactic acid and this together with their production of diacetyl and acetaldehyde are their main contributions to the flavour of cultured milks and fresh cheeses. In matured cheeses, the starter bacteria die out quickly and the rate at which they lyse and release their enzymes into the system has an influence on the rate at which free amino acids are formed. Rennet alone is mainly responsible for the formation of large, medium and small peptides but, without interaction with other enzymes, is capable of producing only methionine, histidine, glycine, serine and glutamic acid at quantifiable levels. Free amino acids in Cheddar cheese are mainly the result of microbial peptidase activity. These amino acids, together with the products of glycolysis, form substrates for secondary flora, the nature of which, in many cases, determines the cheese variety. They also form substrates for enzymes from the milk, e.g. the production of H₂S appears to be dependent on milk enzymes. Methionine, which is released by rennet, is further metabolized by starter enzymes with the production of methanethiol which plays a major role in cheese flavour possibly as a potentiator for other flavours. —Dicarbonyls, particularly methylglyoxal and diacetyl, and bacteria which can produce them, appear to play a crucial role in the formation of cheese flavour, both the desirable flavour of full-fat cheese and the meaty-brothy off-flavour of low-fat cheese. Although, theoretically, there are many compounds in cheese which could react purely chemically to form flavour compounds, these reactions are also mediated by enzymes in the cheese system and it seems unlikely that straight out chemical reactions play a major role in the production of cheese flavour. The role of the secondary flora is likely to be much more important than that of chemical reactions. Particularly in Cheddar and Emmental it has been shown that good quality cheeses have a low oxidation-reduction potential. This is more likely to be an indicator for the establishment of the anaerobic conditions required for the flavour forming reactions to proceed than an active causal agent of flavour formation. The function of glutathione is more likely to be as some sort of facilitator in enzyme reactions than as an agent for the reduction of oxidation-reduction potential. The ability of bacteria to accumulate glutathione from their media is likely to be one of the indicators of flavour generating capacity. Suitable selected strains of adjunct bacteria increase the rate and intensity of formation of Cheddar cheese flavour but unsuitable adjuncts can also cause off-flavours.     

Inactivation of lactococcal aromatic aminotransferase prevents the formation of floral aroma compounds from aromatic amino acids in semi-hard 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 -ketoacid acceptor for transamination is the first limiting factor for amino acid conversion to aroma compounds in cheese. In the presence of -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.     

Biochemistry of cheese ripening

Rennet-coagulated cheeses are ripened for periods ranging from about two weeks to two or more years depending on variety. During ripening, microbiological and biochemical changes occur that result in the development of the flavour and texture characteristic of the variety. Biochemical changes in cheese during ripening may be grouped into primary (lipolysis, proteolysis and metabolism of residual lactose and of lactate and citrate) or secondary (metabolism of fatty acids and of amino acids) events. Residual lactose is metabolized rapidly to lactate during the early stages of ripening. Lactate is an important precursor for a series of reactions including racemization, oxidation or microbial metabolism. Citrate metabolism is of great importance in certain varieties. Lipolysis in cheese is catalysed by lipases from various source, particularly the milk and cheese microflora, and, in varieties where this coagulant is used, by enzymes from rennet paste. Proteolysis is the most complex biochemical event that occurs during ripening and is catalysed by enzymes from residual coagulant, the milk (particularly plasmin) and proteinases and peptidases from lactic acid bacteria and, in certain varieties, other microorganisms that are encouraged to grow in or on the cheese. Secondary reactions lead to the production of volatile flavour compounds and pathways for the production of flavour compounds from fatty acids and amino acids are also reviewed.     

