Technical note: Direct enzyme immunoassay of progesterone in bovine milk whey

A simple extraction-free or direct quantitative ELISA for progesterone in bovine milk whey was developed. Whey samples are easy to collect, transport, and store. This method also allows for monitoring progesterone levels in cattle, which is important in reproductive management. The assay was designed to cover the concentration range 0.05 to 2 ng/mL, and the sensitivity of the method was 1.5 pg/mL. The intra- and interassay coefficients of variation were 8 and 12%, respectively. A high correlation (r = 0.90) between ELISA and radioimmunoassay measurements of progesterone in the same milk whey samples was obtained. The method can be easily applied in practice because samples can be stored at room temperature (22 to 26°C) for 4 d. Moreover, because analysis requires milk coagulation, that process can be initiated during transport by standard mail services to the laboratory. Upon arrival at the laboratory, whey can be kept refrigerated for 1 wk before analysis. This tool is useful for monitoring luteal activity of dairy cows.     

ELISA法测定牛奶中黄曲霉毒素M1不确定度评定

目的本实验对酶联免疫法检测牛奶中黄曲霉毒素M1含量的测定结果进行不确定度的评定,确保检测结果的准确性及可靠性。方法实验分析了酶联免疫法测定牛奶中黄曲霉毒素M1的不确定度的分量及其来源,并通过计算各分量的不确定度得出检测结果的合成标准不确定度。结果酶联免疫法测定牛奶中黄曲霉毒素M1的浓度为45.741 pg/m L,扩展不确定度为11.704 pg/m L,置信水平P=95%,k=2。结论不确定度的主要来源为测量的重复性、试剂盒的灵敏度、标准曲线拟合,而酶标仪测定OD值、ELISA法操作过程中导致的不确定度可以忽略不计。选用酶联免疫法检测黄曲霉毒素M1时增加平行样的测定、注意试剂盒的灵敏度、保持标准曲线的稳定性对于提高酶联免疫法检测的准确性和可靠性具有较强的实用价值。     

The relationship of flow cytometry results with classical measures of bacterial counts in raw refrigerated milk

Bulk milk was collected from 100 farms throughout the year and analysed after storage for either 24, 48 or 72 h, using flow cytometry. The total bacterial counts obtained by two methods – flow cytometry and standard plate count were compared and the conversion relationship between them was assessed: the results showed no effect of the age of the samples relationship between these two methods.     

Near‐infrared spectroscopy and data analysis for predicting milk powder quality attributes

Near‐infrared (NIR) spectroscopy is a rapid analytical method for food products. In this study, NIR spectroscopy, data pretreatment techniques and multivariate data analysis were used to predict fine particle size fraction, dispersibility and bulk density of various milk powder samples, which are believed to have a significant impact on milk powder quality. Predictive models using partial least‐squares (PLS) regression were developed using NIR spectra and milk powder physical and functional properties, and it was concluded that the PLS models predicted milk powder quality with an accuracy of 88‐90 per cent.     

Characterisation of Macedonian white‐brined cheese: Effect of raw or heat‐treated caprine milk

This research analysed the use of raw and pasteurised milk in the production of Macedonian white caprine milk cheese. Pasteurisation resulted in a considerable decrease in the nitrogen fractions, urea‐polyacrylamide gel electrophoresis of caseins, peptide profiles and volatiles during the maturation period. Forty‐five volatile components were detected, including twelve acids, fourteen esters, six ketones, three alcohols, four terpenes and six other compounds. It was deduced that pasteurisation of milk for the manufacture of artisanal white‐brined cheese delayed the evolution of volatiles, which are related to the unique flavour of the cheese.     

Effects of 1 year long‐term freezing with different preservatives on milk cholesterol,progesterone and lactoferrin determination

This study was performed to determine the effects of long‐term freezing on milk composition and to evaluate the possible effects on analysis of four preservative substances (Azidiol, Kathon CG, NaOH and Thiomersal) on the determination of milk cholesterol, progesterone and lactoferrin concentration. Collected milk was separated in aliquots, stored at 20 °C in 10mL plastic vials and analysed after 1, 6 and 12 months. It could be shown that the preservatives are not equally appropriate for all analysing methods used in this experiment. Preservatives and storage conditions for milk samples have to be carefully selected during the study design depending on the parameters to be measured.     

