Microbial composition and viability of natural whey starters used in PDO Comté cheese-making.

Natural whey starters (NWS) are cultures with undefined multiple-strains species commonly used to speed up the fermentation process of cheeses. The aim of this study was to explore the diversity and the viability of Comté cheese NWS microbiota. Culture-dependent methods, i.e. plate counting and genotypic characterization, and culture-independent methods, i.e. qPCR, viability-qPCR, fluorescence microscopy and DNA metabarcoding, were combined to analyze thirty-six NWS collected in six Comté cheese factories at two seasons. Our results highlighted that NWS were dominated by Streptococcus thermophilus (ST) and thermophilic lactobacilli. These species showed a diversity of strains based on Rep-PCR. The dominance of Lactobacillus helveticus (LH) over Lactobacillus delbrueckii (LD) varied depending on the factory and the season. This highlighted two types of NWS: the type-ST/LD (LD > LH) and the type-ST/LH (LD < LH). The microbial composition varied depending on cheese factory. One factory was distinguished by its level of culturable microbial groups (ST, enterococci and yeast) and its fungi diversity. The approaches used to estimate the viability showed that most NWS cells were viable. Further investigations are needed to understand the microbial diversity of these NWS.

Integration of Multiomic Data to Characterize the Influence of Milk Fat Composition on Cantal-Type Cheese Microbiota.

A previous study identified differences in rind aspects between Cantal-type cheeses manufactured from the same skimmed milk, supplemented with cream derived either from pasture-raised cows (P) or from cows fed with maize silage (M). Using an integrated analysis of multiomic data, the present study aimed at investigating potential correlations between cream origin and metagenomic, lipidomic and volatolomic profiles of these Cantal cheeses. Fungal and bacterial communities of cheese cores and rinds were characterized using DNA metabarcoding at different ripening times. Lipidome and volatolome were obtained from the previous study at the end of ripening. Rind microbial communities, especially fungal communities, were influenced by cream origin. Among bacteria, Brachybacterium were more abundant in P-derived cheeses than in M-derived cheeses after 90 and 150 days of ripening. Sporendonema casei, a yeast added as a ripening starter during Cantal manufacture, which contributes to rind typical aspect, had a lower relative abundance in P-derived cheeses after 150 days of ripening. Relative abundance of this fungus was highly negatively correlated with concentrations of C18 polyunsaturated fatty acids and to concentrations of particular volatile organic compounds, including 1-pentanol and 3-methyl-2-pentanol. Overall, these results evidenced original interactions between milk fat composition and the development of fungal communities in cheeses.

Compilation of data on model cheeses composition, rheological and sensory properties, from six research projects exported from the BaGaTel database.

This paper presents data on model cheeses extracted from the BaGaTel database. The data are issued from 6 different research projects in which data on composition, rheological and sensory properties were collected. The manufacturing of the 68 different samples is described. For each model cheese, data are available on final composition (lipid, protein, water, sodium), rheological properties (uniaxial compression), sensory profile analysis (texture, taste, aroma) and for some cheeses chewing activity and in vivo sodium release were also measured. The material and methods used are detailed. Scatter plots of representation of the values for each variable and each project are plotted. Pearson correlations between variables are given for specific subsets of data. The dataset is hosted in an open access data repository. This dataset will allow a comparison of sensory properties of cheeses varying in lipid, protein water and salt content and can be used for the reformulation of cheeses made with a low salt and fat content to follow food-related health recommendations, whilst fulfilling good sensory qualities.

Use of autochthonous mesophilic lactic acid bacteria as starter cultures for making Pecorino Crotonese cheese: Effect on compositional, microbiological and biochemical attributes.