Enzymes involved in flavour formation by bacteria isolated from the smear population of surface-ripened cheese

Twenty-five bacterial isolates recovered from the surface population of smear-ripened cheese were assigned phenotypically as Brevibacterium spp ., Corynebacterium spp. and Aureobacterium spp. using the Biolog GP2 microplate system and database. The range and activity of hydrolytic enzymes involved in the formation of cheese flavour constituents were monitored in cell-free lysates of the isolates. Esterase activity and the presence of a range of enzymes involved in amino acid release and breakdown was confirmed in all strains examined although there were pronounced interspecies and strain differences in the level of activity detected. Peptidolytic activities present in the smear bacteria included dipeptidyl peptidase and aminopeptidases that cleaved various N-terminal amino acids including proline. Subsequent breakdown of the released aromatic and branched-chain amino acids was mediated by α-keto acid dependent aminotransferase action and several of the isolates were able to form thiols from sulphur-containing amino acid precursors. It was confirmed that the enzymic activity of the smear population could be manipulated by the use of defined starter cultures comprising selected combinations of smear isolates. The hydrolytic activities of the smear bacteria are involved in the generation of cheese flavour compounds and the enzyme profile is thus an important selection criterion for strains to be evaluated for use in defined surface smear preparations.     

Accelerating cheese proteolysis by enriching Lactococcus lactis proteolytic system with lactobacilli peptidases

The knowledge available on the genetics and proteolytic system of lactic acid bacteria makes it possible to genetically engineer starters with increased proteolytic properties. Our objective was to identify the best available strains capable of accelerating or modulating casein proteolysis during cheese ripening.To attain this goal, we used Lactococcus lactis strains expressing 5 different Lactobacillus peptidases to ripen a cheese model. At the end of ripening, free amino acids were quantitatively and qualitatively analysed.We identified the mixture of prolidase, PepQ, and X-prolyl dipeptidyl peptidase, PepX, as well as the peptidase PepW as the most efficient peptidases to increase, up to 3-fold, the overall level of amino acids at the end of ripening. The levels of threonine, asparagine, glycine, methionine, valine, glutamine, isoleucine and proline in particular increased (more than 3.5 fold). Grouping the amino acids produced according to the specific aroma compounds that each may give rise to following an enzymatic or chemical conversion, revealed that expression of PepW or PepX and PepQ increased the amounts of all groups of amino acids while expression of PepQ or PepN increased more especially those of aromatic amino acids/proline and glutamic acid, respectively.The combination of increased proteolysis and conversion of amino acids into aroma compounds now needs to be tested. In addition, the role of proline and its derived compounds in the overall flavour of cheese should be investigated.     

Enzymology of lactococci in relation to flavour development from milk proteins

Although the individual chemical compounds which impart flavour profiles to cheese and milk protein hydrolysates have not been defined, it is clear that the sequential breakdown of these proteins to small hydrophillic peptides and to free amino acids is a prerequisite to balanced flavour formation. This paper will review and discuss current knowledge of specificity, stability and availability of enzymes in lactococci in relation to flavour and taste compounds, which can be derived from peptide sequences and amino acid residues present in the proteins in milk.     

Lactic acid bacteria decarboxylation reactions in cheese

Fermentation in cheese comprises oxidation-reduction of carbohydrates to yield organic acids, alcohols and carbon dioxide. Furthermore, organic acid and amino acid metabolism produces a series of compounds that positively or negatively affect final cheese quality. Under the strong selective pressure of the acidic environment of cheese ripening, lactic acid bacteria have developed multiple stress-resistant strategies, including decarboxylase and deiminase reactions that play a main physiological role during the ripening process of cheese production. The control of the expression and activity of these enzymes is one active strategy for intracellular acid-base homeostasis. This review covers relevant pathways and aspects related to gene regulation of gene clusters present in starter or non-starter lactic acid bacteria that are involved in sensory changes such as flavour development. From the point of view of food safety the main decarboxylation pathways that lead to the formation of biogenic amines are described.     

Arginine metabolism in sugar deprived Lactococcus lactis enhances survival and cellular activity, while supporting flavour production

Flavour development in cheese is affected by the integrity of Lactococcus lactis cells. Disintegrated cells enhance for instance the enzymatic degradation of casein to free amino acids, while integer cells are needed to produce specific flavour compounds from amino acids. The impact of the cellular activity of these integer cells on flavour production remains to be elucidated. In this study we investigated whether lactose-deprived L. lactis cells that use arginine as an alternative energy source can extend cellular activity and produce more specific flavours. In cheese experiments we demonstrated that arginine metabolising cells survived about 3 times longer than non-arginine metabolising cells, which suggests prolonged cellular activity. Cellular activity and flavour production of L. lactis was further studied in vitro to enable controlled arginine supplementation. Comparable with the results found in cheese, the survival rates of in vitro incubated cells improved when arginine was metabolised. Furthermore, elongated cellular activity was reflected in 3-4-fold increased activity of flavour generating enzymes. The observed prolonged cellular activity resulted in about 2-fold higher concentrations of typical Gouda cheese flavours. These findings provide new leads for composing starter cultures that will produce specific flavour compounds.     

