beta-lactoglobulin hydrolysis. 1. Peptide composition and functional properties of hydrolysates obtained by the action of plasmin, trypsin, and Staphylococcus aureus V8 protease.

beta-Lactoglobulin (betaLg) was subjected to limited hydrolysis by trypsin, plasmin, and endoproteinase from Staphylococcus aureus V8 (S.aur.V8) to degrees of hydrolysis (DH) of 1, 2, and 4%. The several hydrolysates had different peptide compositions (determined by reversed-phase HPLC and gel-permeation chromatography [GPC]). GPC under nondenaturing, denaturing, and denaturing plus reducing conditions showed that the peptides formed were linked by hydrophobic interactions or by disulfide bonds or were not linked at all. At very low protein concentration, some differences in emulsion-forming properties were observed: only the plasmin hydrolysates could form emulsions with a uniform particle-size distribution. The emulsions formed with S.aur.V8 hydrolysates had poor emulsion-stabilizing properties. Some hydrolysates showed increased foam-forming properties in comparison with the intact protein. All foams formed were stable. Overall, the plasmin hydrolysate (DH4) contained relatively much larger molecules and/or hydrophobic molecules. Many molecules were disulfide-linked peptides. This hydrolysate also had the best functional properties.     

Steady-state kinetics and thermodynamics of the hydrolysis of beta-lactoglobulin by trypsin

Hydrolysis of beta-lactoglobulin (in an equimolar mixture of the A and B variant) by trypsin in neutral aqueous solution [pH 7.7 at 25 degrees C, ionic strength 0.08 (NaCl)] was followed by capillary electrophoresis and thermodynamic parameters derived from a Michaelis-Menten analysis of rate data obtained at 10, 20, 30, and 40 degrees C for disappearance of beta-lactoglobulin. Enthalpy of substrate binding to the enzyme and the energy of activation for the catalytic process were found to have the values, DeltaH(bind) = -28 +/- 4 kJ mol(-)(1) and E(a) = 51 +/- 18 kJ mol(-)(1), respectively. Thus, beta-lactoglobulin shows an enthalpy of activation for free substrate reacting with free enzyme of about 21 kJ mol(-)(1), corresponding to a transition state stabilization of 60 kJ mol(-)(1) when compared to acid-catalyzed hydrolysis. The catalytic efficiency of trypsin in hydrolysis of beta-lactoglobulin is increased significantly by temperature; however, this effect is partly counteracted by a weaker substrate binding resulting in an increase by only 25%/10 degrees C in overall catalytic efficiency.     

Investigation of enzymatic hydrolysis conditions on the properties of protein hydrolysate from fish muscle (Collichthys niveatus) and evaluation of its functional properties

This study was carried out to investigate the enzymatic hydrolysis conditions on the properties of protein hydrolysate from fish muscle of the marine fish species Collichthys niveatus. About 160 fish samples were tested, and the analyzed fish species was found to be a lean fish with low fat (1.77 ± 0.01%) and high protein (16.76 ± 1.21%). Fish muscle of C. niveatus was carefully collected and hydrolyzed with four commercial enzymes: Alcalase, Neutrase, Protamex, and Flavourzyme under the conditions recommended by the manufacturers. Among the tested proteases, Neutrase catalyzed the hydrolysis process most effectively since the hydrolysate generated by Neutrase has the highest content of sweet and umami taste amino acids (SUA). The effect of hydrolysis conditions was further optimized using response surface methodology (RSM), and the optimum values for temperature, pH, and enzyme/substrate ratio (E/S ratio) were found to be 40.7 °C, 7.68, and 0.84%, respectively. Finally, the amino acid composition of the hydrolysate was analyzed by AccQ·Tag derivatization and HPLC-PDA determination. Major amino acids of the muscle of C. niveatus were threonine, glutamic acid, phenyalanine, tryptophan, and lysine, accounting for respectively 10.92%, 10.85%, 10.79%, 9.86%, and 9.76% of total amino acid content. The total content of essential amino acids was 970.7 ng·mL(-1), while that of nonessential amino acids was 709.1 ng·mL(-1). The results suggest that the fish muscle and its protein hydrolysate from C. niveatus provide a versatile supply of the benefits and can be incorporated as supplements in health-care foods.     

