发酵对酸肉蛋白质结构的影响

以猪背最长肌为原料制备酸肉,研究蛋白质结构在发酵中的变化。结果表明,随发酵时间延长,蛋白质静电作用力逐渐减弱,疏水相互作用和二硫键作用显著增强后呈小幅度波动变化。蛋白紫外吸收结果显示,肌原纤维蛋白芳香族氨基酸残基偏向更加疏水的环境移动,肌浆蛋白酪氨酸、色氨酸残基微环境极性增加。内源荧光图谱显示,荧光强度总体呈下降趋势且相对发酵0 d时发生红移,表明色氨酸发生了氧化降解,其残基主要暴露到极性环境中。拉曼光谱分析表明,随发酵进行,蛋白质α-螺旋减少,β-折叠增多,发酵110 d酸肉蛋白二级结构可能发生了重排现象。结果表明长时间发酵可使酸肉蛋白质二级结构和三级结构发生变化,这些变化可从蛋白质结构水平上解释发酵对酸肉品质的影响。     

Oxidation of sarcoplasmic proteins during processing of Cantonese sausage in relation to their aggregation behaviour and in vitro digestibility

The physicochemical changes of sarcoplasmic proteins, especially oxidation behaviour, were measured to determine their mechanism of action on in vitro protein digestibility during Cantonese sausage processing. The results indicated that carbonyl level increased (p<0.05) during the process. The fluorescence loss of tryptophan residues was a direct consequence of the oxidative degradation. All the parameters of protein aggregation were highly (p<0.05) correlated with carbonyl level and protein surface hydrophobicity (H(0)), indicating that protein oxidation and thermal denaturation could induce protein aggregation, leading to secondary structural changes. The analysis of in vitro digestibility showed no correlation between pepsin activity and protein oxidation, due to the biphasic response of sarcoplasmic proteins toward proteolysis. However, a highly significant (p<0.05) correlation was observed with trypsin and α-chymotrypsin activity, indicating that protein oxidation induced the changes in H(0), protein aggregation and secondary structure, which further influenced in vitro digestibility.     

EFFECTS OF SODIUM DODECYL SULFATE,GUANIDINE HYDROCHLORIDE,UREA, AND HEAT ON DENATURATION OF SULFUR RICH PROTEIN IN SOYBEANS (GLYCINE MAX L.)1

Soybean sulfur‐rich protein (SRP) has a stable hydrophobic interior that is resistant to denaturation and unfolding. About half of the tryptophan residues in native SRP are inaccessible to acrylamide quenching. Maximum unfolding, as assessed by fluorescence quenching experiments, was afforded by 2 M urea, 6 M guanidine HCl and 30 nun moist heat. Sodium dodecyl sulfate (SDS) did not improve tryptophan accessibility to acrylamide quenching. SRP tryptophan residues were minimally accessible to the iodide quenching indicating a nonionic environment around the majority of the tryptophan residues in SRP.     

Charakterisierung pankreatischer Casein-Plasteine

Characterization of pancreatic casein plasteins. In the course of the plastein reaction hydrophobic peptides concentrate mainly in the aggregates (plasteins), whilst hydrophilic peptides remain in solution (supernatant). Liquid chromatographic and sequence analytical studies of pancreatic casein plasteins have shown that the aggregates consist mainly of the free amino acids tyrosine, phenylalanine and tryptophan. Plasteins contain, in addition, short-chain peptides, particularly from the C-terminal of β-casein. Characterization of the functional properties of the plasteins has shown clearly that aggregation of the short-chain peptides and free amino acids is brought by non-covalent, hydrophobic and ionogenic interactions. In the supernatants resulting from the plastein reaction caseinophosphopeptide sequences, in particular from α_s-casein, were determined.     

