阴离子交换树脂分离酪蛋白糖巨肽工艺条件优化

采用离子交换树脂从乳清粉中分离酪蛋白糖巨肽(casein glycomacropeptide,CGMP),筛选适于分离CGMP的离子交换树脂并考察静态吸附过程中吸附pH值、吸附时间、缓冲液浓度等因素对CGMP分离效果的影响。乳清粉溶液在pH5.1时离心除杂后与201×4树脂混合,吸附条件为吸附pH3.9、吸附时间1h、缓冲液浓度0.02mol/L、洗脱液为0.5mol/L pH4.0的氯化钠溶液、洗脱时间4h;100g乳清粉利用此工艺条件可得1.45g唾液酸含量10.4%(以蛋白质计)的CGMP。该工艺方法分离过程简单、纯化效果好、唾液酸含量高,适用于工业化生产。     

Purification of Glycomacropeptide from Caseinate Hydrolysate by Gel Chromatography and Treatment with Acidic Solution

ABSTRACT: Glycomacropeptide (GMP) was purified from chymosin-hydrolyzed caseinate solution by the procedure involving: (1) gel chromatography on Sephacryl S-200 at pH 7.0 to obtain a crude GMP fraction; (2) addition of acidic solution, pH 3.5 to the crude glycomacropeptide to precipitate contaminating protein and/or peptide; and (3) re-chromatography of the material soluble in the acidic solution on Sephacryl S-200 at pH 3.5. The purified GMP accounted for 5.3% of dry weight of caseinate hydrolysate, and 0.7% of dry weight of sodium caseinate powder. The preparation was of considerably high purity with its amino-acid composition showing only traces (each < 1 residue / peptide) of arginine, histidine, phenylalanine, and tyrosine, the amino acids that do not occur in GMP.     

Charged Ultrafiltration Membranes Increase the Selectivity of Whey Protein Separations

ABSTRACT:  Ultrafiltration is widely used to concentrate proteins, but fractionation of one protein from another is much less common. This study examined the use of positively charged membranes to increase the selectivity of ultrafiltration and allow the fractionation of proteins from cheese whey. By adding a positive charge to ultrafiltration membranes, and adjusting the solution pH, it was possible to permeate proteins having little or no charge, such as glycomacropeptide, and retain proteins having a positive charge. Placing a charge on the membrane increased the selectivity by over 600% compared to using an uncharged membrane. The data were fit using the stagnant film model that relates the observed sieving coefficient to membrane parameters such as the flux, mass transfer coefficient, and membrane Peclet number. The model was a useful tool for data analysis and for the scale up of membrane separations for whey protein fractionation.     

Influence of milk pre-heating conditions on casein–whey protein interactions and skim milk concentrate viscosity

The viscosity of concentrates (50–55% total solids) prepared from skim milk heated (5 min at 80 or 90 °C) at pH 6.5 and 6.7 was examined. The extent of heat-induced whey protein denaturation increased with increasing temperature and pH. More denatured whey protein and κ-casein were found in the serum phase of milk heated at higher pH. The viscosity of milk concentrates increased considerably with increasing pH at concentration and increasing heating temperature, whereas the distribution of denatured whey proteins and κ-casein between the serum and micellar phase only marginally influenced concentrate viscosity. Skim milk concentrate viscosity thus appears to be governed primarily by volume fraction and interactions of particles, which are governed primarily by concentration factor, the extent of whey protein denaturation and pH. Control and optimization of these factors can facilitate control over skim milk concentrate viscosity and energy efficiency in spray-drying.     

