Investigation of the microbiota associated with ungerminated and germinated Norwegian barley cultivars with focus on lactic acid bacteria.

The microbial community of ungerminated and germinated barley grains from three different cultivars grown at four different locations in Norway was investigated by culture dependent and culture independent methods. Lactic acid bacteria (LAB) was focused in this study and was isolated from germinated barley. The number of LAB ranged between 2.8 and 4.6 log cfu/g in ungerminated grains and between 4.9 and 6.3 log cfu/g in germinated grains. In total 66 out of 190 isolates were Gram+, catalase-negative and presumptive LAB. The LAB isolates were by 16S rRNA sequencing identified to be Carnobacterium maltaromaticum (6), Lactococcus lactis (2), Enterococcus sp. (1) and Leuconostoc sp. (57). Germination significantly influenced the bacterial composition. Regarding the different cultivars and growth places no significant difference in bacterial composition was seen. The most abundant bacterial genus was Pantoea (18.5% of the total sequences), followed by Rhizobium (10.1%) and Sphingomonas (9.9%). Fungal composition was significantly influenced by the germination process and the cultivation place, but no significant difference in fungal composition was detected between the 3 cultivars. The most abundant fungal genera were Cryptococcus (43.8% of all the sequences), Cladosporium (8.2%), Pyrenophora (7.4%) and Vagicola (6.3%). This study revealed knowledge of barley grain associated microbes of Norwegian barley that can be useful to control the malt quality. Germination affected both bacterial and fungal microbiota composition. No difference in bacterial microbiota composition was seen regarding cultivars and cultivation place, however, the fungal microbiota composition was significantly influenced by the cultivation place. Differences in fungal community of ungerminated and germinated barley samples of different geographical locations were more pronounced than differences in bacterial communities.

Short communication: High-throughput sequencing approach to investigate Italian artisanal cheese production.

In this study, high-throughput sequencing (HTS) was used to investigate the microbiota of Robiola di Roccaverano production, an artisanal Protected Designation of Origin soft cheese made with raw goat milk by addition of a natural milk starter (NMS), from the Piedmont region of Italy. Different steps of production of Robiola di Roccaverano cheese at one artisanal dairy were monitored. Matched samples of milk, NMS, curd, and 5-d and 15-d matured cheeses were collected at different periods of the year. The DNA sequences obtained by HTS belonged to 5 phyla: Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Tenericutes. In milk, Proteobacteria and Firmicutes were mainly found, and several operational taxonomic units (OTU) belonging to contaminant bacteria such as Pseudomonas, Serratia, and Staphylococcus were observed. However, in NMS, curd, and 5- and 15-d cheeses, Firmicutes were principally observed where OTU of Lactococcus lactis were predominant, followed by Leuconostoc mesenteroides OTU. The results of the analysis showed high bacterial diversity in milk samples compared with NMS, curd, and 5- and 15-d cheeses, suggesting strong action of NMS in driving the characteristics of the final products.

Investigation of genomic characteristics and carbohydrates' metabolic activity of Lactococcus lactis subsp. lactis during ripening of a Swiss-type cheese.

Genetic diversity and metabolic properties of Lactococcus lactis subsp. lactis were explored using phylogenetic, pan-genomic and metatranscriptomic analysis. The genomes, used in the current study, were available and downloaded from the GenBank which were primarily related with microorganisms isolated from dairy products and secondarily from other foodstuffs. To study the genetic diversity of the microorganism, various bioinformatics tools were employed such as average nucleotide identity, digital DNA-DNA hybridization, phylogenetic analysis, clusters of orthologous groups analysis, KEGG orthology analysis and pan-genomic analysis. The results showed that Lc. lactis subsp. lactis strains cannot be sufficiently separated into phylogenetic lineages based on the 16S rRNA gene sequences and core genome-based phylogenetic analysis was more appropriate. Pan-genomic analysis of the strains indicated that the core, accessory and unique genome comprised of 1036, 3146 and 1296 genes, respectively. Considering the results of pan-genomic and KEGG orthology analyses, the metabolic network of Lc. lactis subsp. lactis was rebuild regarding its carbohydrates' metabolic capabilities. Based on the metatranscriptomic data during the ripening of the Swiss-type Maasdam cheese at 20 °C and 5 °C, it was shown that the microorganism performed mixed acid fermentation producing lactate, formate, acetate, ethanol and 2,3-butanediol. Mixed acid fermentation was more pronounced at higher ripening temperatures. At lower ripening temperatures, the genes involved in mixed acid fermentation were repressed while lactate production remained unaffected resembling to a homolactic fermentation. Comparative genomics and metatranscriptomic analysis are powerful tools to gain knowledge on the genomic diversity of the lactic acid bacteria used as starter cultures as well as on the metabolic activities occurring in fermented dairy products.

