Enzymic differentiation between curds of heated and raw milk II. Milk peroxidase

Unlike phosphatase, peroxidase, once destroyed by heating of milk, is not regenerated by the acidity or microbial growth of curd formation or by reasonable ageing. This reveals that curd-forming micro-organisms do not have or form peroxidase. The test was repeated in various heating conditions of milk. Peroxidase is destroyed even when the temperature of milk is gradually raised to 80 °C (in 3 min). Less-heated milk, e.g. pasteurised milk and its corresponding curd, retain this enzyme. A positive test in the curd of raw milk is not affected by reasonable ageing or increased acidity but by excessive fermentation (secondary fermentation or putrefaction) which again can be halted by addition of formalin. The peroxidase test, therefore, appears to be a test for differentiating curds of heated and unheated milk.     

Dephosphorylation of bovine casein by milk alkaline phosphatase.

The pH of optimum activity of alkaline phosphatase from cow's milk depended on the substrate, being 10-1 for rho-nitrophenylphosphate, 8-6 for phosphoserine, 8-0 for phosvitin and 6-8 for casein. Individual casein components were dephosphorylated more rapidly than mixtures of alphas- and beta-caseins or of alphas-, beta-and kappa-caseins and micellar casein. Mixtures of 2 components involving kappa-casein were more readily dephosphorylated than alphas- and beta-casein mixtures. At pH 6-8, lactose, whey proteins and phosphate ions had an inhibitory effect. beta-Lactoglobulin had an inhibitory effect only when the pH of the reaction was lower than the optimum pH value of the enzyme. Mg2+ and Zn2+ were not inhibitory. The optimum conditions for dephosphorylation of casein are described.     

Effect of high pressure carbon dioxide on alkaline phosphatase activity and quality characteristics of raw bovine milk

Novel food processing techniques would always be pursuits of researchers and food industry to avoid unfavorable thermal effects, especially in dairy and milk processing. In this study, effects of high pressure carbon dioxide (HPCD) on the activity of alkaline phosphatase (ALP) and main quality indices of raw bovine milk at 20 MPa using a batch system were investigated. A complete inactivation of ALP activity as exposure to HPCD treatment at 50 °C and 20 MPa for 50 min was observed. The protein and lactose content of HPCD-treated bovine milk hold steady, while pH value and total solids content decreased, turbidity and average particle size increased significantly (p < 0.05). Although a significant decrease of viscosity (p < 0.05) was observed, the Newtonian flow behavior of raw bovine milk did not alter. More obvious change of quality characteristics of raw bovine milk were observed as subjected to HPCD treatment at higher temperature or treated for longer period. Therefore, a compromise between controlling endogenous enzymes (and/or spoilage and pathogenic microorganisms) and retention of original/fresh like quality of foods should be introduced due to the nature of HPCD processing. It's suggested to keep raw bovine milk with low ALP activity and great quality treated with a batch HPCD apparatus at 20 MPa and 50 °C for 20 min.     

The development of heat-resistant phosphatase activity in raw milk

The detection of phosphatase activity is used as a legal test to determine whether milk has been adequately pasteurized or whether it has been contaminated with raw milk. Occasional failures of the Milk Regulations phosphatase test were experienced by a processing dairy from stored silo milk. Trials demonstrated that the phosphatase was heat resistant and associated with a pasteurization-sensitive, psychrotrophic organism isolated from one supply. The standard total viable count at 30°C (TVC) of the supply was satisfactory at 4.59 log cfu/ml; however, the psychrotrophic count at 7°C was much higher at 5.61 log cfu/ml. The test milk after storage produced sufficient heat-resistant phosphatase activity to give a test failure when the psychrotrophic count reached about 7.09 log cfu/ml or greater. The occurrence of a failure was dependent on the initial numbers of psychrotophic bacteria, the amount of dilution with other milks and the storage time before processing. The psychrotrophic count of the test milk and the count of the phosphatase-producing isolate were found to increase by approximately one log cfu/ml each day on storage at 4°C. This investigation has shown that a phosphatase failure may indicate the development of microbial phosphatase rather than a process failure. A retest after laboratory pasteurization of any sample failing the test will assist in identifying any microbially produced heat-resistant phosphatase activity. While a TVC at 30°C will normally be expected to count most types of psychrotrophic organisms, this investigation has shown that on this occasion it did not detect specific psvchrotophic organisms which had contaminated the milk.     