Technical note: Comparison of chromatographic profile of glycomacropeptide from cheese whey isolated using different methods

Glycomacropeptide (GMP) has heterogeneous carbohydrates, and this attributes to its various biological activities. In this study, we compared the chromatographic profiles of GMP isolated by three methods (trichloracetic acid fractionation, ethanol precipitation, and ultrafiltration) from whey protein isolate (WPI). Seven sharp heterogeneous GMP peaks were eluted from GMP prepared by ethanol precipitation and ultrafiltration using Mono Q anionic chromatography, while only 5 peaks were seen in TCA treated sample. The TCA pretreatment recovered only sialo-GMP (glycosylated) and eliminated all contaminated proteins; however, the recovery rate was the lowest (6.7% of the initial WPI). Ethanol precipitation recovered 20.4% of GMP from WPI and 75.7% was glycosylated, but the heating process might lead to degradation of glycosidic residues. Ultrafiltration was found to be the most effective in recovering GMP. The recovery rate was 33.9% with 81.6% sialo-GMP. We concluded that carbohydrate profile of GMP varied widely and depended on the isolation method. Based on the high recovery of sialo-GMP, the combination of ultrafiltration and anionic chromatography might be a suitable and practical approach on an industrial scale.     

十六烷基三甲基溴化铵和乳清蛋白结合的研究

寻找新型絮凝剂,研究其与蛋白粒子结合的物化因素,实现蛋白分离及对乳清的合理利用。研究不同因素如十六烷基三甲基溴化铵(CTAB)浓度、pH 值、温度、蛋白质量浓度、离子强度对蛋白-CTAB 复合物形成的影响,获得乳清蛋白与CTAB 结合的最佳物化条件。结果表明：CTAB 添加量是影响蛋白-CTAB 结合的最大因素;不同pH 值对CTAB- 蛋白的影响差异不显著;不同pH 值条件下,温度对CTAB- 蛋白结合影响显著;在0～200mmol/L 范围内,离子强度越高,结合的络合物越少。蛋白质量浓度越大,结合的络合物越多,蛋白回收率越大。     

Whey Protein Isolate and Glyco-macropeptide Recovery from Whey Using Ion Exchange Chromatography

ABSTRACT: Cation exchange was used to recover whey protein isolate (WPI) from sweet whey, and the effluent was fed to an anion exchanger to recover glycomacropeptide (GMP). Nearly all of the major whey proteins (α-lactalbu-min, β-lactoglobulin, immunoglobulin G, and serum albumin) and about half of the total Kjeldahl nitrogen (TKN) were recovered by the cation exchanger. No GMP was recovered by the cation exchanger. The anion exchanger recovered nearly all of the GMP from the effluent of the cation exchanger, accounting for about half of the remaining TKN. This process is the first to simultaneously manufacture WPI and GMP from a single stream of whey, increasing the value obtained from whey.     

A Rapid Immunoturbidimetric Method for Whey Proteins in Nonfat Dry Milk and Buttermilk

An immunoturbidimetric assay, using antibodies to whole bovine whey, was developed for the rapid screening of whey proteins in nonfat dry milk and buttermilk. Milk samples are heat-treated prior to analysis to denature the whey proteins for a more uniform response to antibodies. Of the whey proteins tested, the assay is most sensitive to bovine serum albumin and the least sensitive to β-lactoglobulin. Precision of the method is about 4% coefficient of variation with a minimum detectable level of 3% whey protein concentrate added to nonfat dry milk. Results are compared with electrophoretic analysis of whey proteins and high performance size exclusion chromatography of the glycomacropeptide from rennet whey.     

Microfiltration of whey proteins adsorbed on yeast cells

Soluble whey proteins (WPs), adsorbed on yeast cells, were recovered by a crossflow microfiltration (MF) technique using a cellulose nitrate membrane with a pore size of 0.45 μm. The crossflow velocity was 1.5 m s^?1 with a transmembrane pressure of 200 kPa at 25 °C. A series of protein rejections occured at various pH values ranging from 2 to 8. WPs adsorbed more on to yeast cells at low pH (pH < 4) than at high pH values, probably because they were positively charged at low pH. It was also shown that permeate flux increased and Modified Membrane Fouling Index values decreased at low pH levels. When the yeast concentration was 50 g L^?1, the flux decreased five times compared with that in the absence of yeast. Protein recovery increased with increasing yeast concentrations. The highest protein recovery was found to be 85% at a yeast concentration of 50 g L^?1 at a steady state flux rate of 10^?6 m s^?1 at 25 °C. When diluted solutions of whey were used, the same rejection of protein, adsorbed on yeast cells, was achieved at ten times lower amounts of yeast cells. This technique not only provides for the recovery of protein but also may give rise to the direct use of yeast cells, which are rich in protein, in the baking industry. WPs absorbed by yeast cells can be used to produce nutritionally rich products in areas where yeasts have been already used.     