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.

Genetics of Lactococci.

Lactococcus lactis is the best characterized species among the lactococci, and among the most consumed food-fermenting bacteria worldwide. Thanks to their importance in industrialized food production, lactococci are among the lead bacteria understood for fundamental metabolic pathways that dictate growth and survival properties. Interestingly, lactococci belong to the Streptococcaceae family, which includes food, commensal and virulent species. As basic metabolic pathways (e.g., respiration, metal homeostasis, nucleotide metabolism) are now understood to underlie virulence, processes elucidated in lactococci could be important for understanding pathogen fitness and synergy between bacteria. This chapter highlights major findings in lactococci and related bacteria, and covers five themes: distinguishing features of lactococci, metabolic capacities including the less known respiration metabolism in Streptococcaceae, factors and pathways modulating stress response and fitness, interbacterial dialogue via metabolites, and novel applications in health and biotechnology.

High-throughput screening for texturing Lactococcus strains.

In the food industry, lactic acid bacteria (LAB) are used in dairy fermentations, extending the shelf life by lowering the pH and also affecting taste and texture of the fermented milk. The texture of fermented milk is an important quality parameter, affecting consumer acceptance. Finding LAB providing desired texture of a product is time consuming and laborious when using standard methods for measuring texture, e.g. rheology measurements. Screening of 986 Lactococcus lactis strains resulted in few strains with the ability to enhance texture, demonstrating the necessity of implementation of high-throughput screening methods. A high-throughput screening assay was developed, combining small-scale 96-well microtiter plates and pressure measurements during liquid handling, e.g. aspiration, to find strains that give good texture in fermented milk. Only about 1% of the strains were found to enhance milk texture. Two of the texturing strains belong to L. lactis subsp. lactis, which are the first texturing strains from this subsp. reported. Mining for eps gene clusters responsible for exocellular polysaccharide production was performed, as polysaccharide production can contribute positively to fermented milk texture. Comparative genomics approach revealed four types of texturing L. lactis strains with diverse eps gene clusters.

Composition of lactic acid bacteria during spontaneous curly kale (Brassica oleracea var. sabellica) fermentation.

The present work is the first report on spontaneous fermentation of curly kale and characteristics of autochthonous lactic acid bacteria (LAB). Our results indicate that curly kale fermentation is the new possibility of the technological use of this vegetable. Bacteria representing ten different species were isolated from three phases of curly kale fermentation and identified by MALDI-TOF mass spectrometry and 16S rRNA gene sequencing. Among them, four species were identified as Lactobacillus spp. (Lb. plantarum 332, Lb. paraplantarum G2114, Lb. brevis R413, Lb. curvatus 154), two as Weissella spp. (W. hellenica 152, W. cibaria G44), two as Pediococcus spp. (P. pentosaceus 45AN, P. acidilactici 2211), one as Leuconostoc mesenteroides 153, and one as Lactococcus lactis 37BN. The functional properties of isolates, i.e. acid, NaCl and bile salt tolerance, enzyme activities, adhesion to hydrocarbons, and antibiotic resistance, were examined. Among the tested strains, Lb. plantarum 332, Lb. paraplantarum G2114, P. pentosaceus 2211, and Lb. brevis R413 exhibited the best hydrophobicity value and high tolerance to bile salts, NaCl, and low pH.

