Abstract
During cheese processing and ripening, bacteria develop as colonies. Substrates and metabolites must then diffuse either from or into the colonies. Exploring how the inner cells of the colony access the substrates or get rid of the products leads to study the diffusion of solutes inside bacterial colonies immobilized in cheese. Diffusion limitations of substrates within the bacterial colony could lead to starvation for the cells in the center of the colony. This study aimed at better understands ripening at the colony level, by investigating how diffusion phenomena inside colonies vary depending on both the physicochemical properties of the solutes and Lactococcus lactis strain. Dextrans (4, 70, and 155 kDa) and milk proteins (BSA, lactoferrin and αS1-casein) of different sizes and physicochemical properties were chosen as model of diffusing solutes, and two L. lactis strains presenting different surface properties were immobilized as colonies in a model cheese. Diffusion of solutes inside and around colonies was experimentally followed by time-lapse confocal microscopy. Dextran solutes diffused inside both lactococci colonies with a non-significantly different effective diffusion coefficient, which depended mainly on size of the solute. However, whereas flexible and neutral hydrophilic polymers such as dextran can diffuse inside colonies whatever its size, none of the three proteins investigated in this study could penetrate inside lactococci colonies. Therefore, the diffusion behavior of macromolecules through bacterial colonies immobilized in a model cheese did not only depends on the size of the diffusing solutes, but also and mainly on their physicochemical properties. Milk caseins are probably first hydrolyzed by the cell wall proteases of L. lactis and/or other proteases present in the cheese, and then the generated peptides diffuse inside colonies to be further metabolized into smaller peptides and amino acids by all the cells located inside the colonies.
Highlights
During cheese making, regardless of the cheese type, bacteria are immobilized in the curd during the coagulation step, and grow as colonies spread within the cheese curd
The percentages of bacterial adhesion to the chloroform, an acidic solvent and electron acceptor, were not significantly different between the two L. lactis strains, with values inferior to 20% (Table 2). These results are in agreement with values of adhesion to chloroform obtained on various L. lactis strains by Ly et al (2006) and Giaouris et al (2009)
It has been previously shown that the expression of the major cell wallanchored protease was responsible for altering L. lactis surface physicochemical properties, shifting the cell envelope from a hydrophilic surface to an extremely hydrophobic one, going along with an increase of negative charges at the cell surface (Habimana et al, 2007)
Summary
Regardless of the cheese type, bacteria are immobilized in the curd during the coagulation step, and grow as colonies spread within the cheese curd. They are a major actor of ripening which gives the cheese its final sensorial properties During ripening, they are responsible for the proteolysis, the milk protein breakdown, leading to peptides and amino acids. It is highly probable that, on one hand, milk proteins have to diffuse from the cheese matrix (a fat-protein network) into the colony to reach the bacterial cells in the center of the colony. Proteolysis end-products (small peptides and amino acids) have to diffuse from the bacterial colonies into the cheese matrix. If diffusion limitations occur inside the bacterial colony, gradients of concentration of both nitrogen sources (low concentrations in the center of the colony) and nitrogen end-products (high concentrations in the center of the colony) may be generated and may affect the metabolic activity of microbial cells, and the kinetics of the ripening process. If lysis occurs at the center of the colony, it is very important to know if bacterial enzymes could diffuse out of the colony to determine how far from the colony proteolytic enzymes could diffuse outside the colony, in the cheese matrix
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