生物发光快速测定生乳菌落总数的方法

为消除利用ATP生物发光法测定生乳菌落总数时非细菌ATP对测定结果的干扰,建立了一种样品前处理方法。利用ATP生物发光法对经过前处理的生乳样品进行检测,结果表明,生乳菌落总数对数值与生乳细菌ATP发光对数值呈现较好的线性关系(R2=0.982),相关程度为显著相关(P<0.01),说明该前处理方法能够有效排除非细菌ATP的干扰,有利于提高ATP生物发光法定量测定生乳菌落总数准确性。     

高效液相色谱法测定母乳及牛乳中总核苷酸含量

建立高效液相色谱测定母乳和牛乳中总核苷酸含量和组成的分析方法。总核苷酸包括游离核苷酸、游离核苷、核苷聚合物及加合物等不同来源的可利用核苷总量。利用酶解法释放出母乳中的核苷,以硼化聚丙烯酰胺凝胶（Affi-Gel 601）作为固相萃取介质进行样品净化,用反相高效液相色谱仪检测,内标法定量,检测结果以核苷酸计。且针对母乳样品的特性,增加高温灭活步骤进行样品前处理,以检测母乳中核苷酸的天然含量及组成。对所建方法进行全面验证,结果表明其具有良好的线性,相关系数（r2）均在0.999以上;母乳中各核苷酸的检出限和定量限分别在0.18～0.45 mg/L和0.60～1.51 mg/L之间;母乳基质中3 种不同加标水平下总核苷酸的回收率均在99%～108%之间,连续3 d 6 次检测结果的相对标准偏差在0.8%～7.8%之间,显示了良好的准确性及精密度。然后进一步利用所建检测方法,评估母乳及5 种市面常见的牛乳中的核苷酸总量及组成用以验证该方法的适用性。该方法的灵敏度、准确性及精密度均可满足对母乳和牛乳样品中天然的核苷酸含量及组成的测定要求。本研究为今后大样本量调查母乳及牛乳样品中核苷酸的含量和组成提供了更加科学准确的方法。     

Routine testing of producers' milk supplies for compositional quality using randomly selected samples

In a study over a number of months, involving over 500 milk producers in three locations, and representing bulk milk collection from cans as well as mobile and stationary refrigerated bulk milk tanks, the precision of various random sampling and testing frequencies in estimating the fat, protein and lactose content of milk was evaluated. In addition, the traditional procedure of estimating the composition of milk supplies from chemically preserved composites was compared to values obtained by analysis of fresh samples. While sampling and testing every milk collection gave the most precise estimate of milk composition for each producer, it was found that for producers using refrigerated bulk milk tanks with ex-farm collection, four random samples per month gave a precision of ±4% (or ±0.14% fat approx) for monthly fat content and ±l.2% (or ±0.04% fat approx) for annual fat content. Due to lower variability the precision for both protein and lactose estimation was much higher. The sample compositing procedure, while capable of a relatively high level of precision, did not always achieve this in practice and tended to underestimate the constituents in milk, especially protein and lactose.     

Rapid and accurate determination of malate,citrate, pyruvate and oxaloacetate by enzymatic reactions coupled to formation of a fluorochromophore: Application in colorful juices and fermentable food (yogurt,wine) analysis

A fluorometric-coupled reaction for the accurate and rapid determination malate, citrate, pyruvate and oxaloacetate is presented. The method was found useful for an accurate and rapid determination of these metabolites in low volumes of milk, yogurt, apple and lemon juice and wines without considerable pretreatment. In particular, this method was found valuable in characterising the outcome of maloactic acid fermentation (MLF) in wine and outlined for the first time fundamental differences in MLF between red and white wines. Thus, this method has merit in analysing these substances in heterogeneous, opaque and colorful foods.     

Metal analysis in difficult materials with platform furnace Zeeman-atomic absorption spectroscopy. 2. Direct determination of cadmium and lead in milk

A procedure is described for the direct determination of cadmium and lead in whole milk, skim milk, condensed milk, and human milk. Using a Perkin-Elmer 5000 Z instrument with HGA 500 and L'vov platform and by application of oxygen ashing at approx. 600 degrees C, determination limits of approx. 0.02 microgram/l and 0.7 microgram/l for cadmium and lead, respectively, are attainable. Day-to-day precision is 10% for 0.1 microgram/l of cadmium and 2 micrograms/l of lead. Accuracy control at least at higher levels was possible with DPASV after wet digestion. The contents found with this procedure in cows milk are at the lowest limit of very recent literature data, i.e. on average at 0.05 microgram/l for cadmium and 2 microgram/l for lead for samples from nonpolluted regions. The results indicate that milk does not contribute significantly to heavy metal exposure of man.     

