Effect of Beta Cyclodextrin on the Reduction of Cholesterol in Ewe's Milk Manchego Cheese.

Beta-cyclodextrin (ß-CD) is a cyclic oligosaccharide consisting of seven glucose units and is produced from starch using cyclodextrin glycotransferase enzymes to break the polysaccharide chain and forming a cyclic polysaccharide molecule. The use of ß-CD in food research for reduction of cholesterol is increasing due to its affinity for non-polar molecules such as cholesterol. The aim of this study was to evaluate the feasibility of using ß-CD in cholesterol removal from pasteurized ewe's milk Manchego cheese and evaluate the effect on the main components of the milk, lipids, and flavor characteristics. Approximately 97.6% cholesterol reduction was observed in the cheese that was treated using ß-CD. Physicochemical properties (fat, moisture and protein) were not changed by the ß-CD treatment, except the soluble nitrogen and non-protein nitrogen that showed slight differences after the treatment. The amount of the different components of the lipid fraction (fatty acids, triglycerides and phospholipids) were similar in cheeses treated and not treated with ß-CD. Flavor compound and short chain free fatty acids were not mostly significantly influenced by the effect of the ß-CD. ß-CD molecules are edible and nontoxic and as a result they can be used safely for cholesterol removal processing in cheese manufacturing. Therefore, the present study suggests that ß-CD treatment is an effective process for cholesterol removal from Manchego cheese while preserving its properties.

Proteomic comparison of equine and bovine milks on renneting.

Rennet-induced coagulation of bovine milk is a complex mechanism in which chymosin specifically hydrolyzes κ-casein, the protein responsible for the stability of the casein micelle. In equine milk, this mechanism is still unclear, and the protein targets of chymosin are unknown. To reveal the proteins involved, the rennetability of equine milk by calf chymosin was examined using gel-free and gel-based proteomic analysis and compared to bovine milk. RP-HPLC analysis of bovine and equine milks showed the release of several peptides following chymosin incubation. The hydrolyses of equine and bovine casein by chymosin were different, and the major peptides produced from equine milk were identified by mass spectrometry as fragments of ß-casein. Using two-dimensional electrophoresis, equine ß-casein was confirmed as the main target of calf chymosin over 24 h at 30 °C and pH 6.5. The gel-based analysis of equine milk discriminated between the different individual proteins and provided information on the range of isoforms of each protein as a result of post-translational modifications, as well as positively identified for the first time several isoforms of κ-casein. In comparison to bovine milk, κ-casein isoforms in equine milk were not involved in chymosin-induced coagulation. The intensity of equine ß-casein spots decreased following chymosin addition, but at a slower rate than bovine κ-casein.

Comparison of the level of residual coagulant activity in different cheese varieties.

The coagulant retained in cheese curd is a major contributor to proteolysis during ripening. The objective of this study was to quantify residual coagulant in 9 cheese varieties by measuring its activity on a synthetic heptapeptide (Pro-Thr-Glu-Phe-[NO2-Phe]-Arg-Leu) assayed using reversed-phase HPLC. The level of residual coagulant activity was highest in Camembert cheese, probably due to its low pH at whey drainage and the high moisture content of the cheese, followed in order by Feta=Port du Salut=Cheddar>Gouda>Emmental=Parmigiano Reggiano=low-moisture part-skim Mozzarella=Mozzarella di Bufala Campana. The high cooking temperature (50-54 degrees C) used during the manufacture of Emmental and Parmigiano Reggiano cheeses and the cooking and stretching step in hot water during the manufacture of Mozzarella cheese may be the reasons for the lowest residual coagulant activity in these cheeses. The level of residual coagulant activity was higher in Feta cheese made from milk concentrated by ultrafiltration than in conventional Feta.

Compositional, microbiological, biochemical, volatile profile and sensory characterization of four Italian semi-hard goats' cheeses.

Four semi-hard Italian goats' milk cheeses, Flor di Capra (FC), Caprino di Cavalese (CC), Caprino di Valsassina (CV) and Capritilla (C), were compared for compositional, microbiological, biochemical, volatile profile and sensory characteristics. Mean values for the gross composition in part differed between cheeses. At the end of ripening, cheeses contained 7.98-8.51 log10 cfu/g of non-starter lactic acid bacteria. Lactobacillus paracasei, Lb. casei and Lb. plantarum were dominant in almost all cheeses. As shown by the Principal Component Analysis of RP-FPLC data for the pH 4.6-soluble fractions and by the determination of free amino acids, secondary proteolysis of CC and CV mainly differed from the other two cheeses. A total of 72 volatile components were identified by steam distillation-extraction followed by gas chromatography-mass spectrometry. Free fatty acids and esters qualitatively and quantitatively differentiated the profile of CV and CC, respectively. The lowest concentrations of volatile components characterized FC. Descriptive sensory analysis using 17 flavour attributes was carried out by a trained panel. Different flavour attributes distinguished the four goats' cheeses and relationships were found with volatile components, biochemical characteristics and technology.