Screening of adjunct cultures and their application in ester formation in Camembert-type cheese.

The ethanol content and esterase and alcohol acyltransferase activities are the limiting factors in the synthesis of ethyl esters in Camembert-type cheeses. This study aimed to investigate the effects of alcohol, esterase and alcohol acyltransferase activities on ethyl ester formation in Camembert-type cheeses. Five experimental cheeses were prepared with three adjunct cultures with different enzyme activities and two levels of ethanol content (400 or 800 µg/g). The cheeses were aged for 4 weeks and analysed weekly for basic physicochemical, textural, volatile and sensory properties. The results showed that both the enzyme activity and ethanol content were limiting factors in the synthesis of ethyl esters in the Camembert-type cheeses. Variation in the esterase synthesis activity was observed among lactic acid bacteria, and the starter culture Lactococcus lactis MA 14 LYO distinguished itself through its high acidifying and esterase hydrolysis abilities. The addition of CCFM 12, a lactic acid bacteria strain with high esterase and alcohol acyltransferase activity, along with 400 or 800 µg/g of ethanol, notably enhanced the generation of ethyl esters and the corresponding fruity flavour, without causing dramatic changes in the basic physicochemical indices and microbial profile. In addition, cohesiveness was influenced by the addition of 400 and 800 µg/g of ethanol, and more resilience with 800 µg/g of ethanol had been found. The results showed that the addition of CCFM12 with 400 and 800 µg/g of ethanol may be applied in the production of Camembert cheese to enhance its fruity flavour.

Inhibition of herpes simplex virus 1 (HSV-1) and poliovirus (PV-1) by bacteriocins from lactococcus lactis subsp. lactis and enterococcus durans strains isolated from goat milk.

Bacteriocins have unusual inhibitory activity, including antiviral properties, and this can be exploited to give alternative applications. Semi-purified bacteriocins of six lactic acid bacteria (LAB) strains isolated from goat milk (two Lactococcus lactis: GLc03 and GLc05, and four Enterococcus durans: GEn09, GEn12, GEn14 and GEn17) were tested for cytotoxicity in Vero cells (50% Cytotoxicity Concentration: CC50), and for their antiviral activities against herpes simplex virus 1 (HVS-1) and poliovirus (PV-1). Semi-purified bacteriocins presented low cytotoxicity, with CC50 varying from 256.2 µg/mL (GLc05) to 1084.5 µg/mL (GEn14). CC10 was determined for all isolates (GLc03: 36.9 µg/mL; GLc05: 51.2 µg/mL; GEn09: 88.1 µg/mL; GEn12: 99.9 µg/mL; GEn14: 275 µg/mL; and GEn17: 62.2 µg/mL) and considered for antiviral activity assays. Antiviral activity before virus adsorption was recorded against PV-1 for GLc05 (4.9%), GEn09 (3.4%), GEn12 (24.7%) and GEn17 (23.5%), and against HSV-1 for GEn12 (27.9%), GEn14 (58.7%) and GEn17 (39.2%). Antiviral activity after virus adsorption was identified against PV-1 for GLc05 (32.7%), GEn09 (91.0%), GEn12 (93.7%) and GEn17 (57.2%), and against HSV-1 for GEn17 (71.6%). The results obtained indicate the potential of some bacteriocins, particularly those produced by E. durans strains investigated in the present study, in viral inhibition and their application as new antiviral agents.

Finding the Needle in the Haystack-the Use of Microfluidic Droplet Technology to Identify Vitamin-Secreting Lactic Acid Bacteria.