Kinetics of formation and functional properties of conjugates prepared by dry-state incubation of beta-lactoglobulin/acacia gum electrostatic complexes

The formation of conjugates between beta-lactoglobulin and acacia gum based on electrostatic complexes formed at pH 4.2 was investigated upon dry-state incubation for up to 14 days at 60 degrees C and 79% relative humidity (RH). By means of SEC-HPLC and RP-HPLC, it was shown that the beta-lactoglobulin incubated alone was able to form polymers with molecular masses higher than 200 kDa until 50% of the initial monomeric protein disappeared after 14 days. In the presence of acacia gum at initial protein to polysaccharide weight mixing ratios of 2:1 and 1:2, only 35% of the initial beta-lactoglobulin monomers disappeared after 14 days. Using RP-HPLC, an apparent reaction order of 2 was found for the disappearance of monomeric beta-lactoglobulin both in the presence or absence of acacia gum. However, the reaction rate was faster in the absence of acacia gum. SDS-PAGE electrophoresis with silver staining confirmed the formation of beta-lactoglobulin/acacia gum conjugates. The solubility curves of the incubated beta-lactoglobulin showed a minimum around pH 4-5. By contrast, the minimum of solubility of the beta-lactoglobulin/acacia gum incubated mixtures shifted to lower pH values compared to initial mixtures. The conjugates exhibited higher foam capacity than the incubated protein as well as lower equilibrium air/water surface tension. Conjugation at ratio 1:2 led to increased interfacial viscosity (300 mN s m(-1) at 0.01 Hz) compared to beta-lactoglobulin alone (100 mN s m(-1) at 0.01 Hz), but similar interfacial elasticity (30-40 mN m(-1)). The foam capacity of the conjugates was significantly higher than that of the incubated beta-lactoglobulin as well as foam expansion and drainage time, especially at pH 5.3, i.e., higher than the pH of formation of the conjugates.     

Charge state distribution and hydrogen/deuterium exchange of alpha-lactalbumin and beta-lactoglobulin preparations by electrospray ionization mass spectrometry

Charge state distribution (CSD) and hydrogen/deuterium (H/D) exchange of preparations of alpha-lactalbumin (alpha-Lac) and beta-lactoglobulin (beta-Lg) were investigated using electrospray ionization mass spectrometry (ESI-MS). Storage of alpha-Lac at pH 3 resulted in substantial changes in its CSD, with the emergence of new ion species and shifts toward higher charge state, indicating less stable conformation. ESI spectra of alpha-Lac kept at pH 5.5 for 4 days showed stable conformation; however, extending the storage period resulted in substantial changes in CSD and a decrease in the stability of holo-alpha-Lac (Ca(2+)-bound form). In comparison to apo-alpha-Lac, the relative intensity of holo-alpha-Lac was higher at pH 6.8 but lower at pH 8 during the storage period. beta-Lg showed stable CSD at pH 3, substantial changes at pH 5.5, and minor changes at pH 6.8 and 8 during storage. The H/D exchange results demonstrate that the conformation of holo-alpha-Lac was more stable than that of apo-alpha-Lac and that the conformation of beta-Lg variant B was more stable than that of the beta-Lg variant A. Kinetics of H/D exchange indicated that alpha-Lac and beta-Lg fractions obtained from whey protein preparations have the same or improved conformational stabilities compared to those of alpha-Lac and beta-Lg standards. The presence of four or more hexose residues in alpha-Lac enhanced its conformational stability; the presence of two hexose residues in beta-Lg resulted in a less stable conformation.     