Factors Affecting the Distribution of Lactate Dehydrogenase Between Particulate and Soluble Phases of Homogenized Trout Skeletal Muscle

SUMMARY: Skeletal muscle of brown trout contained one electrophoretically distinguishable lactate dehydrogenase (LDH) isozyme. In homogenates of the muscle, release of the enzyme into the soluble phase was favored by high ionic strength and high pH. DPNH solubilized the enzyme and prevented binding of soluble enzyme to particulate matter at concentrations which contributed only negligibly to the ionic strength of the suspending medium. The other compounds involved in the LDH-catalyzed reaction, DPN⁺, pyruvate, and lactate, were less effective. The effect of the latter two was due chiefly to their contribution to the ionic strength of the medium. Pyruvate, however, used with either DPNH or DPN⁺ exhibited a synergistic activity. Effective solubilization showed remarkable specificity for DPNH and TPNH. Solubilization by DPNH was also dependent on the tissue concentration of the suspending medium. The lower the tissue concentration, the more readily LDH is solubilized by DPNH. In addition to certain metabolic implications, this information may be used to define assay conditions to allow the study of the kinetics of LDH in bound and soluble forms.     

土壤中镉含量测定的前处理方法优化研究

利用标准土壤样品对比实际土壤样品,研究电热板消解法和微波消解法对测定土壤中镉元素的不同差异.将两种不同的土壤消解前处理方法以及结果进行分析比较,其中,电热板消解法根据消解状态不同又可分为两个批次,再针对不同消解方法的优缺点以及分析结果,确保推荐的消解预处理方法更加方便快捷、实用,结果更加准确、精密.通过以上测试结果发现...     

乳与乳制品中总砷测定的不同前处理方法比较

目的比较不同前处理方法的处理效果,选择适合乳与乳制品中总砷测定的前处理方法。方法分别采用湿法消解、干法灰化、微波消解对乳与乳制品样品进行前处理,利用氢化物原子荧光法测定总砷含量。结果 3种前处理方式的检测结果均满足方法学要求,湿法消解、干法灰化和微波消解的回收率分别为81.7%～86.5%,90.0%～94.4%和90.8%～95.6%,精密度分别为8.84%～9.80%,3.22%～4.37%和3.18%～4.82%;检出限分别为0.0042、0.00068、0.0028mg/kg;微波消解法处理的质控样品结果更接近于标准值。结论微波消解法操作简便、准确性好,适合乳制品企业批量产品检测。     

Fluorometric determination of beta-hydroxybutyrate in milk and blood plasma

Determination of β-hydroxybutyrate (BHBA) in blood and milk samples is an important tool in the diagnosis of ketosis in dairy cattle. Apart from semiquantitative cow-side tests, well-established laboratory methods exist for measurements in blood serum or plasma. These spectrophotometric methods are, however, neither convenient nor reliable when transferred to analyses of milk. Due to its nontransparent nature, milk needs extensive pretreatment if traditional analyses are to be used. This paper describes a fluorometric determination of BHBA that is useful without pretreatment in opaque matrices such as milk and in blood plasma. The method is easy to automate, saves labor expenses, and is inexpensive. The analytical accuracy and precision are reliable for intensive as well as large-scale analysis; for example, in-line sampling from automatic milking systems. Analysis of 2500 random milk samples showed a BHBA content ranging from 10 to 631 μM (mean 49 μM). Furthermore, selected samples (n = 295) from diagnosed ketotic animals taken on d −35 to +35 from peak level ranged from 10 to 684 μM (median 79 μM, mean 141 μM). Using the same 1240 blood plasma samples, the fluorometric method was closely correlated with a traditional spectrophotometric method (r = 0.987). Hemolysis of samples does not appear to affect the fluorometric determination of BHBA.     

