反相高效液相色谱法测定牛奶中的主要蛋白质

建立了测定牛奶中的主要蛋白质的反相高效液相色谱法,对前处理方法进行了优化,采用Agilent Zorbax 300SB-C8(250 mm×4.6 mm,5 μm)色谱柱,三氟乙酸-水-乙腈流动相梯度洗脱,214 nm检测,柱温45 ℃,外标法定量.测定κ-CN, αs2-CN, αs1-CN, β-CN, Whey和Igg的线性关系良好,相关系数均大于0 999,加标回收率在74.8%～132.5%之间.大豆蛋白质不影响分离与检测.采用本方法分析了9种牛奶样品中上述蛋白质的含量与总量,结果表明,本方法测定结果准确可靠.不同样品中4种酪蛋白与乳清蛋白的比例基本接近,但是不同品牌之间存在一定差异.     

毛细管区带电泳-红色激光诱导荧光检测磷酸化氨基酸

以自行设计合成的1,7-二甲基-3,5-二苯乙烯基-8-苯基-(4′-氧乙酸-N-羟基琥珀酰亚胺酯)-二氟化硼-二吡咯甲烷(DMDSPAB-OSu)为高活性红色荧光衍生试剂,建立了毛细管区带电泳-激光诱导荧光(CZE-LIF)分离检测磷酸化苏氨酸、磷酸化丝氨酸和磷酸化酪氨酸的新方法。DMDSPAB-OSu具有量子产率高、光稳定性好、荧光不受pH和溶剂影响等优势,且其荧光波长与红色半导体激光检测器匹配,可有效减少生物基质内源性荧光干扰。在0.07 mol·L~(-1)H_3BO_3-Na_2B_4O_7缓冲溶液(pH=8.5)中,DMDSPAB-OSu与3种磷酸化氨基酸的衍生反应室温下仅需5 min,衍生产物可在17.5min内实现基线分离,激光诱导荧光检测检出限(S/N=3)为1.5~3.2nmol·L~(-1),方法用于牛奶酪蛋白样品检测,回收率为91.1%~103.8%。     

代谢组学方法研究奶牛注射氯霉素后牛奶中的生物标志物

建立了高效液相色谱-质谱(HPLC-MS)技术研究牛奶代谢组学的方法.样品用乙腈提取,经浓缩富集,采用HPLC-MS技术研究了注射氯霉素后牛奶中内源性代谢物随时间的变化情况.结果表明,注射氯霉素后,牛奶的代谢指纹图谱中8个谱峰的变化随给药及休药时间呈现一定升降规律;采用偏最小二乘判别分析法(PLS-DA)可将给药组与空白组样品完全分开;鉴定了对组间差异贡献较大的化合物为单羟基十八碳二烯酸(HODE),HODE可以作为注射氯霉素后奶牛体内代谢应激产生的内源性生物标志物.     

胰蛋白酶水解全酪蛋白反应过程中的分析

将高效凝胶排阻 (HPSEC)技术与水解度 (DH)概念相结合 ,对酪蛋白 胰蛋白酶水解体系的酶解反应过程进行分析 ,得到定量表征复杂酶解反应进程和不同DH值时多样性酶解产物相对分子质量分布的二维图线 ;依据蛋白质结构信息 ,结合HPSEC实验谱图 ,对胰蛋白酶作用于酪蛋白时的酶解断裂位点进行剖析 ,初步推断反应历程 ,并得到理论酶解肽段的相对分子质量分布图及酶解物中活性多肽酪蛋白磷酸肽 (CPPs)肽谱。     

胰蛋白酶水解全酪蛋白反应过程中的分析

 将高效凝胶排阻 (HPSEC)技术与水解度 (DH)概念相结合 ,对酪蛋白 胰蛋白酶水解体系的酶解反应过程进行分析 ,得到定量表征复杂酶解反应进程和不同DH值时多样性酶解产物相对分子质量分布的二维图线 ;依据蛋白质结构信息 ,结合HPSEC实验谱图 ,对胰蛋白酶作用于酪蛋白时的酶解断裂位点进行剖析 ,初步推断反应历程 ,并得到理论酶解肽段的相对分子质量分布图及酶解物中活性多肽酪蛋白磷酸肽 (CPPs)肽谱。     

