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Background One of todays’ scientific challenge due to climate change and environmental issues is to find alternatives to the production of molecules of interest from petrochemistry. This study focuses on the understanding of the production of butyrate, hydrogen and CO2 from the oxidation of lactate with acetate in C. tyrobutyricum and thus propose an alternative carbon source to glucose. This species is known to produce more butyrate than the other butyrate-producing Clostridium species due to a lack of solventgenesis phase. The recent discoveries of flavin-based electron bifurcation mechanism as a mode of energy conservation have allowed us to propose a new metabolic scheme for the formation of butyrate from lactate-acetate co-metabolism. Results The search for genes encoding for EtfAB complexes and neighboring genes in the genome of C. tyrobutyricum identified a cluster of genes involved in butyrate formation and another cluster involved in lactate oxidation homologous to Acetobacterium woodii. A phylogenetic approach with other butyrate-producing and/or lactate-oxidizing species based on EtfAB complexes confirms these results with A. woodii and Clostridium kluyveri as references. A metabolic scheme on the production of butyrate, hydrogens and CO2 from the lactate-acetate co-metabolism in C. tyrobutyricum has been constructed. From data of steady-state continuous culture, an in silico metabolic carbon flux analysis model was built and made it possible to show the coherence of the scheme from the carbon recovery, the cofactor ratio and the ATP yield. Conclusions Bioinformatics analyzes have highlighted the use of flavin-based electron bifurcation. A metabolic scheme on the conversion of lactate and acetate has been proposed in C. tyrobutyricum, involving electron-transferring flavoprotein (Etf) complexes specific to the butyrate production and the lactate oxidation. The carbon metabolic fluxes analysis has validated this scheme involving the EtfAB complex at the origin of the oxidation of lactate in C. tyrobutyricum. This study contributes significantly to our understanding of the lactate oxidation metabolic pathways, the utility of the presence of acetate and the role of intracellular redox balance, and opens new perspectives for the production of molecules of interest as butyrate and hydrogen with C. tyrobutyricum.

Franche-Comte is the primary producing region of Protected Designation of Origin cheeses in France. Normally, mid-infrared (MIR) prediction models for cheese-making property (CMP) traits are developed using individual bovine milks. However, considering the requests of all actors in the dairy sector, the present study aimed to assess the feasibility of MIR spectroscopy to develop CMP equations of Montbeliarde herd and dairy vat milks. For this purpose, 22 CMP traits were analyzed on samples collected in 2016 (half in February-March and half in May-June) from 100 commercial herds and 70 dairy vats (55 cheese dairies) located in Franche-Comte. These characteristics included 11 rennet coagulation traits and 8 lactic acidification traits measured in either soft cheese or pressed cooked cheese conditions and 3 laboratory curd yields. Models of MIR prediction for each of the 22 CMP traits were built using partial least squares regression with external validation by dividing the data set into calibration (70%) and validation (30%) sets. We confirmed that the variability of milk traits depends largely on the production scale and is higher for individual milk than for herd milk and even higher for vat milk. The best prediction models were obtained in herd milk samples for curd yields expressed in dry matter or fresh, with a coefficient of determination (R2) in external validation of 0.78 and 0.77, respectively. As with individual milk, these traits are closely related to the gross composition of the milk and therefore easier to predict by MIR spectroscopy. However, these curd yield traits were poorly predicted (R2 = 0.58) in vat milk samples due to their lower variability. In herd milk samples, prediction models of other CMP traits were poorly accurate except for the ratio of the time to obtain a standard firmness to the rennet coagulation time in soft cheese or pressed cooked cheese conditions, which showed R2 > 0.66 in external validation. Such trait is important in qualifying the behavior of milk during cheese production. Prediction models of other CMP traits for either herd or vat milk samples had poor accuracy, and further work is needed to improve their performance.

Les aptitudes du lait à la transformation en fromage sont étroitement liées à sa composition. Ces caractères, difficiles à mesurer directement, ont été prédits à partir des spectres dans le moyen infrarouge (MIR) du lait en race Montbéliarde (projet From’MIR). Cet article rassemble les résultats de l’analyse du déterminisme génétique des aptitudes fromagères et de la composition fine du lait, prédites à partir de six millions de spectres MIR de 400 000 vaches. Ces caractères sont modérément à fortement héritables et les corrélations génétiques entre caractères fromagers (rendements et coagulation) et avec la composition du lait (protéines, acides gras et minéraux) sont élevées et favorables. Des analyses d’association (GWAS) et de réseaux de gènes, réalisées à partir des génotypes imputés pour l’ensemble des variations du génome de 20 000 vaches, permettent d’identifier des gènes et des variants candidats ainsi qu’un réseau de 736 gènes impliqués dans des voies métaboliques et des gènes régulateurs fonctionnellement liés à la composition du lait. Enfin, l’estimation de la précision d’une évaluation génomique montre qu’un modèle de type contrôles élémentaires, incluant les variants détectés par les GWAS et présumés causaux, permet de prédire des valeurs génomiques précises. Nous avons par ailleurs simulé une sélection incluant les aptitudes fromagères qui montre les possibilités de sélectionner efficacement les vaches pour qu’elles produisent un lait plus « fromageable », avec un impact limité sur le gain génétique des caractères actuellement sélectionnés. Ces résultats ont conduit à la mise en place d’un prototype d’évaluation génomique en race Montbéliarde dans la zone AOP Comté en 2019

Projet AbsoFrom.; International audience; The study was designed to compare the effect of diets of equal calcium and fat content from pressed-cooked cheese, butter, palm oil or a chow diet, on blood lipids, faecal fat excretion and body composition in rats. Palm oil intake resulted in higher faecal saturated fatty acid (SFAs) excretion compared with pressed-cooked cheese or butter, showing the importance of the positional distribution of SFAs in triacylglycerol. However, compared with butter, cheese fat intake resulted in higher faecal fat excretion, confirming the importance of the dairy-matrix effect in the excretion of fat. Despite a high consumption of fat, animals of pressed-cooked cheese group did not gain weight (no differences in any groups) and did not accumulate fat compared with the control group, contrary to the situation with rats fed with the same amount of fat provided by vegetable oil and extracted from butter that gained fat mass compared with controls.

