The role of microbe-matrix interactions in dairy starter culture functionality

Lactococcus lactis is one of the lactic acid bacteria species used for fermentation of cheese, quark and buttermilk and determines taste, texture, and the shelf life of a product. Surface properties of microorganisms are determined by the molecular composition of the cell wall and influence the interactions of microbes with their environment. This study was focused on a better understanding of molecular mechanisms involved in interactions between L. lactis and the matrix of fermented dairy products. The results suggest that cell surface alteration should allow modifying starter culture functionality and developing new concepts in formulating fermented products with an altered texture. Another example of a new concept for the application of lactic acid bacteria could be clean label stabilization of oil-in-water emulsions with L. lactis strains via the Pickering stabilization mechanism. In this study we have shown that bacterial cell aggregation is of importance in this respect as it improves the stabilization of oil droplets. In yet another example starter cells could be employed as structure elements such as inert fillers or as structure breakers in fermented food. While the knowledge of the molecular mechanisms determining L. lactis cell surface properties and, subsequently, the microbe-matrix interactions are far from complete, this work and future research on the microbe-matrix interactions certainly holds a potential for improving current manufacturing processes and developing novel products

Transcriptional response of Lactococcus lactis during bacterial emulsification

Microbial surface properties are important for interactions with the environment in which cells reside. Surface properties of lactic acid bacteria significantly vary and some strains can form strong emulsions when mixed with a hydrocarbon. Lactococcus lactis NCDO712 forms oil-in-water emulsions upon mixing of a cell suspension with petroleum. In the emulsion the bacteria locate at the oil-water interphase which is consistent with Pickering stabilization. Cells of strain NCDO712 mixed with sunflower seed oil did not stabilize the oil droplets. This study shows that the addition of either ethanol or ammonium sulfate led to cell aggregation, which subsequently allowed stabilizing oil-in-water emulsions. From this, we conclude that bacterial cell aggregation is important for emulsion droplet stabilization. To determine how bacterial emulsification influences the microbial transcriptome RNAseq analysis was performed on lactococci taken from the oil-water interphase. In comparison to cells in suspension 72 genes were significantly differentially expressed with a more than 4-fold difference. The majority of these genes encode proteins involved in transport processes and the metabolism of amino acids, carbohydrates and ions. Especially the proportion of genes belonging to the CodY regulon was high. Our results also point out that in a complex environment such as food fermentations a heterogeneous response of microbes might be caused by microbe-matrix interactions. In addition, microdroplet technologies are increasingly used in research. The understanding of interactions between bacterial cells and oil-water interphases is of importance for conducting and interpreting such experiments

Influence of lactococcal surface properties on cell retention and distribution in cheese curd

During cheese manufacturing, on average 90% of the starter culture cells are believed to be entrapped in the curd, with the remainder lost in whey. This paper shows that plasmid-cured dairy strains of Lactococcus lactis show cell retention in the curd of 30-72%, whereas over-expression of pili on the lactococcal cell surface can increase cell retention to 99%. Exopolysaccharide production and cell clumping and chaining do not influence cell retention in cheese curd. L. lactis surface alteration also strongly affected the distribution of cells in the cheese matrix: clumping and over-expression of pili led to formation of large cell aggregates embedded in the protein matrix whereas exopolysaccharide expression resulted in cells being surrounding by small serum regions in the protein matrix of the cheese. These results suggest that surface properties of dairy starter cultures strongly determine retention and distribution of the bacteria in cheese curd. (C) 2018 The Authors. Published by Elsevier Ltd

Altering textural properties of fermented milk by using surface-engineered Lactococcus lactis

Lactic acid bacteria are widely used for the fermentation of dairy products. While bacterial acidification rates, proteolytic activity and the production of exopolysaccharides are known to influence textural properties of fermented milk products, little is known about the role of the microbial surface on microbe-matrix interactions in dairy products. To investigate how alterations of the bacterial cell surface affect fermented milk properties, 25 isogenic Lactococcus lactis strains that differed with respect to surface charge, hydrophobicity, cell chaining, cell-clumping, attachment to milk proteins, pili expression and EPS production were used to produce fermented milk. We show that overexpression of pili increases surface hydrophobicity of various strains from 3-19% to 94-99%. A profound effect of different cell surface properties was an altered spatial distribution of the cells in the fermented product. Aggregated cells tightly fill the cavities of the protein matrix, while chaining cells seem to be localized randomly. A positive correlation was found between pili overexpression and viscosity and gel hardness of fermented milk. Gel hardness also positively correlated with clumping of cells in the fermented milk. Viscosity of fermented milk was also higher when it was produced with cells with a chaining phenotype or with cells that overexpress exopolysaccharides. Our results show that alteration of cell surface morphology affects textural parameters of fermented milk and cell localization in the product. This is indicative of a cell surface-dependent potential of bacterial cells as structure elements in fermented foods

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

The role of microbe-matrix interactions in dairy starter culture functionality

Lactococcus lactis is one of the lactic acid bacteria species used for fermentation of cheese, quark and buttermilk and determines taste, texture, and the shelf life of a product. Surface properties of microorganisms are determined by the molecular composition of the cell wall and influence the interactions of microbes with their environment. This study was focused on a better understanding of molecular mechanisms involved in interactions between L. lactis and the matrix of fermented dairy products. The results suggest that cell surface alteration should allow modifying starter culture functionality and developing new concepts in formulating fermented products with an altered texture. Another example of a new concept for the application of lactic acid bacteria could be clean label stabilization of oil-in-water emulsions with L. lactis strains via the Pickering stabilization mechanism. In this study we have shown that bacterial cell aggregation is of importance in this respect as it improves the stabilization of oil droplets. In yet another example starter cells could be employed as structure elements such as inert fillers or as structure breakers in fermented food. While the knowledge of the molecular mechanisms determining L. lactis cell surface properties and, subsequently, the microbe-matrix interactions are far from complete, this work and future research on the microbe-matrix interactions certainly holds a potential for improving current manufacturing processes and developing novel products

Influence of lactococcal surface properties on cell retention and distribution in cheese curd

During cheese manufacturing, on average 90% of the starter culture cells are believed to be entrapped in the curd, with the remainder lost in whey. This paper shows that plasmid-cured dairy strains of Lactococcus lactis show cell retention in the curd of 30-72%, whereas over-expression of pili on the lactococcal cell surface can increase cell retention to 99%. Exopolysaccharide production and cell clumping and chaining do not influence cell retention in cheese curd. L. lactis surface alteration also strongly affected the distribution of cells in the cheese matrix: clumping and over-expression of pili led to formation of large cell aggregates embedded in the protein matrix whereas exopolysaccharide expression resulted in cells being surrounding by small serum regions in the protein matrix of the cheese. These results suggest that surface properties of dairy starter cultures strongly determine retention and distribution of the bacteria in cheese curd. (C) 2018 The Authors. Published by Elsevier Ltd

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