基于LTQ-Orbitrap液相色谱-质谱联用技术的乳源乳清蛋白组分的差异性研究

利用LTQ Orbitrap XL组合型傅立叶变换高分辨质谱系统分析了乳源蛋白主要组分肽指纹图谱。对南方水牛乳与不同来源的乳清蛋白的氨基酸序列研究结果表明,乳清蛋白经酶解后主要为α-乳白蛋白(α-La)和β-乳球蛋白(β-Lg)组分,乳清蛋白肽质指纹谱的分析显示水牛乳与荷斯坦奶乳清蛋白α-La氨基酸发生变异的比率明显少于山羊奶乳清蛋白α-La,说明荷斯坦奶α-La和水牛乳α-La的差异更小,同源性更强;而水牛乳β-Lg与荷斯坦乳β-Lg氨基酸发生变异的部位比率要多于山羊奶,水牛乳β-Lg与山羊奶同源性更强;乳源酪蛋白酶解后的肽段主要组分为αs1-CN,β-CN,κ-N,通过对水牛乳酪蛋白的氨基酸序列的差异性分析,不同品种的乳源酪蛋白的氨基酸序列明显存在差异。与乳清蛋白相比,奶牛品种差异导致乳蛋白发生氨基酸差异现象更显著,酪蛋白的氨基酸序列对比表明,水牛奶酪蛋白与山羊奶酪蛋白比与乳牛酪蛋白的差异更大。     

聚胍基离子液体材料制备及其对蛋白质识别性能评价

磷酸化修饰是蛋白质翻译后修饰中最为重要的修饰之一,蛋白质的磷酸化修饰几乎参与生命活动的每一个环节。因此,制备对磷酸化蛋白具有选择性识别性能的材料在磷酸化蛋白质组学中具有重要意义。本实验首先合成胍基离子液体功能单体,通过沉淀聚合法合成聚胍基离子液体材料。通过傅里叶红外光谱(FT-IR)、扫描电子显微镜(SEM)、热重分析仪(TGA)考察了材料的结构、形貌、热稳定性。结果显示所制备材料为粒径约200 nm的球形颗粒。并以标准磷酸化蛋白(β-酪蛋白)为模型蛋白质,考察了聚胍基离子液体材料的识别性能。研究结果表明:材料对磷酸化蛋白具有较高吸附容量(对 β-酪蛋白的最大吸附量达到599.1 mg/g)、较快的吸附速度(1 h内达平衡),而且对磷酸化蛋白表现出较高的选择性。     

应用双向电泳和质谱联用技术研究乳源蛋白的差异性

应用双向电泳和质谱联用技术,对不同乳源蛋白的差异性进行了研究。根据ImageMaster 2DPlatinum图像分析软件对不同乳源酪蛋白和乳清蛋白的双向电泳(2-DE)图谱进行蛋白斑点的匹配分析,获得21个存在于水牛奶中主要分布在低丰度蛋白区的酪蛋白差异蛋白点和24个存在于水牛奶中乳清蛋白差异蛋白点。这些差异蛋白点经质谱鉴定分析,得到4个属于水牛奶酪蛋白的主要组分和2个与水牛奶中酪蛋白有较高同源性的新组分,同时获得4个属于水牛奶乳清蛋白的主要组分和3个与水牛奶中乳清蛋白有较高同源性的组分。     

超高效液相色谱-四极杆/静电场轨道阱高分辨质谱检测食品中的牛奶过敏原酪蛋白

基于超高效液相色谱-四极杆/静电场轨道阱高分辨质谱系统,建立了食品中牛奶过敏原酪蛋白的快速筛查和定量检测方法。样品经缓冲液提取后,采用5 kD超滤膜去除小分子杂质,得到蛋白质提取液。以数据依赖采集(data-dependent acquisition,DDA)方式获得全扫描质谱图,进行蛋白质定性确证,以平行反应监测(parallel reaction monitoring,PRM)技术对目标特征肽段进行定量分析。针对特征肽段,设计并合成了内标肽和内标物质,以降低基质效应和抵消处理过程中的损失。该方法应用于食品中的α-酪蛋白、β-酪蛋白和κ-酪蛋白的快速筛查和定量检测。结果表明,该方法在5~250μg/L范围内线性关系良好,定量限为0.2~5.5μg/kg,平均回收率在68.8%~104.4%之间,RSD6%。该方法可用于果汁饮料、果酱、面包、早餐谷物中牛奶过敏原酪蛋白的快速筛查和定量分析。     