Survey of raw milk cheeses for microbiological quality and prevalence of foodborne pathogens

Cheese may be manufactured in the United States using raw milk, provided the cheese is aged for at least 60 days at temperatures not less than 35 °F (1.7 °C). There is now increased concern among regulators regarding the safety of raw milk cheese due to the potential ability of foodborne pathogens to survive the manufacturing and aging processes. In this study, 41 raw milk cheeses were obtained from retail specialty shops, farmers’ markets, and on-line sources. The cheeses were then analyzed for the presence of Listeria monocytogenes, Salmonella, Escherichia coli O157:H7, Staphylococcus aureus, and Campylobacter. Aerobic plate counts (APC), coliform and yeast/mold counts were also performed. The results revealed that none of the enteric pathogens were detected in any of the samples tested. Five samples contained coliforms; two of those contained E. coli at less than 10² cfu/g. Three other cheese samples contained S. aureus. The APC and yeast-mold counts were within expected ranges. Based on the results obtained from these 41 raw milk cheeses, the 60-day aging rule for unpasteurized milk cheeses appears adequate for producing microbiologically safe products.     

A novel system for on-farm fertility monitoring based on milk progesterone

Timely identification of a cow's reproduction status is essential to minimize fertility-related losses on dairy farms. This includes optimal estrus detection, pregnancy diagnosis, and the timely recognition of early embryonic death and ovarian problems. On-farm milk progesterone (P4) analysis can indicate all of these fertility events simultaneously. However, milk P4 measurements are subject to a large variability both in terms of measurement errors and absolute values between cycles. The objective of this paper is to present a newly developed methodology for detecting luteolysis preceding estrus and give an indication of its on-farm use. The innovative monitoring system presented is based on milk P4 using the principles of synergistic control. Instead of using filtering techniques and fixed thresholds, the present system employs an individually on-line updated model to describe the P4 profile, combined with a statistical process control chart to identify the cow's fertility status. The inputs for the latter are the residuals of the on-line updated model, corrected for the concentration-dependent variability that is typical for milk P4 measurements. To show its possible use, the system was validated on the P4 profiles of 38 dairy cows. The positive predictive value for luteolysis followed by estrus was 100%, meaning that the monitoring system picked up all estrous periods identified by the experts. Pregnancy or embryonic mortality was characterized by the absence or detection of luteolysis following an insemination, respectively. For 13 cows, no luteolysis was detected by the system within the 25 to 32 d after insemination, indicating pregnancy, which was confirmed later by rectal palpation. It was also shown that the system is able to cope with deviating P4 profiles having prolonged follicular or luteal phases, which may suggest the occurrence of cysts. Future research is recommended for optimizing sampling frequency, predicting the optimal insemination window, and establishing rules to detect problems based on deviating P4 patterns.     

Effects of curd heating with or without previous milk pasteurisation on the microbiological quality and safety of craft‐made ‘Pasta Filata’ Kashkaval cheese curds

An empirical ‘Pasta Filata’ process used for traditional Kashkaval cheese manufacture from raw ewes’ milk improved the hygiene of fresh curd. Coagulase‐positive (RPF+) staphylococci and coliforms declined by 2.4 and 4.5 log units, respectively, and Listeria contamination was minimised. Before Pasta Filata, RPF+ staphylococci exceeded the 5‐log threshold level specified in EC Regulation 1441/2007 in raw milk curds despite the prevalence of indigenous mesophilic lactic acid bacteria. Pasta Filata favoured enterococci in raw milk curds. Pasteurisation enhanced prevalence of Streptococcus thermophilus and Lactococcus lactis starters and microbial quality and safety of pasteurised milk curds before and particularly after Pasta Filata.     

Effect of high‐energy emulsification on properties of commercial low‐temperature pasteurised milk

The present study investigated milk quality through changing physicochemical properties by using high‐energy emulsification (microfluidisation, MF; and ultrasonication, US). The results will aid in making better quality dairy products. MF was more effective in changing the physicochemical properties of milk; compared to US, MF resulted in smaller, monodispersed particles, and MF better inactivated protease activity without changing the pH, lipid oxidation and thermal denaturation of protein during storage periods.     

