牛羊乳蛋白组分比较研究

羊乳被国际营养学界誉为"奶中之王",逐渐被人们列为日常生活的营养保健佳品。该文对羊乳和牛乳的蛋白组分(主要是酪蛋白和乳清蛋白)含量、氨基酸组成及变异体等方面的差异进行了综述,并且对两者酪蛋白胶束的差异进行了比较,为羊乳检验和加工提供理论依据。     

Peptidome comparison following gastrointestinal digesta of bovine versus caprine milk serum

Infant formula is used as a supplement for newborns. Although bovine milk-based infant formulas dominate the market, caprine milk-based infant formula has attracted increasing attention because of its lower allergenicity. This study compared the digestive peptidome of bovine and caprine milk serum proteins by using in vitro infant simulating conditions. The result showed that the degradation pattern of milk proteins was similar, whereas the digestive rates of milk proteins differed between bovine and caprine milks. Several proteins, such as α-lactalbumin (LALBA), β-lactoglobulin (LGB), serum amyloid A protein (SAA1), glycosylation-dependent cell adhesion molecule 1 (GLYCAM1), and lactotransferrin (LTF), released more peptides during digestion of caprine milk serum than during digestion of bovine milk serum; however, more peptides derived from α_S1-casein (CSN1S1) were found in bovine digesta. In addition, antimicrobial-related peptides were mostly only found in caprine intestinal digesta. The results of this study may be useful in understanding the digestion characteristics of milk serum proteins and providing guidance on the improvement of infant formula.     

Beta-lactoglobulin is a thermal marker in processed milk as studied by electrophoresis and circular dichroic spectra

As much of the sterilization process involves heat treatment during the preparation of milk on an industrial scale, the unpredictable measures of the process are an essential issue in determining the quality of the milk. The purpose of the present study was to investigate the major protein change(s) of whey proteins in processed milk and extend the knowledge for future reference in the dairy industry. Using a native polyacrylamide gel electrophoresis, we showed almost a 90% loss and denaturation of beta-lactoglobulin (LG), but not alpha-lactalbumin (LA), in some brands of the processed and dry milks. Immunochemical analysis using Western blotting revealed that part of the loss was attributed to the formation of large multiple forms of LG in the processed product. Such denaturation was presumably associated with the heating procedure used in the process. Essentially, LG was the only major fraction converted to aggregates in milk heated at 95 degrees C for 30 min on 2-dimensional PAGE. The detailed thermal denaturation of purified LG and LA at various temperatures (50 to 95 degrees C) and time (5 to 960 s) were investigated using a circular dichroic analysis. The maximal changes of ellipticity at 205 nm (converting beta-structure to disordered structure) were correlated to heating temperature and time. There were no significant conformational changes of LG at temperatures below 70 degrees C for as long as 480 s. Pronounced and rapid changes occurred between 80 to 95 degrees C in a time-dependent manner. Fifty percent of the maximal changes could be reached within 15 s. In conclusion, the unique chemical and immunochemical loss and conformational changes made LG a superior marker for evaluating the thermal processing of milk. The detailed thermal denaturation curves of LG constructed with its time and temperature in this study provide a valuable reference for the dairy industry. We postulate that heat treatment over 80 degrees C in 15 s may induce a significant denaturation of milk LG.     

Exploration of long noncoding RNA in bovine milk exosomes and their stability during digestion in vitro

