大豆分离蛋白与甜菜果胶静电复合过程的研究

利用葡萄糖酸内酯(GDL)诱导大豆分离蛋白(SPI)与甜菜果胶(SBP)形成静电复合物。在不同SPI/SBP混合比例下,通过动态光散射和紫外可见分光光度计对pH酸化过程中的散射光强和浊度进行测定,跟踪复合物的生成及结构演变。实验结果表明,SPI与SBP可生成具有不同结构和不同稳定性的分子复合物,包括分子内可溶复合物、分子间可溶复合物以及分子间不可溶复合物。不同类型复合物的形成取决于溶液体系的pH和不同SPI/SBP混合比例。该研究系统地建立了SPI与SBP静电复合物的相图。      

Gelation properties of salt-extracted pea protein isolate catalyzed by microbial transglutaminase cross-linking

Gelation is a fundamental functional characteristic of plant proteins. In this paper, a salt-extracted pea protein isolate (PPI) was mixed with microbial transglutaminase (MTG) to produce gels and the gelation properties were studied. When the MTG level increased, the magnitude of both the G′ and G″ moduli also increased, which means the gel strength increased. A second order polynomial equation was used to describe the relationships between the G′, G″ modulus and TG level. It was found that with increased heating and cooling rate at the same MTG level, G′ and G″ tended to decrease, resulting in a weaker gel. This was attributed to the rearrangement time of pea protein molecules; slower heating and cooling rates enabled protein molecules to have more time to rearrange and therefore form a stronger gel. At the same MTG level, higher pea protein concentration resulted in higher G′ and G″ values and a power law relationship was found between G′ and pea protein concentration or G″ and pea protein concentration. Frequency sweep data of PPI show that the MTG treatment resulted in higher G′ values and lower tan delta values, indicative of a stronger more elastic gel. The minimum gelation concentration was found to be 3% (w/v) with 10 U MTG treatment, lower than 5.5% required when no MTG was present. When compared to PPI and soy protein isolate (SPI) with and without 10 U MTG treatment, the gel strength of PPI with MTG was stronger than that of SPI with MTG treatment, whereas the opposite was true without the MTG treatment. SDS-PAGE showed that at the same pea protein concentration, higher MTG level induced more cross-linking as fainter bands were seen on the gel and there was a shift in the relative intensities of the bands in the molecular weight range of 35–100 kDa.     

A novel improvement in whey protein isolate emulsion stability: Generation of an enzymatically cross-linked beet pectin layer using horseradish peroxidase

Extensive research has indicated that the electrostatic attraction between polysaccharides and proteins on the oil-water interface can improve the stability of emulsions. However, this electrostatic effect will be weakened or even eliminated as the solution pH or ionic strength of emulsions change, resulting in the shedding of the polysaccharide layer. We prepared primary oil-in-water emulsions at pH 7.0 using whey protein isolate (WPI) as an emulsifier and then beet pectin was added to form secondary emulsions. After the pH of emulsions was adjusted to 4.0 to promote electrostatic attraction between the beet pectin molecules and the protein-coated droplets, horseradish peroxidase was added to generate a cross-linked beet pectin coating. Results show that stable emulsions coated with WPI and cross-linked beet pectin interfaces could be formed. The sensitivity of the emulsions to the environmental stresses of pH changes, ions addition, thermal processing and freezing was also characterized in this work. Our results support the view that cross-linked beet pectin improves the stability of emulsions and is superior to simple deposition on the surface of lipid droplets. The interfacial engineering technology used in this study could be used to create food emulsions with improved stability to environmental stresses.     

Fabrication and characterization of TiO2/whey protein isolate nanocomposite film

Composite films of whey protein isolate and TiO₂ are formed through three simultaneous processes, i.e., the self-assembly of protein–protein, TiO₂–TiO₂, and the association of protein–TiO₂. All the processes could be controlled by adjusting TiO₂ concentration in the blended system. The self-assembly of protein–protein molecules constituted the main network of the composite film. A low TiO₂ concentration (<0.25%) dispersed the TiO₂ nanoparticles in the protein matrix, reinforced the association of protein–TiO₂, reduced the ability of UVC absorption, and promoted the fluorescence and tensile strength of the composite films. In contrast, a high TiO₂ concentration (>0.25%) enhanced the self-assembly of TiO₂–TiO₂ nanoparticles, brought fluorescent quenching, and produced a decline of the tensile strength and water vapor permeability. The transmittance of the visible, UVA, and UVB lights showed a first order exponential decay relative to the TiO₂ concentration.     

