Controlled Autolysis and Enzyme Release in a Recombinant Lactococcal Strain Expressing the Metalloendopeptidase Enterolysin A

This study concerns the exploitation of the lytic enzyme enterolysin A (EntL), produced by Enterococcus faecalis strain DPC5280, to elicit the controlled autolysis of starter lactococci. EntL, a cell wall metalloendopeptidase secreted by some E. faecalis strains, can kill a wide range of gram-positive bacteria, including lactococci. The controlled expression of entL, which encodes EntL, was achieved using a nisin-inducible expression system in a lactococcal host. Zymographic analysis of EntL activity demonstrated that active enzyme is produced by the recombinant lactococcal host. Indeed, expression of EntL resulted in almost complete autolysis of the host strain 2 h after induction with nisin. Model cheese experiments using a starter strain in addition to the inducible enterolysin-producing strain showed a 27-fold increase in activity with respect to the release of lactate dehydrogenase in the strain overexpressing EntL, demonstrating the potential of EntL production in large-scale cheese production systems. Indeed, the observation that a wide range of lactic bacteria are sensitive to EntL suggests that EntL-induced autolysis has potential applications with a variety of lactic acid bacteria and could be a basis for probiotic delivery systems.     

Proteolytic enzyme activities in Cheddar cheese juice made using lactococcal starters of differing autolytic properties

AIMS: To determine proteolytic enzyme activities released in Cheddar cheese juice manufactured using lactococcal starter strains of differing autolytic properties. METHODS AND RESULTS: The activities of residual chymosin, cell envelope proteinase and a range of intracellular proteolytic enzymes were determined during the first 70 days of ripening when starter lactococci predominate the microbial flora. In general, in cell free extracts (CFE) of the strains, the majority of proteolytic activities was highest for Lactococcus lactis HP, intermediate for L. lactis AM2 and lowest for L. lactis 303. However, in cheese juice, as ripening progressed, released proteolytic activities were highest for the highly autolytic strain L. lactis AM2, intermediate for L. lactis 303 and lowest for L. lactis HP. CONCLUSIONS: These results indicate that strain related differences in autolysis influence proteolytic enzyme activities released into Cheddar cheese during ripening. No correlation was found between proteolytic potential of the starter strains measured in CFE prior to cheese manufacture and levels of activities released in cheese juice. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings further support the importance of autolysis of lactococcal starters in determining the levels of proteolytic activities present in cheese during initial stages of ripening.     

Cheddar cheese cooking temperature induces differential lactococcal cell permeabilization and autolytic responses as detected by flow cytometry: implications for intracellular enzyme accessibility

AIMS: To determine the influence of cheese cooking temperature on autolysis and permeabilization of two lactococcal starter strains in broth and in Cheddar cheese juice during ripening. METHODS AND RESULTS: Flow cytometry (FCM) was used to identify and enumerate intact and permeabilized cells in broth and in Cheddar cheese juice. Levels of intracellular enzyme activities were quantified concurrently. Permeabilized cell numbers increased for both strains in broth following a temperature shift from 32 to 38 degrees C and was accompanied by an increase in the level of accessible intracellular enzyme activities. The relative proportions of intact and permeabilized cell populations, as detected by FCM in cheese juice, changed during 42-day ripening. Permeabilized cell populations increased during ripening for both strains; however, an increase in accessible intracellular enzyme activity was observed only for the highly autolytic strain Lactococcus lactis AM2. CONCLUSIONS: Differences in the autolytic and permeabilization response induced by cooking temperature in two lactococcal strains affects intracellular enzyme accessibility in Cheddar cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the importance of the autolytic and permeabilization properties of lactic acid bacteria starter strains and their impact on cheese ripening.     

Lytic systems in lactic acid bacteria and their bacteriophages

Summary Lytic systems of lactic acid bacteria and their bacteriophages are reviewed with an emphasis on molecular characterization. Details of enzyme biochemistry and the cloning and analysis of lytic genes are presented, with coverage of lactococcal prolate headed bacteriophages, lactococcal isometric bacteriophages, Lactobacillus bacteriophages and lactococcal autolysins. Some comments on the importance of autolysis in cheese ripening are included and the biotechological exploitation of cloned and characterized lytic genes is presented.     