Assessing the cheese-making properties (CMP) of milks with a rapid and cost-effective method is of particular interest for the Protected Designation of Origin cheese sector. The aims of this study were to evaluate the potential of mid-infrared (MIR) spectra to estimate coagulation and acidification properties, as well as curd yield (CY) traits of Montbeliarde cow milk. Samples from 250 cows were collected in 216 commercial herds in Franche-Comte with the objectives to maximize the genetic diversity as well as the variation in milk composition. All coagulation and CY traits showed high variability (10 to 43%). Reference analyses performed for soft (SC) and pressed cooked (PCC) cheese technology were matched with MIR spectra. Prediction models were built on 446 informative wavelengths not tainted by the water absorbance, using different approaches such as partial least squares (PLS), uninformative variable elimination PLS, random forest PLS, Bayes A, Bayes B, Bayes C, and Bayes RR. We assessed equation performances for a set of 20 CMP traits (coagulation: 5 for SC and 4 for PCC; acidification: 5 for SC and 3 for PCC; laboratory CY: 3) by comparing prediction accuracies based on cross-validation. Overall, variable selection before PLS did not significantly improve the performances of the PLS regression, the prediction differences between Bayesian methods were negligible, and PLS models always outperformed Bayesian models. This was likely a result of the prior use of informative wavelengths of the MIR spectra. The best accuracies were obtained for curd yields expressed in dry matter (CYDM) or fresh (CYFRESH) and for coagulation traits (curd firmness for PCC and SC) using the PLS regression. Prediction models of other CMP traits were moderately to poorly accurate. Whatever the prediction methodology, the best results were always obtained for CY traits, probably because these traits are closely related to milk composition. The CYDM predictions showed coefficient of determination (R2) values up to 0.92 and 0.87, and RSy,x values of 3 and 4% for PLS and Bayes regressions, respectively. Finally, we divided the data set into calibration (2/3) and validation (1/3) sets and developed prediction models in external validation using PLS regression only. In conclusion, we confirmed, in the validation set, an excellent prediction for CYDM [R2 = 0.91, ratio of performance to deviation (RPD) = 3.39] and a very good prediction for CYFRESH (R2 = 0.84, RPD = 2.49), adequate for analytical purposes. We also obtained good results for both PCC and SC curd firmness traits (R2 ≥ 0.70, RPD ≥1.8), which enable quantitative prediction.

International audience; Sampling from the vat during cheesemaking may be necessary to study the impact of curd washing, which affects both whey composition and the curd's aqueous phase. The objective of this work was to develop a sieving protocol of curd-whey samples, ensuring enough time for drainage of the free whey, while limiting the loss of the aqueous phase entrapped in the curd grains. We studied the flow rate and lactose content of whey during sieving. Both measurements showed a two-stage dynamic representation of both aqueous phases, the intersection indicating the transition point between the flow of free and entrapped whey.

Lactic acid bacteria (LAB) are important microorganisms in the food industry as functional starters for the manufacture of fermented food products and as probiotics. Redox potential (E

The potential adulteration of kid or lamb rennet with calf rennet is of interest for some Protected Designation of Origin cheeses producers and those looking for a specific cheese typicality. The approach proposed here for the authentication of kid or lamb rennet is based on the immuno-detection of bovine pepsin possibly present in calf rennet in varying quantities. The developed immunoassay (indirect ELISA) used a monoclonal antibody (mAb) raised against bovine pepsin. This mAb was found to be specific as it didn't cross-react with the pepsin of animal species other than bovine (kid, lamb, pig) and with other milk-clotting enzymes (chymosin and microbial enzymes). Adulteration tests were conducted with kid and lamb rennets spiked with a wide range of calf rennet (from 0 to 100% v/v). The presence of bovine pepsin was detected at low levels down to 6 mg/L in kid and lamb rennets. Good linear relationships were obtained between added bovine pepsin concentration and the absorbance values over the range 1.25–120 mg/L. Results showed that indirect ELISA proved to be an interesting tool for testing rennets authenticity targeting bovine pepsin as an indicator of the bovine adulteration of kid and lamb rennets.

6 graph.; Pepsin and chymosin are milk-clotting enzymes found in rennets. They differ in their pH and temperature sensitivities and their milk-clotting activity (MCA)/proteolytic activity ratio which impact cheese technology. Therefore, characterization of rennet should not be limited to its total MCA, but also its enzymatic composition. Monoclonal antibodies against pepsin, obtained from mice immunized with purified pepsin, were characterized. A specific inhibition enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of pepsin in rennets. The limit of quantification was 143 ng pepsin/ mL. The precision within runs ranged from 7.0 to 9.4%, for rennets with low and high pepsin concentrations, respectively. The precision among runs also ranged from 8.8 to 11.4%. Satisfying recovery, from 84.7 to 100.3%, was found with spiked samples. The applicability of the developed inhibition ELISA for pepsin quantification was assessed by analyzing commercial rennets, and compared to the standard chromatographic method 110A of International Dairy Federation. The relation and agreement between methods were evaluated using Deming regression analysis and Bland-Altman plot. A good agreement was found with a fixed bias. By means of bias subtraction, inhibition ELISA could be a reliable alternative for rapid and sensitive determination of pepsin in rennet.