微生物转谷氨酰胺酶催化乳清蛋白聚合研究

采用SDS－PAGE分析，研究了不同条件下微生物转谷氨酰胺酶（MTGase）催化乳清蛋白（WPI）聚合。结果显示，MTGase可催化乳清蛋白的β－乳球蛋白（β-LG）和α－乳清蛋白（α－LA）聚合，形成低聚物或生物聚合物，其中β-LG更易受MTGase的催化，当TGase酶浓度一定时（0.5U/mL),TGase催化WPI聚合的最佳底物质量分数范围为2％－4％，对WPI进行加热预处理，同时添加还原剂，可明显提高MTGase对WPI的催化活性，MTGase催化WIP的最适PH值范围为6．5－7．5，当WPI经预热处理（85℃，15min），同时添加20mmol/L的DTT，TGase催化WPI聚合12h，可使质量分数为92％的β-LG和质量分数为75％的α－LA聚合。     

Enzyme catalyzed oxidative gelation of sugar beet pectin: Kinetics and rheology

Sugar beet pectin (SBP) is a marginally utilized co-processing product from sugar production from sugar beets. In this study, the kinetics of oxidative gelation of SBP, taking place via enzyme catalyzed cross-linking of ferulic acid moieties (FA), was studied using small angle oscillatory measurements. The rates of gelation, catalyzed by horseradish peroxidase (HRP) (EC 1.11.1.7) and laccase (EC 1.10.3.2), respectively, were determined by measuring the slope of the increase of the elastic modulus (G′) with time at various enzyme dosages (0.125–2.0 U mL⁻¹). When evaluated at equal enzyme activity dosage levels, the two enzymes produced different gelation kinetics and the resulting gels had different rheological properties: HRP (with addition of H₂O₂) catalyzed a fast rate of gelation compared to laccase (no H₂O₂ addition), but laccase catalysis produced stronger gels (higher G′). The main effects and interactions between different factors on the gelation rates and gel properties were examined in response surface designs in which enzyme dosage (0.125–2.0 U mL⁻¹ for HRP; 0.125–10 U mL⁻¹ for laccase), substrate concentration (1.0–4.0%), temperature (25–55 °C), pH (3.5–5.5), and H₂O₂ (0.1–1.0 mM) (for HRP only) were varied. Gelation rates increased with temperature, substrate concentration, and enzyme dosage; for laccase catalyzed SBP gelation the gel strengths correlated positively with increased gelation rate, whereas no such correlation could be established for HRP catalyzed gelation and at the elevated gelation rates (>100 Pa min⁻¹) gels produced using laccase were stronger (higher G′) than HRP catalyzed gels at similar rates of gelation. Chemical analysis confirmed the formation of ferulic acid dehydrodimers (diFAs) by both enzymes supporting that the gelation was a result of oxidative cross-linking of FAs.     

Stabilization of oil-in-water emulsions by enzyme catalyzed oxidative gelation of sugar beet pectin

