The Secreted Esterase of Propionibacterium freudenreichii Has a Major Role in Cheese Lipolysis

Free fatty acids are important flavor compounds in cheese. Propionibacterium freudenreichii is the main agent of their release through lipolysis in Swiss cheese. Our aim was to identify the esterase(s) involved in lipolysis by P. freudenreichii. We targeted two previously identified esterases: one secreted esterase, PF#279, and one putative cell wall-anchored esterase, PF#774. To evaluate their role in lipolysis, we constructed overexpression and knockout mutants of P. freudenreichii CIRM-BIA1^T for each corresponding gene. The sequences of both genes were also compared in 21 wild-type strains. All strains were assessed for their lipolytic activity on milk fat. The lipolytic activity observed matched data previously reported in cheese, thus validating the relevance of the method used. The mutants overexpressing PF#279 or PF#774 released four times more fatty acids than the wild-type strain, demonstrating that both enzymes are lipolytic esterases. However, inactivation of the pf279 gene induced a 75% reduction in the lipolytic activity compared to that of the wild-type strain, whereas inactivation of the pf774 gene did not modify the phenotype. Two of the 21 wild-type strains tested did not display any detectable lipolytic activity. Interestingly, these two strains exhibited the same single-nucleotide deletion at the beginning of the pf279 gene sequence, leading to a premature stop codon, whereas they harbored a pf774 gene highly similar to that of the other strains. Taken together, these results clearly demonstrate that PF#279 is the main lipolytic esterase in P. freudenreichii and a key agent of Swiss cheese lipolysis.     

Alicyclobacillus acidocaldarius Thermophilic Esterase EST2's Activity in Milk and Cheese Models

The aim of this work was to investigate the behavior of thermophilic esterase EST2 from Alicyclobacillus acidocaldarius in milk and cheese models. The pure enzyme was used to compare the EST2 hydrolytic activity to the activity of endogenous esterase EstA from Lactococcus lactis. The results indicate that EST2 exhibits 30-fold-higher esterase activity than EstA. As EstA has thioesterase activity, EST2 was assayed for this activity under the optimal conditions determined for EstA (namely, 30°C and pH 7.5). Although it is a thermophilic enzyme, EST2 exhibited eightfold-higher thioesterase activity than EstA with S-methyl thiobutanoate. The abilities of EST2 and EstA to synthesize short-chain fatty acid esters were compared. Two methods were developed to do this. In the first method a spectrophotometric assay was used to monitor the synthesis of esters by the pure enzymes using p-nitrophenol as the alcohol substrate. The synthetic activities were also evaluated under conditions that mimicked those present in milk and/or cheese. The second method involved evaluation of the synthetic abilities of the enzymes when they were directly added to a model cheese matrix. Substantial ester synthesis by EST2 was observed under both conditions. Finally, esterase and thioesterase activities were evaluated in milk using the purified EST2 enzyme and in the model cheese matrix using a strain of L. lactis NZ9000 harboring the EST2 gene and thus overproducing EST2. Both the esterase and thioesterase activities measured in milk and in the cheese matrix were much greater than the activities of the controls.     

Alicyclobacillus acidocaldarius thermophilic esterase EST2's activity in milk and cheese models

The aim of this work was to investigate the behavior of thermophilic esterase EST2 from Alicyclobacillus acidocaldarius in milk and cheese models. The pure enzyme was used to compare the EST2 hydrolytic activity to the activity of endogenous esterase EstA from Lactococcus lactis. The results indicate that EST2 exhibits 30-fold-higher esterase activity than EstA. As EstA has thioesterase activity, EST2 was assayed for this activity under the optimal conditions determined for EstA (namely, 30 degrees C and pH 7.5). Although it is a thermophilic enzyme, EST2 exhibited eightfold-higher thioesterase activity than EstA with S-methyl thiobutanoate. The abilities of EST2 and EstA to synthesize short-chain fatty acid esters were compared. Two methods were developed to do this. In the first method a spectrophotometric assay was used to monitor the synthesis of esters by the pure enzymes using p-nitrophenol as the alcohol substrate. The synthetic activities were also evaluated under conditions that mimicked those present in milk and/or cheese. The second method involved evaluation of the synthetic abilities of the enzymes when they were directly added to a model cheese matrix. Substantial ester synthesis by EST2 was observed under both conditions. Finally, esterase and thioesterase activities were evaluated in milk using the purified EST2 enzyme and in the model cheese matrix using a strain of L. lactis NZ9000 harboring the EST2 gene and thus overproducing EST2. Both the esterase and thioesterase activities measured in milk and in the cheese matrix were much greater than the activities of the controls.     