Clostridium tyrobutyricum is the main bacterial species leading to the late blowing defect, a major cause of spoilage in semihard and hard cheeses. This study reports the complete genome sequencing, assembly, and annotation of C. tyrobutyricum strain Cirm BIA 2237, formerly called CNRZ 608, isolated from silage.

International audience; Cheese-making properties of pressed cooked cheeses (PCC) and soft cheeses (SC) were predicted from mid-infrared (MIR) spectra. The traits that were best predicted by MIR spectra (as determined by comparison with reference measurements) were 3 measures of laboratory cheese yield, 5 coagulation traits, and 1 acidification trait for PCC (initial pH; pH0PPC). Coefficients of determination of these traits ranged between 0.54 and 0.89. These 9 traits as well as milk composition traits (fatty acid, protein, mineral, lactose, and citrate content) were then predicted from 1,100,238 MIR spectra from 126,873 primiparous Montbéliarde cows. Using this data set, we estimated the corresponding genetic parameters of these traits by REML procedures. A univariate or bivariate repeatability animal model was used that included the fixed effects of herd × test day × spectrometer, stage of lactation, and year × month of calving as well as the random additive genetic, permanent environmental, and residual effects. Heritability estimates varied between 0.37 and 0.48 for the 9 cheese-making property traits analyzed. Coagulation traits were the ones with the highest heritability (0.42 to 0.48), whereas cheese yields and pH0 PPC. had the lowest heritability (0.37 to 0.39). Strong favorable genetic correlations, with absolute values between 0.64 and 0.97, were found between different measures of cheese yield, between coagulation traits, between cheese yields and coagulation traits, and between coagulation traits measured for PCC and SC. In contrast, the genetic correlations between milk pH0 PPC and CY or coagulation traits were weak (−0.08 to 0.09). The genetic relationships between cheese-making property traits and milk composition were moderate to high. In particular, high levels of proteins, fatty acids, Ca, P, and Mg in milk were associated with better cheese yields and improved coagulation. Proteins in milk were strongly genetically correlated with coagulation traits and, to a lesser extent, with cheese yields, whereas fatty acids in milk were more genetically correlated with cheese yields than with coagulation traits. This study, carried out on a large scale in Montbéliarde cows, shows that MIR predictions of cheese yields and milk coagulation properties are sufficiently accurate to be used for genetic analyses. Cheese-making traits, as predicted from MIR spectra, are moderately heritable and could be integrated into breeding objectives without additional phenotyping cost, thus creating an opportunity for efficient improvement via selection.

International audience; In a previous study, we identified candidate causative variants located in 24 functional candidate genes for milk protein and fatty acid composition in Montbéliarde, Normande, and Holstein cows. We designed these variants on the custom part of the EuroG10K BeadChip (Illumina Inc., San Diego, CA), which is routinely used for genomic selection analyses in French dairy cattle. To validate the effects of these candidate variants on milk composition and to estimate their effects on cheesemaking properties, a genome-wide association study was performed on milk protein, fatty acid and mineral composition, as well as on 9 cheesemaking traits (3 laboratory cheese yields, 5 coagulation traits, and milk pH). All the traits were predicted from midinfrared spectra in the Montbéliarde cow population of the Franche-Comté region. A total of 194 candidate variants located in 24 genes and 17 genomic regions were imputed on 19,862 cows with phenotypes and genotyped with either the BovineSNP50 (Illumina Inc.) or the EuroG10K BeadChip. We then tested the effect of each SNP in a mixed linear model including random polygenic effects estimated with a genomic relationship matrix. We confirm here the effects of candidate causative variants located in 17 functional candidate genes on both cheesemaking properties and milk composition traits. In each candidate gene, we identified the most plausible causative variant: 4 are missense in the ALPL, SLC26A4, CSN3, and SCD genes, 7 are located in 5'UTR (AGPAT6), 3' untranslated region (GPT), or upstream (CSN1S1, CSN1S2, PAEP, DGAT1, and PICALM) regions, and 6 are located in introns of the SLC37A1, MGST1, CSN2, BRI3BP, FASN, and ANKH genes.

International audience; The effects on milk, butter and hard cooked cheese of new n-3 fatty acid (FA) sources in cows' corn silage-based diets – microalgae DHA Gold® (ALG), sieved extruded linseed (SEL) and their combination (SEL/ALG) – were assessed. Butter processing from ALG and SEL/ALG cream was not possible. Butter from SEL had less yellow colour and tended to be softer, with less rancid flavour and odour but stronger cream flavour. For SEL/ALG, cheese yield before brining was higher, but fat content in dry matter and curd dry matter were lower. For ALG and SEL/ALG, cheeses were evaluated as less coloured, more open, less firm, less elastic, more grainy and spicy. Cheese volatile compounds were affected by cows’ diet; higher concentrations of compounds from FA oxidation and amino acid catabolism were observed with the addition of microalgae DHA Gold®. Flavour profiles of cheeses were slightly affected by this addition, but no off-flavour was perceived.

Analytical Methods; Lactoferrin is an iron-binding cationic glycoprotein (pI = 8.7) beneficial for mammal health, especially udder and milk preservation. A new simple two-step method of quantification was developed. Lactoferrin in 1 mL of bovine skim milk was first adsorbed onto 100 mg of macroporous sulfonated-resin at pH 6.8 by rotary stirring for 90 min at 20–25 °C. After washing the resin, lactoferrin was desorbed using 1 mL of 2 M NaCl containing phenylalanine as a dilution marker, then fully resolved and quantified by RP-HPLC at 220 nm using a wide-bore C4 silica column. This robust, inexpensive and flexible method improves selectivity (no protein interference) and sensitivity compared to previous HPLC methods. In-laboratory validation demonstrated its linearity (25 to 514 µg Lf mL(−1)), accuracy (110 to 98% recovery), and precision (< 4%), which were comparable to immunobased methods. The results for individual raw cow’s milk were strongly correlated with results using an ELISA test.

