Cooperation between Lactococcus lactis and nonstarter lactobacilli in the formation of cheese aroma from amino acids

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 alpha-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 alpha-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.     

Ability of thermophilic lactic acid bacteria to produce aroma compounds from amino acids

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 alpha-keto acid such as alpha-ketoglutarate (alpha-KG) as the amino group acceptor, and produced alpha-keto acids. Only S. thermophilus exhibited glutamate dehydrogenase activity, which produces alpha-KG from glutamate, and consequently only S. thermophilus was capable of catabolizing amino acids in the reaction medium without alpha-KG addition. In the presence of alpha-KG, lactobacilli produced much more varied aroma compounds such as acids, aldehydes, and alcohols than S. thermophilus, which mainly produced alpha-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 alpha-keto acids by L. delbrueckii subsp. lactis mainly results from the action of an alpha-keto acid decarboxylase, which produces aldehydes that are then oxidized or reduced to acids or alcohols. In contrast, the enzyme involved in the alpha-keto acid conversion to acids in L. helveticus and S. thermophilus is an alpha-keto acid dehydrogenase that produces acyl coenzymes A.     

Purification and characterization of a branched-chain amino acid aminotransferase from Lactobacillus paracasei subsp. paracasei CHCC 2115

AIM: Purification and characterization of an aminotransferase (AT) specific for the degradation of branched-chain amino acids from Lactobacillus paracasei subsp. paracasei CHCC 2115. METHODS AND RESULTS: The purification protocol consisted of anion exchange chromatography, affinity chromatography and hydrophobic interaction chromatography. The enzyme was found to exist as a monomer with a molecular mass of 40-50 kDa. The AT converted isoleucine, leucine and valine at a similar rate with alpha-ketoglutarate as the amino group acceptor; minor activity was shown for methionine. The enzyme had pH and temperature optima of 7.3 and 43 degrees C, respectively, and activity was detected at the pH and salt conditions found in cheese (pH 5.2, 4% NaCl). Hg2+ completely inhibited the enzyme, and the inhibition pattern was similar to that for pyridoxal-5'-phosphate-dependent enzymes, when studying the effect of other metal ions, thiol- and carbonyl-binding agents. The N-terminal sequence of the enzyme was SVNIDWNNLGFDYMQLPYRYVAHXKDGVXD, and had at the amino acid level, 60 and 53% identity to a branched-chain amino acid AT of Lact. plantarum and Lactococcus lactis, respectively. CONCLUSIONS: The results suggest that Lact. paracasei subsp. paracasei CHCC 2115 may contribute to development of flavour in cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work contribute to the knowledge of transamination performed by cheese-related bacteria, and in the understanding and control of amino acid catabolism and the production of aroma compounds.     

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

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.     

Lactobacillus casei and Lactobacillus plantarum initiate catabolism of methionine by transamination

AIMS: To study the ability of Lactobacillus casei and Lact. plantarum strains to convert methonine to cheese flavour compounds. METHODS AND RESULTS: Strains were assayed for methionine aminotransferase and lyase activities, and amino acid decarboxylase activity. About 25% of the strains assayed showed methionine aminotransferase activity. The presence of glucose in the reaction mixture increased conversion of methionine to 4-methylthio-2-ketobutanoate (KMBA) and 4-methylthio-2-hydroxybutanoate (HMBA) in all strains. The methionine aminotransferase activity in Lact. plantarum and Lact. casei showed variable specificity for the amino group acceptors glyoxylate, ketoglutarate, oxaloacetate and pyruvate. None of the strains showed methionine lyase or glutamate and methionine decarboxylase activities. CONCLUSION: The presence of amino acid converting enzymes in lactobacilli is strain specific. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work suggest that lactobacilli can be used as adjuncts for flavour formation in cheese manufacture.     

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.     