HYDROLYSIS OF LACTOSE IN ACID WHEY BY LACTASE BOUND TO POROUS GLASS PARTICLES IN TUBULAR REACTORS

Partially purified lactases (β-galactosidase, EC3.2.1.23) from Aspergillus niger were covalently bound to acetone-silanized, diazotized porous glass particles (mean pore diam, 86.5 nm; particle diam, 75–125 μm). The temperature (∼55°C) and pH (3.5–4.0) optima were established in acid whey containing 5% total whey solids. Lactose hydrolyzing activity was stable during 43 days of semicontinuous operation at 55°C with reconstituted acid whey (pH = 4.5) at total solids (TS) concentrations varied between 4 and 25% and to which 5 ml/liter toluene had been added to retard microbiological contamination. Kinetic experiments with acid wheys gave results reproducible when assayed by both thin layer chromatography (TLC) and glucose oxidase (GS) procedures, although the TLC method gave systematically higher Values at intermediate conversions and high TS concentrations. The kinetics of lactose hydrolysis by columns of lactase bound to porous glass (LBG) of 1.6 cm diam and lengths of 1, 5 and 10.5 cm showed some evidence for reduction of the rate of lactose hydrolysis by film diffusion resistances. Calculations using correlations for packed beds also suggest the presence of diffusional effects. Lactose was hydrolyzed slightly more rapidly in whey than in deproteinized whey. Lactose hydrolysis rates in both types of reconstituted whey increased as the TS concentrations increased from 4 to 25%. The data did not obey any of a number of integrated reaction rate equations, including a rate equation which accounted for competitive product inhibition of Michaelis enzyme kinetics. Failure of simple models is due in part to diffusional resistances and in part to the large range of concentrations studied. The LBG preparation retained appreciable activity after more than 8 months of frequent use at a wide variety of conditions.     

阳离子交换树脂静态吸附法特异性分离乳清中乳铁蛋白的关键技术

乳铁蛋白(lactoferrin,Lf)是一种对婴幼儿有重要生理功能的活性乳蛋白,掌握Lf特异性分离技术对打破国外技术垄断、降低成本、促进我国婴幼儿配方奶粉发展具有重要意义.该研究以乳清为原料,基于最大吸附量、吸附率、特异吸附为指标,对不同吸附剂做了筛选,探讨了工艺参数对吸附效果的影响及洗脱液离子浓度对洗脱效果的影响,...     

Casein glycomacropeptide pH-driven self-assembly and gelation upon heating

Casein glycomacropeptide (CMP) found in cheese whey is a C-terminal hydrophilic glycopeptide released from κ-casein by the action of chymosin during cheese making. In a previous work a self-assembly model for CMP at room temperature was proposed, involving a first step of hydrophobic assembly followed by a second step of electrostatic interactions which occurs below pH 4.5. The objective of the present work was to study, by dynamic light scattering (DLS), the effect of heating (35–85 °C) on the pH-driven CMP self-assembly and its impact on the dynamics of CMP gelation. The concentration of CMP was 3% w/w for DLS and 12% w/w for rheological measurements. The solutions at pH 4.5 and 6.5 did not show any change in the particle size distributions upon heating. In contrast the solutions at pH lower than 4.5 that showed electrostatic self-assembly at room temperature were affected by heating. The mean diameter of assembled CMP increased by decreasing pH. For all solutions with pH lower than 4.5, the particle size did not change on cooling, suggesting that the assembled CMP forms formed during heating were stable. The gel point determined as G′–G″ crossover, occurred in all systems at 70 °C, but at different times. The rate of self-assembly determined by DLS as well as the rate of gelation increased with increasing temperature and decreasing pH from 4 to 2. Increasing temperature and decreasing pH, the first step of CMP self-assembly by hydrophobic interactions is speed out. All the self-assembled structures and the gels formed at different temperatures were pH-reversible but did not revert to the initial size (monomer) but to associated forms that correspond mainly to CMP dimers.     

