Cell Surface Properties of Lactococcus lactis Reveal Milk Protein Binding Specifically Evolved in Dairy Isolates

Surface properties of bacteria are determined by the molecular composition of the cell wall and they are important for interactions of cells with their environment. Well-known examples of bacterial interactions with surfaces are biofilm formation and the fermentation of solid materials like food and feed. Lactococcus lactis is broadly used for the fermentation of cheese and buttermilk and it is primarily isolated from either plant material or the dairy environment. In this study, we characterized surface hydrophobicity, charge, emulsification properties, and the attachment to milk proteins of 55 L. lactis strains in stationary and exponential growth phases. The attachment to milk protein was assessed through a newly developed flow cytometry-based protocol. Besides finding a high degree of biodiversity, phenotype-genotype matching allowed the identification of candidate genes involved in the modification of the cell surface. Overexpression and gene deletion analysis allowed to verify the predictions for three identified proteins that altered surface hydrophobicity and attachment of milk proteins. The data also showed that lactococci isolated from a dairy environment bind higher amounts of milk proteins when compared to plant isolates. It remains to be determined whether the alteration of surface properties also has potential to alter starter culture functionalities

Enterococcus spp. in Brazilian artisanal cheeses:Occurrence and assessment of phenotypic and safety properties of a large set of strains through the use of high throughput tools combined with multivariate statistics

Traditionally fermented food generally has unique sensorial aspects due to its high microbial diversity, being broadly consumed worldwide. Despite this, some Vancomycin-Resistant-Enterococci (VRE) can occur in these products, which raises questions about their safety. Thus, this study tries to assess information about the occurrence of Enterococcus strains in traditional Brazilian cheese samples collected from the five main regions as well as the characterization of their phenotypic (proteolytic, lipolytic and bacteriogenic potential) and safety (hemolysin activity and growth at commercial VRE selecting media) aspects. A total of 1669 lactic acid bacteria were isolated, of which 605 (36.2%) were identified as Enterococcus by 16sRNA sequencing. Out of these, E. faecium (58.7%) was identified as the dominant species, followed by Enterococcus faecalis (31.8%), Enterococcus durans (4.8%), Enterococcus hermanniensis (1.6%) and Enterococcus gilvus (1.6%). All the data were analyzed by multivariate statistics, and the findings highlighted the presence of Enterococcus as a naturally component of indigenous microbiota of Brazilian artisanal cheeses, mostly in the cheeses of Marajó, Coalho, and Manteiga, possibly as a consequence of their heat resistance and the curd cooking step applied during the process in these regions. The majority of the identified Enterococcus (63.1%) did not produce β-hemolysin and were not resistant to vancomycin. Interestingly some of these strains (21.4%) were able to inhibit Listeria monocytogenes ATCC 35152, mostly isolates from Serrano, Colonial, and Minas artisanal (from Cerrado microregion) cheeses. Also, 10.2 and 11.8% of them have shown moderate to high proteolytic and lipolytic activity, respectively. Anti-listerial activity is of most interest as it can contribute to reduce risk of listeriosis due to dairy products consumption, while proteolysis and lipolysis are responsible for some remarkable features (e. g. flavor, taste, and texture) of fermented foods. In summary, this study represents a contribution towards revealing some phenotypic and safety traits of a large set of strains of Enterococcus strains through the use of multivariate statistics combined with high throughput tools

The bacterial and fungal microbiome of the skin of healthy dogs and dogs with atopic dermatitis and the impact of topical antimicrobial therapy, an exploratory study

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Metabolic Conversions by Lactic Acid Bacteria during Plant Protein Fermentations