NADP‐glutamate dehydrogenase activity in nonstarter lactic acid bacteria: effects of temperature,pH and NaCl on enzyme activity and expression

Aims: To screen the glutamate dehydrogenase (GDH) activity of nonstarter lactic acid bacteria (NSLAB) and to determine the effects of temperature, pH and NaCl values used for cheese ripening on enzyme activity and expression of GDH gene. Methods and Results: A subcellular fractionation protocol and specific enzyme assays were used. The effect of temperature, pH and NaCl on enzyme activity was evaluated. The expression of GDH gene was monitored by real‐time PCR. One selected strain was also used as adjunct starter for cheese making to evaluate the catabolism of free amino acids and the production of volatile organic compounds (VOC) during cheese ripening. The cytoplasm fraction of all strains showed in vitro NADP‐dependent GDH activity. NADP‐GDH activity was markedly strain dependent and varied according to the interactions between temperature, pH and NaCl. Lactobacillus plantarum DPPMA49 showed the highest NADP‐GDH activity under temperature, pH and NaCl values found during cheese ripening. RT‐PCR analysis revealed that GDH expression of Lact. plantarum DPPMA49 was down‐expressed by low temperature (<13°C) and over‐expressed by NaCl (1·87–5·62%). According to NADP‐GDH activity, the highest level of VOC (alcohols, aldehydes, miscellaneous and carboxylic acids) was found in cheeses made with DPPMA49. Conclusions: The results of this study may be considered as an example of the influence of temperature, pH and NaCl on enzyme activity and expression of functional genes, such as GDH, in cheese‐related bacteria. Significance and Impact of the Study: It focuses on the phenotypic and molecular characterization of the NADP‐GDH in lactobacilli under cheese‐ripening conditions. The findings of this study contribute to the knowledge about enzymes involved in the catabolism of amino acids, to be used as an important selection trait for cheese strains.     

Amino acid catabolism in cheese-related bacteria: selection and study of the effects of pH, temperature and NaCl by quadratic response surface methodology

AIMS: To screen the cystathionine lyase and L-methionine aminotransferase activities of cheese-related bacteria (lactococci, non-starter lactobacilli and smear bacteria) and to determine the individual and interactive effects of temperature, pH and NaCl concentration on selected enzyme activities. METHODS AND RESULTS: A subcellular fractionation protocol and specific enzyme assays were used, and a quadratic response surface methodology was applied. The majority of the strains, 21 of 33, had detectable cystathionine lyase activity which differed in the specificity. Aminotransferase activity on L-methionine was observed in only three strains. The cystathionine lyase activities of Lactobacillus reuteri DSM20016, Lactococcus lactis subsp. cremoris MG1363, Brevibacterium linens 10 and Corynebacterium ammoniagenes 8 and the L-methionine aminotransferase activity of Lact. reuteri DSM20016 had temperature and pH optima of 30-45 degrees C, and 7.5-8.0, respectively. As shown by the quadratic response surface methodology these enzymes retained activities in the range of temperature, pH and NaCl concentration which characterized the cheeses from which the bacteria originated. CONCLUSION: The enzyme activities may have a role in flavour development during cheese ripening. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work contribute to the knowledge about the amino acid catabolic enzymes in order to improve cheese ripening.     

Development of a Probiotic Cheddar Cheese Containing Human-Derived Lactobacillus paracasei Strains

Cheddar cheese was manufactured with either Lactobacillus salivarius NFBC 310, NFBC 321, or NFBC 348 or L. paracasei NFBC 338 or NFBC 364 as the dairy starter adjunct. These five strains had previously been isolated from the human small intestine and have been characterized extensively with respect to their probiotic potential. Enumeration of these strains in mature Cheddar cheese, however, was complicated by the presence of high numbers (>10⁷ CFU/g of cheese) of nonstarter lactic acid bacteria, principally composed of lactobacilli which proliferate as the cheese ripens. Attempts to differentiate the adjunct lactobacilli from the nonstarter lactobacilli based on bile tolerance and growth temperature were unsuccessful. In contrast, the randomly amplified polymorphic DNA method allowed the generation of discrete DNA fingerprints for each strain which were clearly distinguishable from those generated from the natural flora of the cheeses. Using this approach, it was found that both L. paracasei strains grew and sustained high viability in cheese during ripening, while each of the L. salivarius species declined over the ripening period. These data demonstrate that Cheddar cheese can be an effective vehicle for delivery of some probiotic organisms to the consumer.     