Previous studies have demonstrated that bovine milk contains mRNA and microRNA that are largely encapsulated in milk-derived exosomes. However, little information is available about long noncoding RNAs (lncRNA) in bovine milk. Increasing evidence suggests that lncRNA are of particular interest given their key role in gene expression and development. We performed a comprehensive analysis of lncRNA in bovine milk exosomes by RNA sequencing. We used a validated human in vitro digestion model to investigate the stability of lncRNA encapsulated in bovine milk exosomes during the digestion process. We identified 3,475 novel lncRNA and 6 annotated lncRNA. The lncRNA shared characteristics with those of other mammals in terms of length, exon number, and open reading frames. However, lncRNA showed higher expression than mRNAs. We selected 12 lncRNA of high-expression abundance and identified them by PCR. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that lncRNA regulate immune function, osteoblastogenesis, neurodevelopment, reproduction, cell proliferation, and cell–cell communication. We also investigated the 12 lncRNA using quantitative real-time PCR to reveal their expression profiles in milk exosomes during different stages of lactation (colostrum 2 d, 30 d, 150 d, and 270 d); their resulting expression levels in milk exosomes showed variations across the stages. A digestion experiment showed that bovine milk exosome lncRNA was resistant to in vitro digestion with different digestive juices, including saliva, gastric juice, pancreatic juice, and bile juice. Taken together, these results show for the first time that cow milk contains lncRNA, and that their abundance varied at different stages of lactation. As expected, bovine milk exosomal lncRNA were stable during in vitro digestion. These findings provide a basis for further understanding of the physiological role of milk lncRNA.     

紫外分光光度法鉴别复原奶和保鲜奶

对保鲜奶和复原奶中未变性乳清蛋白质量浓度的差别进行研究，初步探索了以紫外分光光度法对二者进行快速鉴别的方法。通过对未变性乳清蛋白的吸光度的研究确定了它的最大吸收波长为292nm，可在此波长下测出它的吸光度值并由标准曲线的线性回归方程算得其质量浓度。结果表明，复原奶中的未变性乳清蛋白质量浓度与保鲜奶中未变性乳清蛋白质量浓度差异较大。测量结果与凯氏定氯法的测量结果进行比较，这种方法与凯氏定氯法无显著差异，但此法较为简易，实用性较强。     

Technical note: Flow cytometric identification of bovine milk neutrophils and simultaneous quantification of their viability

The aim of the present study was to develop a flow cytometric procedure for the quantification of the proportion of viable, apoptotic, and necrotic polymorphonuclear neutrophilic leukocytes (PMNL) isolated from both low- and high-somatic-cell-count quarter milk samples. Milk PMNL were differentiated from other cells by indirect fluorescent labeling using a primary anti-bovine granulocyte monoclonal antibody (CH138A) and an Alexa 647-labeled secondary antibody. Polymorphonuclear neutrophilic leukocytes were identified flow cytometrically based on their cytoplasmic granularity and CH138A-positivity. Additional labeling with annexin-V-fluorescein isothiocyanate and propidium iodide was used to determine milk PMNL viability. Thirty milk samples were run in parallel to assess the repeatability of the immunoassay and 6 repeated measurements per sample were performed to assess the instrument stability. Fluorescence microscopic verification of the CH138A staining pattern showed both a high sensitivity (90.9%) and specificity (92.3%). The combination of the side-scatter properties of granulated PMNL and CH138A-Alexa 647 positivity allows the distinction of labeled PMNL from other milk cells and particles that may bind nonspecifically, and from autofluorescent particles present in milk. Quantification of the proportion of PMNL and viable, apoptotic, and necrotic subpopulations in parallel samples gave repeatable results with concordance correlation coefficients varying between 0.93 and 0.99. The average coefficient of variation for repeated measurements in identical samples ranged between 4.2 and 9.7%. In conclusion, this is the first flow cytometric method suited for the simultaneous quantification of viable, apoptotic, and necrotic bovine milk PMNL in a straightforward manner.     