Recent progress of soybean protein foods: Chemistry,technology, and nutrition

Abstract

The most important chemical reactions during the process of soybean protein foods are the intermolecular reactions among the residues exposed on the surface of the protein molecules through the denaturation process. In native soybean protein molecules, most amino acid residues responsible for the reactions—such as cysteine (‐SH), cystine (S‐S), and hydrophobic amino acid residues—are buried in the inside region of the molecule, inaccessible to water. These residues become reactable with each other through the exposure from the inside by heat denaturation during processing. The unique textures of soybean protein foods, such as tofu, kori‐tofu, yuba, and texturized products produced by extruder, etc., are the results of both the intermolecular interchange reaction between the exposed ‐SH and S‐S groups and the intermolecular hydrophobic reaction among the exposed hydrophobic amino acid residues. The exposure of amino acid residues is also important for the hydrolysis of soybean proteins by enzymes, through which soy sauce is produced, because the cleavage of the peptide bonds is carried out after binding between the active sites of the enzymes and the enzyme‐specific amino acid residues exposed through denaturation. These facts indicate the importance of the three‐dimensional structures of soybean protein molecules in the technology of soybean protein foods. Recently great progress has been made in the manufacturing techniques of soybean protein foods, such as soy milk, tofu, abura‐age, textured protein products, and soy sauce. The quality of soy milk and tofu was very much improved by controlling the action of the biologically active substances such as lipoxygenases and β‐glucosidases which are contained in soybeans and responsible for the production of off‐flavor. A new abura‐age, whose texture does not deteriorate during frozen storage or drying, was developed by using soybean protein isolate and oil as materials. A new type of textured protein product was also developed: a deep‐fat‐fried nugget with unique texture and flavor. This product is textured through a twin‐type extruder. For soy sauce manufacturing new biotechnology has been applied on the pilot‐plant scale. This is a system of continuous fermentation through bioreactors with the immobilized whole cells of microorganisms, by which the fermentation term is shortened strikingly. New and important discoveries were made on the nutrition of soybean proteins. According to recent experiments using human beings, the amino acid score of soybean proteins is 100 for persons more than 2 years old, indicating that the nutritive value of soybean proteins is equal to animal proteins. Further, it was elucidated that soybean proteins have cholesterol‐lowering action. A discussion is presented on the future of the soybean protein foods.     

水牛奶酪蛋白胶束结构的荧光光谱研究

利用内源荧光团色氨酸（Trp）和外源荧光探针8-苯胺基-1-萘磺酸（8-anilino-1-naphthalenesulfonic acid,ANS）的荧光特性对水牛奶酪蛋白胶束结构进行研究。结果表明：当蛋白质质量浓度较低时,酪蛋白胶束结构变化不明显,而较高的蛋白质质量浓度会破坏其胶束结构,使其疏水基团暴露;此外,在离子强度较大、pH值较低条件下,对酪蛋白胶束结构影响较大,使酪蛋白疏水基团暴露,酪蛋白微球先膨胀后聚集并形成酪蛋白胶束。     

Pressure denaturation and aggregation of beta-lactoglobulin studied by intrinsic fluorescence depolarization, Rayleigh scattering, radiationless energy transfer and hydrophobic fluoroprobing

beta-Lactoglobulin (beta-lg) in aqueous solution under pressure showed a marked depolarization of intrinsic fluorescence assigned to a gradually increased rotational diffusion of tryptophyl moieties in pressure-unfolded states. The corresponding change in anisotropy provided a new and more accurate method for determining denaturation volume which, for beta-lg in neutral aqueous solution with ionic strength 0.16 (NaCl) at 25 degrees C, was delta V degree = -73 (SE 3) ml mol-1, corresponding to half denaturation at 123 MPa. The pressure unfolding led to exposure of hydrophobic regions to the protein-water interface that could be probed by fluorescence intensity of a beta-lg-1-anilinonaphthalene-8-sulphonic acid (ANS) complex with 1:1 stoichiometry, as determined by Job's method of continuous variation. The unfolding of beta-lg impaired the binding capacity of the inner calyx, with a reduction in binding capacity of 50% at 50 MPa, as shown by decreasing cis-parinaric acid fluorescence, decreasing anisotropy and decreasing radiationless energy transfer from tryptophans to this probe with increasing pressure. The pressure-induced reversible exposure of hydrophobic groups to the protein-water interface may, at least partly, explain the initial aggregation reactions, evident from increased Rayleigh scattering from approximately 50 MPa, prior to irreversible pressure-induced gel formation of beta-lg. Using results from this and previous studies, we propose a three step pressure denaturation model for beta-lg for neutral solution at ambient temperature, including an initial pressure-melted state (up to 50 MPa) with partial collapse of the inner calyx and solvent exposure of the free thiol group, followed by a reversible denaturation with exposure of hydrophobic regions (half denaturation at 123 MPa) and with irreversible denaturation with thiol-disulphide exchange becoming increasingly important at higher pressures. Effects of pressure on beta-lg, as measured by fluorescence depolarization, were found for the reversible denaturation steps to be similar to the effects of chemical denaturants but different with respect to shift in ANS emission maxima.     