The use of selected autochthonous mesophilic lactic acid bacteria as starter cultures was investigated according to the traditional protocol for making Pecorino Crotonose (PC). Leuconostoc mesenteroides subsp. mesenteroides 2A, Lactobacillus casei 23C and Lactobacillus plantarum 18C (Autochthonous Starter, AS1) and Leuc. mesenteroides subsp. mesenteroides 2A, and L. casei 25D and 16A (AS2) were isolated and identified from aged ewes' milk PC cheeses, selected based on several enzymatic activities, and used as starter cultures. As shown by the in vitro kinetic of acidification, selected starter cultures had suitable capabilities to acidify. The manufacture of PC cheeses was carried out at an industrial plant scale. A control cheese (CC) was also made, using commercial starters consisting of mesophilic and thermophilic species. Ripening lasted 105 days at 10 °C. A poly-phasic approach was used to compare cheeses during manufacture and ripening, mainly based on pyrosequencing of the 16S rRNA targeting DNA, proteolysis and volatile component analyses. Compared to CC, both autochthonous starter cultures slightly affected the gross chemical composition of PC cheese. The cell density of thermophilic starters of CC progressively decreased throughout ripening. Plate count and RAPD-PCR showed that the cell number of autochthonous lactobacilli cultures of PC cheeses, made with AS1 and AS2, was almost constant throughout ripening and abundantly higher than that observed in CC. As shown by culture-independent analysis, the OTUs found during ripening varied depending on the manufacture with or without autochthonous starter cultures. The major chemical differences among cheeses were the concentration of free amino acids and the synthesis of some key volatile components (e.g., 2-methyl-1-propanol, 2-methyl-1-butanol, isobutyric, isovaleric, and isocaproic acids). Compared to CC, the use of AS1 positively affected the overall cheese quality.

Milk fat composition modifies the texture and appearance of Cantal-type cheeses but not their flavor.

Although the effects of cow diet on cheese sensory properties have been well documented, the putative interactions between the biochemical and microbial milk components and their respective roles in the development of the sensory properties of cheeses have yet to be explored in depth. The aim of this study was to evaluate the specific contribution of milk fat composition to the formation of cheese sensory properties. Two creams with different fat compositions were obtained from cows fed either pasture or maize silage. Cheeses were manufactured from the same skim milk (identical chemical and microbial composition) with either the pasture- or maize silage-origin pasteurized cream added. The gross composition and microbial composition of milks did not vary with cream origin. In milks and cheeses, the fatty acid (FA) profiles were modified by the origin of the cream. The concentrations of C18:0 and unsaturated FA such as cis-9 C18:1, trans-11 C18:1, C18:3n-3, total conjugated linoleic acids, and mono- and polyunsaturated FA were higher in milks and cheeses with the pasture-origin cream than in those with the maize-origin cream. In contrast, the maize milks and cheeses had higher concentrations of short- and medium-chain saturated FA, C16:0, and C18:2n-6. The level of lipolysis was 11% in the cheese rind and only 0.30% in the cheese core. The rind of pasture cheeses had a higher concentration of free C18:0 and C18:3n-3 and a lower concentration of free C14:0 and free C16:0 than the rind of maize cheeses. The levels of major microbial groups were similar in pasture and maize cheeses at different stages of ripening. The pasture cheeses had a more elastic and creamier texture, a yellower color, and a thinner rind than the maize cheeses, but the odor and aroma of cheeses were not affected by the origin of the cream, despite a few modifications in the balance of volatile compounds from FA catabolism. Based on these results, we conclude that milk fat composition modulated by cow diet had a direct role in the texture of the cheese but no effect on flavor. The high degree of lipolysis in cheese rind, along with the higher concentration of long-chain unsaturated free FA in pasture cheeses may be responsible for antimicrobial activity, which could explain differences in the appearance of cheese rind.

Spatial Distribution of the Metabolically Active Microbiota within Italian PDO Ewes' Milk Cheeses.

