Occurrence of Major Whey Proteins in the pH 4.6 Insoluble Protein Fraction from UHT-Treated Milk

A clear picture of the protein rearrangement in milk following UHT-treatment was drawn by a comparative analysis of the pH 4.6 soluble protein fraction (SPF) and the pH 4.6 insoluble protein fraction (IPF) recovered from raw and UHT-treated milk samples. The two protein fractions were analyzed by mono- or bidimensional gel electrophoresis under reducing and nonreducing conditions, and protein bands were identified by specific immunostaining. Results showed that bovine serum albumin, β-lactoglobulin, and, to a lesser extent, α-lactalbumin coprecipitated with caseins in UHT-treated milk samples at pH 4.6. These proteins were almost exclusively involved in high molecular weight aggregates held together by disulfide bonds. Partition of α-lactalbumin and bovine serum albumin in the protein fractions obtained upon acidification of milk at pH 4.6 was evaluated by competitive immunoassays. The ELISA-based results suggested the possibility of using pH 4.6 insoluble α-lactalbumin and bovine serum albumin, in addition to pH 4.6 insoluble β-lactoglobulin, as indicators of the intensity of the heat treatment applied to milk.     

Mozzarella干酪加工过程中主要理化指标变化及其产率计算

该文目的在于为干酪功能特性、含水率和产率的控制提供理论依据,使用标准化原料乳经低温巴氏杀菌,采用无盐渍新工艺生产Mozzarella干酪,从添加发酵剂开始,到制成新鲜干酪,测定各阶段干酪凝块的温度、水分含量、pH值和滴定酸度及各阶段乳清和凝块的蛋白和脂肪含量,计算Mozzarella干酪的效能和产率。试验结果表明：在排乳清和粉碎加盐阶段,干酪凝块中的水分含量变化幅度最大,排出乳清中的蛋白和脂肪数量明显增加;干酪的脂肪回收率达到92.22%,实际测得的乳清中脂肪的损失率为7.62%;乳蛋白的回收率达到83.60%,仅有16.72%的蛋白质损失于乳清中;在堆酿阶段pH值有一个从6.3(干酪凝块5.5)至5.25的快速下降过程。Mozzarella干酪的实际产率达到12.38%,达到了同类型干酪的产率要求。Mozzarella干酪的理化指标达到了美国全脂Mozzarella干酪的标准。     

Transmission electron microscopy of mozzarella cheeses made from microfluidized milk.

The nanostructure of Mozzarella cheeses prepared from microfluidized milk was compared with that of control cheeses made from untreated milk. Milk heated to 10 or 54 degrees C and containing 1.0 or 3.2% fat was homogenized by microfluidization at 34 or 172 MPa prior to cheesemaking. The effects on the casein particles and fat globules in the cheese were determined by transmission electron microscopy after 1 day and 6 weeks of storage at 4 degrees C. The micrographs showed that electron-dense regions theorized to be casein submicelles rearranged from a homogeneous configuration to a pattern of clusters during the storage period. The nanostructure of the cheeses made from milk processed under the mildest conditions resembled the controls, but otherwise the fat droplets decreased in size and increased in number as the pressure and temperature were increased. The results indicate that both homogenization temperature and pressure affect the nanostructure of Mozzarella cheese.     

Mozzarella干酪成熟中蛋白水解与功能特性的变化

为控制干酪的质量,对Mozzarella干酪成熟过程中蛋白质的水解(测定SDS凝胶电泳和可溶性氮)和未融化干酪的质构变化以及融化干酪功能特性变化进行了研究,干酪成熟过程中由于凝乳酶和乳酸菌酶的作用使蛋白水解,从而使pH 4.6可溶性氮(SN)和12% TCA SN逐渐增加;凝乳酶主要影响酪蛋白的水解范围,乳酸菌及其酶,不但影响酪蛋白的水解范围,而且主要影响酪蛋白的水解深度。干酪中的残留凝乳酶和乳酸菌酶使酪蛋白水解为大分子量的肽段,而乳酸菌酶还可将大分子量的肽段进一步降解为小分子量的肽段和游离氨基酸。由于酪蛋白的水解,使干酪的硬度和弹性下降,融化性和油脂析出性增加,随着小分子量肽和游离氨基酸的增加,干酪的褐变性提高。     

