Diacetylactis Strains Research Articles

Precise Populations’ Description in Dairy Ecosystems Using Digital Droplet PCR: The Case of L. lactis Group in Starters

Lactococcus lactis group (composed of the lactis and cremoris subspecies, recently reassigned as two distinct species) plays a major role in dairy fermentations. Usually present in starter cultures, the two species enable efficient acidification and improve the organoleptic qualities of the final product. Biovar diacetylactis strains produce diacetyl and acetoin, aromas from the citrate metabolization. As these populations have distinct genomic and phenotypic characteristics, the proportions of each other will affect the final product. Today, there is no quantitative test able to distinguish between the two species and the biovar in dairy ecosystems. In this study, we developed a specific, reliable, and accurate strategy to quantify these populations using, species-, and diacetylactis-specific fluorescent probes in digital droplet PCR assays (ddPCR). Species were distinguished based on three single nucleotide polymorphisms in the glutamate decarboxylase gadB gene, and the citD gene involved in citrate metabolism was used to target the biovar. Used in duplex or singleplex, these probes made it possible to measure the proportion of each population. At 59°C, the probes showed target specificity and responded negatively to the non-target species usually found in dairy environments. Depending on the probe, limit of detection values in milk matrix ranged from 3.6 × 103 to 1.8 × 104 copies/ml. The test was applied to quantify sub-populations in the L. lactis group during milk fermentation with a commercial starter. The effect of temperature and pH on the balance of the different populations was pointed out. At the initial state, lactis and cremoris species represent, respectively, 75% and 28% of the total L. lactis group and biovar diacetylactis strains represent 21% of the lactis species strains. These ratios varied as a function of temperature (22°C or 35°C) and acidity (pH 4.5 or 4.3) with cremoris species promoted at 22°C and pH4.5 compared to at 35°C. The biovar diacetylactis strains were less sensitive to acid stress at 35°C. This methodology proved to be useful for quantifying lactis and cremoris species and biovar diacetylactis, and could complete 16S metagenomics studies for the deeply description of L. lactis group in complex ecosystems.

The ability of Lactococcus lactis subsp. lactis bv. diacetylactis strains in producing nisin.

Lactococcus lactis subsp. lactis bv. diacetylactis is a relevant microorganism for the dairy industry because of its role in the production of aromatic compounds. Despite this technological property, the identification of bacteriocinogenic potential of obtained strains can offer the additional positive aspect of biosafety. A panel of 15 L. lactis subsp. lactis bv. diacetylactis strains was characterised for the presence and expression of bacteriocin related genes, and further investigated regarding the nisin operon. Eight strains were positive only for nisA, and one strain (SBR4) presented a full nisin operon, with sequencing that was shown to be similar to nisin Z. Only SBR4 presented inhibitory activity against 16 microbial target strains. The growth curves of selected targets strains confirmed the inhibitory activity of SBR4 and consequently the nisin production. This research has demonstrated the inhibitory potential of L. lactis subsp. lactis bv. diacetylactis strain, SBR4, due to its ability to produce nisin Z. This biopreservative potential, associated to previously characterised technological properties, allow the indication of this strain as a promising candidate to be used by the dairy industry as a starter or adjunct culture.

Technological properties of Lactococcus lactis subsp. lactis bv. diacetylactis obtained from dairy and non-dairy niches.

Lactococcus lactis subsp. lactis bv. diacetylactis strains are often used as starter cultures by the dairy industry due to their production of acetoin and diacetyl, important substances that add buttery flavor notes in dairy products. Twenty-three L. lactis subsp. lactis isolates were obtained from dairy products (milk and cheese) and dairy farms (silage), identified at a biovar level, fingerprinted by rep-PCR and characterized for some technological features. Fifteen isolates presented molecular and phenotypical (diacetyl and citrate) characteristics coherent with L. lactis subsp. lactis bv. diacetylactis and rep-PCR allowed the identification of 12 distinct profiles (minimum similarity of 90%). Based on technological features, only two isolates were not able to coagulate skim milk and 10 were able to produce proteases. All isolates were able to acidify skim milk: two isolates, in special, presented high acidifying ability due to their ability in reducing more than two pH units after 24 h. All isolates were also able to grow at different NaCl concentrations (0 to 10%, w/v), and isolates obtained from peanut and grass silages presented the highest NaCl tolerance (10%, w/v). These results indicate that the L. lactis subsp. lactis bv. diacetylactis isolates presented interesting technological features for potential application in fermented foods production. Despite presenting promising technological features, the isolates must be assessed according to their safety before being considered as starter cultures.