Ti4+固载的纳米复合硅球的制备及其在磷酸化蛋白分离富集中的应用

采用St?ber法,以四乙氧基硅烷(TEOS)和γ-巯丙基三甲氧基硅烷(γ-MPTS)为前驱体合成了硅球纳米粒子,并结合"巯-烯"点击反应和"一锅法",制备得到固载Ti~(4+)的纳米复合硅球。通过红外光谱、透射电镜等方法对材料进行表征,利用蛋白吸附实验以及凝胶电泳等方法探究了该亲和材料对磷酸化蛋白的分离富集效果。结果表明,该纳米复合材料分散性好,粒径均一,对磷酸化蛋白(α-酪蛋白)的最大吸附容量(12.27μmol/g)远大于非磷酸化蛋白(辣根过氧化物酶,1.12μmol/g),且能从牛奶实际样品中分离富集磷酸化蛋白。     

Determination of volatile and non-volatile products of milk fermentation processes using capillary zone electrophoresis and solid phase microextraction coupled to gas chromatography

The aim of the investigations was to develop analytical methods for the determination of selected volatile and non-volatile organic compounds numbering among the final products of milk fermentation. The analyzed compounds were as follows: biacetyl and carboxylic acids (formic, acetic, citric, and lactic). The model yogurt was prepared under controlled conditions in our laboratory by addition of the selected bacteria (Lactobacillus bulgaricus and Streptococcus thermophilus) to the milk sample. The temperature, time, and stirring were controlled during the fermentation process. Factors considered in SPMPE-GC-FID method development included fiber exposure time, salt addition, temperature of extraction, and temperature of desorption. Various SPME fibers, for example with PDMS, CAR/PDMS, PA, and PDMS/DVB coatings, were tested to obtain the highest recovery of the investigated compounds extracted from yogurt samples. Based on these preliminary experiments, qualitative and quantitative analyses for the determination of biacetyl were performed by SPME-GC-FID. Moreover, a capillary zone electrophoresis method was developed for the determination of carboxylic acids in the yogurt samples. The buffer composition as well as deproteinization by acetonitrile were found to have a crucial effect on the analysis.     

Matrix-assisted laser desorption/ionization mass spectrometry for monitoring bacterial protein digestion in yogurt production

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was employed to determine the changes in milk protein profile due to the action of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus, the strains usually employed in yogurt production. Whereas the fermentation with the former shows the digestion of high molecular mass casein with the production of a low molecular mass peptide (3850 Da), the latter does not seem to exhibit any proteolytic activity. Proteolysis becomes relevant when milk is treated with a mixture of the two bacteria, reasonably due to symbiotic phenomena. The analysis of yogurt samples coming from industrial plants indicates increased activity of the two bacteria in the presence of ingredients containing sugars. Copyright 1999 John Wiley & Sons, Ltd.     

Use of Raman spectroscopy to determine the kinetics of chemical transformation in yogurt production

The kinetics of chemical transformation during the yogurt production was obtained using micro-Raman spectroscopy: Raman spectra were obtained as a function of the incubation time in the fermentation process from milk to yogurt. The milk fermentation by the lactic acid bacteria produces morphological, chemical and textural changes. The chemical transformations were followed using micro-Raman spectroscopy, while the aggregation process and some textural properties through the use of dynamic light scattering, viscosity and pH. Samples with three different starter culture concentrations and different incubation temperatures were prepared. The results indicate the presence of two regimes: the first one (primary metabolism) is characterized by an increment in the initial number of bacteria to reach a high concentration according to the conditions of food, temperature and space, together with some initial chemical transformations. The second regime corresponds properly to the fermentation process accelerated by the continuous reduction in pH due to the lactic acid production; this is accompanied by physical and chemical changes where new structures are created. Knowledge of the kinetics of chemical and physical transformations allows having a better control of the final product with an increase in the quality and shelf life of the final product; problems like phase separation, homogeneity, particle size, acidity, etc., can be controlled.     