Effects of high pressure homogenisation of raw bovine milk on alkaline phosphatase and microbial inactivation. A comparison with continuous short-time thermal treatments

Raw whole milk of high microbial quality (58 degrees C), but markedly decreased above 200 MPa when Tin=24 degrees C (T2>60 degrees C). In contrast to inactivation induced by continuous short-time thermal treatments, ALP inactivation induced by HP homogenisation was clearly due to mechanical forces (shear, cavitation and/or impact) in the HP valve and not to the short (<1 s) residence time at temperature T2 in the same valve. Inactivation of the three exogenous microorganisms led to similar conclusions. Homogenisation at 250 MPa or 300 MPa (Tin=24 degrees C) induced a 2-3 log cycle reduction of the total endogenous milk flora and a 1.5-1.8 log cycle reduction of inoculated List. innocua. Higher reduction ratios (2-4 log cycles) were obtained for the two other microorganisms. The highest levels of ALP inactivation corresponded to the highest extents of microbial reduction. Running the milk twice or three times through the homogeniser (recycling), keeping temperature T1 approximately 29 degrees C and pressure=200 MPa, increased homogenisation efficiency.     

Thermal inactivation kinetics of alkaline phosphatase in equine milk

Alkaline phosphatase (ALP) is widely used as an indicator of proper pasteurization in bovine milk. Due to interest in the use of equine ALP as a time/temperature integrator (TTI) for evaluation of the efficacy of thermal processing of equine milk, its inactivation kinetics were evaluated in whole and skimmed equine milk. Experimentally determined decimal reduction times showed that equine ALP is more readily inactivated in equine milk than its bovine counterpart. Thus, considering the required 6 D reduction of pathogens and the rather low enzyme level present in equine milk, equine ALP will not be suitable as indicator for correct pasteurization of equine milk under the conditions currently used in the reference method for the determination of ALP in milk-based products.     

Inactivation kinetics of alkaline phosphatase and lactoperoxidase, and denaturation kinetics of beta-lactoglobulin in raw milk under isothermal and dynamic temperature conditions

A detailed kinetic study of alkaline phosphatase, lactoperoxidase and beta-lactoglobulin was carried out in the context of identifying intrinsic time-temperature indicators for controlling the heat processing of milk. The heat inactivation or denaturation of alkaline phosphatase, lactoperoxidase and beta-lactoglobulin under isothermal conditions was found to follow first order kinetics. Experimental results were analysed using both a two step linear regression and a one step non-linear regression method. Results obtained using the two statistical techniques were comparable, but the 95% confidence interval for the predicted values was smaller when the one step non-linear regression method was used, indicating its superiority for estimating kinetic parameters. Thermal inactivation of alkaline phosphatase and lactoperoxidase was characterized by z values of 5.3 deg C (D60 degrees C = 24.6 min) and 4.3 deg C (D71 degrees C = 38.6 min) respectively. For the denaturation of beta-lactoglobulin we found z values of 7.9 deg C (D7.5 degrees C = 49.9 min) in the temperature range 70-80 degrees C and 24.2 deg C (D85 degrees C = 3.53 min) in the range 83-95 degrees C. Dref and z were evaluated under dynamic temperature conditions. To estimate the statistical accuracy of the parameters, 90% joint confidence regions were constructed.     

乳中碱性磷酸酶活性测定方法研究进展

碱性磷酸酶(ALP,EC 3.1.3.1)是乳中天然存在的一种酶,该酶作为一种热处理强度指指标被广泛应用于评价牛奶巴氏杀菌是否彻底或巴氏杀菌后是否污染有生鲜乳.关于乳中ALP活性的测定不断有新的方法提出,本文对乳中ALP活性的测定方法进行了综述.     