Preparation of nanoparticles from denatured whey protein by pH-cycling treatment

Low temperature cross-linking of denatured whey protein through pH-cycling is proposed to develop nanoparticles with controlled size and properties. Soluble polymers were produced by heating whey protein dispersions at low ionic strength and neutral pH. Nanoparticulation was induced by acidification of diluted polymer dispersions followed by pH neutralization. The effect of aggregation conditions on the physicochemical characteristics and stability of nanoparticles was studied. Nanoparticles with a diameter ranging from 100 to 300 nm were produced depending on the pH of aggregation (5.0, 5.5, 6.0), the added calcium concentration (0, 2.5, 5 mM) and the ageing time at the aggregation pH (0–75 h). The size and the turbidity of nanoparticle dispersions increased with increasing ageing time and calcium concentration. Nanoparticle voluminosity decreased with increasing calcium concentration during pH-cycling, suggesting a more compact and less porous internal structure. The stability of nanoparticles in the presence of different dissociating buffers (EDTA, urea, SDS and DTT) was evaluated and the results showed that whey protein nanoparticles were covalently cross-linked by disulphide bonds.     

Effect of whey protein concentration on the fouling and cleaning of a heat transfer surface

In the studies of fouling and cleaning of heat exchange surfaces in dairy plants, whey protein deposits and heat induced whey protein gels (HIWPG) are considered as suitable model material to simulate the proteinaceous based type “A” milk fouling. Protein concentration of the fouling solution may significantly influence the formation of milk deposits on heat exchange surfaces, hence affecting the cleaning efficiency. In this study, a laboratory produced heat induced whey protein gels (HIWPG) and a pilot plant heat exchanger fouling/cleaning were used to investigate the effect of protein concentration on formation and cleaning of dairy fouling. Here, HIWPGs made from different protein concentrations were formed in capsules and then dissolved in aqueous sodium hydroxide (0.5 wt%). The dissolution rate calculation based on the UV spectrophotometer analysis. In the pilot-scale plant study, whey protein fouling deposits were formed by recirculating whey protein solutions with different concentrations through the heat exchange section in different runs, respectively. The deposit layers were then removed by recirculating aqueous sodium hydroxide (0.5 wt%) and the cleaning efficiency was monitored in the form of the recovery of heat transfer coefficient while both fluid electric conductivity and turbidity were monitored as indications of cleaning completion. It was found that increasing the protein concentration of the HIWPG significantly increased the gel hardness and the dissolution time. In addition, increasing the protein concentration significantly increased both, the amount of the fouling on the pilot-scale plant and the time required to clean the fouling deposit.     

Acid-induced gelation of whey protein polymers: effects of pH and calcium concentration during polymerization

Heating whey protein dispersions (90°C for 15 min) at low ionic strength and pH values far from isoelectric point (pH>6.5) induced the formation of soluble polymers. The effect of mineral environment during heating on the hydrodynamic characteristics and acid-induced gelation properties of polymers was studied. Whey protein dispersions (80 g/l) were denatured at different pH (6.5–8.5) and calcium concentrations (0–4 mm) according to a factorial design. At pH 6.5, the hydrodynamic radius of protein polymers increased with increasing calcium concentration, while the opposite trend was observed at pH 8.5. Intrinsic viscosity results suggested that heating conditions altered the shape of protein polymers. Whey protein polymers were acidified to pH 4.6 with glucono-δ-lactone and formed opaque particulate gels. The storage modulus and firmness of gels were both affected by conditions used to prepare protein polymers. As a general trend, polymers with high intrinsic viscosity produced stronger gels, suggesting a relationship between polymer shape and gel strength.Acid gelation properties of whey protein polymers makes them suitable ingredients for yoghurt applications. Using whey protein polymers to standardize protein content increased yoghurt viscosity to 813 Pa.s while using skim milk powder at same protein concentration increased yoghurt viscosity to 393 Pa.s. Water holding capacity of protein polymers in yoghurt was 19.8 ml/g compared to 7.2 ml/g for skim milk powder protein. Acid gelation properties of whey protein polymers are modulated by calcium concentration and heating pH and offers new alternatives to control the texture of fermented dairy products.     