Real-time TaqMan polymerase chain reaction detection and quantification of cow DNA in pure water buffalo mozzarella cheese: method validation and its application on commercial samples

Mozzarella cheese obtained from buffalo (Bubalus bubalis) milk is a typical Italian product certificated by means of the European Protected Designation of Origin (PDO). Mozzarella cheese can also be obtained from bovine milk or bovine/buffalo milk mixtures, but in this case, it cannot be sold as PDO product, and its label must report the actual ingredients. However, bovine milk in PDO products was frequently detected in the past, suggesting fraudulent addition or accidental contamination. Several methods based on end-point polymerase chain reaction (PCR) have been profitably applied in a large number of tests to detect the presence of undeclared ingredients, also in dairy products. In the present study we report a real-time PCR method able to quantify bovine milk addition to pure buffalo cheese products. We validated a normalized procedure based on two targets: bovine mitochondrial cytochrome b (cyt b) to detect and quantify the bovine DNA and nuclear growth hormone (GH) gene used as a universal reference marker. With the use of this real-time PCR assay, 64 commercial mozzarella di bufala cheese samples purchased at local supermarkets, dairy shops, or directly from cheese manufacturers were analyzed. The results obtained demonstrate that most of the commercial samples were contaminated with bovine milk. Therefore, this assay could be conveniently employed to carry out routine and accurate controls aimed not only to discourage any fraudulent behavior but also to reduce risks for consumer health.     

Prediction of the ripening times of ewe's milk cheese by multivariate regression analysis of capillary electrophoresis casein fractions

The effect of the ripening time on the proteolytic process in cheeses made from ewe's milk during a 139-day ripening period was monitored by the use of capillary electrophoresis of pH 4.6 insoluble fraction. Totals of 18 and 21 peaks were recognized and matched in the electropherograms obtained with a fused-silica capillary and a neutral capillary (hydrophilically coated), respectively. These peaks correspond to intact ovine caseins and their hydrolysis products (alpha(s1)-casein I, alpha(s1)-casein II, alpha(s1)-casein III, alpha(s2)-casein, beta(1)-casein, beta(2)-casein, p-kappa-casein, alpha(s1)-I-casein, gamma(1)-casein, gamma(2)-casein, and gamma(3)-casein). The alpha(s)-caseins (alpha(s1)- and alpha(s2)-casein) displayed similar degradation pattern to one another, but different from those of beta-caseins (beta(1)- and beta(2)-casein). beta-Caseins were very much undergoing lesser degradation during the ripening time than alpha(s)-casein. Finally, partial least-squares regression and principal components regression were used to predict the ripening time in cheeses. The models obtained yielded good results since the root-mean-square error in prediction by cross validation was <8.6 days in all cases.     

Milk excretion of ivermectin and moxidectin in dairy sheep: assessment of drug residues during cheese elaboration and ripening period

Ivermectin (IVM) and moxidectin (MXD) are broad-spectrum endectocide antiparasitic drugs extensively used in food-producing animals. The patterns of IVM and MXD excretion in milk were comparatively characterized following their subcutaneous administration (200 microg.kg(-1) of body weight) to lactating dairy sheep. The relationship between milk excretion and plasma disposition kinetics of both compounds was characterized. A pool of milk collected from all of the animals in each experimental group was used for cheese elaboration. IVM and MXD residual concentrations were assessed during the cheese-making process and ripening period. IVM and MXD concentrations were measured in plasma, milk, and milk product (whey, curd, and cheese) samples using an HPLC-based methodology with fluorescence detection. IVM and MXD were extensively distributed from the bloodstream to the mammary gland, and large quantities, particularly of MXD, were excreted in milk. Residual concentrations of both compounds were recovered in milk up to 30 (IVM) and 35 (MXD) days post-treatment. The total fraction of the administered dose excreted in milk for MXD was significantly higher than that of IVM. During cheese production, the highest residual concentrations of both molecules were measured in the curd. Thirty-four percent of the total drug residue measured in the pooled milk collected from treated sheep was lost during the cheese-making process. The lowest residual concentrations were measured in the whey. IVM and MXD concentrations in the elaborated cheese tended to increase during the ripening period, reaching the highest residual level at 40 days of cheese maturation. The long persistence of milk residual concentrations of MXD and IVM in lactating dairy sheep and the high concentrations found in cheese and other milk-related products should be seriously considered before recommendation of the extralabel use of these antiparasitic drugs in dairy animals.     