The effect of refrigerated storage of raw milk on the physicochemical and microbiological quality of Tunisian semihard Gouda‐type cheese during ripening

A Tunisian semihard Gouda‐type cheese made from milk kept at 4 °C for 24, 48, 72 and 96 h was monitored during 45 days of ripening. The effect of milk refrigeration on the evolution of physicochemical parameters in relation to the quantitative variation of the microbial population during ripening of Gouda‐type cheese was investigated. Microbiological and physicochemical analyses were performed on raw milk and cheese samples after curding, 2, 9, 16, 23, 30, 37 and 45 days of ripening time. The raw milk kept under refrigeration at 4 °C for 96 h showed the highest microbial count and proteolysis level. The duration of storage significantly reduced the cheese yield as a result of important solubilisation casein in proteoses‐peptones. Results of different nitrogenous fractions by Kjeldahl method showed enzymatic hydrolysis products of casein whose intensity depended on the maturing stage as well as the refrigeration time. Besides the evident action of the plasmin, original milk protease, on the hydrolysis of casein in soluble fractions, the proteolysis of cheese caseins is also initiated by proteolytic action of the chymosin and extracellular heat‐resistant proteases notably produced by the same psychrotrophic microflora. Lactic acid bacteria starters that constitute the dominant microflora of this type of cheese are also considered as aroma precursors.     

化学发光酶免疫分析法快速测定牛奶中恩诺沙星的含量

目的 建立快速测定牛奶中恩诺沙星的化学发光酶免疫检测方法.方法 采用竞争法,即将牛奶样品中的恩诺沙星与标记有碱性磷酸酶的恩诺沙星同时与限量的特异性固相恩诺沙星抗体进行竞争结合反应,通过分离未结合的标记抗原,测定标记抗原与抗体复合物化学发光强度,经相应的数学函数计算出待测抗原的含量.根据这一基本原理,利用金刚烷类体系作为化学发光底物,快速地测定牛奶中恩诺沙星残留量.结果 检出限可达239.9 pg/ml,检测范围为350～1 000 pg/ml,批内与批间相对标准偏差均小于15％.结论本方法在抗生素恩诺沙星残留检测及监控等领域有很好的应用前景.     

A multiple lateral flow immunoassay based on AuNP for the detection of 5 chemical contaminants in milk

Melamine (MEL), enrofloxacin (ENR), sulfamethazine (SMZ), tetracycline (TC), and aflatoxin M₁ (AFM₁) are the main chemical contaminants in milk. It is necessary to detect these miscellaneous chemical contaminants in milk synchronously to ensure the safety of the milk. In this study, a multiple lateral flow immunoassay (LFIA) was developed for the detection of MEL, ENR, SMZ, TC, and AFM₁ in milk. Under optimal experimental conditions, the cutoff values were 25 ng/mL for MEL, 1 ng/mL for ENR, 2.5 ng/mL for SMZ, 2.5 ng/mL for TC, and 0.25 ng/mL for AFM₁ in milk samples. The limits of detection of LFIA were 0.173 ng/mL for MEL, 0.078 ng/mL for ENR, 0.059 ng/mL for SMZ, 0.082 ng/mL for TC, and 0.0064 ng/mL for AFM₁. The recovery rates of LFIA in milk were 83.2–104.4% for MEL, 76.5–127.3% for ENR, 96.8–113.5% for SMZ, 107.1–166.6% for TC, and 93.5–130.3% for AFM₁. The coefficients of variation were all less than 15%. As a whole, the developed multiple lateral flow immunoassay showed potential as a highly reliable and excellent tool for the rapid and sensitive screening of MEL, ENR, SMZ, TC, and AFM₁ in milk.     

Instant infusion pasteurisation of bovine milk. I. Effects on bacterial inactivation and physical–chemical properties

Two types of milk, skim milk and non-standardised raw milk, were heat treated using direct heating by instant infusion pasteurisation with treatment temperatures in the range from 72°C to 120°C and with holding times of less than 1 second. Indirect heating by HTST pasteurisation (72°C for 15 seconds) was used for comparison. The inactivation of microorganisms reached at least the same level when using instant infusion pasteurisation compared to HTST pasteurisation. Changes in the physical-chemical properties were observed in the skim milk fractions of instant infusion pasteurised non-standardised milk, whereas for instant infusion pasteurised skim milk less influence from the treatments was observed.     

