Au Québec, plusieurs fromagers cherchent à produire des fromages du terroir, aux caractéristiques uniques, à partir d’un ou de quelques laits dont la composition est influencée par des facteurs génétiques et environnementaux. Ce projet vise à démontrer si des laits du terroir québécois crus et pasteurisés, ainsi que certaines de leurs composantes laitières (perméat (P), caséines (CN), protéines de lactosérum (IPL) et concentrés de bactéries (CB)), obtenues par microfiltration et ultrafiltration du lait, ont un impact sur la croissance et l’activité de microflores laitières. La croissance de Lactococcus lactis subsp. lactis et de Lactococcus lactis subsp. cremoris dans des laits du terroir crus et pasteurisés a été étudiée et le développement de trois souches de Geotrichum candidum a été suivi dans des caillés modèles faits à partir de certains de ces laits. De plus, la croissance de bactéries lactiques dans des mélanges de fractions laitières (P-CN-IPL, P-CN, P-IPL, P et P-CB) d’un lait du terroir et d’un lait industriel crus et pasteurisés a été évaluée. Les résultats ont démontré que l’origine des laits du terroir et le traitement thermique ont influencé le développement de certaines souches de lactocoques, alors que le développement de G. candidum était influencé par son caractère dimorphique. Dans les mélanges laitiers, la croissance des lactocoques était moins élevée dans les mélanges de laits pasteurisés que de laits crus, ce qui est contraire aux observations faites dans les laits entiers. La microfiltration 1,4 µm, lors du retrait de la fraction CB, semble avoir retenu des facteurs de croissance qui se retrouveraient dans les laits pasteurisés et qui favoriseraient la croissance de certaines bactéries lactiques. Ainsi, l’origine du lait, la pasteurisation et la filtration du lait sur membranes ont un impact sur le développement des microflores laitières et il est important pour le maître-fromager de s’adapter à ces changements. / In the province of Quebec, several cheese makers seek to produce local cheeses with unique characteristics, from one or a few milks whose composition is influenced by genetic and environmental factors. The aim of this project was to demonstrate whether raw and pasteurized milk from Québec, and certain milk components (permeate (P), caseins (CN), whey proteins (IPL) and concentrated bacteria (CB)), obtained by using microfiltration and ultrafiltration of milk, have an impact on the growth and activity of dairy microflora. The growth of Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris in the raw and pasteurized local milks was studied and the development of three strains of Geotrichum candidum was followed in curds models made from some of these milks. In addition, the growth of lactic acid bacteria in mixtures of milk fractions (P-CN-IPL, P-CN, P-IPL, P and P-CB) of raw and pasteurized local milk and an industrial milk was evaluated. Results showed that the origin of the local milk and the heat treatment influenced the development of certain strains of Lactococcus, whereas the development of G. candidum was influenced by its dimorphic character. In dairy mixes, the growth of lactococci was lower in pasteurized milk mixtures than in raw milk mixtures, contrary to observations made in whole milk. The microfiltration at 1,4 µm, when removing the CB portion, seems to have retained growth factors that would be found in pasteurized milk and that would promote the growth of certain lactic acid bacteria. In this way, the origin of milk, pasteurization and filtration of milk on membranes have an impact on the development of dairy microflora and it is important for the cheese maker specialist to adapt to these changes.