Italian PDO (Protected Designation of Origin) Fiore Sardo (FS), Pecorino Siciliano (PS) and Pecorino Toscano (PT) ewes' milk cheeses were chosen as hard cheese model systems to investigate the spatial distribution of the metabolically active microbiota and the related effects on proteolysis and synthesis of volatile components (VOC). Cheese slices were divided in nine sub-blocks, each one separately subjected to analysis and compared to whole cheese slice (control). Gradients for moisture, and concentrations of salt, fat and protein distinguished sub-blocks, while the cell density of the main microbial groups did not differ. Secondary proteolysis differed between sub-blocks of each cheese, especially when the number and area of hydrophilic and hydrophobic peptide peaks were assessed. The concentration of free amino acids (FAA) agreed with these data. As determined through Purge and Trap (PT) coupled with Gas Chromatography-Mass Spectrometry (PT-GC/MS), and regardless of the cheese variety, the profile with the lowest level of VOC was restricted to the region identified by the letter E defined as core. As shown through pyrosequencing of the 16S rRNA targeting RNA, the spatial distribution of the metabolically active microbiota agreed with the VOC distribution. Differences were highlighted between core and the rest of the cheese. Top and bottom under rind sub-blocks of all three cheeses harbored the widest biodiversity. The cheese sub-block analysis revealed the presence of a microbiota statistically correlated with secondary proteolysis events and/or synthesis of VOC.

Microbial ecology dynamics reveal a succession in the core microbiota involved in the ripening of pasta filata caciocavallo pugliese cheese.

Pyrosequencing of the 16S rRNA targeting RNA, community-level physiological profiles made with Biolog EcoPlates, proteolysis, and volatile component (VOC) analyses were mainly used to characterize the manufacture and ripening of the pasta filata cheese Caciocavallo Pugliese. Plate counts revealed that cheese manufacture affected the microbial ecology. The results agreed with those from culture-independent approaches. As shown by urea-PAGE, reverse-phase high pressure liquid chromatography (RP-HPLC), and free-amino-acid (FAA) analyses, the extent of secondary proteolysis mainly increased after 30 to 45 days of ripening. VOCs and volatile free fatty acids (VFFA) were identified by a purge-and-trap method (PT) and solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS), respectively. Except for aldehydes, the levels of most of VOCs and VFFA mainly increased from 30 to 45 days onwards. As shown through pyrosequencing analysis, raw cows' milk was contaminated by Firmicutes (53%), Proteobacteria (39%), Bacteroidetes (7.8%), Actinobacteria (0.06%), and Fusobacteria (0.03%), with heterogeneity at the genus level. The primary starter Streptococcus thermophilus dominated the curd population. Other genera occurred at low incidence or sporadically. The microbial dynamics reflected on the overall physiological diversity. At 30 days, a microbial succession was clearly highlighted. The relative abundance of Streptococcus sp. and especially St. thermophilus decreased, while that of Lactobacillus casei, Lactobacillus sp., and especially Lactobacillus paracasei increased consistently. Despite the lower relative abundance compared to St. thermophilus, mesophilic lactobacilli were the only organisms positively correlated with the concentration of FAAs, area of hydrophilic peptide peaks, and several VOCs (e.g., alcohols, ketones, esters and all furans). This study showed that a core microbiota was naturally selected during middle ripening, which seemed to be the main factor responsible for cheese ripening.

Traditional cheeses: rich and diverse microbiota with associated benefits.

The risks and benefits of traditional cheeses, mainly raw milk cheeses, are rarely set out objectively, whence the recurrent confused debate over their pros and cons. This review starts by emphasizing the particularities of the microbiota in traditional cheeses. It then describes the sensory, hygiene, and possible health benefits associated with traditional cheeses. The microbial diversity underlying the benefits of raw milk cheese depends on both the milk microbiota and on traditional practices, including inoculation practices. Traditional know-how from farming to cheese processing helps to maintain both the richness of the microbiota in individual cheeses and the diversity between cheeses throughout processing. All in all more than 400 species of lactic acid bacteria, Gram and catalase-positive bacteria, Gram-negative bacteria, yeasts and moulds have been detected in raw milk. This biodiversity decreases in cheese cores, where a small number of lactic acid bacteria species are numerically dominant, but persists on the cheese surfaces, which harbour numerous species of bacteria, yeasts and moulds. Diversity between cheeses is due particularly to wide variations in the dynamics of the same species in different cheeses. Flavour is more intense and rich in raw milk cheeses than in processed ones. This is mainly because an abundant native microbiota can express in raw milk cheeses, which is not the case in cheeses made from pasteurized or microfiltered milk. Compared to commercial strains, indigenous lactic acid bacteria isolated from milk/cheese, and surface bacteria and yeasts isolated from traditional brines, were associated with more complex volatile profiles and higher scores for some sensorial attributes. The ability of traditional cheeses to combat pathogens is related more to native antipathogenic strains or microbial consortia than to natural non-microbial inhibitor(s) from milk. Quite different native microbiota can protect against Listeria monocytogenes in cheeses (in both core and surface) and on the wooden surfaces of traditional equipment. The inhibition seems to be associated with their qualitative and quantitative composition rather than with their degree of diversity. The inhibitory mechanisms are not well elucidated. Both cross-sectional and cohort studies have evidenced a strong association of raw-milk consumption with protection against allergic/atopic diseases; further studies are needed to determine whether such association extends to traditional raw-milk cheese consumption. In the future, the use of meta-omics methods should help to decipher how traditional cheese ecosystems form and function, opening the way to new methods of risk-benefit management from farm to ripened cheese.