Factors affecting the concentration of sphingomyelin in bovine milk

Sphingomyelin is a phospholipid located in the outer leaflet of the plasma membrane of most cells and is a component of the milk fat globule membrane. Sphingomyelin and its digestion products participate in several antiproliferative pathways that may suppress oncogenesis. Although milk and dairy products are important sources of sphingomyelin in the human diet, little is known about factors that influence sphingomyelin concentrations in milk fat or whether concentrations can be modified via the nutrition of cows. Sphingomyelin concentrations were determined in milk from Holstein and Jersey cows matched for parity and stage of lactation. Sphingomyelin was more concentrated in milk fat from Holstein cows than in milk fat from Jersey cows (1,044 vs. 839 μg/g of fat). Concentrations in whole milk did not differ because of greater milk fat content for milk from Jerseys. Differences between breeds may be related to the greater fat globule size in milk from Jerseys. Sphingomyelin content in whole milk increased with increasing days in milk because of associated increases in milk fat content. Regardless of breed, primiparous cows had greater amounts of stearic acid and less palmitic acid in sphingomyelin than did older cows. The sphingomyelin concentration in milk fat of cows in a commercial Jersey herd was lower for cows in their fourth or greater parity. Sphingomyelin content in whole milk was greater for cows in late lactation because of greater milk fat content. Feed restriction of multiparous Holstein cows to 37% of ad libitum dry matter intake increased milk fat content but did not affect milk sphingomyelin content or milk fat globule size. Supplementation of the diet with 4% soybean oil did not affect milk composition, sphingomyelin content, or milk fat globule size. Milk was sampled seasonally from 7 herds throughout Illinois during a 2-yr period. Sphingomyelin concentration in milk fat was greatest during summer and least during winter, but whole milk concentrations did not vary across seasons. We conclude that 1) sphingomyelin content of milk fat is greater in milk from Holsteins than that from Jerseys, 2) sphingomyelin content in whole milk increases with stage of lactation, and 3) sphingomyelin content of milk fat is greater during summer. However, efforts to produce milk with a greater sphingomyelin content through altering management and nutrition are unlikely to be successful.     

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.     

Considerable variation in the concentration of osteopontin in human milk, bovine milk, and infant formulas

Osteopontin (OPN) is a multifunctional bioactive protein that is implicated in numerous biological processes such as bone remodeling, inhibition of ectopic calcification, and cellular adhesion and migration, as well as several immune functions. Osteopontin has cytokine-like properties and is a key factor in the initiation of T helper 1 immune responses. Osteopontin is present in most tissues and body fluids, with the highest concentrations being found in milk. In the present study, ELISA for human and bovine milk OPN were developed and OPN concentration in human breast milk, bovine milk, and infant formulas was measured and compared. The OPN concentration in human milk was measured to approximately 138 mg/L, which corresponds to 2.1% (wt/wt) of the total protein in human breast milk. This is considerably higher than the corresponding OPN concentrations in bovine milk (∼18 mg/L) and infant formulas (∼9 mg/L). Moreover, bovine milk OPN is shown to induce the expression of the T helper 1 cytokine IL-12 in cultured human lamina propria mononuclear cells isolated from intestinal biopsies. Finally, the OPN concentration in plasma samples from umbilical cords, 3-mo-old infants, and pregnant and nonpregnant adults was measured. The OPN level in plasma from 3-mo-old infants and umbilical cords was found to be 7 to 10 times higher than in adults. Thus, the high levels of OPN in milk and infant plasma suggest that OPN is important to infants and that ingested milk OPN is likely to induce cytokine production in neonate intestinal immune cells.     

Effects of insulin,recombinant bovine somatotropin,and their interaction on insulin-like growth factor-I secretion and milk protein production in dairy cows

