Fine-Tuning of Process Parameters Modulates Specific Metabolic Bacterial Activities and Aroma Compound Production in Semi-Hard Cheese.

The formation of cheese flavor mainly results from the production of volatile compounds by microorganisms. We investigated how fine-tuning cheese-making process parameters changed the cheese volatilome in a semi-hard cheese inoculated with Lactococcus (L.) lactis, Lactiplantibacillus (L.) plantarum, and Propionibacterium (P.) freudenreichii. A standard (Std) cheese was compared with three variants of technological itineraries: a shorter salting time (7 h vs 10 h, Salt7h), a shorter stirring time (15 min vs 30 min, Stir15min), or a higher ripening temperature (16 °C vs 13 °C, Rip16°C). Bacterial counts were similar in the four cheese types, except for a 1.4 log10 reduction of L. lactis counts in Rip16°C cheeses after 7 weeks of ripening. Compared to Std, Stir15min and Rip16°C increased propionibacterial activity, causing higher concentrations of acetic, succinic, and propanoic acids and lower levels of lactic acid. Rip16°C accelerated secondary proteolysis and volatile production. We thus demonstrated that fine-tuning process parameters could modulate the cheese volatilome by influencing specific bacterial metabolisms.

Development of antifungal ingredients for dairy products: From in vitro screening to pilot scale application.

Biopreservation represents a complementary approach to traditional hurdle technologies for reducing microbial contaminants (pathogens and spoilers) in food. In the dairy industry that is concerned by fungal spoilage, biopreservation can also be an alternative to preservatives currently used (e.g. natamycin, potassium sorbate). The aim of this study was to develop antifungal fermentates derived from two dairy substrates using a sequential approach including an in vitro screening followed by an in situ validation. The in vitro screening of the antifungal activity of fermentates derivating from 430 lactic acid bacteria (LAB) (23 species), 70 propionibacteria (4 species) and 198 fungi (87 species) was performed against four major spoilage fungi (Penicillium commune, Mucor racemosus, Galactomyces geotrichum and Yarrowia lipolytica) using a cheese-mimicking model. The most active fermentates were obtained from Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei/paracasei and Lactobacillus plantarum among the tested LAB, Propionibacterium jensenii among propionibacteria, and Mucor lanceolatus among the tested fungi. Then, for the 11 most active fermentates, culture conditions were optimized by varying incubation time and temperature in order to enhance their antifungal activity. Finally, the antifungal activity of 3 fermentates of interest obtained from Lactobacillus rhamnosus CIRM-BIA1952, P. jensenii CIRM-BIA1774 and M. lanceolatus UBOCC-A-109193 were evaluated in real dairy products (sour cream and semi-hard cheese) at a pilot-scale using challenge and durability tests. In parallel, the impact of these ingredients on organoleptic properties of the obtained products was also assessed. In semi-hard cheese, application of the selected fermentates on the cheese surface delayed the growth of spoilage molds for up to 21 days, without any effect on organoleptic properties, P. jensenii CIRM-BIA1774 fermentate being the most active. In sour cream, incorporation of the latter fermentate at 2 or 5% yielded a high antifungal activity but was detrimental to the product organoleptic properties. Determination of the concentration limit, compatible with product acceptability, showed that incorporation of this fermentate at 0.4% prevented growth of fungal contaminants in durability tests but had a more limited effect against M. racemosus and P. commune in challenge tests. To our knowledge, this is the first time that the workflow followed in this study, from in vitro screening using dairy matrix to scale-up in cheese and sour cream, is applied for production of natural ingredients relying on a large microbial diversity in terms of species and strains. This approach allowed obtaining several antifungal fermentates which are promising candidates for dairy products biopreservation.

Lipolysis during ripening of Emmental cheese considering organization of fat and preferential localization of bacteria.

This study followed the progression of lipolysis in Emmental cheese by quantifying the concentrations of individual free fatty acids (FFA) released during ripening in each of the different rooms: 12 days at 12 degrees C, 28 days at 21 degrees C, and 8 days at 4 degrees C. Lipolysis, which corresponded to 1.56% of fat, mainly occurred in the 21 and 4 degrees C rooms, with 68 and 16.5% of total FFA, respectively. The nonselectivity of lipolytic enzymes was evidenced: all fatty acids were released with level of > or =1%. Differential scanning calorimetry experiments showed that the thermal properties of cheese were affected by (i) lipolysis of fat, that is, the monoacylglycerols, diacylglycerols, and FFA that may be localized at the fat/whey interface, and/or by (ii) hydrolysis of high-melting-point triacylglycerols constituted mainly by long-chain saturated fatty acids (e.g., palmitic acid). Analysis of the cheese microstructure was performed using confocal laser scanning microscopy. Fat globules were mainly disrupted after pressing of curd grains, leading to the release of the milk fat globule membrane (MFGM); fat inclusions were surrounded by pockets of whey, delimited by casein strands. Moreover, colonies of bacteria were preferentially localized in situ at the fat/protein interface. This study showed that both the localization of bacteria and the supramolecular organization of fat which was not protected by the MFGM can help the accessibility of milk fat to lipolytic enzymes and then contribute to the quality of cheese.

Survey of bacterial proteins released in cheese: a proteomic approach.

During the ripening of Emmental cheese, the bacterial ecosystem confers its organoleptic characteristics to the evolving curd both by the action of the living cells, and through the release of numerous proteins, including various types of enzymes into the cheese when the cells lyse. In Emmental cheese these proteins can be released from thermophilic lactic acid bacteria used as starters like Lactobacillus helveticus, Lb delbruecki subsp. lactis and Streptococcus salivarius subsp. thermophilus and ripening bacteria such as Propionibacterium freudenreichii. The aim of this study was to obtain a proteomic view of the different groups of proteins within the cheese using proteomic tools to create a reference map. A methodology was therefore developed to reduce the complexity of cheese matrix prior to 2D-PAGE analysis. The aqueous phase of cheese was prefractionated by size exclusion chromatography, bacterial and milk proteins were separated and subsequently characterised by mass spectrometry, prior to peptide mass fingerprint and sequence homology database search. Five functional groups of proteins were identified involved in: (i) proteolysis, (ii) glycolysis, (iii) stress response, (iv) DNA and RNA repair and (v) oxidoreduction. The results revealed stress responses triggered by thermophilic lactic acid bacteria and Propionibacterium strains at the end of ripening. Information was also obtained regarding the origin and nature of the peptidases released into the cheese, thus providing a greater understanding of casein degradation mechanisms during ripening. Different peptidases arose from St thermophilus and Lb helveticus, suggesting that streptococci are involved in peptide degradation in addition to the proteolytic activity of lactobacilli.