Traditional cheeses originate from complex systems that confer on them specific sensory characteristics. These characteristics are linked to various factors of biodiversity such as animal feed, the use of raw milk and its indigenous microflora, the cheese technology, and the ripening conditions, all in conjunction with the knowledge of the cheesemaker and affineur. In Europe, particularly in France, the preservation of traditional cheesemaking processes, some of which have protected designation of origin, is vital for the farming and food industry in certain regions. Among these cheeses, some are made in the Alps or Jura Mountains, including Comté, Beaufort, Abondance, and Emmental, which are made from raw milk. The principle of hard or semihard cooked cheese, produced in the Alps and Jura Mountains, was to make a product during the summer—a period during which the animals feed more and milk production is high—with a shelf life of several months that could be consumed in winter. Today, these traditional cheeses are produced according to a specific approach combining science and tradition in order to better understand and preserve the elements that contribute to the distinctiveness of these cheeses. To address this complex problem, a global approach to the role of the raw milk microflora in the final quality of cheeses was initially chosen. The modifications resulting from the elimination of the raw milk microflora, either by pasteurization or by microfiltration, to the biochemistry of the ripening process and ultimately the sensory quality of the cheeses were evaluated. This approach was achieved mainly with experimental hard cooked cheeses. Other types of traditional cheese made with raw and pasteurized milk are also considered when necessary. Besides the native raw milk microflora, traditional lactic starters (natural or wild starters) also participate in the development of the characteristics of traditional hard and semihard cooked mountain cheeses. After an initial description, their roles are described, mainly for Comté.

In order to determine bovine lactoferrin concentration in cheese, bovine lactoferrin-specific monoclonal antibodies have been raised and an ELISA has been developed to determine lactoferrin concentrations in milk, whey and experimental soft, semi-hard and Swiss-type cheeses made with raw or pasteurised milk. The lactoferrin concentration in cheese was shown to depend on the cheese-making process, with higher values in Swiss-type and semi-hard cheeses than in soft cheeses. Furthermore, Western-blotting analysis of lactoferrin in cheese showed that this protein stayed intact throughout ripening in raw milk cheese, whereas it was partially hydrolysed in cheeses made with pasteurised milk. Based on these observations, we propose that cheese may constitute a natural dairy source of lactoferrin beneficial to health.

The application of defined specific starter strains and their influence on microbiological, biochemical and sensory characteristics were studied during ripening of Venaco cheese, a traditional Corsican raw milk cheese manufactured with goat's or ewe's milk. Three defined starter blends, composed of wild strains of lactic acid bacteria, were tested. The first blend was composed of 2 strains of Lactococcus lactis subsp. lactis (ratio 1:1) and was used as a control. The second was composed of 3 strains, the two Lactococcus strains used in the first starter blend in addition to a strain of Lactobacillus paraplantarum (ratio 2:2:1). The third blend was also composed of 3 strains, the same two lactococci used in the first blend in addition to a strain of Leuconostoc mesenteroides subsp. mesenteroides (ratio 2:2:1). The experiment was carried out, in duplicate, at two cheese dairies. The first dairy transforms raw goat's milk and the second transforms raw ewe's milk. DNA fingerprinting of cheese isolates using the Rep-PCR technique showed that strains inoculated in milk established themselves in cheese. Lactococci and Ln. mesenteroides subsp. mesenteroides strains were present until the end of ripening, while Lb. paraplantarum was detected in cheese only during the first 15 d. Indigenous lactic microflora were found throughout ripening, showing a balance between this microflora and the starter strains. Goat's and ewe's milk cheeses made with Leuconostoc had the highest level of proteolysis, and those made with Lactobacillus, the highest level of lipolysis. These physico-chemical modifications led to significant differences in cheese sensory characteristics assessed by the triangle test., Utilisation de ferments lactiques spécifiques pour la fabrication du fromage Venaco. L'utilisation de levains lactiques sélectionnés spécifiques et leur influence sur les caractéristiques microbiologiques, biochimiques et sensorielles ont été évaluées durant l'affinage du fromage Venaco, un fromage traditionnel corse au lait cru de chèvre ou de brebis. Trois levains définis, composés de souches " sauvages " de bactéries lactiques, ont été testés. Le premier constitué de 2 souches de Lactococcus lactis subsp. lactis (ratio 1:1) était utilisé comme témoin. Le second était composé de 3 souches, la paire de lactocoques utilisée dans le premier ferment associée à une souche de Lactobacillus paraplantarum (ratio 2:2:1). Le troisième ferment comprenait la même paire de lactocoques associée à une souche de Leuconostoc mesenteroides subsp. mesenteroides (ratio 2:2:1). L'expérimentation a été menée dans deux fromageries, la première transformant du lait cru de chèvre, la seconde du lait cru de brebis. Chaque essai a été répété 1 fois. Le typage génétique des isolats collectés des fromages à l'aide de la technique Rep-PCR a montré que les souches inoculées dans le lait s'implantaient dans le fromage. Les souches de lactocoques et de Ln. mesenteroides subsp. mesenteroides étaient présentes jusqu'à la fin de l'affinage, la souche de Lb. paraplantarum jusqu'à 15 j d'affinage. Les bactéries lactiques de la microflore indigène étaient retrouvées durant l'affinage, traduisant un certain équilibre entre cette microflore et les souches du levain. Les fromages de chèvre et de brebis fabriqués avec la souche de lactobacille étaient les plus lipolysés, tandis que l'indice de protéolyse le plus élevé était obtenu avec le ferment contenant la souche de leuconostoc. Ces modifications physico-chimiques conduisaient à des différences significatives des propriétés sensorielles du fromage évaluées par un test triangulaire.