Surface functional properties of native, acid-treated, and reduced soy glycinin. 2. Emulsifying properties

Emulsifying properties of native and chemically modified soy glycinins were studied. The influence of ionic strength, protein sample composition and concentration, and assay conditions on the flocculation-creaming process and coalescence resistance was analyzed. Differences in these emulsifying properties were exhibited by native glycinins, which have a variable content of 4S, 11S, and 15S forms. Structure and functionality of native glycinin were modified by means of combined treatments: mild acidic treatments without heating or with heating at variable time and with or without disulfide bonds reduction. Modified glycinins presented different degrees of deamidation, surface hydrophobicity, and molecular mass. A slight enhancement of emulsifying stability at moderated deamidation degrees was observed. In different protein samples, a positive relationship between the flocculation-creaming rate constant and equilibrium oil volume fraction of emulsions with surface hydrophobicity was detected. A remarkable difference was observed between reduced and nonreduced samples, mainly with respect to behavior at low or high ionic strength.     

Light-scattering study of the structure of aggregates and gels formed by heat-denatured whey protein isolate and beta-lactoglobulin at neutral pH

The structure of aggregates and gels formed by heat-denatured whey protein isolate (WPI) has been studied at pH 7 and different ionic strengths using light scattering and turbidimetry. The results were compared with those obtained for pure beta-lactoglobulin (beta-Lg). WPI aggregates were found to have the same self-similar structure as pure beta-Lg aggregates. WPI formed gels above a critical concentration that varied from close to 100 g/L in the absence of added salt to about 10 g/L at 0.2 M NaCl. At low ionic strength (<0.05 M NaCl) homogeneous transparent gels were formed, while at higher ionic strength the gels became turbid but had the same self-similar structure as reported earlier for pure beta-Lg. The length scale characterizing the heterogeneity of the gels increased exponentially with increasing NaCl concentration for both WPI and pure beta-Lg, but the increase was steeper for the former.     

生长抑素基因疫苗质粒pcS/2SS的构建、表达及免疫*

为构建高免疫原性的生长抑素（somatostatin，SS）DNA疫苗，将SS基因插入到pcS／SS中乙肝表面抗原（HBsAg）S基因第112-113氨基酸残基密码子之间，构建含2拷贝SS的融合表达质粒pcS／2SS。酶切和测序鉴定表明，重组质粒pcS／2SS构建成功。脂质体包裹法将pcS／2SS转染HeLa细胞，ELISA检测表达产物的SS免疫反应原性。结果表明，融合目的基因在HeLa细胞获得表达，S／2SS融合蛋白具有比S／SS融合蛋白更强的SS抗原抗体反应性。将pcS／2SS免疫6只大鼠后，2／3的大鼠产生了抗SS抗体，结果证明，所构建的质粒pcS／2SS可以表达生长抑素，表达产物具有免疫原性。     

Surface functional properties of native, acid-treated, and reduced soy glycinin. 1. Foaming properties

Foaming properties of native and chemically modified glycinin were evaluated. Effects of ionic strength and glycinin composition and concentration on foam formation and stabilization were studied. Glycinin was modified by means of combined treatments: cold or hot acidic treatments, with or without later disulfide bridges reduction. Modified proteins obtained from glycinin present different degrees of dissociation, deamidation, and as consequence, varied surface hydrophobicity and molecular size. Parameters of forming and stabilizing of foam were correlated with both deamidation and dissociation degrees of modified and native glycinin samples. A positive relationship was observed between surface behavior and foaming properties of different protein species. Results show that dissociation, deamidation, and reduction have produced structural changes on glycinin (increased surface hydrophobicity, increased net charge, decreased molecular size) which enhance the adsorption and anchorage of proteins at the air-water interface and, consequently, improve the foam forming and stabilizing capacities.     