Efficient screening technologies aim to reduce both the time and the cost required for identifying rare mutants possessing a phenotype of interest in a mutagenized population. In this study, we combined a mild mutagenesis strategy with high-throughput screening based on microfluidic droplet technology to identify Lactococcus lactis variants secreting vitamin B2 (riboflavin). Initially, we used a roseoflavin-resistant mutant of L. lactis strain MG1363, JC017, which secreted low levels of riboflavin. By using fluorescence-activated droplet sorting, several mutants that secreted riboflavin more efficiently than JC017 were readily isolated from the mutagenesis library. The screening was highly efficient, and candidates with as few as 1.6 mutations per million base pairs (Mbp) were isolated. The genetic characterization revealed that riboflavin production was triggered by mutations inhibiting purine biosynthesis, which is surprising since the purine nucleotide GTP is a riboflavin precursor. Purine starvation in the mutants induced overexpression of the riboflavin biosynthesis cluster ribABGH When the purine starvation was relieved by purine supplementation in the growth medium, the outcome was an immediate downregulation of the riboflavin biosynthesis cluster and a reduction in riboflavin production. Finally, by applying the new isolates in milk fermentation, the riboflavin content of milk (0.99 mg/liter) was improved to 2.81 mg/liter, compared with 0.66 mg/liter and 1.51 mg/liter by using the wild-type strain and the original roseoflavin-resistant mutant JC017, respectively. The results obtained demonstrate how powerful classical mutagenesis can be when combined with droplet-based microfluidic screening technology for obtaining microorganisms with useful attributes.IMPORTANCE The food industry prefers to use classical approaches, e.g., random mutagenesis followed by screening, to improve microorganisms used in food production, as the use of recombinant DNA technologies is still not widely accepted. Although modern automated screening platforms are widely accessible, screening remains as a bottleneck in strain development, especially when a mild mutagenesis approach is applied to reduce the chance of accumulating unintended mutations, which may cause unwanted phenotypic changes. Here, we incorporate a droplet-based high-throughput screening method into the strain development process and readily capture L. lactis variants with more efficient vitamin secretion from low-error-rate mutagenesis libraries. This study shows that useful mutants showing strong phenotypes but without extensive mutations can be identified with efficient screening technologies. It is therefore possible to avoid accumulating detrimental mutations while enriching beneficial ones through iterative mutagenesis screening. Due to the low mutation rates, the genetic determinants are also readily identified.

African fermented dairy products - Overview of predominant technologically important microorganisms focusing on African Streptococcus infantarius variants and potential future applications for enhanced food safety and security.

Milk is a major source of nutrients, but can also be a vehicle for zoonotic foodborne diseases, especially when raw milk is consumed. In Africa, poor processing and storage conditions contribute to contamination, outgrowth and transmission of pathogens, which lead to spoilage, reduced food safety and security. Fermentation helps mitigate the impact of poor handling and storage conditions by enhancing shelf life and food safety. Traditionally-fermented sour milk products are culturally accepted and widely distributed in Africa, and rely on product-specific microbiota responsible for aroma, flavor and texture. Knowledge of microbiota and predominant, technologically important microorganisms is critical in developing products with enhanced quality and safety, as well as sustainable interventions for these products, including Africa-specific starter culture development. This narrative review summarizes current knowledge of technologically-important microorganisms of African fermented dairy products (FDP) and raw milk, taking into consideration novel findings and taxonomy when re-analyzing data of 29 publications covering 25 products from 17 African countries. Technologically-important lactic acid bacteria such as Lactococcus lactis and Streptococcus infantarius subsp. infantarius (Sii), Lactobacillus spp. and yeasts predominated in raw milk and FDP across Africa. Re-analysis of data also suggests a much wider distribution of Sii and thus a potentially longer history of use than previously expected. Therefore, evaluating the role and safety of African Sii lineages is important when developing interventions and starter cultures for FDP in Africa to enhance food safety and food security. In-depth functional genomics, epidemiologic investigations and latest identification approaches coupled with stakeholder involvement will be required to evaluate the possibility of African Sii lineages as novel food-grade Streptococcus lineage.

Comparative and functional genomics of the Lactococcus lactis taxon; insights into evolution and niche adaptation.