Enzyme catalyzed oxidative cross-linking of feruloyl groups can promote gelation of sugar beet pectin (SBP). It is uncertain how the enzyme kinetics of this cross-linking reaction are affected in emulsion systems and whether the gelation affects emulsion stability. In this study, SBP (2.5% w/v) was mixed into an oil-in-water emulsion system (4.4% w/w oil, 0.22% w/w whey protein, pH 4.5). Two separate, identically composed, emulsion systems were prepared by different methods of preparation. The emulsions prepared separately and subsequently mixed with SBP (referred as Mix A) produced significantly larger average particle sizes than the emulsions in which the SBP was homogenized into the emulsion system during emulsion preparation (referred as Mix B). Mix B type emulsions were stable. Enzyme catalyzed oxidative gelation of SBP helped stabilize the emulsions in Mix A. The kinetics of the enzyme catalyzed oxidative gelation of SBP was evaluated by small angle oscillatory measurements for horseradish peroxidase (HRP) (EC 1.11.1.7) and laccase (EC 1.10.3.2) catalysis, respectively. HRP catalyzed gelation rates, determined from the slopes of the increase of elastic modulus (G′) with time, were higher (P < 0.05) than the corresponding laccase catalyzed rates, but the final G′ values were higher for laccase catalyzed gels, regardless of the presence of emulsions or type of emulsion preparation (Mix A or Mix B). For both enzymes, rates of gelation in Mix A were higher (P < 0.05) than in Mix B, and higher stress was needed to break the gels in Mix A than in Mix B at similar enzyme dosage levels. These differences may be related to a lower availability of the feruloyl groups for cross-linking when the SBP was homogenized into the emulsion system during preparation.     

乳清分离蛋白成膜工艺的研究

以乳清分离蛋白(WPI)为主料,通过添加甘油(增塑剂)和半胱氨酸(还原剂),制备乳清分离蛋白膜.同时,对其制备工艺与性能进行了详细分析与测定,从而确定了成膜最佳工艺为:乳清分离蛋白含量为8%,增塑剂添加量为4%.还原剂的添加量为0.6mmol/L.在此工艺条件下测定乳清分离蛋白膜性能:厚度0.101±0.013mm,透明度0.055±0.005.抗拉强度1165.2±20.8g.断裂伸长率70.06%±1.62%,透H2O性17.13±0.63g/m2·h,透O2性3.60±0.08g/m2·d,透CO2性445.56±5.26g/m2·d.     

乳清分离蛋白与大豆分离蛋白混合溶液制备外源式钙交联冷凝胶型微球体

将分别经过热变性处理的乳清分离蛋白和大豆分离蛋白溶液按照2∶1,1∶1和1∶2比例(质量比)混合后,通过外源式添加钙离子的方法,获得钙交联冷凝胶型微球体。检测微球体的蛋白质包埋率、溶解度和溶胀率。结果显示,微球体的蛋白质包埋率较高,均大于99.36%,不同蛋白质比率对蛋白质包埋率没有显著影响。微球体具有一定的水溶性,在缓冲液中的溶解度顺序为pH7.0pH2.5pH4.5,且乳清蛋白质量分数越高,微球体的溶解度越小。微球体在缓冲液中发生不同程度的膨胀,溶胀率顺序为pH2.5pH7.0,而在pH值为4.5缓冲液中发生皱缩,溶胀率为负值,微球体中的乳清蛋白质量分数越高,溶胀率越小。缓冲液中的钙离子会影响微球体的溶解度和溶胀率。基本性质说明外源式钙交联冷凝胶型微球体可以作为热敏性或活性物质的传递载体,应用于相关的食品及医药行业。     

Improved emulsion stabilizing properties of whey protein isolate by conjugation with pectins

Functional properties of glyco-protein conjugates of the anionic polysaccharide pectin with whey protein isolate, obtained by dry heat treatment at 60 °C for 14 days, have been investigated in O/W emulsions containing 20% (w/w) soybean oil and 0.4% (w/w) protein both at pH 4.0 and 5.5. Emulsion stabilizing properties of mixtures and conjugates were compared at five protein to pectin weight ratios by determining changes in droplet size distribution and extent of serum separation with time. The results indicated that the dry heat-induced covalent binding of low methoxyl pectin to whey protein, as shown by SDS-PAGE, led to a substantial improvement in the emulsifying behaviour at pH 5.5, which is near the isoelectric pH of the main protein β-lactoglobulin. At pH 4.0, however, a deterioration of the emulsifying properties of whey protein was observed using either mixtures of protein and pectin or conjugates.The observed effects could be explained by protein solubility and electrophoretic mobility measurements. The protein solubility at pH 5.5 was hardly changed using mixtures of protein and low methoxyl pectin or conjugates, whereas at pH 4.0 it was decreased considerably. Electrophoretic mobility measurements at pH 5.5 revealed a much more pronounced negative charge on the emulsion droplets in the case of protein–pectin conjugates, which clearly indicated that conjugated pectin did adsorb at the interface even at pH conditions above the protein's iso-electric point. Hence, the improved emulsifying properties of whey protein isolate at pH 5.5 upon conjugation with low methoxyl pectin may be explained by enhanced electrosteric stabilization.Comparing two different commercial pectin samples, it was clearly shown that the dextrose content during dry heat treatment of protein–pectin mixtures should be as low as possible since protein–sugar conjugates not only resulted in increased brown colour development, but also gave raise to a largely decreased protein solubility which very badly affected the emulsifying properties.     

