One of the main lactic acid bacterial species found in the kefir grain ecosystem worldwide is Lactobacillus kefiranofaciens, exhibiting strong auto-aggregation capacity and, therefore, being involved ...in the mechanism of grain formation. Its occurrence and dominance in kefir grains of various types of milk and geographical origins have been verified by culture-dependent and independent approaches using multiple growth media and regions of the 16S rRNA gene, respectively, highlighting the importance of their combination for its taxonomic identification. L. kefiranofaciens comprises two subspecies, namely kefiranofaciens and kefirgranum, but only the first one is responsible for the production of kefiran, the water-soluble polysaccharide, which is a basic component of the kefir grain and famous for its technological as well as health-promoting properties. L. kefiranofaciens, although very demanding concerning its growth conditions, can be involved in mechanisms affecting intestinal health, immunomodulation, control of blood lipid levels, hypertension, antimicrobial action, and protection against diabetes and tumors. These valuable bio-functional properties place it among the most exquisite candidates for probiotic use as a starter culture in the production of health-beneficial dairy foods, such as the kefir beverage.
Cheese is characterized by a rich and complex microbiota that plays a vital role during both production and ripening, contributing significantly to the safety, quality, and sensory characteristics of ...the final product. In this context, it is vital to explore the microbiota composition and understand its dynamics and evolution during cheese manufacturing and ripening. Application of high-throughput DNA sequencing technologies have facilitated the more accurate identification of the cheese microbiome, detailed study of its potential functionality, and its contribution to the development of specific organoleptic properties. These technologies include amplicon sequencing, whole-metagenome shotgun sequencing, metatranscriptomics, and, most recently, metabolomics. In recent years, however, the application of multiple meta-omics approaches along with data integration analysis, which was enabled by advanced computational and bioinformatics tools, paved the way to better comprehension of the cheese ripening process, revealing significant associations between the cheese microbiota and metabolites, as well as their impact on cheese flavor and quality.
Staka is a traditional Greek sour cream made mostly from spontaneously fermented sheep milk or a mixture of sheep and goat milk. At the industrial scale, cream separators and starter cultures may ...also be used. Staka is sometimes cooked with flour to absorb most of the fat. In this study, we employed culture-based techniques, amplicon sequencing, and shotgun metagenomics to analyze the Staka microbiome for the first time. The samples were dominated by
or
spp. Most other bacteria were lactic acid bacteria (LAB) from the
and
genera or Gram-negative bacteria from the
,
,
,
-
, and
genera.
,
, or
were the most prevalent genera in the samples, followed by other yeasts and molds like
,
,
,
,
, or
spp. Shotgun metagenomics allowed the species-level identification of
,
,
,
,
,
,
, and
. Binning of assembled shotgun reads followed by recruitment plot analysis of single reads could determine near-complete metagenome assembled genomes (MAGs). Culture-dependent and culture-independent analyses were in overall agreement with some distinct differences. For example, lactococci could not be isolated, presumably because they had entered a viable but not culturable (VBNC) state or because they were dead. Finally, several LAB,
, and
spp. isolates exhibited antimicrobial activities against oral or other pathogenic streptococci, and certain spoilage and pathogenic bacteria establishing their potential role in food bio-protection or new biomedical applications. Our study may pave the way for additional studies concerning artisanal sour creams to better understand the factors affecting their production and the quality.