BACKGROUND: Lactococcus lactis is among the most widely studied lactic acid bacterial species due to its long history of safe use and economic importance to the dairy industry, where it is exploited as a starter culture in cheese production. RESULTS: In the current study, we report on the complete sequencing of 16 L. lactis subsp. lactis and L. lactis subsp. cremoris genomes. The chromosomal features of these 16 L. lactis strains in conjunction with 14 completely sequenced, publicly available lactococcal chromosomes were assessed with particular emphasis on discerning the L. lactis subspecies division, evolution and niche adaptation. The deduced pan-genome of L. lactis was found to be closed, indicating that the representative data sets employed for this analysis are sufficient to fully describe the genetic diversity of the taxon. CONCLUSIONS: Niche adaptation appears to play a significant role in governing the genetic content of each L. lactis subspecies, while (differential) genome decay and redundancy in the dairy niche is also highlighted.

Mesophilic Lactic Acid Bacteria Diversity Encountered in Brazilian Farms Producing Milk with Particular Interest in Lactococcus lactis Strains.

The milk produced in regions with different traditions in Brazil is used for artisanal product production, which is characterized by different sensorial characteristics. This study aimed to identify the bacterial ecosystem of farms located in a traditional dairy region in the state of Minas Gerais and to characterize Lactococcus lactis strains, the species of interest in this study, using a multilocus sequence typing (MLST) protocol and pulsed-field gel electrophoresis (PFGE) technique. Samples were collected from raw milk and dairy environment from six farms. A total of 50 isolates were analyzed using 16S rRNA sequencing and species-specific PCR. Five genera were identified: Lactobacillus, Leuconostoc, Lactococcus, Enterococcus, and Staphylococcus, from ten different species. MLST (with six housekeeping genes) and PFGE (with SmaI endonuclease) were used for the characterization of 20 isolates of Lactococcus lactis from a dairy collection in this study. Both methods revealed a high clonal diversity of strains with a higher discriminatory level for PFGE (15 pulsotypes), compared to MLST (12 ST). This study contributes to the preservation of the Brazilian dairy heritage and provides insights into a part of the LAB population found in raw milk and dairy environment.

Comparative genome-based identification of a cell wall-anchored protein from Lactobacillus plantarum increases adhesion of Lactococcus lactis to human epithelial cells.

Adhesion to host cells is considered important for Lactobacillus plantarum as well as other lactic acid bacteria (LAB) to persist in human gut and thus exert probiotic effects. Here, we sequenced the genome of Lt. plantarum strain NL42 originating from a traditional Chinese dairy product, performed comparative genomic analysis and characterized a novel adhesion factor. The genome of NL42 was highly divergent from its closest neighbors, especially in six large genomic regions. NL42 harbors a total of 42 genes encoding adhesion-associated proteins; among them, cwaA encodes a protein containing multiple domains, including five cell wall surface anchor repeat domains and an LPxTG-like cell wall anchor motif. Expression of cwaA in Lactococcus lactis significantly increased its autoaggregation and hydrophobicity, and conferred the new ability to adhere to human colonic epithelial HT-29 cells by targeting cellular surface proteins, and not carbohydrate moieties, for CwaA adhesion. In addition, the recombinant Lc. lactis inhibited adhesion of Staphylococcus aureus and Escherichia coli to HT-29 cells, mainly by exclusion. We conclude that CwaA is a novel adhesion factor in Lt. plantarum and a potential candidate for improving the adhesion ability of probiotics or other bacteria of interest.

Interactions between Cooccurring Lactic Acid Bacteria in Honey Bee Hives.