Chemometric differentiation of raw and commercial milk by trace elements using principal component analysis.

Nine trace elements (Cr, Mn, Fe, Ni, Cu, Zn, Mo, Cd, and Pb) were determined in the dissolved ash of 36 samples of raw milk. The distribution of the concentration of each element was first investigated by means of a test of normality. The matrix of the correlation between the concentrations of the elements was then used as a starting matrix for principal component analysis. Nine variables were reduced to four principal components, accounting for 75% of the total variance. The biophilic elements Mn-Fe and Cu-Mo were positively associated with the first two principal components, while Cr was correlated to the third and Ni and Cd with the fourth principal component. Pb and Zn are both negatively correlated to the first principal component. Comparison with 42 samples of a commercial milk, by using a two-dimensional plot of the principal component scores, rendered possible the differentiation between raw and commercial milk.     

Detection of alkaline phosphatase by competitive indirect ELISA using immunoglobulin in yolk (IgY) specific against bovine milk alkaline phosphatase

Immunoglobulin in yolk (IgY) (with a titer of 1.3 × 10⁶) specific against bovine milk (BM) alkaline phosphatase (ALP) was obtained by intramuscularly immunizing hens on the thigh and was used as the primary antibody to conduct competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) to determine BMALP in ALPs from BM and Escherichia coli sources. A relationship between the ELISA value and the BMALP level (0.01–10 μg/mL) in whole milk (R² = 0.9019) or in skimmed milk (R² = 0.9402) was observed. The maximal inhibition (%) of BMALP on the microtiter plate by free BMALP at 10 μg/mL whole milk (3.89 mU/μg BMALP) was about 50%, while no inhibition (%) of BMALP by free E. coli ALP at concentrations between 0.01 to 10 μg/mL (60 mU/μg E. coli ALP) was determined. At BMALP levels higher than 0.1 μg/mL, CI-ELISA was proved to be effective in differentiating between BMALP and E. coli ALP and quantifying BMALP in whole milk or skimmed milk in the presence of E. coli ALP with an activity of 0.6 U/mL. Higher inhibition (about 70%) of BMALP on the microtiter plate by free BMALP in diluted (10¹–10⁴ fold) milk samples was observed. The optimal conditions for CI-ELISA in determining BMALP (0.1–10 μg/mL) from ALPs in milk samples were using 10³-fold diluted crude IgY specific against BMALP as primary antibody and 10³-fold diluted goat anti-chicken IgG–ALP conjugate as the secondary antibody.     

Kinetics of thermal inactivation of alkaline phosphatase in bovine and caprine milk and buffer

The thermal inactivation of alkaline phosphatase (ALP) in raw bovine and caprine milk was investigated in the temperature range 54 to 69 °C. To assess the stabilizing effect of milk compounds on ALP, inactivation experiments were also carried out in 0.1 m potassium phosphate buffer, pH 6.6. Each set of inactivation experiments was fitted simultaneously using kinetic models that were based on either one-step or two-step mechanisms. The parameters of the Arrhenius equation showed that the stabilization effect of milk compounds on ALP had an entropic character. They also indicated a different structure of bovine and caprine milk ALPs, which was reflected by a higher stability of the bovine milk enzyme.     

Evaluation of spectrophotometric and fluorometric methods for alkaline phosphatase activity determination in ewe's milk