Molecular, physiological and phylogenetic traits of Lactococcus 936-type phages from distinct dairy environments

Bacteriophage infection of Lactococcus species can cause serious disruption of dairy fermentation processes. The most common isolates from the dairy environment are Siphoviridae lytic 936-type phages. To gain specific knowledge about this group of phages in Polish dairies, we examined 90 isolates from 8 different locations. Based on restriction fragment length polymorphism analysis, coupled with physiological and molecular studies, the isolated phages were divided into 8 distinct groups. Whole-genome sequencing of single representatives from each phage group provided data about their biology and genetic composition. The phages present an overall conserved genome organization. High sequence homology to another Polish isolate, Lactococcus phage bIBB29, indicates their close phylogenetic relatedness to this strain. Such similarity may be suggestive of a general genome conservation among phages persisting in Polish dairies. Comparative genome analyses with other 936-type phages revealed several discriminative traits, including the presence and position of HNH endonuclease genes, varying number of orfs in the early gene region, and a putative TpeX gene. Interestingly, host range of the sequenced phages was restricted to L. lactis subsp. lactis biovar. diacetylactis strains. The results provide new data regarding phages present in the Polish dairy environment and permit analysis of their biology, genome composition and relatedness to other Lactococcus 936-type phages.

Genetic and phenotypic features defining industrial relevant Lactococcus lactis, L. cremoris and L. lactis biovar. diacetylactis strains

Lactococcus lactis strains constitute one of the most important starter cultures for cheese production. In this study, a genome-wide analysis was performed including 68 available genomes of L. lactis group strains showing the existence of two species (L. lactis and L. cremoris) and two biovars (L. lactis biovar. diacetylactis and L. cremoris biovar. lactis). The proposed classification scheme revealed coherency among phenotypic (through in silico and in vivo bacterial function profiling), phylogenomic (through maximum likelihood trees) and genomic (using overall genome sequence-based parameters) approaches. Strain biodiversity for the industrial biovar. diacetylactis was also analyzed, finding they are formed by at least three variants with the CC1 clonal complex as the only one distributed worldwide. These findings and methodologies will help improve the selection of L. lactis group strains for industrial use as well as facilitate the interpretation of previous or future research studies on this diverse group of bacteria.

Draft Genome Sequence of Lactococcus lactis subsp. lactis bv. diacetylactis CRL264, a Citrate-Fermenting Strain.

We report the draft genome sequence of Lactococcus lactis subsp. lactis bv. diacetylactis CRL264, a natural strain isolated from artisanal cheese from northwest Argentina. L. lactis subsp. lactis bv. diacetylactis is one of the most important microorganisms used as starter culture around the world. The CRL264 strain constitutes a model microorganism in the studies on the generation of aroma compounds (diacetyl, acetoin, and 2,3-butanediol) by lactic acid bacteria. Our genome analysis shows similar genetic organization to other available genomes of L. lactis bv. diacetylactis strains.

Draft Genome Sequence of the Moderately Heat-Tolerant Lactococcus lactis subsp. lactis bv. diacetylactis Strain GL2 from Algerian Dromedary Milk.

Lactococcus lactis subsp. lactis bv. diacetylactis GL2 is a moderately thermotolerant lactic acid bacterium isolated from dromedary raw milk. Here, we present the draft genome sequence of this potential new dairy starter strain, which combines thermotolerance and the capacity to metabolize lactose, casein, and citrate.

α-Acetolactate overexpression from glucose in the diacetyl producer Lactococcus lactis ssp. lactis bv. diacetylactis B2103, a natural mutant lacking α-acetolactate decarboxylase