Certain strains of Staphylococcus aureus (S. aureus) are capable of producing enterotoxins. In dairy products, these enterotoxins are the second major cause of foodborne disease in France. The main objective of this study was to type a selection of enterotoxigenic and non-enterotoxigenic strains of S. aureus using a reliable method, pulsed-field gel electrophoresis (PFGE), to explore the genetic relationships between enterotoxin producing strains in vitro and non-enterotoxigenic strains. Ninety-one enterotoxigenic strains of S. aureus, and 20 non-enterotoxigenic strains, were typed by pulsed-field gel electrophoresis after digestion using SmaI endonuclease. This study revealed a specific profile for the enterotoxin D producing S. aureus strains. Enterotoxin D is one of the enterotoxins most frequently isolated in food-poisoning outbreaks, after enterotoxin A, implicating dairy products. Of the 29 PFGE profiles observed, the enterotoxin producing strains of S. aureus were represented by 24 of these profiles. However, no link was found between the different biotypes and the PFGE profiles. Interestingly, all the in vitro enterotoxin D producing strains were clustered in one pulsotype.

Les especes et souches de bacteries lactiques ont ete suivies dans quatre fromages commerciaux de Comte fabriques dans trois fromageries differentes en caracterisant genetiquement des isolats a neuf stades de fabrication et d'affinage. Elles ont ete aussi suivies dans les laits crus et les cultures de levains correspondants. Dix especes ont ete identifiees : Streptococcus thermophilus, Lactobacillus helveticus, Lactobacillus delbrueckii, Enterococcus sp., Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus plantarum, Pediococcus acidilactici et Lactobacillus zeae. Les quatre premieres especes ont ete apportees par les cultures de levains, alors que les trois suivantes l'ont ete par le lait cru. Les six premieres especes etaient dominantes et chacune representee dans chaque fromage par huit souches dominantes et jusqu'a 42 souches sous-dominantes. Lb. paracasei etait l'espece presentant la plus grande diversite de souches, avec de 11 a 15 souches differentes par fromage, suivie par Lb. rhamnosus, avec de 1 a 7 souches, et les autres especes, avec de 1 a 5 souches. Des cinetiques de croissance ont pu etre etablies pour les souches et especes dominantes. Le profil des souches dominantes, mais non des especes, et la dynamique des bacteries lactiques etaient specifiques a chaque fromage. La flore lactique etait complexe pour chaque fromage, en terme de nombre d'especes presentes et de cinetiques de croissance differentes observees.

Cheese made from raw milk represents an important proportion of the traditional cheeses, particularly in South European countries. Besides destruction of pathogenic bacteria, the most significant changes in milk relevant to cheesemaking, which are induced by pasteurization are: • a partial elimination of the milk microorganisms which may grow in cheese during ripening, • a partial or total activation or inhibition of the plasmin/plasminogen complex, cathepsin D, lipoprotein lipase and alkaline phosphatase. Enzymes from psychrotrophic bacteria, acid phosphatase and xanthine oxidase, which may be active during ripening, withstand pasteurization., • a slight (7%) denaturation of serum proteins and little or no modification of the cheesemaking properties (coagulation, acidification by lactic acid bacteria). From experimental work carried out on several cheese varieties, comparing pasteurized or microfiltered milk and raw milk cheeses, it was found that facultatively heterofermentative lactobacilli, Micrococcaceae , enterococci, and propionibacteria in Swiss-type cheese, are found at higher levels in raw milk cheese. The main biochemical modification of cheese during ripening concerns the nature and extent of proteolysis. Although there is no clear trend in the breakdown of α s1 - and β -caseins, milk pasteurization leads to a significant decrease of the amount of small peptides and free amino acids and to different HPLC profiles. Experiments carried out with sensory analysis show that, in all cases, pasteurized or microfiltered milk cheeses have received lower flavour intensity scores than raw milk cheeses. From this review, it is concluded that the indigenous milk microflora, with its diversity of species and strains, appears to be mainly responsible of the specific sensory properties of raw milk cheeses.

International audience; Experimental mini-cheeses were made from raw (Ra), microfiltered (MF), pasteurized (Pa) (72 degrees C?, 30 s) or pasteurized mixed with microfiltration retentate (PR) milk to study the influence of the indigenous microflora and pasteurization on the quality of Swiss-type cheese. To estimate biochemical transformations during cheese ripening, several methods were used: nitrogen fractionation (water-soluble fraction and phosphotungstic acid (PTA)-soluble faction), urea-polyacrylamide gel electrophoresis of caseins, reverse phase liquid chromatography of the water-soluble fraction, lactate and volatile fatty acids. Microbial populations were also enumerated. At the end of ripening, in comparison with MF and Pa milk cheeses, Pa and PR milk cheeses exhibited higher overall aroma intensity and pungency, characteristics which correlated with higher populations of facultatively heterofermentative lactobacilli (10(8) cfu g(-1)), propionibacteria (10(8) cfu g(-1)), and enterococci (10(6) cfu g(-1)) These cheeses had high levels of PTA-soluble N and acetic, propionic and isovaleric acids. MF and Pa milk cheeses, although somewhat different from one another, were very different from the two other types of cheese. Pa milk cheese had a lower pH than MF milk cheese, and contained a higher proportion of gamma-caseins due to the activation of plasmin. Moreover, Pa milk cheese was more acidic. but demonstrated a higher overall aroma intensity. The addition of raw milk flora (retentate) to Pa milk restored almost all the biochemical and sensory characteristics of Ra milk cheese measured in this study.