Feta is the most renowned protected designation of origin (PDO) white brined cheese produced in Greece. The fine organoleptic characteristics and the quality of Feta rely on, among other factors, its ...overall microbial ecosystem. In this study, we employed 16S rDNA and internal transcribed spacer (ITS) amplicon sequencing, as well as shotgun metagenomics, to investigate the microbiome of artisanal homemade and industrial Feta cheese samples from different regions of Greece, which has very rarely been investigated. 16S rDNA data suggested the prevalence of the Lactococcus genus in the homemade samples, while Streptococcus and Lactobacillus genera prevailed in the industrial control samples. Species identification deriving from shotgun metagenomics corroborated these findings, as Lactococcus lactis dominated two homemade samples while Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus were found to be dominating one industrial sample. ITS data revealed a complex diversity of the yeast population among the samples analyzed. Debaryomyces, Kluyveromyces, Cutaneotrichosporon, Pichia, Candida, and Rhodotorula were the major genera identified, which were distributed in a rather arbitrary manner among the different samples. Furthermore, a number of potential metagenome-assembled genomes (MAGs) could be detected among assembled shotgun bins. The overall analysis of the shotgun metagenomics supported the presence of different foodborne pathogens in homemade samples (e.g., Staphylococcus aureus, Listeria monocytogenes, Enterobacter cloacae, and Streptococcus suis), but with low to very low abundances. Concluding, the combination of both amplicon sequencing and shotgun metagenomics allowed us to obtain an in-depth profile of the artisanal homemade Feta cheese microbiome.
In the present study we investigated the incidence of bacteriocins produced by 236 lactic acid bacteria (LAB) food isolates against pathogenic or opportunistic pathogenic oral bacteria. This set of ...LAB contained several strains (≥17%) producing bacteriocins active against food-related bacteria. Interestingly only Streptococcus macedonicus ACA-DC 198 was able to inhibit the growth of Streptococcus oralis, Streptococcus sanguinis and Streptococcus gordonii, while Lactobacillus fermentum ACA-DC 179 and Lactobacillus plantarun ACA-DC 269 produced bacteriocins solely against Streptococcus oralis. Thus, the percentage of strains that were found to produce bacteriocins against oral bacteria was ~1.3%. The rarity of bacteriocins active against oral LAB pathogens produced by food-related LAB was unexpected given their close phylogenetic relationship. Nevertheless, when tested in inhibition assays, the potency of the bacteriocin(s) of S. macedonicus ACA-DC 198 against the three oral streptococci was high. Fourier-transform infrared spectroscopy combined with principal component analysis revealed that exposure of the target cells to the antimicrobial compounds caused major alterations of key cellular constituents. Our findings indicate that bacteriocins produced by food-related LAB against oral LAB may be rare, but deserve further investigation since, when discovered, they can be effective antimicrobials.
The microflora of four batches of traditional Greek Graviera cheese was studied at 5 weeks of ripening, and 200 lactic acid bacteria (LAB) isolates were phenotypically characterized and screened for ...antilisterial bacteriocins. The cheeses were also analyzed for organic acids by high-performance liquid chromatography and for the potential presence of 25 known LAB bacteriocin genes directly in cheese and their microbial consortia by PCR. All batches were safe according to the European Union regulatory criteria for Listeria monocytogenes, Salmonella, enterobacteria, and coagulase-positive staphylococci. The cheese flora was dominated by nonstarter Lactobacillus casei/paracasei (67.5%) and Lactobacillus plantarum (16.3%) strains, whereas few Streptococcus thermophilus (3.8%), Lactococcus lactis subsp. lactis (0.6%), and Leuconostoc (1.9%) organisms were present. Enterococcus faecium (9.4%) and Enterococcus durans (0.6%) were isolated among the dominant LAB from two batches; however, enterococci were present in all batches at 10- to 100-fold lower populations than mesophilic lactobacilli. Sixteen E. faecium isolates produced antilisterial enterocins. In accordance, enterocin B gene was detectable in all cheeses and enterocin P gene was present in one cheese, whereas the consortia of all cheeses contained at least two of the enterocin A, B, P, 31, L50A, and L50B genes. Plantaricin A gene was also amplified from all cheeses. Mean concentrations of lactic, acetic, citric, and propionic acids in the ripened cheeses exceeded 1.5% in total, of which approximately 0.9% was lactate. Thus, organic acid contents constitute an important hurdle factor for inhibiting growth of pathogens in traditional Graviera cheese products, with LAB bacteriocins, mainly enterocins, potentially contributing to increased cheese safety.