Preparation of antioxidant enzymatic hydrolysates from alpha-lactalbumin and beta-lactoglobulin. Identification of active peptides by HPLC-MS/MS

We have investigated the antioxidant activity of hydrolysates from whey proteins bovine alpha-lactalbumin (alpha-La) and beta-lactoglobulin A (beta-Lg A) by commercial proteases (pepsin, trypsin, chymotrypsin, thermolysin, and Corolase PP). Corolase PP was the most appropriate enzyme to obtain antioxidant hydrolysates from alpha-La and beta-Lg A (ORAC-FL values of 2.315 and 2.151 micromol of Trolox equivalent/mg of protein, respectively). A total of 42 peptide fragments were identified by HPLC-MS/MS in the beta-Lg A hydrolysate by Corolase PP. One of the sequences (Trp-Tyr-Ser-Leu-Ala-Met-Ala-Ala-Ser-Asp-Ile) possessed radical scavenging (ORAC-FL value of 2.621 micromol of Trolox equivalent/micromol of peptide) higher than that of butylated hydroxyanisole (BHA). Our results suggest that whey protein hydrolysates could be suitable as natural ingredients in enhancing antioxidant properties of functional foods and in preventing oxidation reaction in food processing.     

Effects of hydration,lipids, and temperature on the binding of the volatile aroma terpenes by beta-lactoglobulin powders

The binding properties of dry proteins are relatively poorly known. Many proteins are present in emulsions and suspensions and also in dry forms. This is particularly true of dairy proteins, which are often stored and sold in powdered form. In the present work, the binding of three terpenes (alpha-terpinene, gamma-terpinene, and terpinolene), which belong to the basic aroma components, and of decane by powdered beta-lactoglobulin (BLG) was studied at different hydration levels (0.05-0.40 g of H(2)O/g of protein) and temperatures (298 and 309.5 K), in the presence or absence of lipids and small concentrations of ethanol. Vapor sorption isotherms were determined for these systems by a static method of headspace gas chromatographic analysis. A cooperative effect of hydrophobic hydration was observed for the binding of aroma terpenes and decane by the solid BLG. The temperature increase from 298 to 309.5 K reduced the observed hydration threshold of BLG by 0.05-0.08 g of H(2)O/g of protein. Lipids (1.2% w/w) in hydrated BLG gave at least a 2-fold increase in its binding affinity for the hydrocarbons studied, and synergic effects of the hydration and lipid on this affinity were observed.     

Effect of heat treatment on the circular dichroism spectra of bovine beta-lactoglobulin A, B, and C.

Dilute solutions of beta-lactoglobulin (beta-Lg) A, B, and C were heated in phosphate buffer at temperatures between 40 and 94 degrees C for 10 min, cooled, and analyzed using near-UV and far-UV circular dichroism (CD). The decrease in near-UV CD intensity at 293 nm (Deltaepsilon(293)) could be analyzed in terms of a two-state model, and the stability was beta-Lg C > beta-Lg A > beta-Lg B on the basis of the midpoint temperatures for samples heated at pH 6.7 and 7.4. However, the slopes of the curves at the midpoint temperature for variant A were generally less than those for beta-Lg B and beta-Lg C, indicating that the substitution of Val (beta-Lg A) for Ala (beta-Lg B or beta-Lg C) at position 118 had altered the entropic contribution to unfolding of the protein. The changes in CD at 270 nm (Deltaepsilon(270)), an index of significant alteration to disulfide bond dihedral angles, occurred at higher temperatures than those for the Deltaepsilon(293) results. The far-UV CD showed some small changes as a consequence of heat treatment, and the shifts at 205 nm ([theta](205)) fitted a two-state model. Plotting the changes in both Deltaepsilon(293) and [theta](205) against the loss of nativelike and sodium dodecyl sulfate-monomeric protein (assessed by polyacrylamide gel electrophoresis) showed a strong 1:1 relationship between Deltaepsilon(293) or [theta](205) and the loss of nativelike beta-Lg. These results indicated that the initial irreversible stage in the heat-induced aggregation of beta-Lg (nativelike monomer to unfolded monomer) altered the chirality of the environment of Trp(19) and modified the secondary structure of beta-Lg slightly. The differences in the behavior of variants A-C were explicable on the basis of generalized electrostatic and hydrophobicity effects as well as specific amino acid effects.     