Summary - During the ripening of raw milk 8wiss-type minicheeses, the microbial ecosystem changed, and the quantitative and qualitative levels of microflora evolved constantly. The starter lactic acid bacteria population, ie, thermophilic streptococci and thermophilic lactobacilli, decreased regularly from 108 to 105 cfu/g. The propionibacteria and the facultatively heterofermentative lactobacilli, which were in very low numbers before ripening, increased rapidly to reach their final level after 6 weeks (approximately 108 cfu/g). The other microflora (Micrococcaceae and enterococci) remained at a subdominant level, or were even undetectable during ripening (coliforms, spores of butyric acid bacteria). Indigenous microflora enumerated in microfiltered milk cheeses evolved identically, except that they started at a lower level. Moreover, the starter bacteria decreased faster. Facultatively heterofermentative lactobacilli seemed to be the most important non-starter lactic acid bacteria. Eighty-eight colonies were isolated on FH agar du ring ripening, and characterized by means of the API system. The species identified evolved from a great diversity in the beginning to Iwo major subspecies: L paracasei subsp paracasei 1 and 3. However, the method used to detect this microflora did not allow us to evaluate the nature of the subdominant lactobacilli, although certainly many other species could be isolated. Swiss-type minicheese / microflora / facultatively heterofermentative lactobacilli / bacterial growth

The oxidoreduction potential (E h) is of growing interest in dairy research and the dairy industry. Lactic acid bacteria are able to decrease the E h to considerable reducing values, and it is an important feature of the technological characteristics of lactic starters and probiotics. Reducing E h is also necessary for the development of the characteristic flavour of certain fermented dairy products such as cheeses. Moreover, data comparisons and interpretations are often difficult due to an absence of standardised expression of the results. The objective of this work was to propose a complete procedure for the use of combined and half-cell platinum (Pt) sensors to obtain stable, repeatable and reproducible E h measurements and to standardise data. Three measurement systems, each made up of a sensor, cable and interface, were compared in tap water. A control chart was used to calculate a confidence interval (CI = 20 mV) to verify whether the process was under control. This complete procedure was then applied to the measurement of E h in milk samples to characterise both pure strains of lactic acid bacteria and complex mixed starters, to study the impact of a process parameter (oxygen) on starter activity, to follow E h during cheese making and to measure E h gradients in cheeses. This manuscript presents a procedure for the measurement of E h as well as the feasibility and interest of this procedure in the dairy chain.

Proteolytic milk-clotting enzymes are extracted from various sources (animals, plants, fungi) and processed according to various methods that are specific to each manufacturer or cheese-maker. Chemical composition and polypeptide patterns of 24 milk-clotting preparations from animal and fungal sources: 10 commercial rennets, 9 artisanal calf rennets, 2 recombinant chymosin preparations and 3 microbial preparations, were compared in order to identify differences according to both their manufacturing process and their source. The preparation from Cryphonectria parasitica had the highest ammonia and small peptide content. Commercial rennets and preparations from Rhizomucor miehei had the highest NaCl and pH values while artisanal rennets had the lowest and recombinant chymosins were intermediate. In comparison with the other commercial preparations, commercial rennets had the highest variability in chemical composition and their polypeptide profiles showed numerous protein bands ranging from 15 kDa to 197 kDa, like artisanal rennets. Protein composition of commercial rennets revealed the presence of bovine serum albumin, either native or degraded, and degraded chymosin. The results indicated that the source of coagulating enzymes and the conditions applied for enzyme extraction led to specific chemical compositions, polypeptide patterns and protein composition which are described in this article. (C) 2012 Elsevier Ltd. All rights reserved.

The objective of this work was to study the microbial diversity of natural whey starters (NWS), with a special focus on Lactococcus strains, during 10 successive days of backslopping. Each day, whey samples were enumerated for their Lactococcus, Enterococcus, Leuconostoc and Lactobacillus levels. The diversity and dynamics of the Lactococcus population in whey were investigated using phenotypic methods such as acidifying aptitude and enzymatic activities. Molecular approach using REP-PCR, ERIC-PCR, plamid profiles, phages and prophages research and PFGE was also applied. Lactococci were the main population in whey. Strains from raw milk were sub-dominant. Based on PFGE and phenotypic results lactococci in whey displayed a more heterogeneous phenotype and pulsotype which may reflect greater variations than previously observed within starter. No phages and prophages were spotted on. Plasmids did not seem to be exchanged from strain to strain. The backslopping practice seemed to allow the strains of the starter to rapidly acquire a specificity of their own. The changes observed presumed a slow adaptation of the strains to the “back- slopping environment”. The study of NWS diversity constitutes an important step for the comprehension of acidification defects that recurrently occur in cheesemaking technologies using the backslopping practice.