To secure a sustainable food supply for the rapidly growing global population, great efforts towards a plant-based diet are underway. However, the use of plant proteins comes with several challenges, such as improvement or removal of undesired flavours, and generation of desired texture properties. Fermentation holds large potential to alter these properties, but compared to dairy fermentations, our knowledge on strain properties in different plant-based substrates is still limited. Here, we explored different lactic acid bacteria for their ability to grow, produce flavour compounds, or remove off-flavour compounds from different plant proteins. For this, 151 LAB strains from dairy and non-dairy origins were cultured in plant protein plus coconut oil emulsions supplemented with glucose. Pea, chickpea, mung, fava, and soybean proteins were used in the study and bacterial strains for screening included the genera Streptococcus, Lactococcus, Lactobacillus, and Leuconostoc. Efficient, high throughput, screening on plant proteins was developed and strains were assessed for their ability to (i) acidify and decrease the pH; (ii) express key enzymes involved in the formation of amino acid derived flavours, which included PepN (aminopeptidase N), PepXP (X-prolyl dipeptidyl peptidase), EstA (esterase), BcAT (branched chain aminotransferase), CBL (cystathione beta lyase), and ArAT (aromatic aminotransferase); and (iii) improve the overall aroma profile by generating dairy/cheesy notes and decreasing off flavours. Suitable screening conditions were determined, and highlighted the importance that a sufficient heat treatment must be applied to samples containing plant proteins, prior to fermentation, as an outgrowth of spore forming Bacillus cereus was observed if the material was only pasteurised. Enzyme activities for strains measured in rich broth vs. a buffered protein solution showed little-to-no correlation, which illustrated the importance of screening conditions to obtain predictive enzyme measurements. Aroma formation analysis allowed to identify strains that were able to increase key aromas such as diacetyl, acetoin, 2- and 3-methyl butanol, and 2,3-pentanedione, as well as decrease the off-flavours hexanal, pentanal, and nonanal. Our findings illustrate the importance of strain specific differences in the assessed functionalities and how a methodical approach to screening LAB can be applied to select suitable microorganisms that show promise in fermentation of plant proteins when applied in non-dairy cheese applications

Metabolic Conversions by Lactic Acid Bacteria during Plant Protein Fermentations

To secure a sustainable food supply for the rapidly growing global population, great efforts towards a plant-based diet are underway. However, the use of plant proteins comes with several challenges, such as improvement or removal of undesired flavours, and generation of desired texture properties. Fermentation holds large potential to alter these properties, but compared to dairy fermentations, our knowledge on strain properties in different plant-based substrates is still limited. Here, we explored different lactic acid bacteria for their ability to grow, produce flavour compounds, or remove off-flavour compounds from different plant proteins. For this, 151 LAB strains from dairy and non-dairy origins were cultured in plant protein plus coconut oil emulsions supplemented with glucose. Pea, chickpea, mung, fava, and soybean proteins were used in the study and bacterial strains for screening included the genera Streptococcus, Lactococcus, Lactobacillus, and Leuconostoc. Efficient, high throughput, screening on plant proteins was developed and strains were assessed for their ability to (i) acidify and decrease the pH; (ii) express key enzymes involved in the formation of amino acid derived flavours, which included PepN (aminopeptidase N), PepXP (X-prolyl dipeptidyl peptidase), EstA (esterase), BcAT (branched chain aminotransferase), CBL (cystathione beta lyase), and ArAT (aromatic aminotransferase); and (iii) improve the overall aroma profile by generating dairy/cheesy notes and decreasing off flavours. Suitable screening conditions were determined, and highlighted the importance that a sufficient heat treatment must be applied to samples containing plant proteins, prior to fermentation, as an outgrowth of spore forming Bacillus cereus was observed if the material was only pasteurised. Enzyme activities for strains measured in rich broth vs. a buffered protein solution showed little-to-no correlation, which illustrated the importance of screening conditions to obtain predictive enzyme measurements. Aroma formation analysis allowed to identify strains that were able to increase key aromas such as diacetyl, acetoin, 2-and 3-methyl butanol, and 2,3pentanedione, as well as decrease the off-flavours hexanal, pentanal, and nonanal. Our findings illustrate the importance of strain specific differences in the assessed functionalities and how a methodical approach to screening LAB can be applied to select suitable microorganisms that show promise in fermentation of plant proteins when applied in non-dairy cheese applications

Cell Surface Properties of Lactococcus lactis Reveal Milk Protein Binding Specifically Evolved in Dairy Isolates

Surface properties of bacteria are determined by the molecular composition of the cell wall and they are important for interactions of cells with their environment. Well-known examples of bacterial interactions with surfaces are biofilm formation and the fermentation of solid materials like food and feed. Lactococcus lactis is broadly used for the fermentation of cheese and buttermilk and it is primarily isolated from either plant material or the dairy environment. In this study, we characterized surface hydrophobicity, charge, emulsification properties, and the attachment to milk proteins of 55 L. lactis strains in stationary and exponential growth phases. The attachment to milk protein was assessed through a newly developed flow cytometry-based protocol. Besides finding a high degree of biodiversity, phenotype-genotype matching allowed the identification of candidate genes involved in the modification of the cell surface. Overexpression and gene deletion analysis allowed to verify the predictions for three identified proteins that altered surface hydrophobicity and attachment of milk proteins. The data also showed that lactococci isolated from a dairy environment bind higher amounts of milk proteins when compared to plant isolates. It remains to be determined whether the alteration of surface properties also has potential to alter starter culture functionalities