We investigated the effects of the oxygen supply rate on the activity of pyruvate metabolic pathways and their end products, the lactatedehydrogenase (LDH), pyruvateformiatelyase (PFL), pyruvatedehydrogenase (PDH) and acetolactatesynthase (ALS) pathways, in the Lactococcus lactis ssp. lactis bv. diacetylactis strain B2103/74. We found that this culture, apart from inactivated α-acetyldecarboxylase, also possesses a unique natural capacity to overexpress α-acetolactate (AL) up to 25–28 mM. Our search for similar properties among the diacetilicus bv. strains showed that this ability is quite rare. We identified a single additional strain, 7590 from the National Russian Collection of Industrial Microorganisms (NRCIM-7590), which displayed a similar capacity. However, unlike B2103/74, NRCIM-7590 has an active α-acetolactate decarboxylase and therefore can only produce acetoin. AL overexpression took place under conditions of intense aeration (KLa ≥ 90–120 h−1), and the composition of the medium played a decisive role in AL productivity. We found that AL overproduction is determined by a diversion of a portion of pyruvate flow from the LDH to the PDH and ALS pathways. We further found that all additional pyruvate, supplied from LDH, is utilized exclusively by the ALS pathway because of the restricted capacity of the PDH pathway. This shift in pyruvate metabolism in the B2103/74 strain, from LDH to PDH and ALS pathways, is associated with the initiation of an oxidation reaction that reduces oxygen to H2O and sequesters NADH from the LDH pathway in the process. A specific manifestation of this reaction in B2103/74 and NRCIM-7590 cultures, which results in a profound shift of the pyruvate metabolism towards the production of α-acetolactate, is due to the function of a potent oxidative system that shifts 75–80% of NADH flow from LDH to the oxidative pathway, resulting in the regeneration of NAD+. The nature of this oxidative system is not known. Based on our studies, we propose that the structure of the newly discovered oxidative system is similar to a simple transmembrane electron transport chain.

New insights into Lactococcus lactis diacetyl- and acetoin-producing strains isolated from diverse origins

Lactococcus lactis subsp. lactis biovar diacetylactis strains are used in the dairy industry for generating acetoin and notably diacetyl which imparts a high level of buttery flavor notes.A collection of domesticated and environmental strains was screened for the production of diacetyl or acetoin (D/A), and citrate fermentation. Unexpectedly, both domesticated and environmental strains produced D/A. Domesticated strains belonging to the currently named “biovar diacetylactis” metabolized citrate and produced large amounts of D/A during early growth. They harbored the citP plasmid gene encoding citrate permease and a chromosomal region citM-citI-citCDEFXG involved in citrate metabolism. In these strains, citrate consumption was identified as the major determinant of aroma production. Environmental strains, specifically UCMA5716 and A12, produced as much D/A as the CitP+ strains, though at slightly lower rates. UCMA5716 was found to contain the citM-citI-citCDEFXG cluster but not the citP gene. A12 had neither. In these strains, production rate of D/A was linearly correlated with pyruvate synthesis rate. However, the correlation factor was strain-dependent, suggesting different modes of regulation for pyruvate rerouting towards fermentation end-products and flavors.This work highlights the genetic and metabolic differences between environmental and domesticated strains. The introduction of environmental strains into industrial processes could considerably increase the diversity of starters, enhancing the delivery of new technological properties.

Gene-Specific PCR Amplification of Technologically Important Lactococcal Genes

ABSTRACTA set of gene-specific PCR techniques were used to characterize lactococcal cultures from the LBB collection. Initially the species identification of lactococci was confirmed by targeting the glutamate decarboxylase gene (gadB). PCR amplification of the genes for membrane proteinase (prtP) and citrate permease (citP) was used to select strains which grow rapidly in milk and/or ferment citrate. The prtP+ and citP+ phenotype was confirmed using differentiating microbiological media. It was proved that the citP + phenotype was always associated with strains of L. lactis ssp. lactis biovar. diacetylactis—an important aroma forming variety of dairy lactococci. In all diacetylactis strains the citP gene was localized on a 8.2 kbp plasmid. The lactococcal cultures were also tested for the presence of nisin synthesis (nisA/Z) genes. Potentially lysogenic lactococcal cultures which carry in their genome the gene of prophage integrase (int) were detected. From the tested strains nearly one third were int-positive. The thermal induction of prophage activity was demonstrated for the int+ strain L. lactis ssp. lactis LBB.C1/6.

Characterization of bacteriocin from Lactococcus isolated from traditional Algerian dairy products

Mesophilic strains of lactic acid bacteria belonging to the genus Lactococcus were isolated from traditional dairy products and selected for their ability to produce antimicrobial substances such as bacteriocins. Six strains: Lactococcus lactis ssp. lactis (strains Lc l1, Lc l3, Lc l5), Lactococcus lactis ssp. cremoris (strains Lc c4, Lc c6) and Lactococcus lactis ssp. lactis biovar diacetylactis (strain Lc d2) were selected for their antagonistic activities against different groups of microorganisms. The most active strain, Lactococcus lactis ssp. lactis (Lc l5), produced its inhibitory substance (Bac5) in M17 broth presenting an important inhibition mainly against Listeria monocytogenes. It had no effect on its producer strain. These properties suggested that the inhibitory substance could be considered as a bacteriocin-like substance. SDS-PAGE analyses indicated that it was a homogeneous protein of higher molecular weight of 67 kDa. The bacteriocin was stable over a wide range of pH and temperature; it was active after autoclaving and was not affected by storage at −20°C for 6 months. Moreover, it was sensitive to proteolytic enzymes confirming the proteinaceous nature of the inhibitory substance.