Diversity of the lactic acid bacterium and yeast microbiota in the switch from firm- to liquid-sourdough fermentation.

Four traditional type I sourdoughs were comparatively propagated (28 days) under firm (dough yield, 160) and liquid (dough yield, 280) conditions to mimic the alternative technology options frequently used for making baked goods. After 28 days of propagation, liquid sourdoughs had the lowest pH and total titratable acidity (TTA), the lowest concentrations of lactic and acetic acids and free amino acids, and the most stable density of presumptive lactic acid bacteria. The cell density of yeasts was the highest in liquid sourdoughs. Liquid sourdoughs showed simplified microbial diversity and harbored a low number of strains, which were persistent. Lactobacillus plantarum dominated firm sourdoughs over time. Leuconostoc lactis and Lactobacillus brevis dominated only some firm sourdoughs, and Lactobacillus sanfranciscensis persisted for some time only in some firm sourdoughs. Leuconostoc citreum persisted in all firm and liquid sourdoughs, and it was the only species detected in liquid sourdoughs at all times; it was flanked by Leuconostoc mesenteroides in some sourdoughs. Saccharomyces cerevisiae, Candida humilis, Saccharomyces servazzii, Saccharomyces bayanus-Kazachstania sp., and Torulaspora delbrueckii were variously identified in firm and liquid sourdoughs. A total of 197 volatile components were identified through purge and trap-/solid-phase microextraction-gas chromatography-mass spectrometry (PT-/SPME-GC-MS). Aldehydes, several alcohols, and some esters were at the highest levels in liquid sourdoughs. Firm sourdoughs mainly contained ethyl acetate, acetic acid, some sulfur compounds, and terpenes. The use of liquid fermentation would change the main microbial and biochemical features of traditional baked goods, which have been manufactured under firm conditions for a long time.

In vivo sodium release and saltiness perception in solid lipoprotein matrices. 1. Effect of composition and texture.

Reducing the sodium content in foods is complex because of their multidimensional sensory characteristics and the multifunctionality of sodium chloride. The aim of this study was to elucidate how food composition may influence in-mouth sodium release and saltiness perception. Lipoprotein matrices (LPM) were produced using milk constituents and characterized by means of rheological measurements, texture, and taste sensory profiles. Texture and taste perceptions were affected differently by variations in the salt level, dry matter, and fat contents. Composition and textural changes also modified temporal sodium release and saltiness perception recorded in five subjects, but the effects varied as a function of the salt content. The water content mainly appeared to influence the amount of sodium released, whereas saltiness perception was mainly related to fat content. Elasticity, coating, and granularity were found to be correlated with temporal sodium release and/or saltiness parameters.

Simplification of a complex microbial antilisterial consortium to evaluate the contribution of its flora in uncooked pressed cheese.