Gamma-irradiation influence on the structure and properties of calcium caseinate-whey protein isolate based films. Part 2. Influence of polysaccharide addition and radiation treatment on the structure and functional properties of the films

The influence of gamma-irradiation (32 kGy) followed by the addition of polysaccharides (potato starch, soluble potato starch, and sodium alginate) and heating on the properties of the films based on calcium caseinate (CC)-whey proteins isolate (WPI) and the gels formed with CaCl(2) was evaluated. Radiation induced an improvement of the mechanical and barrier properties of all films. The polysaccharides' effect on the irradiated and non-irradiated CC-WPI gels could be predicted as the sum of their separate effects on CC and on WPI, apart from the alginate interaction with the irradiated CC-WPI. The better properties of the films achieved after admixing polysaccharides to the formerly irradiated protein solution correspond to the smaller strength of gels. Properties of the films and gels prepared using the irradiated proteins and alginate differed depending on whether alginate was admixed before or after irradiation. Results were related to the protein structure, interaction with polysaccharides, and the film's microstructure.     

Synthesis and structure determination of covalent conjugates formed from the sulfury-roasty-smelling 2-furfurylthiol and di- or trihydroxybenzenes and their identification in coffee brew

Recent investigations demonstrated that the reaction of odor-active thiols such as 2-furfurylthiol with thermally generated chlorogenic acid degradation products is responsible for the rapid aroma staling of coffee beverages. To get a clear understanding of the molecular mechanisms underlying this aroma staling, the existence of putative phenol/thiol conjugates needs to be verified in coffee. The aim of the present study was therefore to synthesize such conjugates for use as reference substances for LC-MS screening of coffee. To achieve this, catechol, 3-methyl-, 4-methyl-, and 4-ethylcatechol, pyrogallol, hydroxyhydroquinone, 5-O-caffeoylquinic acid, and caffeic acid, respectively, were reacted with 2-furfurylthiol in the presence of iron(III) chloride and air oxygen. After purification, the structures of 25 phenol/thiol conjugates were identified by means of LC-MS/MS and 1D/2D NMR experiments. Using these compounds as reference materials, four conjugates, namely, 3-((2-furylmethyl)sulfanyl)catechol, 3-((2-furylmethyl)sulfanyl)-5-ethylcatechol, 4-((2-furylmethyl)sulfanyl)hydroxyhydroquinone, and 3,4-bis((2-furylmethyl)sulfanyl) hydroxyhydroquinone, were identified for the first time in coffee brew by means of HPLC-MS/MS(MRM). These findings clearly demonstrate catechol, 4-ethylcatechol, and hydroxyhydroquinone as the primary thiol trapping agents involved in the aroma staling of coffee beverages.     

Quercetin-dependent inhibition of nitration induced by peroxidase/H2O2/nitrite systems in human saliva and characterization of an oxidation product of quercetin formed during the inhibition

Local pH in the oral cavity can decrease to below 7 at the site where acid-producing bacteria are proliferating. Effects of pH on nitration of 4-hydroxyphenylacetic acid were studied using dialyzed human saliva. Dialyzed saliva nitrated 4-hydroxyphenylacetic acid to 4-hydroxy-3-nitrophenylacetic acid in the presence of nitrite and H(2)O(2). The rate of the nitration was dependent on pH, and the maximal rate was observed between pH 5.5 and 7.2. The optimum pH seemed to reflect rates of formation of nitrogen dioxide and 4-hydroxyphenylacetic acid radicals. Quercetin inhibited the nitration. The quercetin-dependent inhibition might be due to scavenging of nitrogen dioxide and 4-hydroxyphenylacetic acid radicals, which were formed by salivary peroxidase-dependent oxidation of nitrite and 4-hydroxyphenylacetic acid, respectively, and competition with nitrite and 4-hydroxyphenylacetic acid for peroxidase in saliva. An oxidation product of quercetin was formed during inhibition of the nitration by quercetin. The oxidation product was identified as 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone. This component could also be oxidized by salivary peroxidase and nitrogen dioxide radicals. The oxidation products were 2,4,6-trihydroxyphenylglyoxylic and 3,4-dihydroxybenzoic acids. On the basis of the results, the significance of quercetin for inhibition of nitrogen dioxide formation and for scavenging of nitrogen dioxide radicals in the oral cavity is discussed.     