Cell surface properties affect colonisation of raw milk by lactic acid bacteria at the microstructure level

Cell localisation in the matrix microstructure of fermented food is important for microbial activity and ripening. To evaluate the importance of physicochemical factors in these phenomena, the physicochemical behaviour of four strains of lactic acid bacteria were investigated after inoculation of raw milk or milk fractions. To discriminate between physicochemical and metabolic properties, media were (i) rapidly acidified to avoid cell division or (ii) bacteria were allowed to ferment milk. To evaluate the impact of milk components, cells were dispersed in protein or fatty globule fractions. From two Lactococcus lactis subsp. lactis biovar. diacetylactis strains selected from their different surface properties investigated in previous studies, after rapid acidification one was encountered in fatty globules and protein gels whereas the other was found in serum and on the surface of globules. After fermentation, the first one formed colonies in the lipid phase or next to it in the whey and the second formed colonies in the protein gel. Strains forming long chains (Lactobacillus bulgaricus and Streptococcus thermophilus) were always found in contact with fatty globules, where they developed. Our results suggest that localisation and subsequent spatial colonisation depend on the physicochemical properties of cell surfaces, on the structure in chains as well as on the metabolic ability to develop on proteins or lipids.

Production of fresh Cheddar cheese curds with controlled postacidification and enhanced flavor

Cheddar cheese in curd form is very popular in eastern Canada. It is retailed immediately after cheese manufacturing and can be maintained at room temperature for 24h to provide better texture and mouthfeel. Subsequently, the cheese curds must be stored at 4°C. The shelf life is generally 3 d. In this study, Cheddar cheese curds were produced by adding a high diacetyl flavor-producing strain (Lactococcus diacetylactis) to a thermophilic-based starter. The objective was to achieve both postacidification stability to increase the shelf life and enhanced flavor. The addition of L. diacetylactis increased processing time but did not affect cheese composition or the evolution of proteolysis and texture. During cheese manufacturing, streptococci became the dominant microflora in all cheeses, whereas populations of Lactococcus cremoris and L. diacetylactis decreased. During cheese storage, viable counts of L. diacetylactis and Streptococcus thermophilus increased but the counts of L. cremoris decreased. During cheese manufacturing and storage, the concentrations of lactic acid and diacetyl increased rapidly in cheeses produced with L. diacetylactis. Citric acid and galactose contents remained high in cheese made without L. diacetylactis. Sensory evaluation indicated that cheeses containing the L. diacetylactis strain were more flavorful and also had less sourness and could be stored at 4°C for up to 7 d.

Activation of the Diacetyl/Acetoin Pathway in Lactococcus lactis subsp. lactis bv. diacetylactis CRL264 by Acidic Growth

Lactococcus lactis subsp. lactis bv. diacetylactis strains are aroma-producing organisms used in starter cultures for the elaboration of dairy products. This species is essentially a fermentative microorganism, which cometabolizes glucose and citrate to yield aroma compounds through the diacetyl/acetoin biosynthetic pathway. Our previous results have shown that under acidic growth Lactococcus bv. diacetylactis CRL264 expresses coordinately the genes responsible for citrate transport and its conversion into pyruvate. In the present work the impact of acidic growth on glucose, citrate, and pyruvate metabolism of Lactococcus bv. diacetylactis CRL264 has been investigated by proteomic analysis. The results indicated that acid growth triggers the conversion of citrate, but not glucose, into alpha-acetolactate via pyruvate. Moreover, they showed that low pH has no influence on levels of lactate dehydrogenase and pyruvate dehydrogenase. Therefore, the influence of external pH on regulation of the diacetyl/acetoin biosynthetic pathway in Lactococcus bv. diacetylactis CRL264 has been analyzed at the transcriptional level. Expression of the als, aldB, aldC, and butBA genes encoding the enzymes involved in conversion of pyruvate into aroma compounds has been investigated by primer extension, reverse transcription-PCR analysis, and transcriptional fusions. The results support that this biosynthetic pathway is induced at the transcriptional level by acidic growth conditions, presumably contributing to lactococcal pH homeostasis by synthesis of neutral compounds and by decreasing levels of pyruvate.