Evaluation of APHA and AOAC methods for phosphatase in cheese

Varieties of market cheese were analyzed for alkaline phosphatase by the modified rapid colorimetric method of the American Public Health Association (APHA) and the official AOAC method, 16.304-16.306. In the APHA method, 5 g cheese (pH less than 7.0) is macerated with 2 mL 1:1 carbonate buffer, or 2 mL water (for cheese with pH greater than 7.0). Addition of 0.1 mL magnesium acetate (1 mg magnesium) to test portions of cheese extracts yielded reproducible and quantitative recovery of added phosphatase. In the AOAC method, macerating 0.5 g cheese with 1 mL borate buffer before adding milk phosphatase improved recovery among cheeses. Addition of magnesium ion increased phosphatase activity in some cheeses. Phosphatases in blue mold-ripened and Swiss cheeses were inactivated by heat faster than was milk phosphatase, yet milk phosphatase added to various soft cheeses was completely inactivated at 60 degrees C for 10 min. The lability of phosphatase was due to the heat-denaturing effect of NaCl present in finished cheeses. Some Mexican style soft cheeses contained both heat-labile and heat-stable phosphatases. These data suggest that the phosphatase test to differentiate milk and microbial phosphatases on the basis of repasteurization and analysis of cheese is no longer valid.     

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.     

Use of recombinant cyprosin in the manufacture of ewe's milk cheese

A recombinant cyprosin from the cardoon (Cynara cardunculus L.) was assayed and compared with calf rennet in batches of ewes' milk cheese by determining different chemical, biochemical, and microbiological parameters over 4 months of ripening. There were no differences between the two types of coagulants in most chemical parameters, a(w), and pH. Proteolysis was more marked and rapid in cheese containing recombinant cyprosin as coagulant, the soluble nitrogen content of which was significantly higher (p < 0.01) than that of the cheese obtained with animal rennet; at the end of ripening the nonprotein nitrogen of cheese produced with recombinant cyprosin was slightly higher (p > 0.05) as compared with that in cheeses obtained with animal rennet. Microbial counts in the milk used for making cheese were high in most of the groups analyzed. Despite slight differences in counts, the main microbial groups analyzed were similar in cheese produced with both types of coagulants throughout ripening.     

Direct enzyme-linked immunosorbent assay for determining aflatoxin M1 at picogram levels in dairy products

Protocols for detecting picogram quantities of aflatoxin M1 in dairy products were established. Milk samples were subjected to a reverse phase Sep-Pak C18 cartridge treatment before analysis by an enzyme-linked immunosorbent assay (ELISA) according to previously published procedures. M1 in yogurt, brick cheddar, and ripened Brie cheese was extracted by a modified Pons method, subjected to a normal phase silica cartridge treatment, and analyzed by ELISA. The detection limits for M1 in milk, yogurt, cheddar, and Brie were 10, 10, 50, and 25 ppt (ng/kg), respectively. Recovery for M1 added to these products was in the range 70-110%. Good agreement was found for M1 levels in several naturally contaminated milk samples analyzed by both ELISA and liquid chromatography.     

Effect of processing and storage on the stability of flaxseed lignan added to dairy products

This study investigated the effects of processing and storage on the stability of purified, flaxseed-derived secoisolariciresinol diglucoside (SDG) added to milk prior to the manufacture of different dairy products. We analyzed the effect of high-temperature pasteurization, fermentation, and milk renneting as well as storage on the stability of SDG added to milk, yogurt, and cheese. Also, the stability of SDG in whey-based drinks was studied. Added SDG was found to withstand the studied processes well. In edam cheese manufacture, most of the added SDG was retained in the whey fraction and 6% was found in the cheese curd. SDG was also relatively stable in edam cheese during ripening of 6 weeks at 9 degrees C and in yogurt during storage of 21 days at 4 degrees C. Up to 25% of added SDG was lost in whey-based drinks during storage of 6 months at 8 degrees C. We conclude that SDG can be successfully supplemented in dairy-based products.     

