Specificity of disulfide bond formation during thermal aggregation in solutions of beta-lactoglobulin B and kappa-casein A

Heat-induced (90 degrees C, 10 min, pH 6.7) intermolecular disulfide bond formation in 1:1 mixtures of beta-lactoglobulin B (beta-Lg) and kappa-casein A (kappa-CN) was studied by enzymatic digestion with trypsin or glu-C, reverse-phase HPLC, and MALDI-TOF-MS. Observed masses were compared to theoretically calculated masses of disulfide-bonded peptide dimers and trimers, and the number of different masses matching peptide combinations involving each bond was used as a measure of confidence of identification. The beta-Lg cysteine residues 121 or 119 were involved in bonds with both cysteines of kappa-CN and all cysteines of beta-Lg. This agrees with the supposed initiatory role of beta-C121 in heat-induced SH/SS interchange. The largest numbers of matches corresponded to bonds linking beta-C119/C121 with kappa-C11 or with beta-C66. Multiple matches were recorded for beta-C119/C121 bonding with beta-C119/C121, with beta-C160, or with kappa-C88. However, beta-C106 was observed only in bonds with beta-C119/C121 and did not appear to bond to kappa-CN, suggesting it remains buried in the core of the protein.     

Effect of milk concentration on the irreversible thermal denaturation and disulfide aggregation of beta-lactoglobulin

The kinetics of beta-lactoglobulin (beta-LG) denaturation in reconstituted skim milk samples of various concentrations (9.6-38.4% total solids) over a wide temperature range (75-100 degrees C) was studied. The thermal denaturation of beta-LG had a reaction order of 1.5 at all milk solids concentrations and at all temperatures. The rate of denaturation of beta-LG was markedly dependent on the milk solids concentration and the heating temperature. At 75 degrees C, the thermal denaturation of beta-LG was retarded at higher milk solids concentrations. However, this retardation was less pronounced at higher temperatures so that a similar rate of denaturation was observed at all milk solids concentrations at 100 degrees C. From an examination of the level of disulfide-aggregated beta-LG, it was evident that most, but not all, of the denatured beta-LG was involved in disulfide-aggregated complexes, either with other denatured whey proteins or with the casein micelles. As with beta-LG denaturation, the rate of disulfide aggregation of beta-LG was markedly dependent on the milk solids concentration.     

Reduced immunogenicity of beta-lactoglobulin by conjugation with carboxymethyl dextran differing in molecular weight

To reduce the immunogenicity of beta-lactoglobulin (beta-LG), two beta-LG-carboxymethyl dextran (CMD) conjugates (Conj. 40 and Conj. 162) were prepared by using water-soluble carbodiimide (EDC). The molar ratios of beta-LG to CMD in Conj. 40 and Conj. 162 were 8:1 and 7:1, respectively. Each conjugate maintained approximately 50% of the retinol binding activity of beta-LG. Structural analyses by intrinsic fluorescence, CD spectra, and ELISA with monoclonal antibodies indicated that the surface of beta-LG in each conjugate was covered by CMD without great disruption of native conformation. By conjugation with CMD, the antibody response to beta-LG was reduced in BALB/c, C3H/He, and C57BL/6 mice, which was eminent in Conj. 162. The results of B cell epitope scanning using overlapping synthesized peptides showed that the linear epitope profiles of the conjugates were similar to those of beta-LG, whereas the antibody response to each epitope was reduced, which was eminent in Conj. 162. It was concluded that conjugation with CMD of higher molecular weight is effective in reducing the immunogenicity of beta-LG and that masking of epitopes by CMD is responsible for the reduced immunogenicity.     

Steric effects governing disulfide bond interchange during thermal aggregation in solutions of beta-lactoglobulin B and alpha-lactalbumin

Intermolecular disulfide bond formation in pure beta-lactoglobulin (beta-Lg) B and in its 1:1 mixture with alpha-lactalbumin (alpha-La), heated at 85 degrees C for 10 min in solutions of low and high (100 mM NaCl) ionic strength and pH 6.0, was studied by reverse-phase HPLC and MALDI-TOF mass spectrometry. Disulfide bonding between beta-Lg monomers was more extensive than reported in the literature for a temperature of 68.5 degrees C, including formation of trimers connected by two of the three adjacent cysteines, C106/C119/C121. The participation of the different thiol groups in disulfide bonds appeared to depend on their location in the native structure, with surface-located cysteines more involved than internally located ones. This also applied to alpha-La-beta-Lg interactions, where the predominant participants were the surface-located alphaC111, alphaC120, alphaC61, and alphaC6. The least active participant was alphaC28, suggesting that it becomes sterically inaccessible during unfolding of the protein. High ionic strength apparently promoted disulfide bonding. The order of cysteine participation at the high ionic strength was similar to that at low ionic strength, with fewer native-location bonds observed and a lower activity of some groups, such as beta-C106/C119/C121 and alphaC61.     