International audience; 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.

aeres : ACL; International audience; Although redox potential is very rarely taken into account in food fermentation it could be as influential as pH on bacterial activities. Lactococcus lactis is already known to exhibit a powerful reducing activity in milk but its reduction activity was shown to occur prior to its acidification activity with a potential interaction between these two lactococcal activities. Therefore, acidification lag-type phase could be shortened by decreasing the redox potential of milk before inoculation. As the redox potential is highly dependent on the dissolved oxygen level, our objective was to study their separate and combined influences on acidification and growth kinetics of pure L. lactis strains in milk. Results showed that high level of dissolved oxygen is significantly more influential on growth, and even more on acidification kinetics, than initial decreased redox potential of milk. Reduction of milk was drastic and mostly due to bacterial activity. The redox potential of milk only dropped when dissolved oxygen was entirely consumed. When there was no dissolved oxygen from the beginning, L. lactis immediately decreased the redox potential of milk and acidified afterwards. When the level of dissolved oxygen was initially high, acidification and reduction of milk occurred at the same time. Acidification kinetics was then biphasic with a slower rate during the aerobic stage and a faster rate during the anaerobic stage. The seven strains tested demonstrated diversity in both their acidification kinetics and their adaptation to high level of dissolved oxygen, independent of their growth kinetics. To conclude, we have shown that the level of dissolved oxygen in milk has a dramatic influence on acidification kinetics and could be used to control acidification kinetics in dairy industries.

This study investigated the genotypic characteristics of selected and wild homofermentative thermophilic lactobacilli strains during ripening of Comte cheeses, made into two cheese plants. Both amplification and restriction analysis of the 16S rRNA gene (PCR-ARDRA) and classical biochemical tests were used to identify isolates. Diversity within homofermentative lactobacilli was not found in their species composition since the same two species Lactobacillus helveticus and L. delbrueckii susbp. lactis were isolated from cheeses. In cheeses made with natural whey starter, it appeared that the most likely sources of L. helveticus and L. delbrueckii susbp. lactis were the starter and raw milk, respectively. The examination of RAPD profiles of lactobacilli strains revealed 19 RAPD groups among 50 isolates, which were different from selected starter strains. Using RAPD, REP-PCR, and PFGE to identify selected starter strains during cheese ripening, we showed that L. helveticus decreased quickly while L. delbrueckii susbp. lactis sustained high viability during ripening. The use of selected L. delbrueckii susbp. lactis strains diminished the genetic diversity among strains isolated from cheese, probably in preventing the raw milk microflora from growing.

La caracterisation microbiologique et biochimique des fromages de type suisse, et l'etude de facteurs influencant l'affinage et la qualite des fromages ont ete principalement basees sur des resultats experimentaux obtenus sur l'emmental, le comte et le beaufort. Au cours de l'affinage du comte, le nombre de bacteries lactiques thermophiles a diminue continuellement et celui des bacteries non levains a augmente. Les lactobacilles heterofermentaires facultatifs (FHL) et les bacteries propioniques (PAB) sont passes de 10 3 -10 4 a 10 8 ufc.g -1 , pour atteindre rapidement un plateau a la fin de la periode de pre-affinage pour les FHL, et plus tard en cave chaude pour les PAB. Les enterocoques sont restes a 10 3 -10 4 tout au long de l'affinage. Pendant l'affinage, Lactobacillus helveticus provenant uniquement du levain a rapidement disparu. L. delbrueckii ssp. etaient majoritaires dans le lait et le fromage. Plusieurs genotypes ont ete identifies pour L. delbrueckii ssp. lactis. Parmi les FHL, L. paracasei ssp. paracasei etaient majoritaires, avec trois biotypes identifies par RAPD ; L. plantarum et L. rhamnosus ont ete egalement isoles tout au long de l'affinage, Streptococcus thermophilus etait present dans tous les echantillons de lait et a ete trouve dans le fromage jusqu'a un mois. La diversite genetique etait plus grande pour les lactobacilles que pour S. thermophilus. Dans des mini-fromages modeles de comte. 68 % des souches de PAB appartenaient a Propionibacterium freudenreichii. Les differentes especes et sous-especes de PAB ont evolue differemment pendant l'affinage, avec d'importantes interactions entre LAB et le developpement des PAB, que ce soit au niveau des especes ou des souches. L'hydrolyse globale des caseines en fin d'affinage, et plus particulierement le nombre de petits peptides et d'acides amines libres (FAA), a augmente dans l'ordre suivant : emmental < comte < beaufort. En fin d'affinage, l'emmental avait le niveau de caseines natives et de α s1 -I-CN le plus eleve et celui de γ-CN le plus faible. La cartographie RP-HPLC de la fraction peptidique soluble dans l'eau a montre des differences importantes entre les trois varietes de fromages. La pasteurisation (Pa) ou la microfiltration (MF) du lait a redu it le nombre de microorganismes non-levains au stade initial de l'affinage, mais des niveaux en fin d'affinage identiques a ceux des fromages au lait cru (Ra) ont ete observes pour FHL et PAB. La pasteurisation a augmente l'activite de la plasmine (PLM) et induit une hydrolyse concomitante de β-CN. Les fromages Ra contenaient une importante quantite de FAA, avec des aromes plus intenses et plus diversifies que les fromages Pa ou MF. Le chauffage du melange caille/lactoserum a entraine l'activation du plasminogene en PLM. En augmentant la temperature de chauffage, l'activite de la chymosine diminue et la concentration et l'activite de la PLM augmentent, Ces resultats expliquent le niveau plus important de degradation de β-CN dans le comte et le beaufort que dans l'emmental, Les conditions d'affinage utilisees pour l'emmental augmentent la degradation de α s1 -CN.