Rapid and simple estimation of microbiological quality of raw milk using chromogenic Limulus amoebocyte lysate endpoint assay

A rapid chromogenic Limulus amoebocyte lysate (LAL) endpoint assay for the enumeration of total mesophilic microbial loads and coliforms was investigated as a means to assess the microbiological quality of raw milk. For experiment 1, raw milk samples (n = 25) were stored in a refrigerator (2 +/- 2 degrees C) and then analyzed at regular intervals (1, 5, 10, and 15 days). For experiment 2, fresh raw milk samples (n = 50) were tested to determine the utility of the LAL assay for fresh raw milk. The sample was diluted threefold in a 96-well microtiter plate with pyrogen-free water and assayed with a chromogenic LAL kit to find a final reaction point. The LAL results were compared with standard plate counts (SPC) and coliform counts determined by conventional plating methods. The results of the LAL assay were strongly correlated to conventional SPC (r2 = 0.93; n = 100) and were highly correlated to coliforms (r2 = 0.74; n = 100). A highly significant linear relationship (r2 = 0.82; n = 50) was also observed between the predicted SPC based on the LAL value and the actual SPC. The results of LAL testing were classified into one of seven contamination groups. The data set for SPC was effectively differentiated using the LAL technique (P < 0.01). The chromogenic LAL assay was found to be a rapid (within 16 min) and simple (not requiring specific instruments) method for monitoring microbial levels in raw milk. This method may be successfully implemented to rapidly determine highly microbial contaminated raw milk (> 3.0 log10 CFU/ml of SPC).     

Multiplex polymerase chain reaction as a mastitis screening test for Staphylococcus aureus,Streptococcus agalactiae,Streptococcus dysgalactiae and Streptococcus uberis in bulk milk samples

Effective diagnostic tools for screening herds for mastitis pathogens are important in development and monitoring of mastitis control programmes. A multiplex polymerase chain reaction (PCR) assay for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis was used in preliminary studies to assess its applicability as an alternative method for monitoring mastitis caused by these organisms at the herd level. PCR was used to detect the presence of these organisms in bulk milk samples. Correlations with bulk milk somatic cell counts (BMCC), total bacteria counts and thermoduric bacteria counts were evaluated. A total of 176 bulk milk samples were collected from 42 herds on five consecutive occasions at approx. 10-d intervals. Str. uberis was the most common organism in these bulk milk samples. There was no relationship between presence of either Staph. aureus, Str. dysgalactiae or Str. uberis and BMCC, total bacteria counts or thermoduric bacteria counts. However, presence of Str. agalactiae was associated with high BMCC and total bacteria counts. The results of this study show that regular analysis of bulk milk using this multiplex PCR assay may be a useful tool for monitoring herd status with respect to Str. agalactiae, but is of less value for monitoring occurrence of Staph. aureus, Str. dysgalactiae and Str. uberis. Further investigations are needed to clarify the relationship between positive PCR results and the prevalence of infected cows in the herd.     

Prediction of fatty acid profiles in cow,ewe, and goat milk by mid-infrared spectrometry

Mid-infrared (MIR) spectrometry was used to estimate the fatty acid (FA) composition in cow, ewe, and goat milk. The objectives were to compare different statistical approaches with wavelength selection to predict the milk FA composition from MIR spectra, and to develop equations for FA in cow, goat, and ewe milk. In total, a set of 349 cow milk samples, 200 ewe milk samples, and 332 goat milk samples were both analyzed by MIR and by gas chromatography, the reference method. A broad FA variability was ensured by using milk from different breeds and feeding systems. The methods studied were partial least squares regression (PLS), first-derivative pretreatment + PLS, genetic algorithm + PLS, wavelets + PLS, least absolute shrinkage and selection operator method (LASSO), and elastic net. The best results were obtained with PLS, genetic algorithm + PLS and first derivative + PLS. The residual standard deviation and the coefficient of determination in external validation were used to characterize the equations and to retain the best for each FA in each species. In all cases, the predictions were of better quality for FA found at medium to high concentrations (i.e., for saturated FA and some monounsaturated FA with a coefficient of determination in external validation >0.90). The conversion of the FA expressed in grams per 100 mL of milk to grams per 100 g of FA was possible with a small loss of accuracy for some FA.     