In contrast to the honey bee gut, which is colonized by a few characteristic bacterial clades, the hive of the honey bee is home to a diverse array of microbes, including many lactic acid bacteria (LAB). In this study, we used culture, combined with sequencing, to sample the LAB communities found across hive environments. Specifically, we sought to use network analysis to identify microbial hubs sharing nearly identical operational taxonomic units, evidence which may indicate cooccurrence of bacteria between environments. In the process, we identified interactions between noncore bacterial members (Fructobacillus and Lactobacillaceae) and honey bee-specific "core" members. Both Fructobacillus and Lactobacillaceae colonize brood cells, bee bread, and nectar and may serve the role of pioneering species, establishing an environment conducive to the inoculation by honey bee core bacteria. Coculture assays showed that these noncore bacterial members promote the growth of honey bee-specific bacterial species. Specifically, Fructobacillus by-products in spent medium supported the growth of the Firm-5 honey bee-specific clade in vitro. Metabolic characterization of Fructobacillus using carbohydrate utilization assays revealed that this strain is capable of utilizing the simple sugars fructose and glucose, as well as the complex plant carbohydrate lignin. We tested Fructobacillus for antibiotic sensitivity and found that this bacterium, which may be important for establishment of the microbiome, is sensitive to the commonly used antibiotic tetracycline. Our results point to the possible significance of "noncore" and environmental microbial community members in the modulation of honey bee microbiome dynamics and suggest that tetracycline use by beekeepers should be limited.

Evaluation of Lactococcus lactis Isolates from Nondairy Sources with Potential Dairy Applications Reveals Extensive Phenotype-Genotype Disparity and Implications for a Revised Species.

Lactococcus lactis is predominantly associated with dairy fermentations, but evidence suggests that the domesticated organism originated from a plant niche. L. lactis possesses an unusual taxonomic structure whereby strain phenotypes and genotypes often do not correlate, which in turn has led to confusion in L. lactis classification. A bank of L. lactis strains was isolated from various nondairy niches (grass, vegetables, and bovine rumen) and was further characterized on the basis of key technological traits, including growth in milk and key enzyme activities. Phenotypic analysis revealed all strains from nondairy sources to possess an L. lactis subsp. lactis phenotype (lactis phenotype); however, seven of these strains possessed an L. lactis subsp. cremoris genotype (cremoris genotype), determined by two separate PCR assays. Multilocus sequence typing (MLST) showed that strains with lactis and cremoris genotypes clustered together regardless of habitat, but it highlighted the increased diversity that exists among "wild" strains. Calculation of average nucleotide identity (ANI) and tetranucleotide frequency correlation coefficients (TETRA), using the JSpecies software tool, revealed that L. lactis subsp. cremoris and L. lactis subsp. lactis differ in ANI values by ∼14%, below the threshold set for species circumscription. Further analysis of strain TIFN3 and strains from nonindustrial backgrounds revealed TETRA values of <0.99 in addition to ANI values of <95%, implicating that these two groups are separate species. These findings suggest the requirement for a revision of L. lactis taxonomy.

From field to fermentation: the origins of Lactococcus lactis and its domestication to the dairy environment.

Lactococcus lactis is an organism of substantial economic importance, used extensively in the production of fermented foods and widely held to have evolved from plant strains. The domestication of this organism to the milk environment is associated with genome reduction and gene decay, and the acquisition of specific genes involved in protein and lactose utilisation by horizontal gene transfer. In recent years, numerous studies have focused on uncovering the physiology and molecular biology of lactococcal strains from the wider environment for exploitation in the dairy industry. This in turn has facilitated comparative genome analysis of lactococci from different environments and provided insight into the natural phenotypic and genetic diversity of L. lactis. This diversity may be exploited in dairy fermentations to develop products with improved quality and sensory attributes. In this review, we discuss the classification of L. lactis and the problems that arise with phenotype/genotype designation. We also discuss the adaptation of non-dairy lactococci to milk, the traits associated with this adaptation and the potential application of non-dairy lactococci to dairy fermentations.

Purification and characterization of α-acetolactate decarboxylase (ALDC) from newly isolated Lactococcus lactis DX.