Factors affecting the retention of rennet in cheese curd.

The coagulant retained in cheese curd is a major contributor to proteolysis during ripening. The objective of this study was to quantify the effects of several milk-related factors and parameters during cheese manufacture on the retention of coagulant in cheese curd. The amount of coagulant retained in curd was determined by its activity on a synthetic heptapeptide (Pro-Thr-Glu-Phe-[NO2-Phe]-Arg-Leu) using reversed-phase HPLC. The retention of chymosin in cheese curd increased significantly when the pH of milk was reduced at rennet addition below pH 6.1, the pH at whey drainage below pH 5.7, or the average casein micelle size in milk and when the ionic strength of milk was increased. The casein content of milk and the quantity of chymosin added to milk had no significant effect on the retention of chymosin in curd; the quantity of coagulant bound per gram of casein remained unchanged.

Aggregation of rennet-altered casein micelles at low temperatures.

The rennet-induced coagulation of bovine milk at 10 degrees C was investigated. The rate of change of absorbance at 600 nm was higher in milk renneted at 30 degrees C than that at 10 degrees C. The amount of casein sedimented on centrifuging skim milk at 5000g for 1 h at 10 degrees C increased with time after renneting. The viscosity of milk at 10 degrees C at low shear rates did not change significantly until 10 h after rennet addition, but it increased markedly after 20 h. Smaller particles in milk at 10 degrees C disappeared slowly over 36 h after rennet addition and aggregated into larger particles. These results suggested that casein micelles in milk aggregate at low temperatures. Reasons for the slow aggregation of milk renneted at 10 degrees C were investigated by inhibiting chymosin activity by pepstatin A. It is likely that beta-casein, or its hydrolysis, plays a role in aggregation of rennet-altered casein micelles at low temperatures.

Response of Lactobacillus helveticus PR4 to heat stress during propagation in cheese whey with a gradient of decreasing temperatures.

The heat stress response was studied in Lactobacillus helveticus PR4 during propagation in cheese whey with a gradient of naturally decreasing temperature (55 to 20 degrees C). Growth under a gradient of decreasing temperature was compared to growth at a constant temperature of 42 degrees C. Proteinase, peptidase, and acidification activities of L. helveticus PR4 were found to be higher in cells harvested when 40 degrees C was reached by a gradient of decreasing temperature than in cells grown at constant temperature of 42 degrees C. When cells grown under a temperature gradient were harvested after an initial exposure of 35 min to 55 degrees C followed by decreases in temperature to 40 (3 h), 30 (5 h 30 min), or 20 degrees C (13 h 30 min) and were then compared with cells grown for the same time at a constant temperature of 42 degrees C, a frequently transient induction of the levels of expression of 48 proteins was found by two-dimensional electrophoresis analysis. Expression of most of these proteins increased following cooling from 55 to 40 degrees C (3 h). Sixteen of these proteins were subjected to N-terminal and matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses. They were identified as stress proteins (e.g., DnaK and GroEL), glycolysis-related machinery (e.g., enolase and glyceraldehyde-3-phosphate dehydrogenase), and other regulatory proteins or factors (e.g., DNA-binding protein II and ATP-dependent protease). Most of these proteins have been found to play a role in the mechanisms of heat stress adaptation in other bacteria.

Influence of ethanol on the rennet-induced coagulation of milk.

The influence of ethanol on the rennet-induced coagulation of milk was studied to investigate potential synergistic effects of these two mechanisms of destabilisation on the casein micelles. Addition of 5% (v/v) ethanol reduced the rennet coagulation time (RCT) of milk, whereas higher levels of ethanol (10-20%, v/v) progressively increased RCT. The temperature at which milk was coagulable by rennet decreased with increasing ethanol content of the milk. The primary stage of rennet coagulation, i.e., the enzymatic hydrolysis of kappa-casein, was progressively slowed with increasing ethanol content (5-20%, v/v), possibly due to ethanol-induced conformational changes in the enzyme molecule. The secondary stage of rennet coagulation, i.e., the aggregation of kappa-casein-depleted micelles, was enhanced in the presence of 5-15% ethanol, the effect being largest at 5% ethanol. Enhanced aggregation of micelles is probably due to an ethanol-induced decrease in inter-micellar steric repulsion. These results indicate an interrelationship between the effects of ethanol and chymosin on the casein micelles in milk, which may have interesting implications for properties of dairy products.