A complex microbial consortium derived from raw milk and composed of populations classified in 4 groups (lactic acid bacteria (A), Gram positive catalase positive bacteria (B), Gram negative bacteria (C) and yeasts (D)) can contribute to the inhibition of Listeria monocytogenes in the core of an uncooked pressed cheese. To identify what groups may be involved in the inhibition, the consortium was simplified by successively omitting one group at a time. Pasteurized milk was inoculated with these more or less complex consortia and their effects on L. monocytogenes count, pH, acids and volatile compounds in the core of uncooked pressed cheese were evaluated. The growth of L. monocytogenes was the highest in cheeses prepared with pasteurized milk and only St. thermophilus. Inhibition in other cheeses was expressed by comparison with growth in these ones. All the consortia containing both lactic acid bacteria (group A) and Gram positive catalase positive bacteria (group B)--ABCD, ABD, ABC, AB--were more inhibitory than those containing lactic acid bacteria on its own (A) or associated only with yeasts (AD) or/and Gram negative (ADC). Consortia without lactic acid bacteria were weakly inhibitory or had no effect. Gram positive catalase positive bacteria alone were not inhibitory although most of the species became established in the cheeses. The Lactobacillus population (Lb. casei, Lb. plantarum, Lb. curvatus and Lb. farciminis) was predominant in cheeses (9 log CFU/g) with a higher count than Leuconostoc (7 log CFU/g) and Enterococcus (7 log CFU/g). Lactobacillus counts were negatively correlated with those of L. monocytogenes (r=-0.84 at 18 days) and with the level of D-lactic acid. There was no correlation between L. monocytogenes and Leuconostoc or Enterococcus counts. Complex consortium ABCD and AB not only had a stronger inhibitory power in cheeses than consortium AD, they were also associated with the highest levels of L-lactic and acetic acids. All cheeses inoculated with lactic acid bacteria differed from those without by higher levels of ethyl formiate, pentane and alcohols (2-butanol, 2-pentanol), and lower levels of ketones (2-hexanone, 2,3-butanedione) and aldehydes (2-methyl-butanal). Levels of 2-methyl-butanal, 2-butanol and 2-pentanol were higher in ABCD and AB cheeses than in AD cheeses. Beside their contribution to the inhibition, their effect on cheese flavour must be evaluated.

Distribution and mobility of phosphates and sodium ions in cheese by solid-state 31P and double-quantum filtered 23Na NMR spectroscopy.

The feasibility of solid-state magic angle spinning (MAS) (31)P nuclear magnetic resonance (NMR) spectroscopy and (23)Na NMR spectroscopy to investigate both phosphates and Na(+) ions distribution in semi-hard cheeses in a non-destructive way was studied. Two semi-hard cheeses of known composition were made with two different salt contents. (31)P Single-pulse excitation and cross-polarization MAS experiments allowed, for the first time, the identification and quantification of soluble and insoluble phosphates in the cheeses. The presence of a relatively 'mobile' fraction of colloidal phosphates was evidenced. The detection by (23)Na single-quantum NMR experiments of all the sodium ions in the cheeses was validated. The presence of a fraction of 'bound' sodium ions was evidenced by (23)Na double-quantum filtered NMR experiments. We demonstrated that NMR is a suitable tool to investigate both phosphates and Na(+) ions distributions in cheeses. The impact of the sodium content on the various phosphorus forms distribution was discussed and results demonstrated that NMR would be an important tool for the cheese industry for the processes controls.

Effect of mesophilic lactobacilli and enterococci adjunct cultures on the final characteristics of a microfiltered milk Swiss-type cheese.

The effect of four associations of adjunct cultures composed of mesophilic lactobacilli and enterococci, either solely or combined, on the microbiological, biochemical and sensory characteristics of Swiss-type cheese made using microfiltered cows' milk and supplemented with propionibacteria was studied. The global pattern of growth was similar to that generally observed in raw milk cheese and interactions between microflora were highlighted during ripening. Enterococci, which negatively affected the survival of streptococci starters, seemed to play a limited role in the formation of volatile compounds, probably due to their low levels throughout ripening. On the contrary, mesophilic lactobacilli, which affected the evolution of propionibacteria, enterococci and L. delbrueckii subsp. lactis starter counts, modified free amino acid content, production of volatile compounds and organoleptic properties of mature cheese. This population appeared to be of major importance in the formation of cheese flavor as it was positively related to numerous potential flavor compounds such as alcohols and their corresponding esters, acetaldehyde and 4-methyl-4-heptanone. The original mesophilic lactobacilli present in milk could play an important role in the sensorial diversity of raw milk Swiss-type cheeses such as Comte.