Donkey Milk Fermentation by Lactococcus lactis subsp. cremoris and Lactobacillus rhamnosus Affects the Antiviral and Antibacterial Milk Properties

Background: Milk is considered an important source of bioactive peptides, which can be produced by endogenous or starter bacteria, such as lactic acid bacteria, that are considered effective and safe producers of food-grade bioactive peptides. Among the various types of milk, donkey milk has been gaining more and more attention for its nutraceutical properties. Methods: Lactobacillus rhamnosus 17D10 and Lactococcus lactis subsp. cremoris 40FEL3 were selected for their ability to produce peptides from donkey milk. The endogenous peptides and those obtained after bacterial fermentation were assayed for their antioxidant, antibacterial, and antiviral activities. The peptide mixtures were characterized by means of LC-MS/MS and then analyzed in silico using the Milk Bioactive Peptide DataBase. Results: The peptides produced by the two selected bacteria enhanced the antioxidant activity and reduced E. coli growth. Only the peptides produced by L. rhamnosus 17D10 were able to reduce S. aureus growth. All the peptide mixtures were able to inhibit the replication of HSV-1 by more than 50%. Seventeen peptides were found to have 60% sequence similarity with already known bioactive peptides. Conclusions: A lactic acid bacterium fermentation process is able to enhance the value of donkey milk through bioactivities that are important for human health.     

Examination of the swelling of heat protective hydrocolloids by DSC

While the basic fermented (sour) milk products, such as yogurt and kefir can be produced only in live flora version, the post heat-treatment is preferred in their flavored variations to increase the storage time. Casein being in sour coagulum precipitates during heat-treatment; therefore protective colloids surrounding the protein should be used to prevent it. Protective colloids are plant extracts, the most known of them are pectin and amylopectin. Basic requirement of protective colloid effect is the lower swelling temperature of hydrocolloid than the temperature of precipitation of sour coagulum. In this work we have examined the precipitation of sour coagulum as a function of the type of lactic acid bacteria cultures applied during fermentation as well as the swelling of heat protective plant hydrocolloids as a function of the composition (mainly of sugar content) of medium. To investigate the precipitation of fermented coagulum skimmed milk was fermented with mesophilic butter culture, thermophilic yogurt culture as well as with exopolysaccharide (EPS)-producing Prebiolact-2 culture. Precipitation was indicated in the increase of great extent of viscosity. Amylopectin was dispersed into aqueous solution of pH 4.5, the saccharose concentration of which was changed during the investigation of the swelling of heat protective hydrocolloids. A definite exothermic peak was assigned to the swelling of hydrocolloids during the DSC experiments. We could conclude that the precipitation temperature was increasing in the mesophilic-thermophilic-EPS producing microbes line, i.e. the heat stability and swelling temperature of hydrocolloids depend on the saccharose content of aqueous medium and they increase with rising the concentration of saccharose.     

Storage Stability of Antioxidant in Milk Products Fermented with Selected Kefir Grain Microflora

The aim of the study was to assess the antioxidant potential of goat’s milk and whey from goat’s milk fermented with selected bacteria strains from kefir grain (L. plantarum, L. fermentum, L. rhamnosus and L. acidophilus) with regard to fermented cow’s milk with the same bacteria strains. The assessment of antioxidant potential was made by ABTS, DPPH, TPC and FRAP methods. The work also assessed metabolic activity of tested lactic acid bacteria using measurement of electrical impedance changes in the growing medium. The highest values describing the antioxidant potential were found for fermented milk by L. acidophilus. It was also found that the time of cooling storage causes significantly increasing the antioxidant potential of most analyzed samples. Metabolic activity of tested lactic acid bacteria was the highest for cow’s milk. The course of curves for goat’s milk and whey from goat’s milk was similar, which confirms the differences between cow and goat milk.     

Influence of Fermentation Beetroot Juice Process on the Physico-Chemical Properties of Spray Dried Powder

Picking vegetables is, along with salting and drying, one of the oldest ways to preserve food in the world. This is the process of decomposition of simple sugars into lactic acid with the participation of lactic bacteria. The aim of the study was to obtain powders from fermented red beet juice with the highest possible amount of lactic acid bacteria (LAB) and active ingredients. For the analysis, juices were squeezed from the vegetables and two types of fermentation were used: a spontaneous fermentation and a dedicated one. After inoculation, samples were taken for analysis on a daily basis. Extract, pH, total acidity, pigments, and color were measured. In addition, microbiological tests were also carried out. The juices from the fifth day of fermentation was also spray dried, to obtain fermented beetroot powder. Juices from 3–5th day were characterized by a high content of LAB and betanin, had also a low pH, which proves that the lactic fermentation is working properly. The exception was the juice from spontaneous fermentation. According to the observations, the fermentation process did not run properly, and further analysis is needed. The powders were stable; however, results obtained from the pigment content and the LAB content are not satisfactory and require further analysis.     