High pressure-induced changes in bovine milk proteins: a review.

High pressure (HP)-induced changes in the proteins of bovine milk have become an area of considerable research interest in recent years; as a result, there is now a detailed understanding of the effects of HP on casein micelles and whey proteins. HP treatment at pressures >400 or >100 MPa denatures the two most abundant whey proteins, alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg), respectively. The majority of denatured beta-lg in HP-treated milk associates with the casein micelles, although some denatured beta-lg remains in the serum phase or is attached to the milk fat globule membrane; HP-denatured alpha-la is also associated with the milk fat globules. Casein micelles are disrupted on treatment at pressures >200 MPa; the rate and extent of micellar disruption increases with pressure and is probably due to the increased solubility of calcium phosphate with increasing pressure. On prolonged treatment at 250-300 MPa, reassociation of micellar fragments occurs through hydrophobic bonding; this process does not occur at a pressure >300 MPa, leading to considerably smaller micelles in such milk. As a result of HP-induced changes, the size, number, hydration, composition and light-scattering properties of casein micelles in HP-treated milk differ considerably from those in untreated milk.

Effects of high pressure on some constituents and properties of buffalo milk.

In this study, effects of high pressure (HP) on some constituents and properties of buffalo milk were examined. HP treatment at 100-600MPa for 30 min affected casein micelle size only slightly, whereas treatment at 800 MPa increased it by approximately 35%. Levels of non-micellar alpha(S1)and beta-caseins were increased by treatment > or = 250MPa, and were highest after treatment at 400-800MPa. The level of non-micellar calcium increased with increasing pressure up to 600 MPa. The L*-value of the milk decreased gradually with increasing pressure, from approximately 82 for untreated milk to approximately 65 for milk treated at 800 MPa. Milk pH was increased by approximately 0.07 units after treatment at 100-800 MPa, with no significant difference between treatment pressures. Denaturation of alpha-lactalbumin occurred at pressures > or = 400 MPa, and reached >90% after treatment at 800 MPa, whereas beta-lactoglobulin (beta-Ig) was denatured > 100 MPa, reaching approximately 100% after treatment at 400MPa; after treatment > or = 400MPa, all beta-Ig was associated with the casein micelles. The rennet coagulation time of buffalo milk increased with increasing pressure, whereas the strength of the coagulum formed decreased after treatment at 250-800 MPa. Overall, HP treatment affected many constituents and properties of buffalo milk; some of these effects have also been observed in the milk from other species, but the extent of the effects, and the pressure at which they occurred, differed considerably.

Potential applications of high pressure homogenisation in processing of liquid milk.

Studies of the potential of high pressure homogenisation (HPH) for the combined pasteurisation/ homogenisation of raw bovine milk were undertaken. Raw milk was preheated to 45 degrees C and HPH-treated at 150, 200 or 250 MPa; milk outlet temperature at these pressures were 67, 76.8 and 83.6 degrees C, respectively, with a holding time of approximately 20 s. Raw and commercially pasteurized and homogenized (CPH) milk samples were analysed as controls. Fat globules in HPH samples were approximately half the size of those in CPH samples, although differences were not significant (P>0.05). beta-Lactoglobulin was denatured at pressures > or =150MPa, although little denaturation of alpha-lactalbumin was observed. Numbers of psychrotrophic bacteria in raw milk were reduced by 2.73 log cycles by HPH at 150 MPa and were uncountable following HPH at 200 or 250 MPa. Mesophilic bacterial counts were reduced by 1.30, 1.83 and 3.06 log cycles by HPH at 150, 200 or 250 MPa, respectively. No viable Staphylococcus aureus nor coliform cells remained in any HPH milk samples. HPH did not affect the colour of milk and HPH samples did not cream during refrigerated storage. The activities of plasmin, alkaline phosphatase and lactoperoxidase in milk were all greatly reduced by HPH. Pseudomonas fluorescens, inoculated into milk (approximately 10(6) cfu/ml), was reduced to undetectable levels by HPH at 200MPa (milk inlet temperature, approximately 10 degrees C); however, Ps. fluorescens proteinase was quite resistant to HPH under such conditions. Overall, owing to the significant increase in temperature and the possibility of varying the holding time, there may be potential applications for HPH as a novel liquid milk processing technique, combining many advantages of conventional homogenization and pasteurization of milk in a single process.