Interactions of vitamin D3 with bovine beta-lactoglobulin A and beta-casein

It is of nutritional significance to fortify processed dairy products (e.g., cheese, yogurt, and ice cream) with vitamin D3; however, the inherent complexity of these foods may influence the stability and bioavailability of this nutrient. In the present study, the interactions of vitamin D3 with beta-lactoglobulin A and beta-casein were investigated under various environmental conditions (i.e., pH and ionic strength) using fluorescence and circular dichroism spectroscopic techniques. The results indicated that vitamin D3 was bound to both beta-lactoglobulin A and beta-casein depending on the solution conditions. The apparent dissociation constants ranged from 0.02 to 0.29 microM for beta-lactoglobulin A, whereas the beta-casein apparent dissociation constants ranged from 0.06 to 0.26 microM. The apparent mole ratios were also comparable, i.e., 0.51-2.04 and 1.16-2.05 mol of vitamin D3 were bound per mole of beta-lactoglobulin A and beta-casein, respectively. It was concluded that these interactions may strongly influence vitamin D3 stability and, hence, bioavailability in processed dairy products.     

Binding of the pepper alkaloid piperine to bovine beta-lactoglobulin: circular dichroism spectroscopy and molecular modeling study

The pepper alkaloid piperine is a nontoxic, natural dietary compound with a broad range of physiological activity. The present work is the first demonstration of its interaction with a mammalian protein. Circular dichroism (CD) spectroscopy was used to reveal and analyze the binding of piperine to a lipocalin protein. Induced CD spectra measured in pH 7.7 phosphate buffer at 37 degrees C demonstrated reversible, non-covalent association of piperine with bovine beta-lactoglobulin (BLG), the major whey protein in milk. The binding parameters (K(a) approximately 8 x 10(4) M(-1), n = 0.8) determined from the CD titration data showed no significant differences between the piperine binding properties of the two main genetic variants of BLG (A and B). The vanishing extrinsic CD signal obtained upon acidification of the piperine-BLG sample solution (Tanford transition) suggested that the ligand binds in the central hydrophobic cavity of the beta-barrel. The cavity binding concept was further supported by a CD displacement experiment using palmitic acid, the well-known hydrophobic ligand of BLG. Molecular docking calculations showed that piperine can be efficiently accommodated within the calyx of BLG. Additional molecular modeling calculations indicated that the beta-barrel of human tear lipocalin, human serum retinol binding protein, and human neutrophil gelatinase associated lipocalin might also accommodate a piperine molecule.     

Effects of high-pressure processing at low temperature on the molecular structure and surface properties of beta-lactoglobulin

High-pressure processing (HPP) was utilized to induce unfolding of beta-lactoglobulin (beta-LG). beta-Lactoglobulin solutions at concentrations of 0.5 mg/mL, in pH 7.5 phosphate buffer, were pressure treated at 510 MPa for 10 min at either 8 or 24 degrees C. The secondary structure, as determined by circular dichroism (CD), of beta-LG processed at 8 degrees C appeared to be unchanged, whereas beta-LG processed at 24 degrees C lost alpha-helix structure. Tertiary structures for beta-LG, as determined by near-UV CD, intrinsic protein fluorescence spectroscopy, hydrophobic fluorescent probe binding, and thiol group reactivity, were changed following processing at either temperature. The largest changes to tertiary structure were observed for the samples processed at 24 degrees C. Model solutions containing the pressure-treated beta-LG showed significant decreases in surface tension at liquid-air interfaces with values of 54.00 and 51.69 mN/m for the samples treated at 24 and 8 degrees C, respectively. In comparison, the surface tension for model solutions containing the untreated control was 60.60 mN/m. Changes in protein structure during frozen and freeze-dried storage were also monitored, and some renaturation was observed for both storage conditions. Significantly, the sample pressure-treated at 8 degrees C continued to display the lowest surface tension.     