Identification of the possible new odor-active compounds “12-methyltridecanal and its analogs” responsible for the characteristic aroma of ripe Gouda-type cheese

The aroma concentrates of the three maturation stages of Gouda-type cheeses were prepared by combining the solvent extraction and the solvent assisted flavor evaporation techniques. The aroma extract dilution analysis applied to the volatile fraction revealed 31 odorants that were identified or tentatively identified from the 38 odor-active peaks with FD factors between 4³ and 4⁸. By comparison with the FD factors in the three maturation stages of the cheeses, 16 odorants, including 12-methyltridecanal, which is a newly identified odorant from the cheese, increased with the increasing maturation stage of the cheese. In addition, many iso- and anteiso-methyl-branched long-chain aliphatic aldehydes could be identified as the analogs of 12-methyltridecanal, which have a unique odor note. It may be then expected that these aldehydes were able to influence the flavor of the highly ripened Gouda cheese, since these compounds also increased with the increasing maturation stage.     

Genotypic and phenotypic characterization of the dynamics of the lactic acid bacterial population of adjunct-containing Cheddar cheese manufactured from raw and microfiltered pasteurised milk

AIMS: This study investigates the dynamics of the microflora, particularly the lactobacilli, in Cheddar cheese manufactured from raw and microfiltered milk containing different adjunct cultures. METHODS AND RESULTS: Sixteen cheeses - raw milk, adjunct and control cheeses - were manufactured in four trials. Lactobacilli were identified by PCR methods in one trial, and by phenotypic typing for all trials. Numbers of lactobacilli were significantly different at day 1 and 3 months in the control and adjunct-containing cheeses. In the raw milk cheeses, Lactobacillus paracasei was detected throughout ripening, Lact. curvatus at the end, and Lact. plantarum at day 1 only. Lactobacillus strain diversity decreased from raw, control to adjunct cheeses. Enteroccoci and coliform numbers further differentiated raw cheeses from the others. Lactococcal starter numbers also differed in the three cheese types and differences were observed within adjunct cheeses. Although adjunct lactobacilli dominated in the cheese to which they were added, strains with similar phenotypic profiles were also detected on occasions in some of the control cheeses. CONCLUSIONS: The addition of adjunct lactobacilli modified the growth kinetics of both adventitious lactobacilli and starter lactococci during ripening. Appropriate strain tracking is necessary to monitor changes in the population profiles of control and experimental cheeses in trials utilizing adjunct cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Investigations of the role of adjunct strain(s) in cheeses may be complicated by the interactions between the adjunct and the other cheese strains, and effective strain monitoring by genotypic or phenotypic methods is essential if valid comparisons are to be made.     

Predictive formulae for goat cheese yield based on milk composition

Prediction of the yield and quality of different types of cheeses that could be produced from a given type and/or amount of goat milk is of great economic benefit to goat milk producers and goat cheese manufacturers. Bulk tank goat milk was used for manufacturing hard, semi-hard and soft cheeses (N = 25, 25 and 24, respectively) to develop predictive formulae of cheese yield based on milk composition. Fat, total solids, total protein and casein contents in milk and moisture-adjusted cheese yield were determined to establish relationships between milk composition and cheese yield. Soft, semi-hard and hard cheeses in this study had moisture contents of 66, 46 and 38%, respectively, which could be used as reference standards. In soft cheese, individual components of goat milk or a combination of two or three components predicted cheese yield with a reasonably high correlation coefficient (R² = 0.73–0.81). However, correlation coefficients of predictions were lower for both semi-hard and hard cheeses. Overall, total solids of goat milk was the strongest indicator of yield in all three types of cheeses, followed by fat and total protein, while casein was not a good predictor for both semi-hard and hard cheeses. When compared with moisture-adjusted cheese yield, there was no difference (P > 0.05) in predicting yield of semi-hard and hard goat milk cheeses between the developed yield formulae in this study and a standard formula (the Van Slyke formula) commonly used for cow cheese. Future research will include further validation of the yield predictive formulae for hard and semi-hard cheeses of goat milk using larger data sets over several lactations, because of variation in relationships between milk components due to breed, stage of lactation, season, feeding regime, somatic cell count and differences in casein variants.     

Technological properties of Enterococcus faecium isolated from ewe's milk and cheese with importance for flavour development

Eight Enterococcus faecium strains isolated from ewe milk and artisanal cheese from northwest Argentina were screened for biotechnological properties relevant to flavour development. The API ZYM test showed absence of proteases, presence of high amounts of peptidases, and high esterase-lipase activities. Low extracellular proteolytic activity was observed. Most strains produced diacetyl in milk, with E. faecium OvL 214 and OvL 254 being the best producers. Biomass and growth rate increased when citrate was added to the medium, suggesting that these strains could use citrate as a main energy source. After 24 h of incubation, citrate was completely consumed in complex medium supplemented with glucose and citrate. An average of 17% residual citrate was detected in complex media supplemented with citrate. For all strains, esterase activity was detected up to alpha-naphthyl-caproate. They hydrolyzed alpha-naphthyl derivatives of fatty acids in this order: C3 > C6 > C4 > C8 > C2. Post-electrophoretic detection of esterase activities revealed the presence of multiple esterases. Hydrolysis of tributyrin, tricaprylin, and milk fat was observed in cell-free extracts. Enterococcus faecium strains isolated from ewe milk and artisanal cheese from northwest Argentina present the metabolic potential to contribute to cheese flavour development.     