Optimisation of the formulation for barley–milk composite‐based fermented drink

The effect of barley flour concentration, Lactobacillus plantarum NCDC344 (Lp344) and co‐culture (Streptococcus thermophilus 20) inoculum levels on the sensory quality, Lp344 count, β‐glucan content and viscosity of barley–milk composite‐based fermented drink was investigated. A central composite rotatable design of response surface methodology was used for optimisation of the formulation. Of the three formulation variables, barley flour concentration was found to be the most critical as it significantly affected overall acceptability, Lp344 count and β‐glucan content (P < 0.01). The optimised drink rated 7.80 on a 9‐point hedonic scale, and contained 8.59 log cfu/mL of Lp344 cells and 0.144 g/100 g of β‐glucan.     

Rapid on‐site determination of melamine in raw milk by an immunochromatographic strip

A rapid and simple method was established based on gold nanoparticle‐labelled monoclonal antibody probes for the detection of melamine pollution in raw milk. The conditions for conjugation between the antibody and gold nanoparticles were optimised (pH 8.0, antibody concentration 5 μg mL^?1). It gives a single proportional to melamine concentration with a performance time of only 3 min. A practical calibration curve was established with a reader system with limit of detection calculated as 4.47 and 8.34 μg L^?1 with naked eyes. Three structural analogues, atrazine, desethyl‐desisopropyl‐atrazine and cyromazine, were used to test the specificity of the immunochromatographic strip, and small colour changes on the strip test line were found even at the 500 ng mL^?1 spiked level. Fifty raw milk samples were detected with this strip method, and the resulting data coincided well with results from gas chromatography tandem mass spectrometry. The above‐mentioned results showed that this test strip can be used for melamine screening in the daily monitoring of milk.     

Importance of casein micelle size and milk composition for milk gelation

The economic output of the dairy industry is to a great extent dependent on the processing of milk into other milk-based products such as cheese. The yield and quality of cheese are dependent on both the composition and technological properties of milk. The objective of this study was to evaluate the importance and effects of casein (CN) micelle size and milk composition on milk gelation characteristics in order to evaluate the possibilities for enhancing gelation properties through breeding. Milk was collected on 4 sampling occasions at the farm level in winter and summer from dairy cows with high genetic merit, classified as elite dairy cows, of the Swedish Red and Swedish Holstein breeds. Comparisons were made with milk from a Swedish Red herd, a Swedish Holstein herd, and a Swedish dairy processor. Properties of CN micelles, such as their native and rennet-induced CN micelle size and their ζ-potential, were analyzed by photon correlation spectroscopy, and rennet-induced gelation characteristics, including gel strength, gelation time, and frequency sweeps, were determined. Milk parameters of the protein, lipid, and carbohydrate profiles as well as minerals were used to obtain correlations with native CN micelle size and gelation characteristics. Milk pH and protein, CN, and lactose contents were found to affect milk gelation. Smaller native CN micelles were shown to form stronger gels when poorly coagulating milk was excluded from the correlation analysis. In addition, milk pH correlated positively, whereas Mg and K correlated negatively with native CN micellar size. The milk from the elite dairy cows was shown to have good gelation characteristics. Furthermore, genetic progress in relation to CN micelle size was found for these cows as a correlated response to selection for the Swedish breeding objective if optimizing for milk gelation characteristics. The results indicate that selection for smaller native CN micelles and lower milk pH through breeding would enhance gelation properties and may thus improve the initial step in the processing of cheese.     

微生物抑制法快速检测鲜牛乳中红霉素残留

目的利用微生物抑制法快速检测鲜牛乳中红霉素残留。方法将待测的牛奶样品和空白样品经杀菌后接入乳酸菌,经过相同时间发酵后,计算待测牛奶样品发酵前后的pH变化差值与空白样品发酵前后的pH变化差值,从而判断鲜牛奶中红霉素残留的含量。结果经过单因素及正交实验,确定乳酸菌发酵产酸条件如下:接种量0.15 U/L、发酵温度40℃、菌粉溶解时间25 min、发酵时间3.5 h,利用乳酸菌发酵产酸建立了检测鲜牛奶中红霉素残留的标准曲线Y=0.024X-0.013(其中,Y为△pH,X为鲜牛乳样品中的红霉素浓度),线性相关系数R~2=0.9949,检测的灵敏度IC_(50)为29.71μg/L,检测的线性范围为0~50μg/L,加标回收率为103.54%~124.58%,同一批次内检测的变异系数为4.39%~16.46%。结论建立的检测方法可用于筛选鲜牛乳中是否有抗生素残留并进行定量检测。