Precursors of the volatile sulphides in spoiling north sea cod (Gadus morhua)

Quantitative analyses of the free sulphur amino acids, cyst(e)ine, methionine, taurine and glutathione in the muscle of spoiling chill-stored cod, showed that the concentrations of cyst(e)ine and methionine increased until the twelfth day of storage before decreasing rapidly. Only the disappearance of methionine and cyst(e)ine could be correlated with the production of volatile sulphides in the flesh. Taurine, the principal sulphur amino acid present appeared resistant to both microbial and autolytic enzymes, whilst glutathione disappeared before the onset of active bacterial spoilage. The importance of these compounds as odour and/or flavour precursors in white fish is dssed.     

Flavour formation from amino acids by lactic acid bacteria: predictions from genome sequence analysis

Flavour development in dairy fermentations is the result of a series of chemical and biochemical processes during ripening. Starter lactic acid bacteria provide the enzymes involved in the formation of specific flavours. Amino acids, and in particular methionine, the aromatic and the branched-chain amino acids, are major precursors for volatile aroma compounds. The recent sequencing of complete genomes of several lactic acid bacteria (i.e. Lactococcus lactis, Lactobacillus plantarum, Streptococcus thermophilus) is beginning to provide insight into the full complement of proteins that may be involved in flavour-forming reactions, and hence the potential for formation of specific flavour compounds. Examples are given how bioinformatics tools can be used to search in genomes for essential components, such as proteinases, peptidases, aminotransferases, enzymes for biosynthesis of amino acids, and transport systems for peptides and amino acids.     

Meaty flavour compounds formation from the submerged liquid culture of Pleurotus ostreatus

The capacity of oyster mushroom (Pleurotus ostreatus) mycelium to produce meaty flavour compounds in the presence of amino acids and sugars was studied. The submerged liquid culture of P. ostreatus mycelium along with base medium made of defatted soybean powder, sucrose, potassium phosphate, and magnesium sulphate was incubated for 3 days at 25 °C. Cysteine, glutamine, or methionine and fructose, galactose, ribose, or xylose were added to the base medium to study the effect of amino acids and sugars on meaty flavour formation by trained panelists. The flavour compounds were isolated and identified by GC–MS and GC retention time of authentic compounds. The base medium required P. ostreatus, cysteine, ribose, and heat treatment to produce meaty flavour. The sulphur containing heterocyclic compounds such as 2,5‐diethylthiophene, 2‐formyl‐5‐methylthiophene, 3‐ethyl‐2‐formylthiophene, and dimethylformyl thiophene were responsible for meaty flavour. These compounds were formed by non‐enzymatic browning reaction between ribose and cysteine during heat treatment.     

Effect of Browning Reactions on the Formation of Flavour Substances

Nonenzymic browning reactions are usually accompanied by side reactions resulting in the formation of various flavour substances. The amount of volatile flavour compounds is very low in comparison with that of brown pigments. Flavour compounds are formed from intermediary reaction products by several secondary reactions: 1. cyclization of oligosubstituted hydroxylic, thiol, amine, carboxylic and carbonylic derivatives; 2. condensation of volatile carbonylic derivatives with amine, sulphur, or other carbonylic derivatives; 3. Strecker degradation of amino acids and peptides by quinones, osones, and triosones; 4. aldolization of aldehydes, especially in presence of amines; 5. spontaneous or thermic decomposition of macromolecular brown pigments or intermediary Schiff bases; 6. binding of flavour compounds into flavour-neutral or modified-flavour derivatives. By modifying the composition of ingredients or by adjusting the processing and storage conditions it is possible to obtain desirable flavour properties.     