Stabilization of α-Amylase,the Key Enzyme in Carbohydrates Properties Alterations,at Low pH

The present study correlates the mechanism of α-amylase denaturation under acidic condition and its structural stabilization in presence of selected cosolvents (sorbitol, sucrose, trehalose, and glycerol). The objective of the present study was to minimize the enzyme inactivation at lower pH by stabilizing the enzyme structure. The above cosolvents were found to be an effective stabilizer of α-amylase against denaturation at extreme low pH. The optimum activity of α-amylase was found to be in the pH range of 4.5 to 7. Decreasing the pH of enzyme solution below this range results in a decrease in enzyme activity. The intrinsic and ANS fluorescence indicated the gradual unfolding of protein below pH 4 and resulted in exposure of hydrophobic cluster. The pH-induced unfolding also resulted in protein aggregation. The intrinsic and 8-anilinonaphathalene-1-sulphonic acid (ANS) fluorescence, acrylamide quenching, near and far UV-CD spectra of α-amylase at lower pH (pH 2.25) indicated the complete loss of tertiary structure and substantial loss of secondary structure. Cosolvents were shown to prevent the acid induced unfolding of the enzyme to a certain extent and help in retaining the secondary structure of enzyme equivalent to native state. Among all the cosolvents used, sorbitol was proven to be the most efficient stabilizer. At lower pH, in presence of cosolvents the enzyme was shown to retain significant amount of secondary structure and poorly defined tertiary structure. This structure resembles the molten globule of the protein, which has substantial amount of secondary structure but poorly defined tertiary structure.     

Structure,physicochemical, and functional properties of protein isolates and major fractions from cumin (Cuminum cyminum) seeds

In this study, cumin protein isolates (CPI) and major protein fractions were extracted and separated from cumin seeds, their structure, physicochemical, and functional properties were investigated. Albumin (62.29%) and glutelin (25.16%) were the predominant protein fractions of cumin seeds. Glutamic acid (Glu) and aspartic acid (Asp) were the major amino acids of cumin proteins, whereas more hydrophobic and aromatic amino acids were predominantly found in chickpea protein isolates. Electrophoresis profiles indicated that CPI have more disulphide bonds than major protein fractions. The intrinsic fluorescence data revealed that glutelin displayed greater exposure of tyrosine (Tyr) and tryptophan (Trp) residues compared to albumin and CPI. Circular dichroism (CD) data showed CPI presented more α-helix (14.4%) and less β-strand (30.7%) than albumin and glutelin. The atomic force microscope (AFM) profile and hydrodynamic diameter (D_h) determination showed the presence of low particle size in albumin fractions. Differences in the hydrophobicity (H_o) and the zeta-potential (ζ) of CPI, albumin, and glutelin were also observed due to their difference in structure and amino acid composition. Compared with CPI and glutelin, albumin exhibited the highest emulsifying activity (103.67 m²/g) and stability (42.84 min) and the smallest emulsion particle size (4.29 μm). The CPI, albumin and glutelin presented typical U-shaped protein solubility–pH curves, with the lowest solubility at pH 4.0. Rheological investigation demonstrated that CPIs were efficient in forming a gel at 80.6°C, whereas glutelin could form the hardest gel at 92.6°C. The overall results suggested that the cumin proteins can be a promising protein source for the food industry.     