Lytr, a phage-derived amidase is most effective in induced lysis of Lactococcus lactis compared with other lactococcal amidases and glucosaminidases

In the genome of Lactococcus lactis IL1403 five genes encoding peptidoglycan hydrolases are present: four glucosaminidases (acmA, acmB, acmC and acmD) and an endopeptidase (yjgB). Genes for six prophage lysins have also been identified. The genes acmB, acmC, acmD, yjgB and the lysin lytR of prophage r1t and the lytic gene of the prophage bIL309 were cloned downstream of the nisin-inducible promoter in L. lactis NZ9000. Upon nisin induction, increased lysis of the expressing strain was obtained in all cases. Most lysis was obtained with AcmC and the two prophage lysins. Moreover, lysis by AcmB, AcmD and YjgB depends on the presence of the autolysin AcmA. The genes lytR, acmD and acmD in an operon-like structure with acmA were expressed in a foodgrade manner in a L. lactis strain that was used in conjunction with a starter to make Gouda-type cheese. Overexpression of lytR or the combination acmA/acmD resulted in increased cellular lysis in the cheese compared to the reference culture. LytR induced lysis was delayed but eventually resulted in increased lysis in trans of the acidifying L. lactis strain 13 M.

The bacterial and fungal microbiome of the skin of healthy dogs and dogs with atopic dermatitis and the impact of topical antimicrobial therapy, an exploratory study

Canine atopic dermatitis is a genetically predisposed inflammatory and pruritic allergic skin disease that is often complicated by (secondary) bacterial and fungal (yeast) infections. High-throughput DNA sequencing was used to characterize the composition of the microbiome (bacteria and fungi) inhabiting specific sites of skin in healthy dogs and dogs with atopic dermatitis (AD) before and after topical antimicrobial treatment. Skin microbiome samples were collected from six healthy control dogs and three dogs spontaneously affected by AD by swabbing at (non-) predilection sites before, during and after treatment. Bacteria and fungi were profiled by Illumina sequencing of the 16S ribosomal RNA gene of bacteria (16S) and the internally transcribed spacer of the ribosomal gene cassette in fungi (ITS). The total cohort of dogs showed a high diversity of microbes on skin with a strong individual variability of both 16S and ITS profiles. The genera of Staphylococcus and Porphyromonas were dominantly present both on atopic and healthy skin and across all skin sites studied. In addition, bacterial and fungal alpha diversity were similar at the different skin sites. The topical antimicrobial treatment increased the diversity of bacterial and fungal compositions in course of time on both AD and healthy skin

The bacterial and fungal microbiome of the skin of healthy dogs and dogs with atopic dermatitis and the impact of topical antimicrobial therapy, an exploratory study

Canine atopic dermatitis is a genetically predisposed inflammatory and pruritic allergic skin disease that is often complicated by (secondary) bacterial and fungal (yeast) infections. High-throughput DNA sequencing was used to characterize the composition of the microbiome (bacteria and fungi) inhabiting specific sites of skin in healthy dogs and dogs with atopic dermatitis (AD) before and after topical antimicrobial treatment. Skin microbiome samples were collected from six healthy control dogs and three dogs spontaneously affected by AD by swabbing at (non-) predilection sites before, during and after treatment. Bacteria and fungi were profiled by Illumina sequencing of the 16S ribosomal RNA gene of bacteria (16S) and the internally transcribed spacer of the ribosomal gene cassette in fungi (ITS). The total cohort of dogs showed a high diversity of microbes on skin with a strong individual variability of both 16S and ITS profiles. The genera of Staphylococcus and Porphyromonas were dominantly present both on atopic and healthy skin and across all skin sites studied. In addition, bacterial and fungal alpha diversity were similar at the different skin sites. The topical antimicrobial treatment increased the diversity of bacterial and fungal compositions in course of time on both AD and healthy skin