Hydrophilic and hydrophobic peptides produced in cheese by wild Lactococcus lactis strains

AIMS: To study the production of hydrophilic and hydrophobic peptides in cheese by 32 wild Lactococcus lactis strains of different RAPD patterns and to compare them with the peptides produced by lactococcal cells incubated with whole casein. METHOD AND RESULTS: Chromatograms of peptides from cheeses made using each strain as single starter culture were divided into five regions, and strains were classified in three groups by hierarchical cluster analysis of region areas. Thirty out of the 32 wild L. lactis strains produced higher levels of hydrophobic peptides in cheese than on whole casein. CONCLUSIONS: Cheese was a more favourable substrate than whole casein for hydrophobic peptide formation by L. lactis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: New strains of lactococci should be screened for bitterness under cheese conditions, as the formation of hydrophobic peptides may be underestimated in assays with casein as substrate.     

An application in cheddar cheese manufacture for a strain of Lactococcus lactis producing a novel broad-spectrum bacteriocin, lacticin 3147.

Lactococcus lactis DPC3147, a strain isolated from an Irish kefir grain, produces a bacteriocin with a broad spectrum of inhibition. The bacteriocin produced is heat stable, particularly at a low pH, and inhibits nisin-producing (Nip+) lactococci. On the basis of the observation that the nisin structural gene (nisA) does not hybridize to DPC3147 genomic DNA, the bacteriocin produced was considered novel and designated lacticin 3147. The genetic determinants which encode lacticin 3147 are contained on a 63-kb plasmid, which was conjugally mobilized to a commercial cheese starter, L. lactis subsp. cremoris DPC4268. The resultant transconjugant, DPC4275, both produces and is immune to lacticin 3147. The ability of lacticin 3147-producing lactococci to perform as cheddar cheese starters was subsequently investigated in cheesemaking trials. Bacteriocin-producing starters (which included the transconjugant strain DPC4275) produced acid at rates similar to those of commercial strains. The level of lacticin 3147 produced in cheese remained constant over 6 months of ripening and correlated with a significant reduction in the levels of nonstarter lactic acid bacteria. Such results suggest that these starters provide a means of controlling developing microflora in ripened fermented products.     

Use of DNA quantification to measure growth and autolysis of Lactococcus and Propionibacterium spp. in mixed populations

Autolysis is self-degradation of the bacterial cell wall that results in the release of enzymes and DNA. Autolysis of starter bacteria, such as lactococci and propionibacteria, is essential for cheese ripening, but our understanding of this important process is limited. This is mainly because the current tools for measuring autolysis cannot readily be used for analysis of bacteria in mixed populations. We have now addressed this problem by species-specific detection and quantification of free DNA released during autolysis. This was done by use of 16S rRNA gene single-nucleotide extension probes in combination with competitive PCR. We analyzed pure and mixed populations of Lactococcus lactis subsp. lactis and three different species of Propionibacterium. Results showed that L. lactis subsp. lactis INF L2 autolyzed first, followed by Propionibacterium acidipropionici ATCC 4965, Propionibacterium freudenreichii ISU P59, and then Propionibacterium jensenii INF P303. We also investigated the autolytic effect of rennet (commonly used in cheese production). We found that the effect was highly strain specific, with all the strains responding differently. Finally, autolysis of L. lactis subsp. lactis INF L2 and P. freudenreichii ISU P59 was analyzed in a liquid cheese model. Autolysis was detected later in this cheese model system than in broth media. A challenge with DNA, however, is DNA degradation. We addressed this challenge by using a DNA degradation marker. We obtained a good correlation between the degradation of the marker and the target in a model experiment. We conclude that our DNA approach will be a valuable tool for use in future analyses and for understanding autolysis in mixed bacterial populations.     