High pressure-induced denaturation of alpha-lactalbumin and beta-lactoglobulin in bovine milk and whey: a possible mechanism.

In this study, high pressure (HP)-induced denaturation of alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) in dairy systems was examined. In both milk and whey, beta-lg was less baroresistant than alpha-la; both proteins were considerably more resistant to HP-induced denaturation in whey than in milk. HP-induced denaturation of alpha-la and beta-lg increased with increasing proportion of milk in mixtures of milk and whey. Addition of a sulphydryl-oxidising agent, KlO3, to milk or whey increased HP-induced denaturation of beta-lg, but reduced the denaturation of alpha-la. Denaturation of both alpha-la and beta-lg was prevented by adding a sulphydryl-blocking agent, N-ethylmaleimide, to milk or whey prior to HP treatment, highlighting the crucial role of sulphydryl-disulphide interchange reactions in HP-induced denaturation of alpha-la and beta-lg. Removal of colloidal calcium phosphate from milk also reduced HP-induced denaturation of alpha-la and beta-lg significantly. The higher level of HP-induced denaturation of alpha-la and beta-lg in milk than in whey may be the result of the abscence of the casein micelles and colloidal calcium phosphate from whey, which facilitate HP-induced denaturation of alpha-la and beta-lg in milk.

High pressure treatment of bovine milk: effects on casein micelles and whey proteins.

Effects of high pressure (HP) on average casein micelle size and denaturation of alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) in raw skim bovine milk were studied over a range of conditions. Micelle size was not influenced by treatment at pressures <200 MPa, but treatment at 250 MPa increased micelle size by approximately 25%, while treatment at > or = 300 MPa irreversibly reduced it to approximately 50% of that in untreated milk. The increase in micelle size after treatment at 250 MPa was greater with increasing treatment time and temperature and milk pH. Treatment times > or = 2 min at 400 MPa resulted in similar levels of micelle disruption, but increasing milk pH to 7.0 partially stabilised micelles against HP-induced disruption. Denaturation of alpha-la did not occur < or = 400 MPa, whereas beta-lg was denatured at pressures >100 MPa. Denaturation of alpha-la and beta-lg increased with increasing pressure, treatment time and temperature and milk pH. The majority of denatured beta-lg was apparently associated with casein micelles. These effects of HP on casein micelles and whey proteins in milk may have significant implications for properties of products made from HP-treated milk.

Heat shock response in Lactobacillus plantarum.

Heat stress resistance and response were studied in strains of Lactobacillus plantarum. Stationary-phase cells of L. plantarum DPC2739 had decimal reduction times (D values) (D value was the time that it took to reduce the number of cells by 1 log cycle) in sterile milk of 32.9, 14.7, and 7.14 s at 60, 72, and 75 degrees C, respectively. When mid-exponential-phase cells were used, the D values decreased. The temperature increases which caused a 10-fold reduction in the D value ranged from 9 to 20 degrees C, depending on the strain. Part of the cell population treated at 72 degrees C for 90 s recovered viability during incubation at 7 degrees C in sterile milk for 20 days. When mid-exponential- or stationary-phase cells of L. plantarum DPC2739 were adapted to 42 degrees C for 1 h, the heat resistance at 72 degrees C for 90 s increased ca. 3 and 2 log cycles, respectively. Heat-adapted cells also showed increased growth at pH 5 and in the presence of 6% NaCl. Two-dimensional gel electrophoresis of proteins expressed by control and heat-adapted cells revealed changes in the levels of expression of 31 and 18 proteins in mid-exponential- and stationary-phase cells, respectively. Twelve proteins were commonly induced. Nine proteins induced in the heat-adapted mid-exponential- and/or stationary-phase cells of L. plantarum DPC2739 were subjected to N-terminal sequencing. These proteins were identified as DnaK, GroEL, trigger factor, ribosomal proteins L1, L11, L31, and S6, DNA-binding protein II HlbA, and CspC. All of these proteins have been found to play a role in the mechanisms of stress adaptation in other bacteria. Antibodies against GroES detected a protein which was induced moderately, while antibodies against DnaJ and GrpE reacted with proteins whose level of expression did not vary after heat adaptation. This study showed that the heat resistance of L. plantarum is a complex process involving proteins with various roles in cell physiology, including chaperone activity, ribosome stability, stringent response mediation, temperature sensing, and control of ribosomal function. The physiological mechanisms of response to pasteurization in L. plantarum are fundamental for survival in cheese during manufacture.

