Glutamate dehydrogenase activity in lactobacilli and the use of glutamate dehydrogenase-producing adjunct Lactobacillus spp. cultures in the manufacture of cheddar cheese

AIMS: The study was undertaken to investigate the occurrence of glutamate dehydrogenase activity in different species of lactobacilli, and to determine, in a series of cheese-making trials, the effects of glutamate dehydrogenase-producing adjunct cultures on sensory attribute development during the maturation of cheddar cheese. METHODS AND RESULTS: The presence of dehydrogenase activity with glutamate as substrate was monitored in cell lysates of >100 strains from 30 different species of lactobacilli using a qualitative colorimetric plate screening assay. Activity was detectable in 25 of the 29 representative species obtained from culture collections and in 12 of the 13 non-starter species isolated from cheese. There were pronounced interspecies and strain differences in the occurrence, level and pyridine nucleotide specificity of the glutamate dehydrogenase activity detected. Among the non-starter lactobacilli the highest frequency of enzyme occurrence and activity was detected in the Lactobacillus plantarum isolates. The establishment of glutamate dehydrogenase-producing adjunct strains in the predominant population of lactobacilli in the cheese curd affected the formation of a number of volatile compounds in ripening cheddar cheese, while the presence of Lact. plantarum strains, in particular, was associated with an intensification and acceleration of aroma and flavour development during the maturation period. CONCLUSIONS: Glutamate dehydrogenase formation by lactobacilli is a strain-dependent metabolic attribute, and adjunct cultures expressing the activity that are able to proliferate during cheese ripening have a positive impact on the rate of development and the intensity of cheddar cheese aroma and flavour development. SIGNIFICANCE AND IMPACT OF THE STUDY: It has been demonstrated that some strains of glutamate dehydrogenase-producing lactobacilli have potential use as adjunct cultures to accelerate and intensify aroma and flavour formation during the manufacture of cheddar and, by analogy, other similar varieties of cheese. The importance of phenotypic discriminative monitoring of the dominant lactobacilli present during ripening to confirm adjunct establishment and population complexity was highlighted as was the requirement to establish the metabolic attributes of the non-starter population in uninoculated control cheeses in comparative trials.     

Flavour formation by amino acid catabolism

Microbial catabolism of amino acids produces flavour compounds of importance for foods such as cheese, wine and fermented sausages. Lactic acid bacteria are equipped with enzyme systems for using the amino acids in their metabolism and are useful for flavour formation of foods. Branched-chain amino acids (Leu, Ile, Val) are converted into compounds contributing to malty, fruity and sweaty flavours; catabolism of aromatic amino acids (Phe, Tyr, Trp) produce floral, chemical and faecal flavours; aspartic acid (Asp) is catabolised into buttery flavours and sulphuric amino acids (Met, Cys) are transferred into compounds contributing to boiled cabbage, meaty and garlic flavours.     

The effect of alpha-ketoglutaric acid on amino acid utilization by nonstarter Lactobacillus spp. isolated from Cheddar cheese

AIMS: To examine the effect of alpha-ketoglutaric acid (alpha-KG) on the utilization and catabolism of amino acids by strains of nonstarter lactobacilli isolated from Cheddar cheese. METHODS AND RESULTS: The effect of alpha-KG in the growth medium of nonstarter lactobacilli on amino acid metabolism, catabolite levels, peptide hydrolase and aminotransferase activities was examined. The pattern of amino acid utilization, catabolite formation and aminotransferase activity was affected by keto acid. CONCLUSIONS: Amino acid conversion into cheese aroma and flavour compounds by nonstarter lactobacilli is enhanced in the presence of alpha-ketoglutarate. SIGNIFICANCE AND IMPACT OF THE STUDY: Increasing the availability of alpha-ketoglutarate in cheese offers a possible method of reducing the maturation period by accelerating the rate of character compound formation from amino acids by the nonstarter lactobacilli.     

Technological properties of Enterococcus faecium isolated from ewe's milk and cheese with importance for flavour development

Eight Enterococcus faecium strains isolated from ewe milk and artisanal cheese from northwest Argentina were screened for biotechnological properties relevant to flavour development. The API ZYM test showed absence of proteases, presence of high amounts of peptidases, and high esterase-lipase activities. Low extracellular proteolytic activity was observed. Most strains produced diacetyl in milk, with E. faecium OvL 214 and OvL 254 being the best producers. Biomass and growth rate increased when citrate was added to the medium, suggesting that these strains could use citrate as a main energy source. After 24 h of incubation, citrate was completely consumed in complex medium supplemented with glucose and citrate. An average of 17% residual citrate was detected in complex media supplemented with citrate. For all strains, esterase activity was detected up to alpha-naphthyl-caproate. They hydrolyzed alpha-naphthyl derivatives of fatty acids in this order: C3 > C6 > C4 > C8 > C2. Post-electrophoretic detection of esterase activities revealed the presence of multiple esterases. Hydrolysis of tributyrin, tricaprylin, and milk fat was observed in cell-free extracts. Enterococcus faecium strains isolated from ewe milk and artisanal cheese from northwest Argentina present the metabolic potential to contribute to cheese flavour development.     