Inhibition of Listeria monocytogenes by enterocin 4 during the manufacture andripening of Manchego cheese

The inhibitory effect of enterocin 4, a bacteriocin produced by Enterococcus faecalis INIA 4, on Listeria monocytogenes strains Ohio and Scott A during themanufacture and ripening of Manchego cheese was investigated. Raw ewe's milk wasinoculated with ca 10⁵ cfu ml⁻¹ of L.monocytogenes and with 1% of a commercial lactic starter, 1% of an Ent. faecalis INIA 4 culture, or 1% of each culture. Manchego cheeses were manufactured according tousual procedures. Listeria monocytogenes Ohio counts decreased by 3 log units after8 h and by 6 log units after 7 d in cheese made from milk inoculated with Ent. faecalis INIA 4 or with both cultures, whereas no inhibition was recorded after 60 d in cheese made frommilk inoculated with commercial lactic starter. Listeria monocytogenes Scott A wasnot inhibited by enterocin 4 during cheese manufacture, but decreases of 1 log unit after 7 d andof 2 log units after 60 d were achieved in cheese made from milk inoculated with bothcommercial lactic starter and Ent. faecalis INIA 4.     

Antilisterial activity of lactic acid bacteria isolated from rigouta, a traditional Tunisian cheese

AIMS: Screening for lactic acid bacteria (LAB) producing bacteriocins and other antimicrobial compounds is of a great significance for the dairy industry to improve food safety. METHODS AND RESULTS: Six-hundred strains of LAB isolated from 'rigouta', a Tunisian fermented cheese, were tested for antilisterial activity. Eight bacteriocinogenic strains were selected and analysed. Seven of these strains were identified as Lactococcus lactis and produced nisin Z as demonstrated by mass spectrometry analysis of the purified antibacterial compound. Polymerase chain reaction experiments using nisin gene-specific primers confirmed the presence of nisin operon. Plasmid profiles analysis suggests the presence of, at least, three different strains in this group. MMT05, the eighth strain of this antilisterial collection was identified, at molecular level, as Enterococcus faecalis. The purified bacteriocin produced by this strain showed a molecular mass of 10 201.33 +/- 0.85 Da. This new member of class III bacteriocins was termed enterocin MMT05. CONCLUSIONS: Seven lactococcal strains producing nisin Z were selected and could be useful as bio-preservative starter cultures. Additional experiments are needed to evaluate the promising strain MMT05 as bio-preservative as Enterococci could exert detrimental or beneficial role in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: Only a few antibacterial strains isolated from traditional African dairy products were described. The new eight strains described herein contribute to the knowledge of this poorly studied environment and constitute promising strains for fermented food safety.     

A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI

A new food-grade cloning vector for lactic acid bacteria was constructed using the nisin immunity gene nisI as a selection marker. The food-grade plasmid, pLEB590, was constructed entirely of lactococcal DNA: the pSH71 replicon, the nisI gene, and the constitutive promoter P45 for nisI expression. Electroporation into Lactococcus lactis MG1614 with 60 international units (IU) nisin/ml selection yielded approximately 10⁵ transformants/μg DNA. MG1614 carrying pLEB590 was shown to be able to grow in medium containing a maximum of 250 IU nisin/ml. Plasmid pLEB590 was succesfully transformed into an industrial L. lactis cheese starter carrying multiple cryptic plasmids. Suitability for molecular cloning was confirmed by cloning and expressing the proline iminopeptidase gene pepI from Lactobacillus helveticus in L. lactis and Lb. plantarum. These results show that the food-grade expression system reported in this paper has potential for expression of foreign genes in lactic acid bacteria in order to construct improved starter bacteria for food applications. Electronic Publication     

The physiology and biochemistry of the proteolytic system in lactic acid bacteria