Growth of and bacteriocin production by Streptococcus macedonicus ACA-DC 198 were assessed and modeled under conditions simulating Kasseri cheese production. Controlled fermentations were performed ...in milk supplemented with yeast extract at different combinations of temperature (25, 40, and 55°C), constant pH (pHs 5 and 6), and added NaCl (at concentrations of 0, 2, and 4%, wt/vol). The data obtained were used to construct two types of predictive models, namely, a modeling approach based on the gamma concept, as well as a model based on artificial neural networks (ANNs). The latter computational methods were used on 36 control fermentations to quantify the complex relationships between the conditions applied (temperature, pH, and NaCl) and population behavior and to calculate the associated biokinetic parameters, i.e., maximum specific growth and cell count decrease rates and specific bacteriocin production. The functions obtained were able to estimate these biokinetic parameters for four validation fermentation experiments and obtained good agreement between modeled and experimental values. Overall, these experiments show that both methods can be successfully used to unravel complex kinetic patterns within biological data of this kind and to predict population kinetics. Whereas ANNs yield a better correlation between experimental and predicted results, the gamma-concept-based model is more suitable for biological interpretation. Also, while the gamma-concept-based model has not been designed for modeling of other biokinetic parameters than the specific growth rate, ANNs are able to deal with any parameter of relevance, including specific bacteriocin production.
Bacteriocin production by
Enterococcus faecium FAIR-E 198, isolated from Greek Feta cheese, was studied in batch fermentations, under conditions simulating Feta cheese preparation. Maximum enterocin ...activity and growth rate was obtained in de Man–Rogosa–Sharpe (MRS) broth at 37 °C with controlled pH 6.5. The enterocin was produced throughout the growth phase of the microorganism, showing primary metabolite kinetics with a peak activity during the mid-exponential phase. The use of skimmed milk as substrate revealed low enterocin activity. When fermentations were performed in skimmed milk in the presence of rennet, CaCl
2, and a mixed starter culture, no enterocin activity was observed, although the examined strain grew well under the above conditions. Finally, when
E. faecium FAIR-E 198 was applied as adjunct starter in Feta cheese making, no enterocin activity was detected throughout ripening. Results obtained underline the frequently underestimated finding that in vitro production by novel bacteriocinogenic starter or co-cultures is no guarantee for in situ efficiency. It was concluded that the complex food environment thoroughly interferes with bacteriocin production levels.
A total of 129
E. faecium,
E. faecalis and
E. durans strains of food, veterinary and human origin were screened for biochemical properties relevant to their technological performance. Strains ...exhibited low milk acidifying ability and low extracellular proteolytic activity, with food origin and
E. faecalis strains being generally more active. Their peptidase activities were low and mainly specific against glycine-proline- and glutamate-4-nitroanilide, while only food origin and
E. durans strains showed broader substrate specificity. In contrast, their lipolytic activities were relatively higher; food and veterinary origin and
E. faecalis strains were the most lipolytic. The post-electrophoretic detection of esterase activities showed that the esterolytic system of enterococci was rather complex. All species showed strain-to-strain variation in their ability to metabolise citrate and pyruvate, with
E. faecalis strains being generally more active. The main volatile compounds produced in milk were acetaldehyde, ethanol and acetoin; generally,
E. faecalis strains produced the highest concentrations. None of the strains decarboxylated histidine, lysine and ornithine, but the majority produced tyramine from tyrosine, independently of origin and species. In respect of most biochemical properties considered in this study,
E. faecalis strains were generally more active compared to
E. faecium and
E. durans. This was also the case for the isolates of food origin compared to those of veterinary and human origin. Results obtained allow the selection of enterococci strains to be used as adjunct starters in food fermentations. However, a final selection should take into account the potential virulence factors of enterococci.
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