高效液相色谱分析乳清蛋白方法研究

建立测定乳清蛋白中α-乳白蛋白和β-乳球蛋白含量的高效液相色谱分析方法,采用Agilent的ZORBOX Eclipse XDB-C8色谱柱(150 mm×4.6 mm),流动相A为10%乙腈中含0.1%三氟乙酸,流动相B为90%乙腈中含0.1%三氟乙酸。采用梯度洗脱,流速为0.25 mL/min,二极管阵列检测器,检测波长214 nm,柱温40℃。外标法定量,α-La和β-Lg两种组分线性关系良好,相关系数分别为0.993 1和0.990 9,检测限为3μg/mL、8μg/mL。     

高效液相色谱法测定乳清蛋白主要成分的研究

建立了测定乳清蛋白中α-La和β-Lg含量的高效液相色谱分析方法,采用Kromasil C8色谱柱(5μm,250mm×4.6mm),流动相A为0.1%的三氟乙酸-水溶液,流动相B为0.09%的三氟乙酸-乙腈溶液梯度洗脱,流速为0.8mL/min,二极管阵列检测器,检测波长215nm,柱温30℃。外标法定量,α-La和β-Lg两种组分线性关系良好,相关系数分别为0.9998和0.9984,检测限为3μg/mL、10μg/mL。     

高效液相色谱法定量分析茶籽粕中的茶皂素

以脱脂冷榨茶籽饼粕为原料,采用反相ODS填料纯化制备茶皂素标准品,并采用紫外吸收光谱、红外光谱及质谱对该标准品进行鉴定;采用自制标准品为标样,建立一种高效液相色谱法定量分析茶籽饼粕中茶皂素含量的方法。高效液相色谱法条件为反相ODS色谱柱、检测波长215nm、柱温室温、流速1.0mL/min、流动相以乙腈-水体系进行梯度洗脱(0～5min内乙腈体积分数保持为20%,5～10min内流动相乙腈体积分数从20%增至100%,10～15min内流动相乙腈体积分数从100%回至20%)。采用该条件检测得到的茶皂素峰型对称,保留时间合适,杂峰和茶皂素峰分离度较高。     

HPLC检测火腿肠中大豆分离蛋白的方法研究

利用高效液相色谱(HPLC)法测定火腿肠中大豆分离蛋白的含量。色谱条件为:Xb ridgeBEH300 C4色谱柱;蒸发光散射检测器;梯度洗脱;流动相A:0.05%三氟乙酸-HPLC级水溶液,B:0.05%三氟乙酸四氢呋喃溶液;流速1.0 mL/min;检测波长254 nm;柱温30℃;进样量20μL。大豆分离蛋白在1%～9%含量范围内与特征峰面积呈良好的线性关系,回归方程为Y=2 923.1 X-970.85,相关系数R=0.994 2,平均回收率为99.30%,RSD为2.04%。HPLC法准确度、精密度、稳定性、重现性良好,为火腿肠中大豆分离蛋白的定量检测提供了可选择的方法。     

反向高效液相色谱法分离检测乳粉中的酪蛋白

以C8色谱柱为分离柱,利用反相高效液相色谱法,在流速1.0 mL/min,检测波长220 nm,柱温25℃,0.1%TFA水溶液为A流动相;100%乙腈溶液为B流动相的梯度洗脱条件下对乳粉中酪蛋白的4种组分(α_(S1)-酪蛋白、α_(S2)-酪蛋白、β-酪蛋白和κ-酪蛋白)分离检测。结果表明,该法具有良好的线性关系,重复性好,加标回收率高,对全脂牛乳粉、羊乳粉等乳粉中酪蛋白组分的分离测定有很好的效果。     

反相高效液相色谱法测定发酵液中的风味强化肽

采用反相高效液相色谱法对发酵液中的风味强化肽进行定量分析。色谱条件 :色谱柱为日本shimadzu公司的ShimadzuVP ODSC18(5 μm ,1 5 0mm× 4 6mmi d )不锈钢柱 ,流动相为乙腈 水 (含体积分数为 0 1 %的三氟乙酸 )溶液 ,采用线性梯度洗脱方式 ,流动相B(含 0 1 %三氟乙酸的乙腈溶液 )体积分数在 3 0min内由 5 %线性升至 5 0 % ,流速为 1 0mL/min ,检测波长为 2 3 0nm ,室温测定。结果 :RP HPLC法测定浓度在 0 1 2 5～ 2 0 0 0mg/mL时线性关系良好 ,r =0 9998。回收率为 86 2 %～ 1 0 3 4% ,最低检测限量为 1 2 5ng ,该方法精密度高 ,相对标准偏差 (RSD)为1 4%。此方法准确、灵敏、重现性好 ,适合于发酵液这种复杂体系中风味强化肽的定量分析     