Covalent interactions between amino acid side chains and oxidation products of caffeoylquinic acid (chlorogenic acid)

BACKGROUND: Physicochemical properties and digestibility of proteins can be modified by covalent interactions with oxidized phenolic compounds, i.e., quinones. In order to control these interactions in food products, the covalent interactions between quinones from caffeoylquinic acid (CQA) and amino acid side chains were studied with mass spectrometry using N‐terminally protected amino acids. RESULTS: The addition of two molecules of CQA, presumably in the form of a pre‐formed dimer, was observed for lysine, tyrosine, histidine and tryptophan. A monomer of CQA seemed to be able to react with histidine and tryptophan, whereas no interaction with a CQA monomer was observed for lysine and tyrosine. Serine and threonine showed no covalent interactions with CQA. Cross‐linking between CQA and the side chains of two molecules of lysine is likely to occur also in proteins. The results show that protein cross‐linking may also be expected to occur via two tyrosine residues in the absence of other phenolic substrates. The side chains of lysine and tyrosine are more reactive than that of histidine and tryptophan. CONCLUSIONS: These results show that covalent protein modification by oxidized phenolics occurs preferentially via an initial dimerization and encompasses not only lysine and cysteine residues. Copyright © 2008 Society of Chemical Industry     

Pulsed Electric Field-Induced Structural Modification of Whey Protein Isolate

Effects of pulsed electric fields (PEF) on structural modification and surface hydrophobicity were assessed for whey protein isolate (WPI) of protein concentrations (3% and 5%) using fluorescence spectroscopy. The effects of a factorial combination of electric field intensities (12, 16, and 20 kV cm⁻¹) and number of pulses (10, 20, and 30) on the intrinsic tryptophan fluorescence intensity, extrinsic fluorescence intensity, and surface hydrophobicity of WPI were evaluated. PEF treatments of WPI resulted in increases in the intrinsic tryptophan fluorescence intensity and led to 2–4-nm red shifts in emission wavelengths, indicating changes in the polarity of tryptophan residues microenvironment in whey proteins from a less polar to a more polar environment. The extrinsic fluorescence intensity of WPI increased with PEF treatments, but with 2–4-nm blue shifts, indicating partial denaturation of WPI fractions and exposure of more hydrophobic regions under these PEF treatments. Thus, under the conditions studied, PEF treatments of WPI yielded increases in surface hydrophobicity. The study confirmed that PEF treatments resulted in whey protein structure modifications. These results suggested that controlled PEF could be applied to process liquids food including WPI ingredients and modify their structure and function in order to get desired food products.     

Aggregation and structural changes of silver carp actomyosin as affected by mild acidification with d-gluconic acid δ-lactone

The structural changes and aggregation properties of silver carp actomyosin acidified with d-gluconic acid-δ-lactone (GDL) were investigated. Results showed that silver carp actomyosin underwent aggregation and formation of precipitate as indicated by turbidity and centrifugation coupled electrophoresis analysis. Circular dichroism indicated that myosin rod unfolded during acidification, resulting in a gradual decrease in α-helical content. The changes in tertiary structure of actomyosin under acidic conditions were demonstrated by second-derivative UV spectra and intrinsic fluorescence. Tyrosine residues were exposed to the surface of proteins when pH was decreased to 5.5, and were buried inside the protein aggregates with further reduction in pH. In contrast, more tryptophan residues were exposed to the polar environment with decreasing pH. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinking experiments showed that the intensity of myosin heavy chain (MHC) bands decreased sharply with decreasing pH and the actin bands decreased more slowly, suggesting that MHC is the major protein component involved in the non-covalent cross-linking and formation of aggregates during acidification of silver carp actomyosin.     

Quantitative comparison of casein and rapeseed proteolysis by pancreatin

The problem of quantitative comparison of kinetic curves was solved for casein and rapeseed pancreatin hydrolysis in a membrane reactor, which ensured the measurement of proteolysis kinetics for the products with a molecular weight of less than 1000. Coordinates were derived which provided good linearization of kinetic curves and the determination of relative rate constants irrespective of reagent concentrations. E₀/S₀ ratio and time intervals of kinetic measurements. When the relative rate constants of the release of the individual amino acid residues in the low-weight proteolysis products were compared, trypsin-dependent constants (for Lys and Arg residues) were found to be two times less for rapeseed than for casein, and chymotrypsin-dependent constants (for Tyr and Phe residues) were approximately 1.3 times higher for rapeseed than for casein. Statistical analysis demonstrated that the distribution of constants was narrower for rapeseed than for casein. Differences between target (Arg, Lys, Tyr and Phe) and non-target constants of release in the form of peptides and free amino acids, or in the form of free amino acids only, were attributed to the differences in the peptide bond masking for casein and rapeseed proteins. Computer simulation of proteolysis kinetics was performed by PROTEOLYSIS program package to confirm the dependence of rate constant distribution on the state of masking.     