Yeasts are traditionally used as part of the surface microflora in surface-ripened cheeses, where they contribute positively to the flavour of the cheese. The primary objective of this study was to investigate the potential of three dairy yeasts to provide attributes as adjuncts in anaerobically ripened cheeses. Geotrichum candidum (B9001), Yarrowia lipolytica (B9014) and Candida kefyr (B9006), obtained from the Fonterra Co-operative Group Ltd, Palmerston North, New Zealand, were studied. They showed diverse metabolic activities in laboratory media, which were influenced by the growth conditions. The metabolic activities of special interest were the lipase and proteinase activities and the production of volatile compounds, as these are important for cheese ripening and flavour development. Lipase activity (p-nitrophenyl butyrate assay) and proteinase activity (fluorescein isothiocyanate β-casein assay) were determined in three fractions prepared from yeast cultures and designated as extracellular fraction, washed-cell fraction and intracellular fraction. Lipase activity of G. candidum was detected only in the extracellular fraction and increased five fold when induced by safflower oil in a shake culture (0.16 µM/min/mL supernatant at 24 h). Lipase expression was delayed in static cultures. Y. lipolytica showed lipase activity in extracellular, washed-cell and intracellular fractions under all conditions. Static cultures in both glucose and safflower oil media showed higher lipase activity than shake cultures. The lipase activity of Y. lipolytica was higher in the late stationary phase than in the log phase under all conditions tested. The highest lipase activity was detected in a 192 h static culture grown in safflower oil medium (0.13 µM/min/mg dry cell weight, 0.3 µM/min/mg dry cell weight and 4.29 µM/min/mL supernatant in the intracellular, washed-cell and extracellular fractions respectively). C. kefyr did not show any lipase activity (< 0.03 µM/min/mL culture) under any of the growth conditions tested. Proteinase activity was detected in the intracellular fraction of 72 h shake cultures of G. candidum grown in both glucose medium and safflower oil medium (154 and 122 RFU/min/mg dry cell weight respectively) but was not detected in static cultures. Proteinase activity was absent in the Y. lipolytica cultures under all conditions tested (< 10 RFU/min/mL culture). C kefyr showed low proteinase activity (12-74 RFU/min/mL supernatant) in the extracellular fraction only in shake cultures grown in glucose medium. Volatile compounds of the headspace were sampled and analysed using solid phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS). The concentrations of volatile compounds were highest in shake cultures grown in glucose medium for all three yeasts. All yeasts produced several alcohols. Several esters were also detected in the G. candidum and C. kefyr cultures whereas aldehydes were detected only in the G. candidum cultures. G. candidum and Y. lipolytica were selected for cheese production trials because of their active cheese ripening enzymes. These yeasts, grown under different conditions, were added to Cheddar cheese (10 L vat). The yeast adjuncts influenced the cheese ripening by lipolysis [in terms of the production of free fatty acids (FFAs) analysed by gas chromatography-flame ionisation detector (GC-FID)] and the production of volatile compounds (SPME-GC-MS), whereas proteolysis (analysed by size-exclusion high performance liquid chromatography) by yeast enzymes was not obvious. The influence of Y. lipolytica as an anaerobic adjunct to cheese ripening was dependent on the growth conditions used during its propagation in laboratory media. The concentration of total FFAs was very high (37.1 mg/g cheese at 6 months) when a 192 h Y. lipolytica culture grown in safflower oil medium was added to a cheese make, whereas the cultures grown in glucose medium did not have any detectable effect. Addition of G. candidum culture to the cheese curd was more effective than its addition to the cheese milk. Both G. candidum and Y. lipolytica lipase(s) selectively hydrolysed the long-chain unsaturated fatty acids from the milk triglyceride in the cheese environment. Also, Y. lipolytica lipase exhibited some selectivity towards hydrolysis of butyric acid from the milk fat in the cheese. 2-Heptanone, 3-methyl-2-butanone and 2-nonanone were detected (1-10 x 106 relative peak area) only in the cheeses with yeast adjuncts but not in the control cheese. Enhancement of the production of both conjugated linoleic acid (CLA) and ethyl esters in a washed-curd, dry-salted cheese (375 L vat), made with G. candidum, Y. lipolytica, Propionibacterium freudenreichii ssp. shermanii, Lactobacillus fermentum and Lb. rhamnosus, was only partially successful. Higher concentrations of ethyl esters (> five fold; analysed by SPME-GC-MS) were produced in the cheeses made with yeast adjuncts. However, the concentration of total CLA (free plus esterified; analysed by GC-FID) did not increase although a higher concentration of free linoleic acid (> 10 fold), the substrate for CLA synthesis, was produced in the cheeses made with yeast adjuncts. A study of the formation of aromatic volatile compounds by C. kefyr in a medium containing L-phenylalanine (L-phe) showed that the yeast's ability to produce phenyl ethanol, phenyl ethyl acetate and benzaldehydc (analysed by SPME-GC-MS) was enhanced with an increase in the initial L-phe concentration (in the experimental range; analysed by enzymatic assay using phenylalanine ammonia lyase), but the yield was very low (20-27%). The initial concentration of glucose (in the experimental range; analysed by enzymatic assay using Peridochrom glucose reagent) did not affect the production of these aromatic volatile compounds. This study successfully showed that the yeasts G. candidum and Y. lipolytica, when used as anaerobic adjuncts, can influence the ripening and flavour development in Cheddar and washed-curd, dry-salted cheeses. The study also showed the capability of C. kefyr to produce aromatic volatile compounds from amino acid fermentation but the yields need to be increased by further manipulation of the medium components and the culture conditions before this capability can be used commercially.