The orotate transporter encoded by oroP from Lactococcus lactis is required for orotate utilization and has utility as a food-grade selectable marker

A new lactococcal plasmid, pDBORO, was isolated from the Lactococcus lactis subsp. lactis biovar diacetylactis strain DB0410. This plasmid is responsible for the sensitivity of DB0410 to the toxic pyrimidine analogue 5-fluoroorotate. The complete nucleotide sequence has been determined and amounts to 16 404 bp. Of 15 ORFs encountered, three were found to be insertion sequence (IS) elements, identified as two IS946 and one IS982. Two ORFs are incomplete due to the insertion of an IS element in their C-terminal region. Homologues for four ORFs were found in the IL1403 sequence: the copB gene, coding for a copper-potassium-transporting ATPase B, and the ysbA, ysbB and ysbC genes. The structural organization of the pDBORO replication region is highly similar to other theta-replicating plasmids in both the cis- (repA) and trans-acting (repB and orfX) sequences. By plasmid deletion analysis and molecular cloning, a single locus on pDBORO was found to confer sensitivity to 5-fluoroorotate. It was identified as ysbC, but renamed oroP in order to reflect its function. The oroP gene was found to be essential for the utilization of orotate as the sole pyrimidine source in a strain deficient in pyrimidine de novo synthesis. The amino acid sequence encoded by the ORF showed the characteristic features of a membrane protein. Therefore, oroP most probably encodes an orotate transporter. Surprisingly, homologues of oroP could be identified in the genomes of both L. lactis MG1363 and L. lactis IL1403 despite the fact that these strains were unable to significantly utilize orotate. Cloning of oroP in Escherichia coli and Bacillus subtilis showed that the orotate transport phenotype could be transformed to both organisms. The findings presented indicate that oroP can be used as a powerful, food-grade selection/counterselection marker in many different organisms.

Metabolic and Transcriptomic Adaptation of Lactococcus lactis subsp. lactis Biovar diacetylactis in Response to Autoacidification and Temperature Downshift in Skim Milk

For the first time, a combined genome-wide transcriptome and metabolic analysis was performed with a dairy Lactococcus lactis subsp. lactis biovar diacetylactis strain under dynamic conditions similar to the conditions encountered during the cheese-making process. A culture was grown in skim milk in an anaerobic environment without pH regulation and with a controlled temperature downshift. Fermentation kinetics, as well as central metabolism enzyme activities, were determined throughout the culture. Based on the enzymatic analysis, a type of glycolytic control was postulated, which was shared by most of the enzymes during the growth phase; in particular, the phosphofructokinase and some enzymes of the phosphoglycerate pathway during the postacidification phase were implicated. These conclusions were reinforced by whole-genome transcriptomic data. First, limited enzyme activities relative to the carbon flux were measured for most of the glycolytic enzymes; second, transcripts and enzyme activities exhibited similar changes during the culture; and third, genes involved in alternative metabolic pathways derived from some glycolytic metabolites were induced just upstream of the postulated glycolytic bottlenecks, as a consequence of accumulation of these metabolites. Other transcriptional responses to autoacidification and a decrease in temperature were induced at the end of the growth phase and were partially maintained during the stationary phase. If specific responses to acid and cold stresses were identified, this exhaustive analysis also enabled induction of unexpected pathways to be shown.

Characteristics of “Tetilla” pasteurized milk cheese manufactured with the addition of autochthonous cultures

Fourteen batches of Tetilla, a short-ripening cows-milk cheese produced in Galicia (NW Spain), were prepared from pasteurized milk inoculated with autochthonous cultures isolated from Tetilla raw milk cheeses. Two batches were made with an acid-aromatic starter containing Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. lactis var. diacetylactis strains. Twelve batches were made with the same lactococcal starter plus one of lactobacilli, leuconostoc, enterococci or micrococci strains. The microbiological, chemical, biochemical and sensory characteristics of the different batches were studied. Cheese batches made with micrococci strains showed greatest diversity, while cheese batches made with lactococci strains were the most homogeneous. Mean counts of aerobic mesophilic bacteria, lactic acid bacteria and citrate-fermented bacteria were similar in all cheese batches. Coliforms were only detected in five batches and Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and Salmonella spp. were not detected. pH, total solids and fat contents agreed with the specifications of the Appellation of Origin of Tetilla cheese. Ash and protein contents were lower than those observed in Tetilla raw milk cheese. The mean degradation of β-casein in the cheese batches made with starter including lactobacilli was lower than in the remainder, while the degradation of α s1-casein was higher. These cheese batches and those manufactured with the addition of Leuconostoc showed the highest diacetyl-acetoin content. The most desirable sensorial characteristics were achieved in the batches elaborated with acid-aromatic starter with the addition of selected enterococci or micrococci strains.