Succinate production and citrate catabolism by Cheddar cheese nonstarter lactobacilli

AIMS: To identify strains of Cheddar cheese nonstarter lactobacilli that synthesize succinate from common precursors and characterize the biochemical pathways utilized. METHODS AND RESULTS: Whole cell incubations of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus zeae and Lactobacillus rhamnosus, were used to identify strains that accumulated succinate from citrate, l-lactate, aspartic acid or isocitrate. In vivo 13C-nuclear magnetic resonance spectroscopy (13C-NMR) identified the biochemical pathway involved at pH 7.0, and under conditions more representative of the cheese ripening environment (pH 5.1/4% NaCl/13 degrees C). Enzyme assays on cell-free extracts were used to support the pathway suggested by 13C-NMR. CONCLUSIONS: The Lact. plantarum strains studied synthesize succinate from citrate by the reductive tricarboxylic acid (TCA) cycle at either pH 7.0 or pH 5.1/4% NaCl/13 degrees C. Lactobacillus casei, Lact. zeae and Lact. rhamnosus strains lack one or more enzymatic activities present in this pathway, and do not accumulate succinate from any of the four precursors studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of Lact. plantarum strains to milk during cheese manufacture may increase the accumulation of the flavour enhancer succinate.     

Effect of L-malic and citric acids metabolism on the essential amino acid requirements for Oenococcus oeni growth

AIMS: The purpose of this work was to study the effect of L-malic and/or citric acids on Oenococcus oeni m growth in deficient nutritional conditions, and their roles as possible biosynthetic precursors of the essential amino acids. METHODS AND RESULTS: Bacterial cultures were performed in synthetic media. Bacterial growth rate was reduced or annulled when one amino acid was omitted from basal medium, especially for members of aspartate family, except lysine. The organic acids increased or restored the growth rates to the respective reference values. In each medium deficient in one essential amino acid, the L-malic acid utilization was accompanied by an increase of L-lactic acid concentration and accounted for approximately 100%l-malic acid consumed. D-lactic acid formation from glucose decreased in the medium without cysteine. Except for tyrosine, the recovery of glucose-citrate as D-lactic acid was lower than in the complete medium when asparagine, isoleucine or cysteine were excluded. The ethanol and acetate production was not modified. CONCLUSIONS: L-malic and citric acids favoured Oenococcus oeni m growth in nutritional stress conditions. Specifically citric acid was involved in the biosynthesis of the aspartate-derived essential amino acids and glucose in the cysteine biosynthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Such beneficial effect of l-malic and citric acids on amino acids requirements of Oenococcus oeni m have great significance considering the low amino acids concentration in wine.     

Lactobacillus casei metabolic potential to utilize citrate as an energy source in ripening cheese: a bioinformatics approach