The impact of biotechnology on the dairy industry

The review focusses on the use of genetic techniques to manipulate bacteria that are important to the dairy industry. Both classical and molecular approaches have been used to improve strains involved in yoghurt and cheese production. Examples are provided of methods for; increasing efficiency of substrate conversion, regulating the production of flavour enhancing metabolites, and developing starter cultures resistant to bacteriophage and bacteriocin attack. The possible applications of these systems are discussed     

Protease,peptidase and esterase activities by lactobacilli and yeast isolates from Feta cheese brine

AIMS: The study of peptidase, esterase and caseinolytic activity of Lactobacillus paracasei subsp. paracasei, Debaryomyces hansenii and Sacchromyces cerevisiae isolates from Feta cheese brine. METHODS AND RESULTS: Cell-free extracts from four strains of Lact. paracasei subsp. paracasei, four strains of D. hansenii and three strains of S. cerevisiae, isolated from Feta cheese brine were tested for their proteolytic and esterase enzyme activities. Lactobacillus paracasei subsp. paracasei strains had intracellular aminopeptidase, dipeptidyl aminopeptidase, dipeptidase, endopeptidase and carboxypeptidase activities. Esterases were detected in three of four strains of lactobacilli and their activities were smaller with higher molecular weight fatty acids. The strains of yeasts did not exhibit endopeptidase as well as dipeptidase activities except on Pro-Leu. Their intracellular proteolytic activity was higher than that of lactobacilli. Esterases from yeasts preferentially degraded short chain fatty acids. Lactobacilli degraded preferentially beta-casein. Caseinolytic activity of yeasts was higher than that of lactobacilli. CONCLUSIONS: The results suggest that Lact. paracasei subsp. paracasei and yeasts may contribute to the development of flavour in Feta cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: Selected strains could be used as adjunct starters to make high quality Feta 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 -ketoglutarate since this -ketoacid is essential for the first step of the conversion. Indeed, we have demonstrated that adding exogenous -ketoglutarate to cheese curd, as well as using a genetically modified L. lactis strain capable of producing -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 -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.     

Branched chain aldehydes: production and breakdown pathways and relevance for flavour in foods

Branched aldehydes, such as 2-methyl propanal and 2- and 3-methyl butanal, are important flavour compounds in many food products, both fermented and non-fermented (heat-treated) products. The production and degradation of these aldehydes from amino acids is described and reviewed extensively in literature. This paper reviews aspects influencing the formation of these aldehydes at the level of metabolic conversions, microbial and food composition. Special emphasis was on 3-methyl butanal and its presence in various food products. Knowledge gained about the generation pathways of these flavour compounds is essential for being able to control the formation of desired levels of these aldehydes.     

Fermentation of plant-based dairy alternatives by lactic acid bacteria

Ethical, environmental and health concerns around dairy products are driving a fast-growing industry for plant-based dairy alternatives, but undesirable flavours and textures in available products are limiting their uptake into the mainstream. The molecular processes initiated during fermentation by lactic acid bacteria in dairy products is well understood, such as proteolysis of caseins into peptides and amino acids, and the utilisation of carbohydrates to form lactic acid and exopolysaccharides. These processes are fundamental to developing the flavour and texture of fermented dairy products like cheese and yoghurt, yet how these processes work in plant-based alternatives is poorly understood. With this knowledge, bespoke fermentative processes could be engineered for specific food qualities in plant-based foods. This review will provide an overview of recent research that reveals how fermentation occurs in plant-based milk, with a focus on how differences in plant proteins and carbohydrate structure affect how they undergo the fermentation process. The practical aspects of how this knowledge has been used to develop plant-based cheeses and yoghurts is also discussed.     

Acceleration of flavour formation in cheese by a bacteriocin-producing adjunct lactic culture

Bacteriocin-producing Enterococcus faecalis INIA 4 was used as adjunct lactic culture at inoculum levels ranging from 0.003% to 0.10% in the manufacture of semi-hard cheese. Cheese made from milk with 0.003% adjunct culture had the most pronounced proteolysis and developed a characteristic cheese flavour faster than the rest, reaching in 30.4 days the flavour intensity score of a 45-day control cheese made without adjunct culture.     