Hydrophobic grid membrane filter/MUG method for total coliform and Escherichia coli enumeration in foods: collaborative study

Twenty-four laboratories participated in a collaborative study to validate a hydrophobic grid membrane filter (HGMF) method incorporating the use of 4-methylumbelliferyl-beta-D-glucuronide (MUG) for enumeration of total coliform and Escherichia coli bacteria in foods by comparing its performance against the AOAC 3-tube MPN method (46.013-46.016). Raw milk, raw ground poultry, whole egg powder, cheese powder, and ground black pepper were included in the study. The total coliform methods did not differ significantly, except that the 3-tube method detected a significantly higher level of total coliforms than did the HGMF method in the ground black pepper. Conversely, the HGMF/MUG E. coli method detected significantly higher numbers of E. coli present in the egg powder, cheese powder, and ground black pepper samples, while not differing significantly from the 3-tube method for the raw milk and raw ground poultry samples. The overall confirmation rate of MUG-positive colonies isolated using the HGMF method was 99.5%. The hydrophobic grid membrane filter/MUG method has been adopted official first action as an additional method to AOAC official final action method 46.030-46.034.     

Neamin as an immunogen for the development of a generic ELISA detecting gentamicin,kanamycin, and neomycin in milk

A broad-specific ELISA using one antibody preparation for the detection of gentamicin, kanamycin, and neomycin in milk is reported for the first time. For the immunization of rabbits, neamin was used as the generic hapten on the basis of the facts that it is a two-ring fragment of neomycin and, in shape and charge, it resembles parts of kanamycin and gentamicin. Neamin was linked to the preactivated carrier protein keyhole limpet hemocyanin by EDC coupling. The specificity and sensitivity of the polyclonal antibodies for the aminoglycoside antibiotics were tested in a competitive assay using homologous and heterologous conjugates coupled by various conjugation procedures as the ELISA solid phase. In contrast to the homologous assay recognizing only neomycin, the heterologous assay could be used for the detection of the whole subclass of deoxystreptamin antibiotics in buffer and raw milk. Gentamicin, kanamycin, and neomcyin were detected in artificially contaminated undiluted raw milk (without sample pretreatment) with 50% inhibition levels at 9, 21, and 113 ng mL(-)(1), respectively. Neomycin levels were also measured in milk samples obtained from a cow suffering from mastitis and treated with an antibiotic cocktail including neomycin. Levels below the EU maximum residue levels for deoxystreptamin antibiotics could readily be measured in this generic ELISA.     

Development of an immunochromatographic strip test for rapid detection of melamine in raw milk, milk products and animal feed

A simple, rapid and sensitive immunogold chromatographic strip test based on a monoclonal antibody was developed for the detection of melamine (MEL) residues in raw milk, milk products and animal feed. The limit of detection was estimated to be 0.05 μg/mL in raw milk, since the detection test line on the strip test completely disappeared at this concentration. The limit of detection was 2 μg/mL (or 2 μg/g) for milk drinks, yogurt, condensed milk, cheese, and animal feed and 1 μg/g for milk powder. Sample pretreatment was simple and rapid, and the results can be obtained within 3-10 min. A parallel analysis of MEL in 52 blind raw milk samples conducted by gas chromatography-mass spectrometry showed comparable results to those obtained from the strip test. The results demonstrate that the developed method is suitable for the onsite determination of MEL residues in a large number of samples.     