BACKGROUND: Diacetyl (2,3-butanedione) is a common flavor aroma from fermented dairy products. There is a need to screen new microorganisms that can efficiently produce large amounts of diacetyl. RESULTS: A new lactic acid bacterium that produced high concentrations of diacetyl was identified based on Gram staining, microscopic examination and 16S rDNA sequence analysis as Lactococcus lactis DX. Its α-acetolactate decarboxylase (ALDC) was purified using 0.45 g mL(-1) ammonium sulfate precipitation, Sephacryl S-300 and S-200 HR and native-PAGE. The purified ALDC displayed a monomer structure and had a molecular mass of about 73.1 kDa, which was estimated using SDS-PAGE. IR analysis showed that the ALDC had a typical protein structure. The optimal temperature and pH for ALDC activity were 40 °C and 6.5 respectively. The ALDC of L. lactis DX was activated by Fe(2+) , Zn(2+) , Mg(2+) , Ba(2+) and Ca(2+) , while Cu(2+) significantly inhibited ALDC activity. Leucine, valine and isoleucine activated the ALDC. CONCLUSION: A strain that had high ability to produce diacetyl was identified as L. lactis DX. The difference in diacetyl production may be due to the ALDC, which is different from other ALDCs.

Short communication: Genotypic and phenotypic identification of environmental streptococci and association of Lactococcus lactis ssp. lactis with intramammary infections among different dairy farms.

Lactococcus species are counted among a large and closely related group of environmental streptococci and streptococci-like bacteria that include bovine mastitis pathogenic Streptococcus, Enterococcus, and Aerococcus species. Phenotypic and biochemical identification methods can be inaccurate and unreliable for species within this group, particularly for Lactococcus spp. As a result, the incidence of Lactococcus spp. on the farm may have been historically underreported and consequently little is known about the clinical importance of this genus as a mastitis pathogen. We used molecular genetic identification methods to accurately differentiate 60 environmental streptococci and streptococci-like bacteria isolated from cows with high somatic cell count and chronic intramammary infection (IMI; >2 somatic cell scores above 4) among 5 geographically distinct farms in New York and Minnesota that exhibited an observed increase in IMI. These isolates were phenotypically identified as Streptococcus uberis and Streptococcus spp. Genetic methods identified 42 isolates (70%) as Lactococcus lactis ssp. lactis, including all 10 isolates originally phenotypically identified as Streptococcus uberis. Antibiotic inhibition testing of all Lc. lactis ssp. lactis showed that 7 isolates were resistant to tetracycline. In the present study, a predominance of Lc. lactis ssp. lactis was identified in association with chronic, clinical bovine IMI among all 5 farms and characterized antimicrobial resistance for treatment therapies. Routine use by mastitis testing labs of molecular identification methods for environmental streptococci and streptococci-like bacteria can further define the role and prevalence of Lc. lactis ssp. lactis in association with bovine IMI and may lead to more targeted therapies.

Identification of genomic heterogeneity among Lactococcus lactis strains by plasmid profiling, PFGE and 16S rDNA sequence analysis.

Lactococcus lactis strains are used commonly as starters, which contribute to desirable flavour and texture properties known as strain-specific, in dairy industry. Genomic heterogeneity of 30 L. lactis strains originating from Turkey and characterized phenotypically were investigated in this study. Plasmid profiling, PFGE and 16S rDNA sequence analyses were performed to determine the genetic variability of strains. High degree of heterogeneity was detected among the L. lactis strains. Plasmid profiles of strains showed that compared to the plasmid free control strains, namely; L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris MG1614, all tested strains carried one to ten plasmids with molecular size ranging from 1.5 to 41.5kb. The fingerprints of strains obtained by PFGE from digestion with ApaI, SmaI and I-CeuI restriction endonucleases of chromosomal DNA's were compared with each other. All strains out of four were grouped into a large cluster A with at least 44% similarity level. The other four strains formed a minor duster B, distinctively different from major cluster A. PFGE results were confirmed by 16S rDNA sequence analysis and strains included in cluster B were identified as members of different species. These results suggested that morphologic and biochemical methods should be verified by reliable molecular approaches for the purpose of strain typing. Also, PFGE was found suitable to determine genomic differentiations among inter- and intra species.