This trial was designed to test the effects of insulin, recombinant bovine somatotropin (rbST), and their interaction on milk protein and selected blood parameters in dairy cows. Eight Holstein cows (86 +/- 10 d in milk) were divided in two groups and used in two replicates of a Latin square design with four animals, four periods, and four treatments: 1) intravenous infusion of saline, 2) infusion of saline and subcutaneous administration of 40 mg of rbST per day, 3) intravenous infusion of 12 mg of insulin per day coupled with glucose infusion, and 4) rbST administration combined with insulin and glucose infusion. The glucose infusion rate was adjusted to maintain euglycemia. Each experimental period lasted 14 d: treatments were administered during the first 6 d, and no treatment was administered during the following 8-d resting phase. The average daily amount of glucose infusion needed to avoid hypoglycemia was 2.8 kg/cow when only insulin was infused as opposed to 2.2 kg/cow when both insulin and rbST were administered, indicating that either rbST causes a peripheral resistance to insulin or rbST increased liver gluconeogenesis or both. Data from the last 3 d of infusion were analyzed by using the SAS system for mixed models. Percent protein of milk tended to be lower (2.84 vs. 2.79%) and milk urea content was lower (16.6 vs. 14.8 mg/dl) during rbST administration, regardless of insulin infusion. Insulin infusion increased percent protein (2.78 vs. 2.85%) and percent casein (2.36 vs. 2.46%) and decreased milk urea content (17.1 vs. 14.3 mg/dl) regardless of rbST administration. For milk yield, protein yield, casein yield, lactose percent, and lactose yield, there were significant interactions between insulin and rbST administration. For example, casein yield averaged 1.17, 1.12, 1.20, and 1.28 kg/d for saline, insulin, rbST, and insulin combined with rbST, respectively. Similarly, there was a significant interaction between insulin and rbST on IGF-I levels, which were 122.5, 181.3, 342.3, and 492.2 ng/ml for saline, insulin, rbST, and insulin combined with rbST, respectively. In conclusion, these results clearly demonstrated that insulin interacts with bST in early lactation to improve milk protein synthesis and yield in dairy cows. These effects are probably mediated through a combination of bST nutrient mobilization, bST-induced gluconeogenesis, bST-induced insulin peripheral resistance, and bST/insulin synergism on insulin-like growth factor-I secretion and on mammary epithelial tissue.     

牛乳体细胞数与乳蛋白含量相关性的研究

对呼和浩特郊区一牧场30头荷斯坦乳牛进行6个月单个采样，共得427个有效样本，检测乳样中体细胞数、酪蛋白（包括α-酪蛋白、β-酪蛋白、κ-酪蛋白）、乳清蛋白、总蛋白、游离氨基氮、酪蛋白／总蛋白和乳清蛋白／总蛋白。结果表明，乳中总蛋白、游离氨基氮含量及乳清蛋白／总蛋白与SCC呈显著正相关；酪蛋白／总蛋白与SCC呈显著负相关；乳清蛋白含量与SCC呈极显著正相关。酪蛋白、α-酪蛋白／酪蛋白、β-酪蛋白／酪蛋白、κ-酪蛋白／酪蛋白与SCC的相关性不显著。酪蛋白含量与总蛋白含量呈极显著的正相关，与游离氨基氮含量、乳清蛋白／总蛋白呈极显著的负相关。乳清蛋白含量与游离氨基氮、总蛋白含量呈极显著正相关。     

牛乳中乳铁蛋白的分离纯化研究

牛乳经酸沉淀除酪蛋白后,通过超滤、离子交换法分离纯化得到乳铁蛋白,利用考马斯亮蓝、SDS-PAGE电泳定性定量检测。结果表明:每毫升牛乳能提取乳铁蛋白0.22mg,纯度为95%。     

Pseudomonas aeruginosa lectin PA-IIL as a powerful probe for human and bovine milk analysis

Milk composition exhibits species-specific differences depending on genetic, evolutionary, and environmental factors. In addition, commercial milk preparations are also changed by industrial manipulations, including severe heat processing. Cow milk, used as human food, provides important nutrients but lacks some essential components that are present in raw human milk. The present study, which was aimed at comparing infant breastfeeding to cow-based formula nourishment, shows major differences between the human and the commercial cow milk glycans detectable by the lectins PA-IL (galactose-binding) and PA-IIL (fucose and mannose-binding) isolated from the cells of human pathogen Pseudomonas aeruginosa. More than 40 human milk samples, several cow milks, and bovine milk-based infant formulas, were examined using these two lectins. For purposes of comparison, the plant lectins Concanavalin A (Con A), which binds mannose, and Ulex europaeus 1st lectin (UEA-I), which binds fucose, were also used. The most prominent difference was revealed using PA-IIL, which displayed a unique high sensitivity to the human milk fucosylated compounds. PA-IL and UEA-I also exhibited preferential sensitivity to the human milk but considerably lower than that of PA-IIL. Con A was inhibited by human and the other milk preparations examined to the same extent. These findings indicate the superb applicability of PA-IIL for rapid and reliable comparative investigation of milk glycans from human and cow, indicating which glycans could be added to infant formulas in order to enrich them, as well as for verification and quality control of otherwise improved bovine milk-based infant formulas.     