自由基氧化引起乳清蛋白理化性质变化的研究

研究了在羟基自由基氧化体系中,不同H2O2浓度(1～20 mmol/L)及不同FeCl3浓度(0.1～2 mmol/L)对乳清蛋白羰基、巯基、二聚酪氨酸等理化性质的影响.每种氧化条件的氧化时间分别为1,3,5 h..结果表明:氧化显著地影响了乳清蛋白的理化性质,同未氧化的对照组乳清蛋白相比,经过5 h氧化,所有浓度的FeCl3体系中,羰基增加3倍以上;所有条件下的巯基损失均达40%以上;在浓度为20 mmol/L的H2O2或2 mmol/L的FeCl3中,二聚酪氨酸分别增加了5倍和7倍.并且发现,在不同的FeCl3条件下其变化趋势更为迅速.由此可知,氧化极大程度地改变了蛋白的理化性质,并可能导致蛋白结构的改变,进而可能影响其功能性质.     

Isolation and identification of antimicrobial peptides derived by peptic cleavage of whey protein isolate

The antimicrobial potential of whey protein isolate hydrolyzed by gastrointestinal enzymes was determined by attempting to identify and characterize the antimicrobial peptides responsible. While tryptic and chymotryptic hydrolysates did not show antibacterial activity, whey proteins hydrolyzed for 45–90 min by pepsin exhibited significant activity. Fractionation of 60-min hydrolysate by reversed-phase high performance liquid chromatography yielded 5 fractions that were antibacterial, with minimum inhibitory concentrations comprised between 20 and 35 μg/mL. These fractions contained short peptides not previously identified as antimicrobial. Fragment 14–18 (KVAGT) of β-lactoglobulin is very close to a sequence previously identified as antibacterial and is found in antimicrobial sequences of diverse origin. Five other peptides derived from β-lactoglobulin, and one fragment from α-lactalbumin (f117–121, KVGIN), were also identified as antibacterial. The identified peptides do not match pepsin action exactly, indicating modified proteolysis of unknown origin. Protein by-products of the dairy industry offer potential for large-scale production of antimicrobial peptides.     

牛乳清蛋白与甘薯淀粉加热混合凝胶物化及微观结构特性的研究

研究了不同pH和NaCl、CaCl2浓度对WPI凝胶及WPI与甘薯淀粉混合凝胶物化特性和微观结构的影响。结果表明:随着pH的增加,凝胶的硬度和溶解度减小,添加淀粉进一步降低了混合凝胶的溶解度;盐浓度的增加使混合凝胶硬度增加,而蛋白溶解度、持水力和巯基含量则呈下降趋势;添加NaCl和CaCl2的混合凝胶分别呈现出多孔网状和颗粒状结构。     

Improvement of functional properties of whey protein isolate through glycation and phosphorylation by dry heating

Whey protein isolate (WPI) was glycated with maltopentaose (MP) through the Maillard reaction, and the MP-conjugated WPI (MP-WPI) was then phosphorylated by dry heating in the presence of pyrophosphate. Glycation occurred efficiently, and the sugar content of WPI increased approximately 19.9% through the Maillard reaction. The phosphorylation of MP-WPI was enhanced with an increase in the dry-heating time from 1 to 5 d, and the phosphorus content of WPI increased approximately 1.05% by dry heating at pH 4.0 and 85°C for 5 d in the presence of pyrophosphate. The electrophoretic mobility of WPI increased with an increase in the phosphorylation level. The stability of WPI against heat-induced insolubility at pH 7.0 was improved by conjugation with MP alone, and further improved by phosphorylation. Although the emulsifying activity of WPI was barely affected by glycation and phosphorylation, the emulsifying stability of phosphorylated MP-WPI (5 d), was 2.2 times higher than that of MP-WPI. Gelling properties such as hardness, resiliency, and water-holding capacity of heat-induced WPI gel were markedly improved, and the gel was rendered transparent by phosphorylation. The calcium phosphate-solubilizing ability of WPI was enhanced by phosphorylation. These results suggested that phosphorylation by dry heating in the presence of pyrophosphate after conjugation with MP is a useful method for improving the functional properties of WPI.     