高效液相色谱快速分析亚麻籽环肽条件研究

采用高效液相色谱法分析亚麻籽环肽组成及相对含量。以14种亚麻籽环肽色谱峰平均分离度和分析效率作为指标,对不同色谱柱、进样量、梯度洗脱初始体积分数、流速、乙腈体积分数上升速率进行优化,并利用HPLC-ESI-MS进行定性分析。结果表明,高效液相色谱最优条件为:采用Kinetex■色谱柱,水、乙腈为流动相,进样量5μL,梯度洗脱程序为乙腈体积分数从40%以5%/min升至90%,流速1 m L/min。最优条件下亚麻籽环肽14个色谱峰分离度较好,平均分离度为1. 65,分析时间为9. 09 min,分离效率为10. 86。优化后的色谱条件可对市售亚麻籽、亚麻籽油以及亚麻籽粕中环肽组成进行有效分析。     

液相色谱串联质谱检测食品中的黄曲霉毒素

为提高黄曲霉毒素的检测灵敏度,建立快速液相色谱串联质谱(LC-MS/MS)法对食品中的4 种黄曲霉毒素B1、B2、G1、G2 进行定性定量分析。样品粉碎后用体积比为84:16 的乙腈- 水混合液提取,过滤后通过真菌毒素净化柱进样,采用C18 柱分离,0.1% 甲酸溶液和甲醇做流动相,以60:40 比例等度洗脱,质谱在多反应监测(MRM)的正离子模式下进行分析。4 种组分在5min 内完全分离,而且此方法线性关系良好,黄曲霉毒素B1、B2、G1、G2 的检出限分别是0.012、0.009、0.013、0.007μg/kg,平均加标回收率在80%～95% 之间,相对标准偏差小于5%。该方法快速灵敏、准确可靠,其检出限可满足欧盟地区严格的黄曲霉毒素限量标准。     

高效液相色谱法测定牛乳中α-乳白蛋白

利用常规的C18色谱柱的高效液相色谱建立测定牛乳中主要过敏蛋白α-乳白蛋白含量的方法。色谱条件:色谱柱Alltima-C18(4.6 mm×200 mm,5μm),柱温为45℃,UV检测波长215 nm,流动相A为含0.1%三氟乙酸的超纯水,流动相B为乙腈:超纯水:三氟乙酸=400:100:0.5,采用梯度洗脱方法,流速0.8 mL/min。结果表明:α-乳白蛋白的线性范围分别为50～1 000μg/mL(r=0.990 9);α-乳白蛋白平均回收率为97.27%,RSD=0.76%(n=5);该方法简便、准确,适合于乳制品中α-乳白蛋白含量的测定。     

凝胶过滤色谱纯化乳清蛋白降胆固醇肽的研究

从乳清蛋白水解物中纯化降胆固醇活性肽,乳清蛋白经胰蛋白酶水解得到的水解物经超滤粗分离,采用凝胶过滤色谱分离纯化,并对其洗脱流动相种类、pH值和洗脱液浓度进行优化筛选,采用体外检测方法筛选出最佳洗脱条件下的具有胆固醇胶束溶解度抑制活性最高的组分,并用已知分子量的标准蛋白质凝胶层析绘制校正曲线,建立分子量的对数与出峰时间的关系。结果表明,优化的最佳洗脱液为磷酸钠溶液,研究得到了凝胶过滤色谱的校正曲线,在洗脱时间为71 min时得到的组分降胆固醇活性最高,为57.48%,经计算其分子量在2 000~4 700 u范围内。     

Yield and aging of Cheddar cheeses manufactured from milks with different milk serum protein contents

Whey proteins in general and specifically β-lactoglobulin, α-lactalbumin, and immunoglobulins have been thought to decrease proteolysis in cheeses manufactured from concentrated retentates from ultrafiltration. The proteins found in whey are called whey proteins and are called milk serum proteins (SP) when they are in milk. The experiment included 3 treatments; low milk SP (0.18%), control (0.52%), and high milk SP (0.63%), and was replicated 3 times. The standardized milk for cheese making of the low milk SP treatment contained more casein as a percentage of true protein and more calcium as a percentage of crude protein, whereas the nonprotein nitrogen and total calcium content was not different from the control and high SP treatments. The nonprotein nitrogen and total calcium content of the milks did not differ because of the process used to remove the milk SP from skim milk. The low milk SP milk contained less free fatty acids (FFA) than the control and high milk SP treatment; however, no differences in FFA content of the cheeses was detected. Approximately 40 to 45% of the FFA found in the milk before cheese making was lost into the whey during cheese making. Decreasing the milk SP content of milk by 65% and increasing the content by 21% did not significantly influence general Cheddar cheese composition. Higher fat recovery and cheese yield were detected in the low milk SP treatment cheeses. There was more proteolysis in the low milk SP cheese and this may be due to the lower concentration of undenatured β-lactoglobulin, α-lactalbumin, and other high molecular weight SP retained in the cheeses made from milk with low milk SP content.     