In all, 4379 isolates from 35 products, including 24 artisanal cheeses, were surveyed with a view to identifying strains that could be used as starters in commercial dairy fermentations. Of the isolates, 38% were classified as Lactococcus, 17% as Enterococcus, 14% as Streptococcus thermophilus, 12% as mesophilic Lactobacillus, 10% as Leuconostoc and 9% as thermophilic Lactobacillus. Acid production by the isolates varied considerably. Of the 1582 isolates of Lactococcus and 482 isolates of mesophilic Lactobacillus tested, only 8 and 2% respectively produced sufficient acid to lower the pH of milk to < 5·3 in 6 h at 30 °C. In contrast, 53, 32 and 13% of Str. thermophilus, thermophilic Lactobacillus and Enterococcus isolates respectively reduced the pH to 5·3. These isolates were found only in some French, Italian and Greek cheeses. Bacteriocins were produced by 11% of the 2257 isolates tested and 26 of them produced broad-spectrum bacteriocins which inhibited at least eight of the ten target strains used, which included lactic acid bacteria, clostridia and Listeria innocua. The most proteolytic of the 2469 isolates tested were Str. thermophilus from Fontina cheese followed by Enterococcus from Fiore Sardo and Toma cheese and thermophilic Lactobacillus from all sources. Exopolysaccharides were produced by 5·3% of the 2224 isolates tested., The European Union is thanked for partly financing this project under ECLAIR contract CT-91-0064.

Summary - Using the same microfiltered milk, three 'raw milk' Swiss-type minicheeses were made after addition of three different retentates; one, remaining bacteria-free, was used as reference. The experiment was repeated three times at two different seasons. Factorial discriminant analyses were made on biochemical, physico-chemical and microbiological data at 12 weeks (end of warm storage, 18 "C) and 24 weeks (end of cold storage, 7 "C), At each ripening period, 'raw milk' cheeses were easily and significantly separated from the reference cheese. At 12 weeks, the three 'raw milk' cheeses were separated on the basis of propionic fermentation products, and to a lesser extent on proteolysis and levels of facultatively heterofermentative lactobacilli and starter lactic acid bacteria. At 24 weeks, differentiation was mainly based on proteolysis, since volatile fatty acids and lactate concentrations were nearly the same, irrespective of the cheeses. The same conclusions were drawn for win ter and autumn chee ses. Based on sensory evaluations made at the end of ripening, it was also possible to differentiate between the different 'raw milk' cheeses in winter and in autumn. Reference cheeses were always clearly separated from the other cheeses. The qualitative and quantitative importance of the indigenous microf1ora of milk on the biochemical and sensory characteristics of Swiss-type cheeses has been discussed.

La technique DEFT (direct epifluorescent filter technique), a ete employee pour le denombrement des cellules somatiques du lait cru. Quatre-vingt-six echantillons de lait cru ont ete analyses en double par la methode microscopique de reference, la technique DEFT et l'appareil automatique Fossomatic

National audience; Introduction et but de l’étude : L’alimentation de la vache laitière a un impact fort sur la qualité nutritionnelle des produits car il existe une relation entre les acides gras (AG) ingérés et ceux du lait. On peut améliorer la qualité nutritionnelle en introduisant dans l’alimentation des vaches laitières des AG intéressants pour la santé humaine. Il est donc important d’améliorer l'efficacité du transfert des AG de l'aliment des animaux à l'aliment des hommes. Deux voies d'amélioration de l'efficacité de transfert des AG n-3 ont été étudiées : nouvelle technique de traitement des graines de lin et utilisation de micro-algues riches en DHA. Matériel et méthodes : Ces aliments (lin riche LF+ ou pauvre en fibres LF-, micro-algues ALG, mélange micro-algues-lin riche en fibres LF+/ALG) ont été comparés à un régime témoin (ensilage de maïs, concentré céréales et tourteau de soja TEM) ou à ce même régime complété par du lin extrudé LIN. Cette expérimentation a été réalisée selon un schéma en continu sur 48 vaches laitières en milieu de lactation (6 lots de 8 vaches). Une analyse de covariance a été réalisée selon la procédure GLM de SAS. L’effet « traitement » a été testé. Résultats et Analyse statistique : Les traitements ALG et LF+/ALG ont induit une forte diminution de la production laitière (respectivement -2,8 et -6,0 kg/j) et de la teneur en matières grasses du lait (respectivement -17,3 et -17,9 g/kg). Le profil en AG des laits a été modifié : augmentation du pourcentage de C18:3 n-3 avec les traitements LIN, LF-, LF+ et LF+/ALG (respectivement 0,30, 0,28, 0,32 et 0,26 %), augmentation du C22:5 n-3 avec ALG et LF+/ALG (respectivement 0,43 et 0,15 %). Des fabrications beurrières et fromagères de type pâte pressée cuite ont été réalisées avec les laits TEM, LF+, ALG et LF+/ALG. Les paramètres de fabrication n’ont pas été différents entre TEM et LF+. Le beurre LF+ a eu tendance à être moins dur et était moins jaune. Concernant les propriétés sensorielles, le beurre LF+ avait une odeur et une flaveur rances moins prononcées (respectivement -1 et -1,2 points) et une flaveur crème plus intense (0,5 point) que le beurre TEM. Le rendement fromager en frais a été plus élevé avec LF+/ALG (0,52 kg/100 kg par rapport à TEM). Par contre, le rapport gras/sec et l’extrait sec du caillé ont été moins élevés avec ALG et LF+/ALG et le lactosérum était plus riche en matières grasses. Les fromages ALG et LF+/ALG ont été jugés moins colorés, plus ouverts, moins fermes, plus élastiques, plus granuleux et plus piquants. Conclusion : En conclusion, le lin LF+ est très bien valorisé par les vaches laitières. Les laits sont enrichis en C18:3 et sont similaires à ceux du lin « normal ». Ils ont de bonnes aptitudes à la transformation beurrière et fromagère et les produits obtenus ont de bonnes propriétés sensorielles. Les micro-algues ont eu un effet exacerbé : les vaches ont produit moins de lait mais un lait demi-écrémé enrichi en DHA. Des essais seraient nécessaires pour trouver la bonne dose et le bon ratio LF+/ALG afin de valoriser au mieux leur intérêt nutritionnel.