Characterisation of binding of iron to sodium caseinate and whey protein isolate

The fortification of dairy products with iron is an important approach to delivering iron in required quantities to the consumer. The binding of iron (ferrous sulfate) to two commercial milk protein products, sodium caseinate and whey protein isolate (WPI), dissolved in 50 mM HEPES buffer, was examined as a function of pH and iron concentration. Sodium caseinate had more sites (n = 14) than WPI (n = 8) for binding iron, and the affinity of caseinate to bind iron was also higher than that of WPI. These differences were attributed to the presence of clusters of phosphoserine residues in casein molecules, which are known to bind divalent cations strongly. The amount of iron bound to sodium caseinate was found to be independent of pH in the range 5.5–7.0, whereas acidification (pH range 7.0–3.0) caused a marked decrease in the amount of iron bound to WPI.     

Incorporated glucosamine adversely affects the emulsifying properties of whey protein isolate polymerized by transglutaminase

Glucosamine (GlcN) and microbial transglutaminase (Tgase) are used separately or together to improve the emulsifying properties of whey protein isolate (WPI). However, little is known about how the emulsifying properties change when GlcN residues are incorporated into WPI cross-linked by Tgase. We used Tgase as a biocatalyst to cross-link WPI in the presence of GlcN in a liquid system for 12 h at a moderate temperature (25°C). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analyses indicated that protein polymerization and GlcN conjugation occurred simultaneously, phenomena also supported by the loss of free amines (9.4–20.5%). Addition of 5 U Tgase/g protein improved the emulsifying properties of moderately cross-linked WPI polymers. The Tgase-treated WPI polymers had a larger particle size (～2.6-fold) than native WPI, which may have reduced coalescence and flocculation in an oil-in-water emulsion system. However, the incorporation of GlcN residues into WPI, predominantly via enzymatic glycation, partly inhibited the cross-links between the WPI molecules catalyzed by Tgase, reducing the size of the WPI polymers 0.81- to 0.86-fold). Consequently, WPI+GlcN conjugates provided less stability to the emulsion. Moreover, high NaCl concentration (0.2 M) decreased the emulsifying properties of the WPI+GlcN conjugates by neutralizing negative electric charges in the glycoconjugates. However, the improved emulsifying properties of WPI cross-linked by Tgase may be useful in food processing at higher NaCl concentrations due to the formation of the thicker steric barrier at the oil-water interface.     

海藻酸钠-果胶改性复合膜的制备及表征

以海藻酸钠(SA)和柚皮果胶(PEC)为基材,辅以添加甘油和不同种类脂质制备复合膜,通过分析各组分对膜水蒸气透过系数、过氧化值、抗拉强度等指标的影响,确定复合膜的适宜配方:SA与PEC的质量比为10/2,甘油的添加量0.3%,石蜡添加量为0.12%。以CaCl_2、BaCl_2、FeCl_3、Al_2(SO_4)_3不同交联剂对该复合膜进行改性,以CaCl_2为交联剂,CaCl_2浓度3%、交联时间3min条件下制备的海藻酸钠-果胶改性复合膜各项性能提高。以FTIR、SEM、XRD、接触角测定膜的表面结构形态和性质进行表征和测试。研究结果表明海藻酸钠-果胶复合膜各组分间的相容性好,经CaCl_2交联后显著改善膜的阻隔性能、机械性能及抗水性能,海藻酸钠-果胶改性复合膜是一种具有良好发展前景的可降解复合包装膜。