一种乳酸菌多糖对酸乳凝胶的影响机理

以乳酸菌菌株和由粘性乳酸菌分泌的胞外多糖为原料制备不同的酸乳凝胶, 并用电子显微镜和质构仪等手段对其微观结构和质构特性进行了观测. 发现在酸乳体系中, 中性乳酸菌多糖与乳酪蛋白是相斥的, 多糖对酸乳凝胶的影响主要是利用其自身分子形成的空间位垒, 干扰酪蛋白微球之间的相互链接方式, 从而动态影响酪蛋白微球立体网状结构的构建. 提出酸乳乳酸菌胞外多糖对酸乳凝胶结构的影响不仅与其分子大小及结构有关, 还与胞外多糖添加到酸乳体系中的方式、时间、速度、浓度有关. 进一步解释了酸乳制作中, 粘性发酵剂所能达到的效果不容易用直接添加增稠剂的方法替代的原因.     

Reference map of soluble proteins from Streptococcus thermophilus by two-dimensional electrophoresis

Streptococcus thermophilus is a lactic acid bacterium widely used for the production of fermented dairy products. The two-dimensional electrophoresis (2-DE) protein profile was obtained from three independent analyses of 2-DE gels of soluble proteins of the strain PB18. About 270 spots were detected by silver staining and the average molecular weight and isoelectric point of each protein spot were calculated to be 41 600 and 5.2, respectively. Twelve proteins were purified by chromatographic techniques because their concentration was too low for direct sequencing from blots. Eleven were located in the PB18 2-DE profile after silver staining. These preliminary results contribute to the setting up of a two-dimensional image (or reference map) of the proteins from S. thermophilus in order to identify and compare strains of various origin or to follow metabolic process such as stress. Bidimensional autoradiographs of two strains (PB18 and ST105) of S. thermophilus grown in exponential phase at 42 degrees C with [35S]methionine were compared with an image analysis system. Among the eleven located proteins in the 2-DE silver-stained profile, nine were found in PB18 and eight in ST105 autoradiographs. One protein was specific to PB18. The eight proteins could play the role of internal 2-D PAGE markers of p/ and Mr for S. thermophilus.     

Effects of Fermentation and Storage on Bioactive Activities in Milks and Yoghurts

Fermentation of milk enhances its nutritional value through improved bioavailability of nutrients and production of bioactive substances which have biological functions. The goals of this research were to study the effect of fermentation and storage on antioxidant and antimicrobial activities in buffalo, goat and cow milks and yoghurts. Samples of buffalo, goat, cow milks and their yoghurts during their fermentation and storage were determined for proximate analysis and bioactive activities including antioxidant activities of DPPH, ABTS and reducing power assays, and antimicrobial activities against Staphylococcus aureus, Bacillus cereus, Salmonella typhimurium and Escherichia coli. Results showed that buffalo, cow and goat yoghurts had antioxidant activities in all assays and their activities significantly increased during their fermentation. Pasteurization did not affect the antioxidant activities. The activities of all yoghurts remained unchanged after a storage time of 21 days at 4 °C. For the antimicrobial activities, only yoghurts from buffalo, cow and goat milks had the activities, while all milks did not show any activities. However, buffalo yoghurt could inhibit only Gram positive strains (S. aureus and B. cereus), while goat and cow yoghurt inhibited all tested strains. A chemical responsible for antimicrobial activities in yoghurts was lactic acid formed by lactic acid bacteria. However, bioactive peptides produced by protein digestion during milk fermentation by lactic acid bacteria could not be ruled out for antioxidant and antimicrobial activities present. The antimicrobial activities of all yoghurts remained constant during their storage. It is concluded that all yoghurts would retain milk nutrition and bioactive functions during their storage in a refrigerator and may be served as a functional food with benefits from those activities for consumers.