Arginine catabolism by sourdough lactic acid bacteria: purification and characterization of the arginine deiminase pathway enzymes from Lactobacillus sanfranciscensis CB1.

The cytoplasmic extracts of 70 strains of the most frequently isolated sourdough lactic acid bacteria were screened initially for arginine deiminase (ADI), ornithine transcarbamoylase (OTC), and carbamate kinase (CK) activities, which comprise the ADI (or arginine dihydrolase) pathway. Only obligately heterofermentative strains such as Lactobacillus sanfranciscensis CB1; Lactobacillus brevis AM1, AM8, and 10A; Lactobacillus hilgardii 51B; and Lactobacillus fructivorans DD3 and DA106 showed all three enzyme activities. Lactobacillus plantarum B14 did not show CK activity. L. sanfranciscensis CB1 showed the highest activities, and the three enzymes were purified from this microorganism to homogeneity by several chromatographic steps. ADI, OTC, and CK had apparent molecular masses of ca. 46, 39, and 37 kDa, respectively, and the pIs were in the range of 5.07 to 5.2. The OTCs, CKs, and especially ADIs were well adapted to pH (acidic, pH 3.5 to 4.5) and temperature (30 to 37 degrees C) conditions which are usually found during sourdough fermentation. Internal peptide sequences of the three enzymes had the highest level of homology with ADI, OTC, and CK of Lactobacillus sakei. L. sanfranciscensis CB1 expressed the ADI pathway either on MAM broth containing 17 mM arginine or during sourdough fermentation with 1 to 43 mM added arginine. Two-dimensional electrophoresis showed that ADI, OTC, and CK were induced by factors of ca. 10, 4, and 2 in the whole-cell extract of cells grown in MAM broth containing 17 mM arginine compared to cells cultivated without arginine. Arginine catabolism in L. sanfranciscensis CB1 depended on the presence of a carbon source and arginine; glucose at up to ca. 54 mM did not exert an inhibitory effect, and the pH was not relevant for induction. The pH of sourdoughs fermented by L. sanfranciscensis CB1 was dependent on the amount of arginine added to the dough. A low supply of arginine (6 mM) during sourdough fermentation by L. sanfranciscensis CB1 enhanced cell growth, cell survival during storage at 7 degrees C, and tolerance to acid environmental stress and favored the production of ornithine, which is an important precursor of crust aroma compounds.

Biodiversity of the bacterial flora on the surface of a smear cheese.

The bacteria on the surface of a farmhouse smear-ripened cheese at four stages of ripening (4, 16, 23, and 37 days) from inoculated (i.e., deliberately inoculated with Brevibacterium linens BL2) and noninoculated (not deliberately inoculated with B. linens BL2) cheese were investigated. The results show that, contrary to accepted belief, B. linens is not a significant member of the surface flora of smear cheese and no microbial succession of species occurred during the ripening of the cheeses. Of 400 isolates made, 390 were lactate-utilizing coryneforms and 10 were coagulase-negative Staphylococcus spp. A detailed analysis of the coryneforms was undertaken using phenotypic analysis, molecular fingerprinting, chemotaxonomic techniques, and 16S rRNA gene sequencing. DNA banding profiles (ramdom amplified polymorphic DNA [RAPD]-PCR) of all the coryneform isolates showed large numbers of clusters. However, pulsed-field gel electrophoresis (PFGE) of the isolates from the cheeses showed that all isolates within a cluster and in many contiguous clusters were the same. The inoculated and noninoculated cheeses were dominated by single clones of novel species of Corynebacterium casei (50.2% of isolates), Corynebacterium mooreparkense (26% of isolates), and Microbacterium gubbeenense (12.8% of isolates). In addition, five of the isolates from the inoculated cheese were Corynebacterium flavescens. Thirty-seven strains were not identified but many had similar PFGE patterns, indicating that they were the same species. C. mooreparkense and C. casei grew at pH values below 4.9 in the presence of 8% NaCl, while M. gubbeenense did not grow below pH 5.8 in the presence of 5 to 10% NaCl. B. linens BL2 was not recovered from the inoculated cheese because it was inhibited by all the Staphylococcus isolates and many of the coryneforms. It was concluded that within a particular batch of cheese there was significant bacterial diversity in the microflora on the surface.