扫频超声处理时间对β-乳球蛋白酶解制备多肽抗氧化活性的影响

目的:研究不同扫频超声处理时间对β-乳球蛋白酶解制备多肽抗氧化活性的影响。方法:研究不同的超声预处理时间(10,20,30,60,90 min)对β-乳球蛋白表观结构的影响,以及超声波处理对β-乳球蛋白酶解产物的抗氧化活性、氨基酸组成、分子质量分布和疏水性的影响。结论:超声波处理可显著提高β-乳球蛋白酶解产物的DPPH自由基清除率、ABTS·清除能力和Fe2+络合能力。随着超声时间的延长,β-乳球蛋白酶解产物抗氧化活性呈先增加后降低的趋势。扫频超声波处理可以提高β-LG酶解产物的疏水性,并且显著增加多肽中的疏水性氨基酸的含量。扫频超声处理10~60 min有利于分子质量为200~2000 u多肽的生成,从而提高其酶解产物的抗氧化活性。粒径分布表明短时间的超声处理(<30 min)引起β-LG粒径减小,大分子蛋白的结构疏松,分子间的疏水性作用力增加;长时间(60~90 min)的超声处理则引起大分子蛋白聚集,疏水性作用力降低,蛋白颗粒粒径增大。     

EFFECTS OF ACETYLATION ON PHYSICOCHEMICAL PROPERTIES OF J1S SOY PROTEIN

Acetylation of ε-amino groups of lysine residues changed conformation of glycinin isolated from soybean, the extent of which depended upon the degree of modification. Soy glycinin with lysine residue modifications of 0%, 28%, 65%, 85%, and 95% were used for the experiment. Accessibility of tyrosine and tryptophan residues, which were shown to increase as modification percent increased, were detected using uv absorption spectra and fluorescence spectra, respectively. Surface hydrophobicity was found to increase more than two times over native glycinin, when lysine residues were excessively modified to above 95%. Masking of charged lysine residues, exposure of hydrophobic interior, and subunit dissociation should have contributed to the increase. Enthalpy, as obtained from differential scanning calorimetry, dropped from 3.6 callg native protein to 0 callg protein, when lysine residues were acetylated above 65%. implicating complete denaturation. The hydrolytic rates, using α-chymotrypsin, increased initially, then decreased at more than 65% lysine modification.     

Structural modification of soy protein by 13-hydroperoxyoctadecadienoic acid

To reveal the role of primary products of lipid peroxidation during soy protein oxidation process, oxidative modification of soy protein by 13-hydroperoxyoctadecadienoic acid (13-HPODE) generated by lipoxygenase-catalyzed oxidation of linoleic acid was investigated in this article. Incubation of soy protein with increasing concentration of 13-HPODE resulted in generation of protein carbonyl derivatives and loss of protein sulfhydryl groups. Circular dichroism spectra indicated that exposure of soy protein to 13-HPODE led to loss of α-helix structure. Effect of oxidation on tertiary structure was demonstrated by surface hydrophobicity and tryptophan fluorescence. Surface hydrophobicity gradually decreased, accompanied by loss and burial of some tryptophan residues. The results of surface hydrophobicity and tryptophan fluorescence implied that aggregation was induced by oxidation. Size exclusion chromatogram indicated that the extent of aggregation was increased in a 13-HPODE dose-dependent manner. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that non-disulfide linkages were involved in aggregate formation, and β-conglycinin was more vulnerable to 13-HPODE than glycinin.     