The alkaline phosphatase (ALP) activity test has been used since 1935 to assess the effectiveness of pasteurization. Different analytical methods exist for detecting ALP in milk. Unfortunately, there is little information about ALP activity in ewe's milk. The aim of this study was to assess and compare the official European method (spectrophotometric method) and the Fluorophos method (fluorometric method) regarding their use in ewe's milk. Bulk ewe's milk samples were taken from a flock and from three different dairies. A portion of the original sample was pasteurized at 63 degrees C for 30 min in a circulating bath; another portion was heated to and kept at 95 degrees C for about 2 min, and 0.1% (vol/vol) of raw milk was added. The samples obtained were analyzed in duplicate using the spectrophotometric and fluorometric methods. The relation between ALP activity determined by the two methods was characterized by the following equation: Y = 1.34 + 0.0039X (where Y = ALP in microg of phenol per ml of milk and X = ALP in mU/liter; R2 = 91.5%). Precision parameters (repeatability [r], standard deviation of repeatability [s(r)], and relative standard deviation of repeatability [RSDr]) for both methods were calculated. The values of RSDr for the Fluorophos method were 4.30 for pasteurized milk and 2.96 for 0.1% raw milk, close to the value indicated by Rocco in whole cow's milk (RSDr = 4.4). The repeatability for the official method (r = 2.16) was close to that indicated for whole cow's milk (r = 2).     

Proteins recovered from acid whey/skim milk mixtures heated at alkaline pH values

Skim milk and mixtures prepared by combining acid whey with skim milk at volume ratios of 2:1, 1:1, 1:2, 1:3 and 1:4 were adjusted to pH 7.5 and heated at 90°C × 15 min. Protein was isolated from these heated samples by precipitation at pH 4.6 and it was found that 65% of the whey protein was recovered in each case. Non-recovered proteins included the proteose peptones and small quantities of β-lactoglobulin, α-lactalbumin and bovine serum albumin. The solubility of these isolates, which contained from 10–25% whey protein, decreased to > 95% when the whey protein exceeded ˜16%. Further characterization of the isolate, prepared from the 1:1 volume ratio of acid whey and skim milk, showed that ˜50% of the whey protein was insoluble, bound to casein and non-functional while the other ˜50% was complexed with casein and was soluble. The addition of a reducing agent suggests that sulphydryl bonding alone is not responsible for complex formation.     

The differential determination of lysine in heated milk. I.—In vitro methods

Lysine content and availability were determined in a set of milk samples which had sustained increasing heat treatments. Lysine content was measured after acid hydrolysis of the sample (TLV or x-value), lysine availability by an enzymic digestion procedure (ALV-e or y-value) and by two modifications of the fluorodinitrobenzene method: the direct method (ALV-f I or z-value) and Carpenter's corrected straight acid procedure (ALV-f II or v-value). The four procedures gave strongly correlated, but numerically quite different results. The enzymic procedure and Carpenter's chemical method produced very similar results for lysine availability. In all heated samples, lysine content was much higher than lysine availability. The direct fluorodinitrobenzene method yielded values intermediate between those for lysine content and ‘true’ lysine availability. A nomograph is presented which accounts for the different conditions of lysine in heated milk and allows the interconversion of the x, y, z and v-values.     

Distinction between dry and raw milk using monoclonal antibodies prepared against dry milk proteins

It is well established that the heating process during the preparation of dry milk (DMLK) causes structural changes in some milk proteins. However, because such changes are subtle, whether they can be detected by an immunochemical approach remains questionable. The present study attempted to develop a sensitive mAb that might distinguish the DMLK from freshly prepared raw milk. To test this possibility, we immunized mice with commercially prepared DMLK and produced a panel of mAb. From 900 hybridomas screened using an ELISA, 4 clones were found to be specific to DMLK; the other 68 clones recognized both DMLK and raw milk. In contrast to polyclonal antibodies, only the specific mAb could detect the DMLK spiked into the raw milk at as low as 5% in concentration (vol/vol). Western blot analysis shows that these specific mAb were all directed against beta-lactoglobulin (LG) and LG-milk protein conjugates. These mAb reacted with raw milk heated at 95 degrees for 15 min; the reaction with LG-conjugates, however, was abolished when treated with reducing reagent. Thus, results suggests that a new antigenic epitope was exposed in a heating process, and the thio group of LG cross linked with other protein moiety played a provocative role in mAb recognition. A hypothetical model with respect to the interaction between the mAb and DMLK is proposed and discussed.     