Autolysis of selected Lactobacillus helveticus adjunct strains during Cheddar cheese ripening

Cheddar cheeses were manufactured on a pilot scale (500 L vats) with three different Lactobacillus helveticus strains, which showed varying degrees of autolysis, added as adjuncts to the starter. Autolysis of adjunct strains was monitored by reduction in cell numbers, level of intracellular enzymes released into the cheese, and by the consequent changes in the degree of proteolysis and concentration of free amino acids in the cheese. The flavour profiles of the cheeses at 6 months were also determined. Significant variation in viability of the Lb. helveticus strains, which showed a positive correlation with the indicators of autolysis, was observed. However, cheese manufactured with the most autolytic strain did not receive the highest flavour scores. The results indicate that whereas autolysis of adjunct strains is an important factor in Cheddar cheese flavour development, other factors also contribute to the overall flavour improvement observed.     

The development of free amino acids and volatile compounds in cheese ‘Oloumoucké tvarůžky’ (PGI) during ripening

Twenty‐two free amino acid (FAA) concentrations were observed during manufacture (7 days) and ripening period (42 days follow‐up) of Olomoucké tvar??ky (PGI, smear acid cheese). During the ripening period, the amounts of volatile compounds and selected sensory attributes were also analysed. The free amino acids were determined by means of ion‐exchange chromatography, and the volatile compounds were detected and identified using HS‐SPME coupled with GC/MS method. The development of the individual FAA content positively correlated with the ripening period (r = 0.7734–0.9229; P < 0.01). Forty‐six volatile compounds (14 alcohols; 10 esters; 7 ketones; 5 acids; 4 aldehydes; 3 sulphur compounds; 2 phenol compounds; and 1 terpene) were identified. Concentrations of several compounds increased (e.g. 3‐methylbutanal, 2‐butanol, 2‐butanone; P < 0.05) or decreased (e.g. dimethyldisulphide, methanethiol, 2‐phenylethylacetate, methylbutyric acid; P < 0.05) over cheese ageing. The results gave information about the development of sensory active substances and its precursors in Olomoucké tvar??ky during ripening. In conclusion, we found that free amino acid concentrations and sulphur compounds are positive with improved flavour.     

Flavours of cheese products: metabolic pathways, analytical tools and identification of producing strains

Aroma development in cheese products results from the metabolic activities of cheese bacteria, by glycolysis, lipolysis and proteolysis. To respond to the increasing demand for products with improved aroma characteristics, the use of bacterial strains for cheese ripening with enhanced flavour production is seen as promising. In this review, the catabolism of amino acids, presumably the origin of some major aroma compounds, is discussed. The techniques of detection of flavour-producing strains are then presented. Their detection may be achieved either by genotyping, by enzymatic analysis, or by physico-chemical analysis such as HPLC, TLC, GC, and electronic nose.     

Volatile composition and proteolysis in traditionally produced mature Kashar cheese

Twelve samples of raw milk mature Kashar cheese at different stages of ripening were collected from retail outlets. The average pH, moisture, fat-in-dry matter, protein, salt-in-dry matter and titratable acidity contents of the samples were 5.33, 39.39%, 45.20%, 27.33%, 6.62% and 0.65% (as lactic acid), respectively. Indices of proteolysis varied from 10.72% to 23.75% and 7.09% to 12.26% for pH 4.6-soluble and 12% trichloroacetic acid-soluble nitrogen fractions, respectively, and total free amino acid concentrations ranged from 6.36 to 36.03 mg Leu g⁻¹ of cheese. The cheeses were analysed for volatile compounds by Solid Phase Microextraction and Gas Chromatography-Mass Spectrometry (GC-MS). A total of 113 compounds were detected and identified belonging to the following chemical groups: acids (eleven), esters (sixteen), ketones (sixteen), aldehydes (six), alcohols (twenty-seven), sulphur compounds (seven), terpenes (seven) and miscellaneous compounds (twenty-three). The potential effect of each compound on the flavour profile of Kashar cheese is discussed. Acids, esters, ketones and alcohols were found at considerable levels in the samples. Kashar cheeses obtained from different retail outlets displayed some differences in terms of chemical composition, proteolysis and patterns of aroma compounds; and may be attributed to their production technologies and age-related variations.     