华北平原农田厚包气带及含水层反硝化细菌的分离鉴定及作用机制研究

华北平原农田由于长期过量施用氮肥,造成了土壤硝酸盐累积,导致地下水硝酸盐污染日趋严重。微生物的反硝化作用可将土壤中累积的硝酸盐或亚硝酸盐还原为气态产物,是消减厚包气带土壤累积的硝酸盐的重要途径。因此筛选高效反硝化微生物资源,对人工强化厚包气带土壤反硝化脱氮,阻控地下水硝酸盐污染具有重要作用。基于此,本研究采集位于华北平原的中国科学院栾城农业生态系统试验站长期施氮[施氮量为600kg(N)·hm~(-2)·a~(-1)]定位试验0～150m农田厚包气带及含水层土壤样品,从中筛选到62株细菌。16SrRNA基因序列分析表明这62株菌株与变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、厚壁菌门(Firmicutes)中的9个属具有较高的同源性。根据系统发育树的结果,挑选7株亲缘关系较远的菌株进行反硝化潜势试验,结果表明,菌株L71、L13和L103具备反硝化产气能力。电镜观察结果表明,这3株菌均为无鞭毛的杆状细菌,其长度分别为1.0μm、1.5μm和1.5μm,只有L103具有运动能力。此外,菌株L103具有完全反硝化能力,且脱氮能力受到pH的影响,在本试验条件下,菌株L103的反硝化速率高达1.62～2.36g(KNO_3)·d~(-1)·L~(-1),具备实际应用潜力。本研究表明华北平原厚包气带土壤中存在完全反硝化微生物,并可为人工强化治理厚包气带土壤硝酸盐污染提供菌种资源和理论依据。     

The H+/M2+ exchange stoichiometry of calcium and zinc adsorption by ferrihydrite

The specific adsorption of Ca²⁺ and Zn²⁺ by ferrihydrite results in the net release of H⁺. The rate and H⁺/M²⁺ exchange stoichiometry of this reaction were monitored with a pH-stat. A rapid reaction of less than 6 min was followed by a slower reaction which continued at a diminishing rate for at least 2 days. Adsorption of Ca²⁺ at pH 7.8 and Zn²⁺ at pH 5.4 resulted in the net release of 0.92 and 1.70 mol H⁺/mol M²⁺ adsorbed, respectively. For Zn²⁺ adsorption, this stoichiometry was shown to be independent of pH. These estimates agree well with independent estimates based on the pH dependence of adsorption. The difference between the Ca²⁺ and Zn²⁺ stoichiometries was related to the differing acidity of the –OH₂ ligands attached to the adsorbed ions.     

BF2/肽四聚体的构建及其在特异性T细胞检测上的应用

为构建可溶性鸡 组织相容性复合体(MHC)Ⅰ 类分子（BF2）/肽复合物,大量表达并纯化了可生物素化的MHC Ⅰ类分子重链（BF2-BSP）和轻链（Chβ2m）,并合成了鸡传染性支气管炎病毒（IBV）核蛋白N71~78 T细胞表位肽,将其与纯化后的重组蛋白BF2-BSP和Chβ2m在重折叠缓冲液中复性。复性后在快速蛋白液相色谱仪（FPLC）上分离出复性组装成功的BF2/肽单体复合物。将该单体复合物在体外进行生物素化,其产物再次采用FPLC通过分子筛分离出生物素化后的BF2/肽单体复合物;然后将生物素化后的BF2/肽单体复合物与藻红蛋白（PE）标记的链霉亲和素按一定比例反应生成BF2/肽四聚体。为了检测所制备BF2/肽四聚体是否能应用于检测特异性T细胞,从感染IBV H52株10 d后的SPF鸡分离外周血单核细胞（PBMC）,染色后采用流式细胞术检测,结果表明,所制备的BF2/肽四聚体可用于检测IBV N蛋白特异性T淋巴细胞,检测1周龄SPF鸡接种H52后10 d时针对N蛋白的特异性T细胞比率为3.65%。