Abstract: The inability of lactic acid bacteria to synthesize many of the amino acids required for protein synthesis necessitates the active functioning of a proteolytic system in those environments where protein constitutes the main nitrogen source. Biochemical and genetic analysis of the pathway by which exogenous proteins supply essential amino acids for growth has been one of the most actively investigated aspects of the metabolism of lactic acid bacteria especially in those species which are of importance in the dairy industry, such as the lactococci. Much information has now been accumulated on individual components of the proteolytic pathway in lactococci, namely, the cell envelope proteinase(s), a range of peptidases and the amino acid and peptide transport systems of the cell membrane. Possible models of the proteolytic system in lactococci can be proposed but there are still many unresolved questions concerning the operation of the pathway in vivo. This review will examine current knowledge and outstanding problems regarding the proteolytic system in lactococci and also the extent to which the lactococcal system provides a model for understanding proteolysis in other groups of lactic acid bacteria.     

Controlling Listeria monocytogenes in Cottage cheese through heterologous production of enterocin A by Lactococcus lactis

Aims: Enterocin A is an example of a class IIa bacteriocin with potent anti‐listerial activity. This study was initiated with a view to harnessing this activity, through heterologous production by a lactococcal starter strain, to limit levels of Listeria monocytogenes in a food (Cottage cheese). Methods and Results: Plasmid pEnt02 (containing entA, I, T and D genes under the control of a constitutive promoter) was introduced into a Lactococcus lactis strain capable of fermenting lactose. When this bacteriocin‐producing starter was used in combination with a non‐enterocin A producer, thereby compensating for an associated reduction in acid production, during a Cottage cheese fermentation, a decrease in L. monocytogenes (tagged with lux genes for convenience) levels was evident. Conclusions: Enterocin A, heterologously produced by a food grade lactic acid bacteria (LAB), was therefore shown to have potential for use as a biocontrol agent in food. Significance and Impact of the Study: Many of the most active anti‐listerial compounds identified to date are enterocins. However, because of Enterococcus‐associated concerns, the use of these antimicrobials in a food setting has been curtailed. Although enterocins have been heterologously produced in LAB to overcome this problem, this study represents the first occasion upon which the benefits of such heterologous production have been demonstrated in a food context.     

Recombinant Lactococcus starters as a potential source of additional peptidolytic activity in cheese ripening

AIMS: The aim of this study was to modulate the lactococcal proteolytic system for enhancement of the cheese ripening process. METHODS AND RESULTS: The genes encoding PepN, PepC, PepX and PepI peptidases of a highly proteolytic Lactobacillus helveticus strain were transferred into Lactococcus lactis in a food-grade cloning system. A comparison of the relative peptidase activities from the transformants with those from the untransformed host, determined in the conditions of maturing cheese, showed that an increase in peptidase activity could be achieved by introducing a selected peptidase gene from Lact. helveticus into L. lactis. CONCLUSIONS: Recombinant L. lactis starter strains, carrying a peptidase gene from Lact. helveticus, may have an important contribution to the proteolysis of maturing cheese by producing an additional peptidolytic enzyme activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will be of importance in shortening the ripening period and production of special cheeses (e.g. reduced-fat cheeses) with improved characteristics.     

Use of DNA Quantification To Measure Growth and Autolysis of Lactococcus and Propionibacterium spp. in Mixed Populations

Autolysis is self-degradation of the bacterial cell wall that results in the release of enzymes and DNA. Autolysis of starter bacteria, such as lactococci and propionibacteria, is essential for cheese ripening, but our understanding of this important process is limited. This is mainly because the current tools for measuring autolysis cannot readily be used for analysis of bacteria in mixed populations. We have now addressed this problem by species-specific detection and quantification of free DNA released during autolysis. This was done by use of 16S rRNA gene single-nucleotide extension probes in combination with competitive PCR. We analyzed pure and mixed populations of Lactococcus lactis subsp. lactis and three different species of Propionibacterium. Results showed that L. lactis subsp. lactis INF L2 autolyzed first, followed by Propionibacterium acidipropionici ATCC 4965, Propionibacterium freudenreichii ISU P59, and then Propionibacterium jensenii INF P303. We also investigated the autolytic effect of rennet (commonly used in cheese production). We found that the effect was highly strain specific, with all the strains responding differently. Finally, autolysis of L. lactis subsp. lactis INF L2 and P. freudenreichii ISU P59 was analyzed in a liquid cheese model. Autolysis was detected later in this cheese model system than in broth media. A challenge with DNA, however, is DNA degradation. We addressed this challenge by using a DNA degradation marker. We obtained a good correlation between the degradation of the marker and the target in a model experiment. We conclude that our DNA approach will be a valuable tool for use in future analyses and for understanding autolysis in mixed bacterial populations.     