HPLC Determination of Riboflavin in Eggs and Dairy Products

An isocratic HPLC method has been developed to determine riboflavin in eggs, whole milk, 2% fat milk, skim milk, dry milk, yogurt, cottage cheese, and cheddar cheese. The developed method involves acidification, centrifugation, and quantification of riboflavin in the supernatant with HPLC. The HPLC system consisted of a μ Bondapak C₁₈ column, a solvent system of water-methanol-acetic acid (68:32:0.1 v/v), a flow rate of 1.0 ml/min, and a UV detector. The method is simple, rapid, sensitive, and specific for riboflavin. Recoveries of more than 90% were obtained in all samples analyzed.     

USE OF SEMI-AUTOMATIC INSTRUMENTS FOR PROCESS AND PRODUCT CONTROL IN THE DAIRY-FOOD INDUSTRY

An integrated procedure for the rapid determination of total protein and heat treatment classification of skim milk powders by dye binding is described. Total protein values estimated by the Pro-Milk were in good agreement with the macro-Kjeldahl method; the correlation coefficient was +0–98 and the SD of the differences between the two methods was ±0.55, corresponding to a CV of ±1.52 per cent. Residual undenatured whey protein was measured by the Pro-Milk difference (PMD) method on undiluted isoelectric filtrates. These were prepared by addition of acetic acid - sodium acetate buffer to reconstituted skim milk powders. The undenatured whey protein levels thus determined correlated well with the ADMI Kjeldahl procedure. The reproducibility of the PMD method in our laboratory was ±0.010, and satisfactory reports on the reliability of the Pro-Milk procedure have been received from industry. It is recommended that future heat treatment classifications of milk powders should be based on dye binding. Furthermore, it is proposed that milk powders should be heat classified according to the percentage of undenatured whey protein to total protein in the reconstituted milk and expressed as the whey protein ratio (WPR) of the milk powder.     

Rapid detection of the addition of soybean proteins to cheese and other dairy products by reversed-phase perfusion chromatography

The undeclared addition of soybean proteins to milk products is forbidden and a method is needed for food control and enforcement. This paper reports the development of a chromatographic method for routine analysis enabling the detection of the addition of soybean proteins to dairy products. A perfusion chromatography column and a linear binary gradient of acetonitrile-water-0.1% (v/v) trifluoroacetic acid at a temperature of 60°C were used. A very simple sample treatment consisting of mixing the sample with a suitable solvent (Milli-Q water or bicarbonate buffer (pH = 11)) and centrifuging was used. The method enabled the separation of soybean proteins from milk proteins in less than 4 min (at a flow-rate of 3 ml/min). The method has been successfully applied to the detection of soybean proteins in milk, cheese, yogurt, and enteral formula. The correct quantitation of these vegetable proteins has also been possible in milk adulterated at origin with known sources of soybean proteins. The application of the method to samples adulterated at origin also leads to interesting conclusions as to the effect of the processing conditions used for the preparation of each dairy product on the determination of soybean proteins.     

Selenium content of goat milk and its distribution in protein fractions.

This study reports on selenium distribution in goat milk. Skim milk was found to contain the major part (94%) of total milk selenium. The selenium distribution over casein and whey protein fractions depends on the separation method used, but irrespective of these methods, skim milk selenium is mainly associated with the casein fraction (greater than 69%). Approximately 9%, 7% and 24% of selenium is removed by dialysis (molecular cutoff 10-12 kDa) from skim milk, casein and whey respectively, indicating a major association of selenium with milk proteins. This observation is confirmed by selenium analysis of individual caseins and whey proteins isolated through ion-exchange chromatography and gel filtration. Selenium concentrations of the different isolated milk proteins show considerable variation (caseins: 294-550 ng Se/g; whey proteins: 217-457 ng Se/g).     

Improving the structure and rheology of high protein,low fat yoghurt with undenatured whey proteins

The objective of this study was to investigate the effects of whey protein denaturation and whey protein:casein-ratio on the structural, rheological and sensory properties of high protein (8% true protein), low fat (<0.5% fat) yoghurt. Yoghurt milk bases were made by adding undenatured whey proteins from native whey protein concentrate (NWPC) to casein concentrate in different whey protein:casein-ratios. The degree of whey protein denaturation was then controlled by the temperature treatment of the yoghurt milk bases. Addition of NWPC in low (whey protein:casein-ratio 25:75) or medium levels (whey protein:casein-ratio 35:65) in combination with heat treatment at 75 °C for 5 min gave yoghurts with significantly lower firmness, lower storage modulus (G′), and better sensory properties (less coarse and granular and more smooth), compared with corresponding yoghurts produced from yoghurt milk bases heat-treated at 95 °C for 5 min or with control yoghurts (no addition of NWPC).     