3种毛细管电泳方法分析牛乳蛋白的比较

对比毛细管凝胶电泳(CGE)、毛细管未涂层管区带电泳(CZE)和涂层管区带电泳(CZE)在牛乳蛋白分离和定量分析,结果表明在分离条件、分离效果和定量特征上,毛细管涂层区带电泳优于其他两种电泳。应用涂层毛细管区带电泳对不同阶段婴幼儿配方乳粉的蛋白组成进行测定,分离效果较好。     

Existence of functional lingual antimicrobial peptide in bovine milk

The lingual antimicrobial peptide (LAP) belongs to the β-defensin family in cattle and is localized in epithelial cells of alveoli in mammary glands. The aim was to investigate whether LAP is secreted into milk and whether the secreted LAP has antimicrobial activity. Decaseinated bovine skim milk was applied to sample extraction cartridges, and the eluate was used for competitive enzyme immunoassay and Western blotting to test for the presence of LAP in milk. After tricine-SDS PAGE, the gel was stained using the periodic acid-Schiff reaction to examine the possibility of glycosylation of LAP. The eluate obtained from the sample extraction cartridges was subjected to a LAP antibody-coupled affinity column, after which the antimicrobial activity of its eluate against Escherichia coli was investigated with radial diffusion plate assay and colony-forming unit enumeration following the culture of bacteria with the sample. The immunoreactive LAP was detected in the eluate by competitive enzyme immunoassay (optical density = 0.437 ± 0.012 vs. 0.468 ± 0.016). In the Western blotting analysis, immunoreactive bands were seen around 8, 14, and 17 kDa. The bands at 14 and 17 kDa, but not 8 kDa, were periodic acid-Schiff reaction-positive. The eluate of LAP antibody-coupled affinity column had antimicrobial activity against E. coli (cfu/control = 0.17 ± 0.18). These results suggest that bovine milk contains functional LAP-like substances that exert antimicrobial activity.     

Causes of spoilage of thermally processed fish in Morocco

Of 946 cans of fish suspected of being spoiled, which were collected from local stores and canneries in Morocco, 484 cans (51%) had major container defects. Leaking seams at the manufacturer's end and droops and vees on the canner's end were the most prevalent defects (36 and 28%, respectively). Upon microbiological analysis of 256 cans, viable micro-organisms were recovered from 168 cans, of which 72% contained typical leaker spoilage organisms, while 28% contained typical underprocessing spoilage organisms. Thermophilic organisms were found in association with other organisms in half of the cans. The more severe the swell, the greater were the chances of recovering micro-organisms. The scoring system of Davidson & Pflug (1981) indicated that 16% and 71% of the cans had seams of questionable and poor quality, respectively, and these had a potential for leakage. The most probable cause for spoilage was determined: leakage, 80%; non-microbial swells, 10%; underprocessing, 10%.     

Proteomic analysis of the temporal expression of bovine milk proteins during coliform mastitis and label-free relative quantification