离子色谱法测定奶粉中的葡萄糖、蔗糖和乳糖

建立高效阴离子交换色谱-脉冲安培检测法测定奶粉中葡萄糖、蔗糖和乳糖的方法。以METROSEP CARB 1(150mm×4.0mm)阴离子交换柱为分离柱,脉冲安培检测,37mmol/L NaOH溶液为淋洗液,以柠檬酸作为蛋白质沉淀剂。葡萄糖、蔗糖和乳糖的线性范围分别为1～40、1～50、1～50mg/L,相关系数分别0.9966、0.9968、0.9985,检出限分别为0.014、0.091、0.083mg/L,精密度为1.10%～4.96%,回收率为90.13%～104.83%。该方法分析时间短,前处理简单,适用于快速测定奶粉中的葡萄糖、蔗糖和乳糖。     

Conventional tube and microplate Limulus amoebocyte lysate procedures for determination of gram-negative bacteria in milk

A comparison was made of the conventional tube and microplate Limulus amoebocyte lysate assay for detection of gram-negative bacterial lipopolysaccharide in milk. Raw whole milk samples were maintained frozen and portions were examined periodically on alternate days during 13-d storage to evaluate the reproducibility of both Limulus amoebocyte lysate procedures and to determine optimum reaction conditions for the microplate method. One-day-old, raw and locally purchased pasteurized milk samples, held at 7 degrees C, were analyzed during storage to establish the correlation of both procedures with aerobic and modified psychrotrophic plate counts. Vitamin- and mineral-fortified dairy-based products were examined using the microplate Limulus amoebocyte lysate test as a potential indicator of raw material or finished product bacterial quality and possible postprocessing contamination. Statistical analysis of the data collected comparing the conventional tube and the microplate Limulus amoebocyte lysate assay demonstrated no significant difference exists between the methods when either the modified psychrotrophic bacterial count or the aerobic plate count was used to determine gram-negative bacteria in pasteurized or raw milk (P less than .91). The microplate method, which uses half the lysate reagent, was a good indicator of the bacterial quality of milk and fortified dairy products, consistently detecting bacterial levels greater than 10(3) to 10(4)/ml.     

酸性品红-B-R体系共振光散射法测定牛奶中的新霉素

为建立一种快速测定牛奶中新霉素的新方法,以市售超高温灭菌纯牛奶为样品,基于样品中残留新霉素与酸性品红在pH5.50的B-R(Britton-Robinson)缓冲溶液中形成新的缔合物,利用共振光散射技术进行测定。结果表明：其最大共振光散射峰位于625nm波长处,在0.00～2.50μg/mL范围内新霉素质量浓度与体系的相对散射光强度(ΔI)有良好的线性关系,相关系数r=0.9978,检出限为0.013μg/mL;样品测定的RSD小于2.5%(n=5),加标回收率为84.0%～96.7%。此法快速简便、灵敏度较高,用于牛奶中新霉素残留量的测定,结果满意。     

离子色谱安培检测器测定食品中的碘

添加氢氧化钾和硫代硫酸钠溶液于食品样品中,减压干燥后,采用灰化法处理,样液通过离子色谱进行分离,安培检测器检测食品中的碘。该方法优化了前处理条件,检出限为5μg/kg,样品测定的精密度在1.26%～4.54%,加标回收率为85.9%～112.3%。采用所建立的方法分析国家标准物质圆白菜(GBW10014)、菠菜(GBW10015)、奶粉(GBW10017)、鸡肉(GBW10018),测定值与标准值吻合。     