Effects of Trolox and ascorbic acid on the riboflavin photosensitised oxidation of aromatic amino acids

The effects of 0, 100, 250, 500, 750 or 1000 ppm Trolox and ascorbic acid on the singlet oxygen oxidation of aromatic amino acids viz. phenylalanine, tryptophan, tyrosine in the presence of 25 ppm riboflavin were determined by measuring depleted headspace oxygen by gas chromatography and aromatic amino acid content by high performance liquid chromatography. The coefficients of variations (CVs) for headspace oxygen analysis and HPLC analysis of aromatic amino acids were 1.4% and 4.7%, respectively. Samples were stored under light (1000 lux) at 30 °C for 10 h. Both Trolox and ascorbic acid acted as antioxidants. As the concentration of Trolox and ascorbic increased from 0 to 1000 ppm, the head space oxygen depletion increased. This was due to the oxidation of Trolox and ascorbic acid along with amino acids in the presence of riboflavin. High performance liquid chromatography analysis of the samples clearly indicated that both Trolox and ascorbic acid minimised the degradation of phenylalanine, tryptophan, tyrosine significantly (p < 0.05), but did not prevent their oxidation completely. Trolox acted as a better antioxidant than ascorbic acid in protecting phenylalanine, tryptophan and tyrosine. Type-I mechanism was mainly responsible for riboflavin photosensitised degradation of aromatic amino acids.     

氨基酸组成及溶剂环境对淡水鱼胶原蛋白热稳定性能的影响

本文探讨了氨基酸组成、分布以及溶剂环境对鱼源胶原蛋白蛋白热稳定性能的影响。利用不同鱼类的胶原蛋白为实验样本,测定胶原蛋白氨基酸组成和热变性温度,通过相关性分析明确了影响胶原蛋白热变性温度的主要氨基酸种类和分布指标。结果表明,胶原蛋白中脯氨酸羟基化率、碱性氨基酸、带电荷极性氨基酸和总极性氨基酸含量与胶原蛋白热变性温度呈正相关,而亚氨基酸与非极性氨基酸含量与胶原蛋白热变性温度呈负相关(p0.01)。运用逐步多元回归分析方法,建立了脯氨酸羟基化率和带电荷极性氨基酸与胶原蛋白热变性温度之间的相关性数学模型,经验证,该模型能较好的预测鱼源胶原蛋白的热变性温度。在此基础上,进一步研究了胶原蛋白所处溶剂环境对其热稳定性能的影响。结果表明,提高水分子浸润胶原蛋白程度和体系离子强度,或降低体系pH均会显著降低胶原蛋白热稳定性能。     

超声处理对蛋清蛋白结构性质及蛋清液起泡性的影响

研究脉冲式超声处理对蛋清液拉曼光谱、内源性荧光光谱、静态流变学性质、粒径和理化结构的影响,结合起泡性的变化进行相关性分析。研究发现,超声处理能够改变蛋清蛋白二硫键构象,降低蛋清液黏度,减少总巯基含量,对无规卷曲、β-转角含量和酪氨酸残基峰强比（I850/I830）影响较小,蛋清液依旧为假塑性流体。在超声处理15 min内,蛋清蛋白质粒径、表面巯基含量、热变性焓（ΔH）、α-螺旋和β-折叠含量逐渐减少,内源性荧光强度和表面疏水性逐渐增强,色氨酸残基趋向于“暴露的”展开形式;起泡性得到改善,且泡沫体积小,呈均匀紧密的排列,泡沫稳定性小幅降低。随着超声时间的延长,β-折叠含量继续降低,粒径、表面巯基含量、ΔH、α-螺旋含量、色氨酸残基峰强比（I1 363/I1 338）、内源性荧光强度和表面疏水性呈现不同的变化趋势,起泡性逐渐降低,但泡沫稳定性有所升高。相关性分析表明,起泡性与I1 363/I1 338、表面疏水性呈正相关,与ΔH、平均粒径、α-螺旋含量和表面巯基含量呈负相关,泡沫稳定性与表面疏水性呈负相关。     

超高压和巴氏杀菌对蛋清蛋白结构及起泡性能的影响

本实验以市售鲜蛋制备得到的蛋清为原料,分别利用巴氏杀菌和超高压杀菌处理(200～300MPa,2.5～12.5 min),经喷雾干燥后,采用圆二色光谱、傅里叶变换红外光谱、紫外光谱、荧光光谱和动态光散射激光粒度仪等研究超高压杀菌处理对蛋清蛋白的构象、粒径、表面电位、起泡特性的影响.结果 表明,巴氏杀菌和超高压杀菌处理均...