Effect of autochthonous lactic acid bacteria starters on health-promoting and sensory properties of tomato juices.

Strains of Lactobacillus plantarum, Weissella cibaria/confusa, Lactobacillus brevis, Pediococcus pentosaceous, Lactobacillus sp. and Enterococcus faecium/faecalis were identified from raw tomatoes by Biolog System, partial 16S rRNA gene sequence and subjected to typing by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) analysis. Ten autochthonous strains were singly used to ferment tomato juice (TJ) via a protocol which included fermentation at 25 degrees C for 17 h and further storage at 4 degrees C for 40 days. Unstarted TJ and TJ fermented with an allochthonous strain of L. plantarum were used as the controls. All autochthonous strains grew well in TJ reaching cell densities ca. 10,000 and 10 times higher than unstarted TJ and TJ fermented with the allochthonous strain. Viscosity of TJs fermented with autochthonous strains was generally the highest, especially when started with W. cibaria/confusa which synthesized exo-polysaccharides. Overall, unstarted TJ and TJ fermented with the allochthonous strain showed marked decreases of ascorbic acid (ASC), glutathione (GSH) and total antioxidant activity (TTA) during storage. On the contrary, several TJs fermented with autochthonous strains, especially with L. plantarum POM1 and POM 35, maintained elevated values of ASC, GSH and TAA. The variation of color indexes mirrored the above behavior. TJs fermented with the above two autochthonous strains were compared to controls based on volatile components through Purge and Trap or Solid Phase Microextraction Gas Chromatography-Mass Spectrometry (PT or SPME-GC/MS) analysis. As shown by Principal Component Analysis a large number of volatiles belonging to various chemical classes markedly differentiated TJs fermented with autochthonous strains with respect to controls.

Cell-cell communication in sourdough lactic acid bacteria: a proteomic study in Lactobacillus sanfranciscensis CB1.

The mechanisms of cell-cell communication in Lactobacillus sanfranciscensis CB1 were studied. The highest number of dead/damaged cells of L. sanfranciscensis CB1 was found in cocultures with Lactobacillus plantarum DC400 or Lactobacillus brevis CR13 when the late stationary phase of growth (18 h) was reached. 2-DE analysis was carried out. Almost the same proteins were induced in all three cocultures at the mid-exponential phase of growth (7 h). The number of induced proteins markedly increased at 18 h, especially when L. sanfranciscensis CB1 was cocultured with L. plantarum DC400 or L. brevis CR13. Nineteen overexpressed proteins were identified. These proteins had a central role in stress response mechanisms and LuxS-mediated signalling was involved in the regulation of most of them. The luxS and metF genes were partially sequenced in L. sanfranciscensis CB1. RT-PCR showed that the expression of luxS gene decreased from 7 to 12 h. It was highest in cocultures with L. plantarum DC400 and L. brevis CR13. 2(3H)dihydrofuranone-5ethyl and 2(3H)dihydrofuranone-5pentyl were identified as presumptive signalling molecules when L. sanfranciscensis CB1 was cocultured with L. brevis CR13 and, especially, L. plantarum DC400. The synthesis of other volatile compounds and peptidase activities were also influenced by the type of microbial cocultures.

Tentative hierarchization of the influence of milk properties and technological practices on rheological properties of Abondance cheese.

An attempt at classifying the influence of different characteristics of milk and cheesemaking on the rheological properties of Abondance cheese is presented. Abondance is a traditional farmhouse French hard cheese with protected denomination of origin (PDO). Thirty-nine cheeses made from unpasteurized cows' milk were sampled. Spline partial least squares regression was used to relate milk properties and cheesemaking practices to rheological properties of the six-month-old cheeses. These properties were the deformability modulus and the strain and stress at fracture measured by compression. Milk properties and technological practices had overall the same degree of relationship with the rheological properties investigated: plasminogen-derived activity in milk and mineral-protein equilibrium, on the one hand, and brining and resting, on the other hand. However, acidification kinetics and 1-d pH, which result from both milk properties and technological practices, showed the strongest relationships with rheological characteristics. Factors that were most appropriate for modelling Abondance rheological properties are discussed.