Quantification of beta casein in milk and cheese using an optical immunosensor

beta-Casein was quantified in milk and cheese, using an optical immunosensor, based on surface plasmon resonance (SPR) measurement. The assay consists of a two-step sandwich strategy, with two anti-beta-casein antibodies directed against each extremity of the casein. This strategy permits only native beta-casein to be quantified and not its degradation products. The calibration curve was obtained with a reference milk powder of known beta-casein concentration. The analysis time per sample was less than 10 minutes. The antibody-coated surface could be used for more than 250 determinations. The detection limit was established at 85 ng x mL(-)(1) and the intra- and inter-assay variation coefficients were 2.6 and 6.2% respectively. The method was applied to raw milk to quantify intact beta-casein, with no pretreatment of the sample. A second application was realized with cheese, to follow the proteolysis of beta-casein during ripening.     

凝乳方式对奶油干酪品质的影响

凝乳是干酪加工的关键环节,对奶油干酪的品质具有重要影响。通过对奶油干酪理化成分、产率、涂抹性的测定,比较了酶凝、酸凝、酸-酶互作凝乳3种方式对奶油干酪涂抹性的影响。结果表明,随着酸化程度的加深,奶油干酪的脂肪含量和蛋白质含量逐渐减少,含水率显著增加,校正产率、脂肪回收率和酪蛋白回收率降低。有酶高酸组(RHA)涂抹性最佳,其剪切功、屈服应力都分别为最小(19.23N·s、195.67Pa),与传统酸凝型奶油干酪(NR)相比,RHA的剪切功和屈服应力分别降低了17.04%和27.88%,干酪涂抹性得到明显改善。这为涂抹型奶油干酪的研发提供了技术基础。     

Effect of pH on the thermal denaturation of whey proteins in milk

The effect of pH on thermal denaturation of four main whey protein fractions in skim milk was examined by gel permeation FPLC. On heating skim milk at 80 degrees C for 0.5-20.0 min over the pH range 5.2-8.8, the extent of denaturation, based on loss of solubility at pH 4.6, increased with heating time and was usually in the order immunoglobulins > serum albumin/lactoferrin > beta-lactoglobulin > alpha-lactalbumin. Rates of denaturation of the immunoglobulins and the serum albumin/lactoferrin fraction were highest at the lower end of this pH range, whereas those of beta-lactoglobulin and alpha-lactalbumin increased over most of the pH range. The effects of pH, addition of Ca, and reduction of disulfide bonds on the rates of the unfolding and aggregation stages of denaturation are discussed.     

Changes of astringent sensation of soy milk during tofu curd formation

The effect of isoflavone on soy milk and tofu astringency was investigated, and no consistency was found between an undesirable astringent taste and isoflavone contents. Isoflavone-enriched extract (approximately 39% isoflavones) showed no astringency. Soybean foods having high amounts of isoflavones showed less astringency. About 80% of isoflavones exist freely in both soy milk and tofu, but 55% of phytates (which play an important role in the formation of the tofu curd network) exist freely in the soy milk, and 6-13%, on the basis of coagulation, existed freely in the tofu curds. A 1% potassium phytate solution at pH 7 showed the very same astringency as soy milk; however, calcium phytate at the same concentration and pH showed no undesirable sensation. Thus, it is assumed that the astringent characteristics caused by phytic ions in soy milk are lost upon conversion of phytic ions to their insoluble salt forms during soy milk coagulation.     

Straightforward process for removal of milk fat globule membranes and production of fat-free whey protein concentrate from cheese whey

A straightforward method for the separation of milk fat globule membrane (MFGM) and production of fat-free whey protein concentrate/isolate from cheese whey has been developed. Lowering of the conductivity of the whey from its initial value of about 5600 μS cm(-1) to about 2000-500 μS cm(-1) via diafiltration with water caused selective precipitation of MFGM when incubated for 30 min at pH 4.2 and 35 °C. The whey proteins remained soluble in the supernatant under these conditions. Experimental evidence suggested that precipitation of MFGM at pH 4.2 was not due to a nonspecific effect of lowering of the conductivity of the whey but due to the specific effect of removal of Ca2+ from the whey. The lipid content of whey protein isolate obtained by this process was <0.2%, and the protein loss was <14%. The method provides an industrially feasible process for the production of fat-free whey protein concentrate/isolate. The MFGM, which is reported to contain bioactive/nutraceutical lipids and proteins, is a valuable byproduct of the process.