Hydrophobic probe binding of beta-lactoglobulin in the native and molten globule state induced by high pressure as affected by pH,KIO(3) and N-ethylmaleimide

High hydrostatic pressure (HHP) at 500 MPa and 50 degrees C induces beta-LG into the molten globule state. Retinol, cis-parinaric acid (CPA), and 1-anilino-naphthalene-8-sulfonate (ANS) fluorescence from pH 2.5 to 10.5 in the presence of the native and molten globule states of beta-LG indicate that retinol binds to beta-LG in the calyx, CPA at the surface hydrophobic site, and ANS in multiple hydrophobic sites. HHP treatment results in a decrease of beta-LG affinity for retinol and CPA, suggesting conformational changes in the calyx and surface hydrophobic site of beta-LG during HHP treatment. beta-LG treated by HHP in the presence of N-ethylmaleimide (NEM) retains retinol affinity, suggesting that NEM protects the calyx conformation of beta-LG during HHP treatment. HHP treatment of beta-LG in the presence of KIO(3) exhibits a great decrease of CPA affinity compared to HHP-treated beta-LG in the absence of KIO(3), suggesting the formation of non-native disulfide bonding at the CPA binding site.     

玉米粮堆霉变发热过程中的温湿度场变化规律研究

为模拟储粮粮堆局部含水率偏高引起的霉变发热现象,进而研究此现象中温、湿度场的变化规律,该文在试验仓内湿基含水率14.0%的玉米粮堆中心加入湿基含水率18.2%的玉米,在30℃室内储藏40 d。试验粮堆由于霉变引起自发热。试验过程中,通过计算玉米粮堆中垂面内高温区和高湿区的面积变化,从而揭示玉米粮堆霉变发热过程中温、湿度场的变化规律。试验结果表明,粮堆中垂面高湿区面积缓慢扩大,高温区面积开始扩大缓慢,但在与周围粮温最高温差升至3.7℃后,面积扩大速率加快,且高温区与高湿区面积的当量半径r与温度差?T成正比,此正比关系经过了粮库浅圆仓的验证。这为进一步定量分析粮食仓储过程中的高温区和高湿区扩散提供了依据。     

Characterization and in vitro digestibility of bovine beta-lactoglobulin glycated with galactooligosaccharides

Galactooligosaccharides (GOS) are well-known prebiotic ingredients which can form the basis of new functional dairy products. In this work, the production and characterization of glycated beta-lactoglobulin (beta-LG) with prebiotic GOS through the Maillard reaction under controlled conditions ( a(w) = 0.44, 40 degrees C for 23 days) have been studied. The extent of glycation of beta-LG was evaluated by formation of furosine which progressively increased with storage for up to 16 days, suggesting that the formation of Amadori compounds prevailed over their degradation. RP-HPLC-UV, SDS-PAGE, and IEF profiles of beta-LG were modified as a consequence of its glycation. MALDI-ToF mass spectra of glycated beta-LG showed an increase of up to approximately 21% in its average molecular mass after storage for 23 days. Moreover, a decrease in unconjugated GOS (one tri-, two tetra-, and one pentasaccharide) was observed by HPAEC-PAD upon glycation. These results were confirmed by ESI MS. The stability of the glycated beta-LG to in vitro simulated gastrointestinal digestion was also described and compared with that of the unglycated protein. The yield of digestion products of glycated beta-LG was lower than that observed for the unglycated protein. The conjugation of prebiotic carbohydrates to stable proteins and peptides could open new routes of research in the study of functional ingredients.     

Influence of binding of sodium dodecyl sulfate, all-trans-retinol, and 8-anilino-1-naphthalenesulfonate on the high-pressure-induced unfolding and aggregation of beta-lactoglobulin B