Levels of cystathionine gamma lyase production by Geotrichum candidum in synthetic media and correlation with the presence of sulphur flavours in cheese

Geotrichum candidum is a cheese-ripening agent with the potential to produce sulphur flavour compounds in soft cheeses. We aimed to develop an alternative test for predicting the aromatic (sulphur flavours) potential of G. candidum strains in soft cheese. Twelve strains of G. candidum with different levels of demethiolase activity (determined by a chemical method) in YEL-met (yeast extract, lactate methionine) medium were studied. We investigated cgl (cystathionine gamma lyase) gene expression after culture in three media - YEL-met, casamino acid and curd media - and then carried out sensory analysis on a Camembert cheese matrix. We found no correlation between demethiolase activity in vitro and cgl gene expression. Sensory analysis (detection of sulphur flavours) identified different aromatic profiles linked to cgl expression, but not to demethiolase activity. The RT-PCR technique described here is potentially useful for predicting the tendency of a given strain of G. candidum to develop sulphur flavours in cheese matrix. This is the first demonstration that an in vitro molecular approach could be used as a predictive test for evaluating the potential of G. candidum strains to generate sulphur compounds in situ (Camembert cheese matrix).     

New insights into physiology and metabolism of Propionibacterium freudenreichii

Dairy propionibacteria are Actinobacteria, mainly isolated from dairy environments. Propionibacterium freudenreichii has been used for a long time as a ripening culture in Swiss-type cheese manufacture, and is more and more considered for its potent probiotic effects. This review summarises the knowledge on the main P. freudenreichii pathways and the main features explaining its hardiness, and focuses on recent advances concerning its applications as a cheese ripening agent and as a probiotic for human health. Propionibacteria have a peculiar metabolism, characterised by the formation of propionic acid as main fermentation end-product. They have few nutritional requirements and are able to use a variety of carbon substrates. From the sequence of P. freudenreichii CIRM-BIA1^T genome, many pathways were reconstituted, including the Wood-Werkman cycle, enzymes of the respiratory chain, synthesis pathways for all amino acids and many vitamins including vitamin B₁₂. P. freudenreichii displays features allowing its long-term survival. It accumulates inorganic polyphosphate (polyP) as energy reserve, carbon storage compounds (glycogen), and compatible solutes such as trehalose. In cheese, P. freudenreichii plays an essential role in the production of a variety of flavour compounds, including not only propionic acid, but also free fatty acids released via lipolysis of milk glycerides and methyl-butanoic acids resulting from amino acid degradation. P. freudenreichii can exert health-promoting activities, such as a bifidogenic effect in the human gut and promising immunomodulatory effects. Many P. freudenreichii properties involved in adaptation, cheese ripening, bio-preservation and probiotic effects are highly strain-dependent. The elucidation of the molecular mechanisms involved is now facilitated by the availability of genome sequence and molecular tools. It will help in the selection of the most appropriate strain for each application.     

Partial purification and characterization of two aminotransferases from Lactococcus lactis subsp. cremoris B78 involved in the catabolism of methionine and branched-chain amino acids

Transamination of methionine and other amino acids followed by conversion of the resulting α-keto acids by enzymes from the mesophilic starter organism Lactococcus lactis subsp. cremoris B78 was studied. Two aminotransferases, displaying activity towards methionine, were partially purified and characterized. The enzymes most likely were branched-chain aminotransferases, since their activity towards valine, leucine and isoleucine was even higher than towards methionine. The enzymes, AT-A and AT-B, both showed a molecular mass of approximately 75 kDa and consisted of two identical subunits, each with a molecular mass of approximately 40 kDa. AT-A and AT-B also had a broad substrate specificity for the amino-group acceptor, α-ketoglutaric acid being the preferred cosubstrate. The enzymes catalyzed the conversion of methionine to 4-methylthio-2-ketobutyric acid, which was subsequently converted to methanethiol and dimethyldisulphide. The formation of these and other volatile sulfur compounds is considered to play an important role in the development of cheese flavour. Both AT-A and AT-B had a rather high optimum temperature, 45–50°C, and a pH optimum of 8. However, under simulated cheese-ripening conditions (10–15°C and pH 5.2–5.4) sufficient activity remained for conversion of methionine.