The chemistry of some microbially induced flavor defects in milk and dairy foods

The “malty” flavor defect that commonly develops in raw milk produced in certain areas of North America and Europe has long been known to be due to the metabolic activity of Streptococcus lactis var. maltigenes. The identification of the aldehydes and alcohols responsible for this flavor defect and the mechanisms involved in their formation from amino acids are discussed. Pseudomonas fragi, a common psychrophilic recontaminant, is responsible for development of “fruity” flavors in processed dairy products by virtue of the organisms ability to hydrolyze milk fat and esterify certain of the lower fatty acids with ethanol. A similar esterase is present in certain lactic cultures used in the manufacture of cheddar cheese. The “musty potato” aroma first described in eggs and milk and other dairy products due to the growth of Pseudomonas graveolens (Pseudomonas taetrolens) continues to be reported as a defect in eggs and carcass meats. Pseudomonas perolens has been found to produce a similar aroma in spoiling fish. Vapors entrained from milk and fish tissue cultures of these organisms, collected on porous polymer traps and analyzed by GLC-alkali flame and GLC-MS systems, revealed both organisms produce 2-methoxy-3-alkylpyrazines. 2-Methyoxy-3-isopropylpyrazine was found to be responsible for the musty potato aroma. A possible mechanism for the formation of pyrazines is discussed.     

Optimized enrichment for the detection of Escherichia coli O26 in French raw milk cheeses

Aims: Our main objective was to optimize the enrichment of Escherichia coli O26 in raw milk cheeses for their subsequent detection with a new automated immunological method. Methods and Results: Ten enrichment broths were tested for the detection of E. coli O26. Two categories of experimentally inoculated raw milk cheeses, semi‐hard uncooked cheese and ‘Camembert’ type cheese, were initially used to investigate the relative efficacy of the different enrichments. The enrichments that were considered optimal for the growth of E. coli O26 in these cheeses were then challenged with other types of raw milk cheeses. Buffered peptone water supplemented with cefixim–tellurite and acriflavin was shown to optimize the growth of E. coli O26 artificially inoculated in the cheeses tested. Despite the low inoculum level (1–10 CFU per 25 g) in the cheeses, E. coli O26 counts reached at least 5·10⁴ CFU ml^?1 after 24‐h incubation at 41·5°C in this medium. Conclusions: All the experimentally inoculated cheeses were found positive by the immunological method in the enrichment broth selected. Significance and Impact of the Study: Optimized E. coli O26 enrichment and rapid detection constitute the first steps of a complete procedure that could be used in routine to detect E. coli O26 in raw milk cheeses.     

Biosynthesis of flavors by Penicillium roqueforti

The development of the unique flavor of blue type cheese depends on the concerted action of numerous enzymes of Penicillium roqueforti involved in protein and lipid metabolism. Protease(s) by degrading casein modify the texture and background flavor of the ripening cheese. Lipase by hydrolyzing milk triglycerides provides flavorful fatty acids and precursors of methyl ketones. The enzyme complex involved in the partial oxidation of free fatty acids and the properties of β-ketoacyl decarboxylase which generates the major flavor components of blue cheese are discussed. Fermentation of P. roqueforti for the rapid production of methyl ketones is briefly reviewed.     

Nonspecific esterases of Mus musculus

Seventeen genes controlling the expression of carboxylic ester hydrolases, commonly known as esterases, have been identified in the mouse Mus musculus. Seven esterase loci are found on chromosome 8, where two clusters of esterase loci occur. It seems probable that the genes within these clusters have arisen from a common ancestral gene by tandem duplication. Close linkage of esterase genes is also found in the rat, rabbit, and prairie vole. Some mouse esterases appear to be homologous with certain human esterases. The function of these nonspecific enzymes is still unknown.     

Effects of aging on functional properties of caprine milk made into Cheddar- and Colby-like cheeses

The effects of cheese milk obtained at three times during lactation (weeks 4–5, 12–15, and 21–23) and cheese storage (up to 16 or 24 weeks) on meltability, sliceability, and color changes upon heating (232 °C for 5 min, high baking temperature, HT, or 130 °C for 75 min, low baking temperature, LT) of caprine milk cheeses were evaluated. The cheeses were manufactured from milk from Alpine goats and based on the procedures of Cheddar and Colby cheese manufacture. In Cheddar-like cheese, the sliceability (force required to slice sample) was at its highest when the cheese was made with milk from weeks 12–15 into lactation. Color change was variable although it tended to be lowest in cheese made at weeks 4–5 into lactation. In Colby-like cheeses, meltability was at its highest and sliceability was very poor (after 8 weeks of aging) when made with milk obtained later in lactation. Color changes were variable at the two different baking temperatures. As expected during aging, the meltability of the cheeses increased and the force required to slice the cheeses decreased with the significant changes occurring within the first 16 weeks for Cheddar-like and the first 8 weeks for Colby-like cheeses. The color changes upon heating were variable for aged Cheddar-like cheeses and did not change significantly for aged Colby-like cheeses. Color changes were highly correlated with proteolysis occurring during storage. Cheese milk obtained at different times of lactation and aging of the cheese impact the functional properties of caprine milk cheeses and will affect their optimal utilization.