Technological optimization of manufacture of probiotic whey cheese matrices

In attempts to optimize their manufacture, whey cheese matrices obtained via thermal processing of whey (leading to protein precipitation) and inoculated with probiotic cultures were tested. A central composite, face-centered design was followed, so a total of 16 experiments were run using fractional addition of bovine milk to feedstock whey, homogenization time, and storage time of whey cheese as processing parameters. Probiotic whey cheese matrices were inoculated with Lactobacillus casei LAFTIL26 at 10% (v/v), whereas control whey cheese matrices were added with skim milk previously acidified with lactic acid to the same level. All whey cheeses were stored at 7 °C up to 14 d. Chemical and sensory analyses were carried out for all samples, as well as rheological characterization by oscillatory viscometry and textural profiling. As expected, differences were found between control and probiotic matrices: fractional addition of milk and storage time were the factors accounting for the most important effects. Estimation of the best operating parameters was via response surface analysis: milk addition at a rate of 10% to 15% (v/v), and homogenization for 5 min led to the best probiotic whey cheeses in terms of texture and organoleptic properties, whereas the best time for consumption was found to be by 9 d of storage following manufacture.     

Evaluation of Alternative Methods to Increase Calcium Retention in Cottage Cheese Curd

The effect of several alternative methods including addition of rennet, addition of carrageenan and use of 2:1 (v/v) preconcentrated skim milk by ultrafiltration (UF) upon calcium retention, yield, composition and sensory properties of dry curd cottage cheese was investigated. Although each of the processing methods resulted in the manufacture of dry curd cottage cheese with different compositions and properties, none of them was satisfactory for increasing calcium retention. Added carrageenan bound additional whey proteins, added rennet interfered with curd syneresis and whey expulsion during cooking and use of UF preconcentrated skim milk resulted in an increase in yield, total solids and protein of the curd.     

TWO-DIMENSIONAL ANALYSIS OF SKIM MILK PROTEINS USING PREPARATIVE ISOELECTRIC FOCUSING FOLLOWED BY POLYACRYLAMIDE GEL ELECTROPHORESIS

ABSTRACT A preparative isoelectric focusing (IEF) method was applied to separate skim milk proteins using the Rotofor device in a pH 3–10 gradient containing 4 M urea/1% triton X-100. Each of the 20 fractions obtained from the Rotofor device was then analyzed by polyacrylamide gel electrophoresis (PAGE). Both urea-PAGE and SDS-PAGE were used to separate purified caseins and skim milk resulting in comparable two-dimensional patterns. The major bovine caseins (αs1, αs2, β, K-casein) were resolved better on urea-PAGE. The αs1-and β-casein were focused at pH ～ 4.5 and 4.8, respectively, whereas αs2-caseins focused as several bands at pH 6.2–6.8. The A variant of K-casein focuses at Fractions 6–9 which is slightly more acidic than the B variant that focuses at Fractions 7–13. No sample pretreatment was necessary to analyze skim milk proteins and urea-PAGE clearly resolved bands of all major caseins and whey proteins. Preparative isoelectric focusing followed by PAGE was found to be a useful and powerful method to analyze milk proteins in two-dimensions. This technique facilitates the analysis of the relative amounts of proteins in milk, as well as simplifies the detection of changes and foreign proteins in milk.     

胶体金免疫层析法快速检测配方羊奶粉中的牛β-乳球蛋白

建立了一种可快速检测配方羊奶粉中牛β-乳球蛋白(β-lactoglobulin,β-lg)的胶体金免疫层析检测方法。通过杂交瘤技术制备β-lg单克隆抗体(monoclonal antibody,mAb),半抑制浓度(50% inhibiting concentration,IC₅₀)为5.87 μg/mL。将胶体金标记的β-lg mAb包被于金标垫,β-lg和山羊抗小鼠IgG标记于硝酸纤维素膜(nitrocellulose membrane,NC膜)分别作为检测线(T线)和质控线(C线),开发了可检测β-lg的免疫层析试纸条。该试纸条对β-lg的检测限(limit of detection,LOD)值为50 μg/mL,与其他基质成分均未产生有效交叉反应,对全脂山羊乳粉中掺杂脱脂牛奶粉(nonfat skim milk,NFSM)、脱盐乳清粉(desalted whey powder,DWP)和乳清蛋白粉(whey protein powder,WPP)的LOD值分别为5%、5%和0.1%。运用该方法对9个市售配方羊奶粉进行分析,检测结果与商品化酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)试剂盒一致。该方法前处理快速简单,5 min即可裸眼判定结果,可用于配方羊奶粉商品的现场快速检测。