The discovery of biomarkers in milk indicative of local inflammation or disease in the bovine mammary gland has been hindered by the extreme biological complexity of milk, the dynamic range of proteins in the matrix that renders the identification of low-abundance proteins difficult, and the challenges associated with quantifying changes during disease in the abundance of proteins for which no antibody exists. The objectives of the current study were to characterize the temporal expression of milk proteins following Escherichia coli challenge and to evaluate change in relative abundance of identified proteins using a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) label-free semiquantitative approach. Liquid chromatography-MS/MS conducted on whey from milk samples collected just before infusion with E. coli and at 12, 18, 24, 36, 48, and 60 h following infection resulted in the identification of the high- to medium-abundance proteins α_S1-, α_S2- β-, and κ-caseins and the whey proteins serum albumin, β-lactoglobulin, and α-lactalbumin. Additionally, a select number of lower abundance markers of inflammation were also identified, including lactoferrin, transferrin, apolipoprotein AI, fibrinogen, glycosylation-dependent cell adhesion molecule-1, peptidoglycan recognition receptor protein, and cyclic dodecapeptide-1. Normalized peptide counts for each protein identified were used to evaluate temporal changes in milk proteins following infection. For comparison with relative protein abundance determined using proteomic-based methods, changes in serum albumin, lactoferrin, and transferrin in milk during disease were also measured using ELISA. Label-free, proteomic-based quantification revealed relative changes in milk proteins that corresponded to expression profiles generated by ELISA. The results indicate that label-free LC-MS/MS methods are a viable means of tracking changes in relative protein abundance in milk during disease. Despite the identification of primarily abundant milk proteins, the results indicate that, with further refinement, LC-MS/MS could be used to evaluate temporal changes in proteins related to host response for which no antibody or ELISA currently exists.     

毛细管电泳法对乳及乳制品中乳源蛋白的研究

采用毛细管电泳方法对原料乳、市售鲜奶、不同厂家的巴氏灭菌乳、不同厂家和产地超高温灭菌乳(UHT)、调味乳、乳酸饮料、复原乳、酸奶、奶粉中蛋白成分进行检测。选择聚乙烯醇涂层毛细管,采用柠檬酸缓冲体系,在紫外检测214nm、分离电压20kV条件下对乳及乳制品中的α一乳白蛋白(α-La)、β一乳球蛋白(β-Lg)、α-酪蛋白(α-CN)、β-酪蛋白(β-CN)和k-酪蛋白(k-CN)进行分离测定。结果表明:五种蛋白的含量在原料乳(巴氏灭菌乳、市售鲜奶)、UHT乳、酸奶、调味乳、乳酸饮料、复原乳中依次降低,而UHT乳含量随保质期的增加而减少,奶粉中蛋白质含量因其适应人群而有差异。乳及乳制品中蛋白质的含量与其存在形式、产地及加工工艺相关。     

Proteomic analysis of differentially expressed proteins in bovine milk during experimentally induced Escherichia coli mastitis

The objectives of the current study were to profile changes in protein composition using 2-dimensional gel electrophoresis on whey samples from a group of 8 cows before and 18 h after infection with Escherichia coli and to identify differentially expressed milk proteins by peptide sequencing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry post source decay. Only proteins present in whey fractions of all 8 cows were sequenced to avoid reporting a protein response unique to only a subset of infected cows. Despite the overwhelming presence of casein and β-lactoglobulin, the low abundance proteins transthyretin, lactadherin, β-2-microglobulin precursor, α-1-acid glycoprotein, and complement C3 precursor could be identified in whey samples from healthy cows. Whey samples at 18 h postinfection were characterized by an abundance of serum albumin, in spots of varying mass and isoelectric point, as well as increased transthyretin and complement C3 precursor levels. Also detected at 18 h postinoculation were the antimicrobial peptides cathelicidin, indolicidin, and bactenecin 5 and 7, and the proteins β-fibrinogen, α-2-HS-glycoprotein, S100-A12, and α-1-antiproteinase. Most notable was the detection of the acute phase protein α-1-acid glycoprotein in mastitic whey samples, a result not previously reported. In contrast to methods used in previous proteomic analyses of bovine milk, the methods used in the current study enabled the rapid identification of milk proteins with minimal sample preparation. Use of a larger sample size than previous analyses also allowed for more robust protein identification. Results indicate that examination of the protein profile of whey samples from cows after inoculation with E. coli could provide a rapid survey of milk protein modulation during coliform mastitis and aid in the identification of biomarkers of this disease.