AIMS: To identify potential pathways for citrate catabolism by Lactobacillus casei under conditions similar to ripening cheese. METHODS AND RESULTS: A putative citric acid cycle (PCAC) for Lact. casei was generated utilizing the genome sequence, and metabolic flux analyses. Although it was possible to construct a unique PCAC for Lact. casei, its full functionality was unknown. Therefore, the Lact. casei PCAC was evaluated utilizing end-product analyses of citric acid catabolism during growth in modified chemically defined media (mCDM), and Cheddar cheese extract (CCE). Results suggest that under energy source excess and limitation in mCDM this micro-organism produces mainly L-lactic acid and acetic acid, respectively. Both organic acids were produced in CCE. Additional end products include D-lactic acid, acetoin, formic acid, ethanol, and diacetyl. Production of succinic acid, malic acid, and butanendiol was not observed. CONCLUSIONS: Under conditions similar to those present in ripening cheese, citric acid is converted to acetic acid, L/D-lactic acid, acetoin, diacetyl, ethanol, and formic acid. The PCAC suggests that conversion of the citric acid-derived pyruvic acid into acetic acid, instead of lactic acid, may yield two ATPs per molecule of citric acid. Functionality of the PCAC reductive route was not observed. SIGNIFICANCE AND IMPACT OF THE STUDY: This research describes a unique PCAC for Lact. casei. Additionally, it describes the citric acid catabolism end product by this nonstarter lactic acid bacteria during growth, and under conditions similar to those present in ripening cheese. It provides insights on pathways preferably utilized to derive energy in the presence of limiting carbohydrates by this micro-organism.     

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.     

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.     

Preliminary characterization of lactic acid bacteria isolated from Zlatar cheese

AIMS: Isolation, characterization and identification of lactic acid bacteria (LAB) from artisanal Zlatar cheese during the ripening process and selection of strains with good technological characteristics. METHODS AND RESULTS: Characterization of LAB was performed based on morphological, physiological and biochemical assays, as well as, by determining proteolytic activity and plasmid profile. rep-polymerase chain reaction (PCR) analysis and 16S rDNA sequencing were used for the identification of LAB. PCR analysis was performed with specific primers for detection of the gene encoding nisin production. Strains Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Enterococcus faecium and Enterococcus faecalis were the main groups present in the Zlatar cheese during ripening. CONCLUSIONS: Temporal changes in the species were observed during the Zlatar cheese ripening. Mesophilic lactobacilli are predominant microflora in Zlatar cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study we determined that Zlatar cheese up to 30 days old could be used as a source of strains for the preparation of potential starter cultures in the process of industrial cheese production. As the Serbian food market is adjusting to European Union regulations, the standardization of Zlatar cheese production by using starter culture(s) based on autochtonous well-characterized LAB will enable the industrial production of this popular cheese in the future.     

The enantioselective participation of (S)- and (R)-diaminovaleric acids in the formation of delta-aminolevulinic acid in cyanobacteria.

Although it is recognized that 4,5-diaminovaleric acid, formed from glutamate 1-semialdehyde, functions as the intermediate in the last step of delta-aminolevulinic acid formation from glutamate, the enantioselectivity of the participating glutamate 1-semialdehyde aminotransferase for 4,5-diaminovaleric acid has remained unknown. In the present work the involvement of (S)- and (R)-4,5-diaminovaleric acids, newly available by organic synthesis, was investigated, using glutamate 1-semialdehyde aminotransferase from Synechococcus. The preferred enantiomer was (S)-4,5-diaminovalerate. In experiments on the transformation of (S)-4,5-diaminovalerate to delta-aminolevulinate it was found that glutamate 1-semialdehyde aminotransferase was unusual among aminotransferases in that the common amino acceptors pyruvate, oxaloacetate, alpha-ketoglutarate were inactive, while 4,5-dioxovaleric acid could be utilized as a sluggish amino acceptor in place of glutamate 1-semialdehyde. In conclusion, glutamate 1-semialdehyde aminotransferase is highly but not absolutely enantioselective for (S)-4,5-diaminovaleric acid, and 4,5-dioxovaleric acid can function as amino acceptor not because of a physiological role in the C5 pathway of delta-aminolevulinic acid formation, but because of its structural resemblance to glutamate 1-semialdehyde.     