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.     

Metabolic engineering of lactic acid bacteria: overview of the approaches and results of pathway rerouting involved in food fermentations.

Lactic acid bacteria such as Lactococcus lactis are the microorganisms of choice for performing metabolic engineering in relation to food fermentation. These bacteria are used extensively in food fermentations, they have a simple and therefore controllable metabolism and the molecular genetics of these food bacteria is well-developed. There have been recent successes in metabolic engineering in these lactic acid bacteria, including examples of changes in both primary metabolism (diacetyl and alanine) and secondary metabolism (exopolysaccharides and flavour).     

Determination of aspartase activity in dairy Propionibacterium strains

Propionic acid bacteria (PAB) are important as starter cultures for the dairy industry in the manufacture of Swiss-type cheeses, in which they are involved in the formation of eyes and are responsible for the typical flavour and aroma. These characteristics are mainly due to the classical propionic acid fermentation, but also the conversion of aspartate to fumarate and ammonia by the enzyme aspartase and the subsequent reduction of fumarate to succinate, which occur in dairy Propionibacterium freudenreichii ssp. shermanii and ssp. freudenreichii starter strains. Additionally, the metabolism of free amino acids may be partly responsible for secondary fermentation and the subsequent split defects in cheese matrix. Here a method for aspartase activity was established and a number of dairy propionibacteria belonging to P. freudenreichii ssp. shermanii and freudenreichii were screened for this enzyme activity. A wide range of aspartase activity could be found in PAB isolates originating from cheese. The majority, i.e. 70% of the 100 isolates tested, showed very low levels of aspartate activity.     

Production of volatile compounds in liquid cultures by six strains of coryneform bacteria

Summary The aromatic profiles of four strains of Brevibacterium linens, one strain of Brevibacterium sp. and one strain of Microbacterium sp. were determined with some pure cultures of these microorganisms in standard trypcase soy liquid medium, which enabled four of these six strains to produce flavour compounds of ripened cheese. Thirty-two flavour compounds were identified by gas chromatography and mass spectrometry. The identified flavour compounds included the following: fatty acids, alcohols, methylketones, sulphur compounds, aromatic compounds and a pyrazine. Some important differences were found among the six strains studied. The four strains of B. linens had similar flavour profiles. Their typical flavour was probably due to dimethyltrisulphide. The two other strains did not appear to produce this compound. Three strains produced significant amounts of the floral aromas phenylethanol and phenylpropanone.Offprint requests to: J.-M. Belin     

1H HRMAS-NMR based metabolic fingerprints for discrimination of cheeses based on sensory qualities

In this study, the ¹H HRMAS-NMR (High-resolution Magic Angle Spinning-Nuclear Magnetic Resonance) spectra of 52 cheese samples obtained from the South Korean dairy farms were evaluated for their metabolic profiling and intensities associating with the sensory qualities. The NMR profiles displayed a broad range of compounds comprising amino acids, carbohydrates, organic acids, and phospholipids. Afterwards, the cheese samples were categorized into three groups (more likeness - G1, moderate likeness - G2, less likeness - G3), in relating to their sensory scores. The NMR data of the samples were later investigated through multivariate statistical tools to define the variations in metabolic fingerprints of every cheese sample and their intensities hailing in individual sensory groups. The unsupervised PCA employing all cheese samples unveiled the uniqueness in metabolite profiles of the brown and cheddar type cheeses (outliers). Moreover, Gouda and other types of cheeses displayed samples positioning in respective of their metabolite profiles. The pairwise comparison of sensory groups in the supervised models perceived better separation in OPLS-DA than PLS-DA. The corresponding VIP (PLS-DA) and loading (OPLS-DA) plots revealed amino acids and organic acids (lactate, citrate) as significant variables. The discrimination of G 1 Gouda type of cheeses against G 2 and G 3 was highly associated with their citrate levels. Further investigation using heatmaps displayed clear differentiation between each sensory group in terms of the levels of amino acids, lactate, citrate, phospholipids, and glycerol, conveying these variations are likely due to proteolytic and metabolic processes in cheese ripening. This study concluded that ¹H HRMAS-NMR metabolite profile of the Korean cheeses is consistence with their sensory qualities. Further, the candidate metabolites identified in this study confers their potential application as biomarkers in cheese industries for faster and effective validation of sensory characteristics.     