Biosensor assay for determination of haptoglobin in bovine milk

Despite more than 30 years of research into mastitis diagnostics, there are few alternatives to the somatic cell count (SCC) in practical use for identification of cows with subclinical mastitis. Mastitis is not only an animal welfare problem, but also affects the yield, composition and technological properties of milk. Hence, dairy cooperatives give farmers a premium quality payment to encourage low SCC although there is no clear scientific data defining the level of SCC in bulk tank milk that is associated with additional benefits in terms of milk quality. Recent research on alternative markers for inflammatory reactions in the lactating cow, e.g. in mastitis, includes investigations of the acute phase protein, haptoglobin (Hp). So far, the content of Hp in milk has mainly been studied in relation to mastitis diagnostics, with little attention given to its importance for milk composition and technological properties. At present, Hp in milk is measured using ELISA, but this technique is not suitable for routine large-scale analysis. In recent years, optical biosensor technology has been used for automated and rapid quantitative analysis of different components in milk, but so far not for analysis of acute phase proteins. The aim of the present study was to develop a rapid and sensitive biosensor method to determine Hp in milk. An affinity sensor assay based on the interaction between Hp and haemoglobin was developed using surface plasmon resonance (SPR) biosensor technology. The assay was used to analyse Hp in composite milk samples from cows without any clinical signs of mastitis and quarter milk samples with a weak to strong reaction in the California Mastitis Test (CMT). A commercial ELISA for determination of Hp in milk was used for comparison. The limit of detection (LOD) of the biosensor assay was determined as 1.1 mg/l. Within-assay and between-day variations were determined both with bulk tank milk spiked with human Hp and with composite milk samples containing bovine Hp. Coefficients of variation varied between 3.6 and 8.6% at concentrations between 4.0 and 12 mg/l, respectively. Agreement between the results obtained by the biosensor assay and the ELISA was satisfactory; however, the results obtained by the biosensor were generally lower than the results obtained by the ELISA. Possible explanations for this observation are discussed.     

离子色谱法检测硝酸盐亚硝酸盐前处理方法的研究

目的 利用离子色谱法检测乳及乳制品中亚硝酸盐、硝酸盐,在实际检测中用该国标方法在前处理样品时,存在沉淀样品不完全进而影响检测准确性、重复性不理想的弊端,为提高检测的准确性,缩小检测偏差,需对国标方法中样品的前处理改进。方法 样品经乙腈沉淀脂肪蛋白质后,采用相应的方法提取和净化,以氢氧化钾溶液为淋洗液,阴离子交换柱分离,电导检测器检测。以保留时间定性,外标法定量。结果 该方法克服了乳及乳制品GB5009.33-2010第三法检测检出限高及检测用时长,精密度低的的弊端。结论本方法适合于乳及乳制品定量测定。     

Double-antibody based immunoassay for the detection of β-casein in bovine milk samples

The concentration of casein (CN) is one of the most important parameters for measuring the quality of bovine milk. Traditional approach to CN concentration determination is Kjeldahl, which is an indirect method for determination of total nitrogen content. Here, we described a double-antibody based direct immunoassay for the detection of β-CN in bovine milk samples. Monoclonal antibody (McAb) was used as capture antibody and polyclonal antibody (PcAb) labelled with horseradish peroxidase (HRP) as detection antibody. With the direct immunoassay format, the linear range of the detection was 0.1–10.0 μg mL⁻¹. The detection limit was 0.04 μg mL⁻¹. In addition, the concentration of β-CN in real bovine milk samples has been detected by the developed immunoassay. There was a good correlation between the results obtained by the developed technique and Kjeldahl method from commercial samples. Compared to the traditional approach, the advantage of the assay is no need of time-consuming sample pretreatment.     

Analytic validation of an infrared milk urea assay and effects of sample acquisition factors on milk urea results

The objective of this study was to determine if milk samples, as they are routinely collected by Ontario Dairy Herd Improvement, would yield accurate milk urea results with an infrared assay. This investigation involved analytic validation of the infrared assay and assessment of the effect of DHI routine sample acquisition factors on milk urea results. Analytic validation of an automated milk urea assay was performed by assessing the relative accuracy and precision of milk urea results produced by the Fossomatic 4000 Milk Analyzer, an infrared method of analysis, compared with the Eurochem test, an accepted reference method. Results indicated that, when interpreted at the group level, milk urea results between the infrared method and the reference test were in good agreement. The two tests shared a similar and high level of precision. Milk urea concentrations obtained from composite (metered) milk samples, and not quarter stripping samples, were most representative of concurrent serum urea concentrations. The addition of bronopol preservative did not result in a numerically important change in milk urea concentrations. Storage of preserved metered milk samples for up to 4 d at either room temperature or by refrigeration, or for up to 3 d by freezing, did not result in changes in milk urea concentrations. We concluded that milk samples, as they are routinely collected and handled by DHI, are suitable for measurement of milk urea concentrations with the infrared method of analysis if data are interpreted at the group level.