Effect of leaving milk trucks empty and idle for 6 h between raw milk loads

The US Pasteurized Milk Ordinance (PMO) allows milk tanker trucks to be used repeatedly for 24 h before mandatory clean-in-place cleaning, but no specifications are given for the length of time a tanker can be empty between loads. We defined a worst-case hauling scenario as a hauling vessel left empty and dirty (idle) for extended periods between loads, especially in warm weather. Initial studies were conducted using 5-gallon milk cans (pilot-scale) as a proof-of-concept and to demonstrate that extended idle time intervals could contribute to compromised raw milk quality. Based on pilot-scale results, a commercial hauling study was conducted through partnership with a Pacific Northwest dairy co-op to verify that extended idle times of 6 h between loads have minimal influence on the microbiological populations and enzyme activity in subsequent loads of milk. Milk cans were used to haul raw milk (load 1), emptied, incubated at 30°C for 3, 6, 10, and 20 h, and refilled with commercially pasteurized whole milk (load 2) to measure cross-contamination. For the commercial-scale study, a single tanker was filled with milk from a farm known to have poorer quality milk (farm A, load 1), emptied, and refilled immediately (0 h) or after a delay (6 h) with milk from a farm known to have superior quality milk (farm B, load 2). In both experiments, milk samples were obtained from each farm's bulk tank and from the milk can or tanker before unloading. Each sample was microbiologically assessed for standard plate count (SPC), lactic acid bacteria (LAB), and coliform counts. Selected isolates were assessed for lipolytic and proteolytic activity using spirit blue agar and skim milk agar, respectively. The pilot-scale experiment effectively demonstrated that extended periods of idle (>3 h) of soiled hauling vessels can significantly affect the microbiological quality of raw milk in subsequent loads; however, extended idle times of 6 h or less would not measurably compromise milk quality in subsequent loads in commercial tankers. Current tanker sanitation practices appear to be sufficient for maintaining raw milk SPC, LAB, and coliform levels, which are important measures of milk quality.     

原料乳中体细胞数与UHT乳中酪蛋白成分的关系研究

研究了原料乳中体细胞数与15批次UHT乳样本中酪蛋白成分之间的关系。将原料乳巴氏杀菌后进行超高温处理。分别于8,30,60,90和120 d采集贮藏于室温条件下的UHT乳样本,并使用高效液相色谱法对酪蛋白成分进行分析。体细胞数范围1.97×105～8×105 mL-1。体细胞数与原料乳或UHT乳中的κ-酪蛋白质量浓度之间没有相关性(P<0.05)。原料乳中αs2-酪蛋白和β-酪蛋白与体细胞数呈负相关(P<0.05)。UHT乳中,αs1-酪蛋白(P<0.05)和β-酪蛋白(P<0.05)与体细胞数在贮藏第8天呈负相关,αs2-(P<0.01)与体细胞数在贮藏第60天呈负相关。结果表明,原料乳中体细胞数较高与β-酪蛋白和αs-酪蛋白的大量水解有关,并且可能导致UHT乳在贮藏期内出现质量问题。     

Development of a monoclonal antibody-based immunoassay for specific quantification of bovine milk alkaline phosphatase

The detection of alkaline phosphatase (ALP) activity is used as a legal test to determine whether milk has been adequately pasteurized or recontaminated with raw milk. However, a wide variety of microorganisms produce both heat labile and heat stable ALPs which cannot be differentiated from the milk ALP by current enzymatic methods. Monoclonal antibodies specific of the bovine milk ALP were obtained in mice from a raw bovine milk ALP preparation. Coated in microtitre plates, these antibodies specifically capture the bovine milk ALP from dairy products. After washing, the enzymatic activity of the captured ALP is revealed by adding p-nitrophenyl-phosphate as a substrate. This simple immunoassay does not react with ALPs of intestinal or bacterial origin and, once optimized, was found to be the first immunoassay suitable to detect raw milk in boiled milk down to a 0.02% dilution. Moreover, in contrast with competitive indirect ELISA formats, the capture immunoassay does not require purified ALP.