Formation of flavour compounds in the Maillard reaction

This paper discusses the importance of the Maillard reaction for food quality and focuses on flavour compound formation. The most important classes of Maillard flavour compounds are indicated and it is shown where they are formed in the Maillard reaction. Some emphasis is given on the kinetics of formation of flavour compounds. It is concluded that the essential elements for predicting the formation of flavour compounds in the Maillard reaction are now established but much more work needs to be done on specific effects such as the amino acid type, the pH, water content and interactions in the food matrix. It is also concluded that most work is done on free amino acids but hardly anything on peptides and proteins, which could generate peptide- or protein-specific flavour compounds.     

The arthrobacter arilaitensis Re117 genome sequence reveals its genetic adaptation to the surface of cheese

Arthrobacter arilaitensis is one of the major bacterial species found at the surface of cheeses, especially in smear-ripened cheeses, where it contributes to the typical colour, flavour and texture properties of the final product. The A. arilaitensis Re117 genome is composed of a 3,859,257 bp chromosome and two plasmids of 50,407 and 8,528 bp. The chromosome shares large regions of synteny with the chromosomes of three environmental Arthrobacter strains for which genome sequences are available: A. aurescens TC1, A. chlorophenolicus A6 and Arthrobacter sp. FB24. In contrast however, 4.92% of the A. arilaitensis chromosome is composed of ISs elements, a portion that is at least 15 fold higher than for the other Arthrobacter strains. Comparative genomic analyses reveal an extensive loss of genes associated with catabolic activities, presumably as a result of adaptation to the properties of the cheese surface habitat. Like the environmental Arthrobacter strains, A. arilaitensis Re117 is well-equipped with enzymes required for the catabolism of major carbon substrates present at cheese surfaces such as fatty acids, amino acids and lactic acid. However, A. arilaitensis has several specificities which seem to be linked to its adaptation to its particular niche. These include the ability to catabolize D-galactonate, a high number of glycine betaine and related osmolyte transporters, two siderophore biosynthesis gene clusters and a high number of Fe(3+)/siderophore transport systems. In model cheese experiments, addition of small amounts of iron strongly stimulated the growth of A. arilaitensis, indicating that cheese is a highly iron-restricted medium. We suggest that there is a strong selective pressure at the surface of cheese for strains with efficient iron acquisition and salt-tolerance systems together with abilities to catabolize substrates such as lactic acid, lipids and amino acids.     

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 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.     

Assessment of industrial lipases for flavour development in commercial Idiazabal (ewe's raw milk) cheese

Three commercial lipases (lipases A1 and A2 were pregastric lipases and lipase A3 was a fungal lipase) from three different manufacturers were studied to develop the characteristic pungent flavour of Idiazabal (sheep's raw milk) cheese in experimental productions (50 L vats), both in summer and in winter. In the experimental productions, all lipases significantly increased the concentration of total free fatty acids (FFA), both after 90 and 180 days of ripening. Lipases A1 and A2 increased primarily the concentration of short-chain FFA (C4, C6 and C10), whereas C16:0 and C18:1 were the main FFA obtained with lipase A3. Cheeses made with no lipase had the lowest concentrations of total FFA, with C16:0 and C18:1 as the major FFA. The percent of short-chain FFA was 70–80% of the total for lipases A1 and A2, but 30–40% in cheeses made with lipase A3 or with no lipase. Sensory analysis was performed after 90 and 180 days of ripening. Cheeses made with lipases A1 or A2 or with lamb rennet paste (which contains pregastric lipase and was used as control for sensory analysis) had significantly higher scores in 6 of the 22 attributes analyzed (odour and flavour intensity, sharp odour, pungent flavour and rennet odour and flavour) than cheeses made with lipase A3 or with no lipase added. Lipase A1 was selected to conduct commercial cheese productions (200 L vats) made by artisan cheese makers adding 94 lipase units (LU)/50 L (high amount) or 8 LU/50 L (low amount). Control cheeses were made with lamb rennet paste having comparable amounts of lipolytic activity. After 90 and 180 days of ripening, cheeses made with the same amount of lipase (either as lipase A1 or present in lamb rennet paste) were indistinguishable from each other, both from the sensory and analytical points of view (comparable amounts of total FFA, and percent FFA composition). Those with low amount of lipase were rated as ‘mild’, whereas those with high amount of lipase were rated as ‘strong’. An industrial production (5000 L vat) with 100 LU/50 L yielded cheeses of ‘intermediate strength’ and intermediate levels of lipolysis. A linear correlation was observed between percent of short-chain FFA and the score for pungent flavour, for all amounts of lipase used in this study. We conclude that lipase A1 is an adequate commercial lipase to develop the characteristic flavour of Idiazabal cheese, both in artisan and industrial fabrications.