Effect of inoculum composition and low KCl supplementation on the biological and rheological stability of an immobilized-cell system for mixed mesophilic lactic starter production.

Two strains of Lactococcus lactis subsp. lactis (L. lactis KB and KBP) and one of L. lactis subsp. lactis biovar. diacetylactis (L. diacetylactis MD) were immobilized separately in kappa-carrageenan-locust bean gum gel beads. Continuous fermentations were carried out in supplemented whey permeate in a 1-L pH-controlled stirred tank reactor inoculated with a 30% (v/v) bead inoculum and a bead ratio of 55:30:15 for KB, KBP, and MD, respectively. The process demonstrated a high productivity and microbial stability during the 7-week continuous culture. Compared with previous experiments carried out with an inoculum bead ratio of 33:33:33 for KB, KBP, and MD beads, respectively, the modification of the inoculum bead ratio had apparently little effect on free and immobilized, total and specific populations. A dominant behavior of L. diacetylactis MD over the other strains of the mixed culture was observed both with free-cell populations in the effluent and with immobilized-cell populations. Additional experiments were carried out with other strain combinations for continuous inoculation-prefermentation of milk. The data also confirmed the dominance of L. diacetylactis during long-term continuous immobilized-cell fermentations. This dominance may be tentatively explained by the local competition involved in the development of the bead cross-contamination and in citrate utilization by L. diacetylactis strains. The gel beads demonstrated a high rheological stability during the 7-week continuous fermentation even at low KCl supplementation of the broth medium (25 mM KCl).

Characterization of the Lactic Acid Bacteria in Ewe's Milk and Cheese from Northwest Argentina

Indigenous lactic acid bacteria in ewe's milk and artisanal cheese were studied in four samples of fresh raw milk and four 1-month-old cheeses from the provinces of northwest Argentina. Mean growth counts on M17, MRS, and MSE agar media did not show significant differences (P < 0.05) in raw milk and cheeses. Isolates of lactic acid bacteria from milk were identified as Enterococcus (48%), lactococci (14%), leuconostocs (8%), and lactobacilli (30%). All lactococci were identified as Lactococcus lactis (subsp. lactis and subsp. cremoris). Lactobacilli were identified as Lactobacillus plantarum (92%) and Lactobacillus acidophilus (8%). Enterococci (59%) and lactobacilli (41%) were isolated from cheeses. L. plantarum (93%), L. acidophilus (5%), and Lactobacillus casei (2%) were most frequently isolated. L. lactis subsp. lactis biovar diacetylactis strains were considered as fast acid producers. L. lactis subsp. cremoris strains were slow acid producers. L. plantarum and L. casei strains identified from the cheeses showed slow acid production. The majority of the lactobacilli and Lactococcus lactis strains utilized citrate and produced diacetyl and acetoin in milk. Enzyme activities (API-ZYM tests) of lactococci were low, but activities of L. plantarum strains were considerably higher. The predominance of L. plantarum in artisanal cheese is probably important in the ripening of these cheeses due to their physiological and biochemical characteristics.

Diacetyl and alpha-acetolactate overproduction by Lactococcus lactis subsp. lactis biovar diacetylactis mutants that are deficient in alpha-acetolactate decarboxylase and have a low lactate dehydrogenase activity.

Lactococcus lactis subsp. lactis biovar diacetylactis strains are utilized in several industrial processes for producing the flavoring compound diacetyl or its precursor alpha-acetolactate. Using random mutagenesis with nitrosoguanidine, we selected mutants that were deficient in alpha-acetolactate decarboxylase and had low lactate dehydrogenase activity. The mutants produced large amounts of alpha-acetolactate in anaerobic milk cultures but not in aerobic cultures, except when the medium was supplemented with catalase, yeast extract, or hemoglobin.