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.     

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.     

Developing applications for lactococcal bacteriocins

While much of the applied research carried out to date with bacteriocins has concerned nisin, lactococci produce other bacteriocins with economic potential. An example is the two component bacteriocin lacticin 3147, which is active over a wide pH range and has a broad spectrum of activity against Gram-positive bacteria. Since the genetic determinants for lacticin 3147 are encoded on a large self-transmissible plasmid, the bacteriocin genes may be conveniently transferred to different lactococcal starters. The resulting food-grade strains can then be used to make a significant impact on the safety and quality of a variety of fermented foods, through the inhibition of undesirable microflora. The bacteriocin is heat stable so it can also be used as an ingredient in a powdered form such as a spray-dried fermentate. Given the observation that lacticin 3147 is effective at physiological pH, there is also considerable potential for biomedical applications. Field trials have demonstrat ed its efficacy in the prevention of mastitis infections in dairy cows. In contrast to lacticin 3147, the lactococcin bacteriocins A, B and M have a narrow spectrum of activity limited to lactococci. Strains which produce these inhibitors can be exploited in the acceleration of cheese ripening by assisting the premature lysis of starter cultures.     

Biogenic amine-forming microbial communities in cheese

The aim of this study was to screen two cheese starter cultures and cheese-borne microbial communities with the potential to produce biogenic amines in cheese during ripening. Bacteria of the genera Enterococcus and Lactobacillus and coliform bacteria were isolated from Dutch-type semi-hard cheese at the beginning of the ripening period. Statistically significant counts of bacterial isolates were screened for the presence of specific DNA sequences coding for tyrosine decarboxylase (tyrDC) and histidine decarboxylase (hDC) enzymes. The PCR analysis of DNA from 14 Enterococcus and 3 Lactobacillus isolates confirmed the presence of the targetted DNA sequences. Simultaneously, 13 tyrDC- and 3 hDC-positive isolates were grown in decarboxylase screening medium and this was followed by HPLC analysis of the produced tyramine and histamine. Conventional and molecular taxonomic analyses of the above-mentioned isolates identified the following species: Enterococcus durans (7 strains), Enterococcus faecalis (3 strains), Enterococcus faecium (1 strain), Enterococcus casseliflavus (3 strains), Lactobacillus curvatus (1 strain), Lactobacillus lactis (1 strain) and Lactobacillus helveticus (1 strain). All of the above Enterococcus and two of the Lactobacillus strains originated from contaminating microbial communities. The L. helveticus strain, which was tyrosine decarboxylase-positive and exhibited tyramine production, originated from starter culture 1 used for cheese production. Comparison of partial tyrDC sequences of positive Enterococcus isolates revealed 89% sequence similarity, and that of hDC-positive Lactobacillus isolates revealed 99% sequence similarity.     

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 impact of lactic acid bacteria on cheese flavor

Abstract Chesse flavor is a manifestation of complex interactions of volatile and non-volatile flavor-active compounds plus tactual perception. Numerous agents, including lactic acid bacteria, procece the flavou sensations. The effect of lactic acid bacteria is more dominant in cheese varieties with limited growth of secondary flora. This review describes the indirect and direct impacts of lactic acid bacteria in cheese with emphasis on carbohydrate fermentation, changes in oxidation-reduction potential, interactions with non-starter bacteria, autolysis, proteolytic and peptidolytic activities, transport of metabolites and flavor production.