Diversity of amino acid converting enzymes in wild lactic acid bacteria

A total of 156 lactic acid bacteria isolates belonging to the genera Lactococcus, Lactobacillus and Leuconostoc were analysed for the amino acid converting enzymes aminotransferases, glutamate dehydrogenase, and α-ketoisovalerate decarboxylase. All isolates showed aminotransferase activity towards phenylalanine (substrate for the aromatic aminotransferase AraT) and isoleucine (substrate for the branched-chain aminotransferase BcaT). Although there was a high variability inter- and intra-species, the lactococcal strains showed the highest values for both aminotransferase activities. Moreover, α-ketoisovalerate decarboxylase (Kivd) activity was only found in lactococcal isolates, although at low relative numbers (16%). On the other hand, glutamate dehydrogenase (Gdh) activity values were highest in facultative heterofermentative lactobacilli (FHL) and the activity was found at high relative numbers (50%) in leuconostocs. Results showed a high variability in amino acid convertase activities within the wild LAB isolates assayed, therefore the utilisation in the dairy industry of new strains with high flavour-forming abilities could be a powerful tool to enhance cheese aroma development.     

A lacticin 481-producing adjunct culture increases starter lysis while inhibiting nonstarter lactic acid bacteria proliferation during Cheddar cheese ripening

AIMS: The main aim of this study was to exploit a lacticin 481 producing strain, Lactococcus lactis CNRZ481, as an adjunct for Cheddar cheese manufacture, to increase starter cell lysis and control nonstarter lactic acid bacteria (NSLAB) proliferation in cheese. METHODS AND RESULTS: Lactococcus lactis CNRZ481 was exploited as an adjunct to L. lactis HP for the manufacture of Cheddar cheese at pilot scale (450 l). In these trials, inclusion of the adjunct strain did not compromise acid production by L. lactis HP and cheese was successfully manufactured within 5 h. Experimental cheese exhibited levels of lactate dehydrogenase (LDH) up to five-fold higher than control cheese and a significant reduction in NSLAB growth was also observed throughout the ripening period. CONCLUSIONS: The aims of the study were accomplished as (i) greater enzyme release was achieved through lacticin 481-induced lysis which was associated with an improved flavoured cheese as assessed by a commercial grader and (ii) NSLAB growth was controlled, thus reducing the risk of off-flavour development. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of lacticin 481-producing adjuncts for cheese manufacture may prove beneficial for manufacturers who aim to achieve faster ripening through premature and elevated intracellular enzyme release while minimizing inconsistencies in cheese quality because of NSLAB activity.     

Molecular analysis of the role of two aromatic aminotransferases and a broad-specificity aspartate aminotransferase in the aromatic amino acid metabolism of Pyrococcus furiosus

The genes encoding aromatic aminotransferase II (AroAT II) and aspartate aminotransferase (AspAT) from Pyrococcus furiosus have been identified, expressed in Escherichia coli and the recombinant proteins characterized. The AroAT II enzyme was specific for the transamination reaction of the aromatic amino acids, and uses a-ketoglutarate as the amino acceptor. Like the previously characterized AroAT I, AroAT II has highest efficiency for phenylalanine (k(cat)/Km = 923 s(-1) mM(-1)). Northern blot analyses revealed that AroAT I was mainly expressed when tryptone was the primary carbon and energy source. Although the expression was significantly lower, a similar trend was observed for AroAT II. These observations suggest that both AroATs are involved in amino acid degradation. Although AspAT exhibited highest activity with aspartate and alpha-ketoglutarate (k(cat) approximately 105 s(-1)), it also showed significant activity with alanine, glutamate and the aromatic amino acids. With aspartate as the amino donor, AspAT catalyzed the amination of alpha-ketoglutarate, pyruvate and phenyl-pyruvate. No activity was detected with either branched-chain amino acids or alpha-keto acids. The AspAT gene (aspC) was expressed as a polycistronic message as part of the aro operon, with expression observed only when the aromatic amino acids were absent from the growth medium, indicating a role in the biosynthesis of the aromatic amino acids.