Protease-Induced Aggregation and Gelation of Whey Proteins

Aggregation and gelation of whey proteins induced by a specific protease from Bacillus licheniformis was revealed by turbidimetry, size exclusion chromatography, dynamic light scattering and rheology. The microstructure of the gel was examined by transmission electron microscopy. During incubation of 12% whey protein isolate solutions at 40°C and pH 7, the major whey proteins were partly hydrolyzed and the solution gradually became turbid due to formation of aggregates of increasing size. The viscosity of the hydrolysate simultaneously increased and eventually a gel formed. The gel had a particulate type of microstructure. We hypothesized that the aggregates forming the gel were held together by noncovalent interactions.     

超滤技术在干酪生产中的应用

对超滤技术在干酪生产中的应用作以综述.原料乳通过超滤,脂肪、细菌、酪蛋白和乳清蛋白等大分子颗粒被浓缩,其成分及缓冲能力等的变化对干酪加工成熟过程均产生了重要的影响.超滤技术应用于乳清可以生产乳糖、乳清粉、乳清蛋白浓缩物、乳清蛋白分离物甚至β-乳球蛋白、α-乳白蛋白、糖巨肽及乳铁蛋白等高附加值的产品.     

Proliferation and intracellular glutathione in Jurkat T cells with concentrated whey protein products

The effect of two whey protein concentrates (WPCs) and three whey protein isolates (WPIs) on the growth and intracellular glutathione concentration of Jurkat T cells was determined. Standard RPMI 1640 media containing foetal calf serum with no WPC or WPI supplementation was used as the control, while supplementation with N-acetyl cysteine—a known glutathione promoter—was included as a positive control. Both WPCs lowered the cell count-adjusted glutathione concentration following a 24 h incubation period and one significantly (p<0.05) increased cell proliferation. Only one of the three WPIs significantly (p<0.05) inhibited cell proliferation although its composition with respect to β-lactoglobulin, glycomacropeptide, α-lactalbumin, IgG, proteose peptone and BSA content was almost identical to another WPI, as determined by HPLC. Based on co-migration with standards under two different modes of chromatography, lactoferrin was detected in the WPI showing the inhibitory effect at a level of 0.4 mg mL⁻¹, but not in any of the other concentrated whey protein products. None of the whey protein products tested increased cell-adjusted intracellular glutathione concentration.     

Distribution and stability of aflatoxin M1 during production and storage of yoghurt.

Yoghurt from cow's milk artificially contaminated with aflatoxin M1 (AFM1) at levels of 0.050 and 0.100 g l(-1) was fermented to reach pHs 4.0 and 4.6. Yoghurt fermented to pH 4.6 was also used for preparing strained yoghurt. Yoghurts were stored at 4 degrees C for up to 4 weeks. Analysis of AFM1 in milk, yoghurt, strained yoghurt and yoghurt whey was carried out using immunoaffinity column extraction and liquid chromatography coupled with fluorometric detection. AFM1 levels in yoghurt samples showed a significant decrease (p < 0.01) compared with those initially added to milk. Growth of culture lactic acid bacteria was not affected in the AFM1 contaminated yoghurts, with the exception of Streptococcus thermophilus that showed a significantly (p < 0.01) lower increase in the yoghurt containing the toxin at high concentration. Following fermentation, AFM1 was significantly lower (p < 0.01) in yoghurts with pH 4.0 than in yoghurts with pH 4.6 at both contamination levels. During refrigerated storage, AFM1 was rather more stable in yoghurts with pH 4.6 than with pH 4.0. The percentage loss of the initial amount of AFM1 in milk was estimated at about 13 and 22% by the end of the fermentation, and 16 and 34% by the end of storage for yoghurts with pHs 4.6 and 4.0, respectively. The percentage distribution ratio of AFM1 in strained yoghurt/yoghurt whey of the initial toxin present in the yoghurt was about 90/10 and 87/13 for the lower and the higher contamination levels, respectively.