Bovine beta-lactoglobulin B (beta-LG) is susceptible to pressure treatment, which unfolds it, allowing thiol-catalyzed disulfide bond interchange to occur, facilitating intermolecular bonding (both noncovalent and disulfide). In the present study, beta-LG was mixed with sodium dodecyl sulfate (SDS), all-trans-retinol (retinol), or 8-anilino-1-naphthalenesulfonate (ANS) on a 1:1.1 molar basis, and aliquots were held at pressures between 50 and 800 MPa for 30 min at pH 7.2 and 20 degrees C. Polyacrylamide gel electrophoresis (PAGE) showed that beta-LG alone (control) was converted into a non-native monomer and a series of dimers, trimers, etc., at pressures beyond 100 MPa; SDS inhibited the formation of non-native species up to 200 MPa, and neither retinol nor ANS inhibited the formation of the non-native species as effectively as SDS. At pressures beyond 350 MPa, SDS ceased to have any inhibitory effect, but both ANS and retinol showed significant inhibition. The near- and far-UV CD patterns and the ANS fluorescent data were consistent with the PAGE data, but the retinol fluorescent data did not show sufficient change to interpret. The results suggested that there were three discernible structural stages. In Stage I (0.1-150 MPa), the native structure is stable; in Stage II (200-450 MPa), the native monomer is reversibly interchanging with non-native monomers and disulfide-bonded dimers; and in Stage III (>500 MPa), the free CysH in non-native monomer and dimer interacts with -S-S- bonds to produce high molecular weight aggregates of beta-LG. SDS inhibited the Stage I to Stage II transition at 200 MPa, and ANS and retinol inhibited the Stage II to Stage III transition at 600 MPa.     

Impacts of land use change on soil aggregation and aggregate stabilizing compounds as dependent on time

Land use change is known to strongly affect soil aggregation and aggregate stabilizing compounds. In this study we wanted to gain insight into the temporal and spatial dimension of this process. Therefore, we studied water-stable aggregates, total organic carbon (TOC), carbohydrates and glomalin-related soil protein (GRSP) in a 110-year chronosequence of Stagnosols, which have been converted from pasture to cropland at different times in history. To describe the temporal dimension, the measured concentrations were approximated by an exponential decay function. The spatial dimension was assessed by analyzing the distribution of TOC, carbohydrates, GRSP, and ¹⁴C among the different aggregate-size fractions over the course of 110 years of cropland use. It was found that the TOC concentration decreased monoexponentially (R² = 0.92) from 195.2 t ha⁻¹ to 45.13 t ha⁻¹ in the first depth interval (0-20 cm) during the first 110 years after the conversion, and reached a new equilibrium 23 (±5) years after land use change. Carbohydrates and GRSP obtained a new equilibrium after 14 (±6) and 56 (±5) years in the same depth interval. The mean-weight diameter (MWD) of the water-stable aggregates reached a new equilibrium 33 (±2) years after the land use conversion. With respect to the spatial dimension we found that TOC, carbohydrates, and GRSP showed higher concentrations in the macroaggregates than in the microaggregates. The ratios of the distribution of TOC, carbohydrates and GRSP among macro- and microaggregates did not change significantly during the 110 years of arable use of the soils. The average age of the organic carbon in the different aggregate-size fractions analyzed by its ¹⁴C concentration showed of a wide range from 65 (±25) to 251 (±30) years, and did not change significantly during the cultivation of the sites. Thus, we conclude that water-stable aggregates and the concentrations of TOC, carbohydrates and GRSP reacted towards land use change at different speeds and reached a new equilibrium between 14 (±6) and 56 (±5) years after the conversion of land use. Secondly, we found, that the spatial distribution of aggregate stabilizing compounds was not significantly changed during the first 110 years of cultivation.     

水稻苗期响应光强日变化的分子行为生态初探

本研究以常规优质稻"佳辐占"为材料,应用差异蛋白组学的方法研究福建省光照最强月份(7月份)水稻秧苗功能叶响应光强日变化的分子机制.结果表明:共有7个蛋白质发生表达丰度的日变化,经质谱分析与功能鉴定,预测了6个蛋白质,分另4是叶绿体光合系统Ⅰ反应中心亚基Ⅱ-类蛋白(蛋白点1)、核苷单磷酸激酶b(蛋白点2),含Lon保守区的蛋白酶类蛋白(蛋白点3)、推定的还原酶(蛋白点4)、分子伴侣(蛋白点5)和倍半萜环化酶1(蛋白点6),其中,除蛋白点2、3在午问表达丰度增强之外,其余均下降,揭示了水稻功能叶蛋白质响应光强日变化的分子行为生态学特性.     

Influence of binding of sodium dodecyl sulfate, all-trans-retinol, palmitate, and 8-anilino-1-naphthalenesulfonate on the heat-induced unfolding and aggregation of beta-lactoglobulin B

Heat treatment of bovine beta-lactoglobulin B (beta-LG) causes it to partially unfold and aggregate via hydrophobic association and intra- and interprotein disulfide bonds. The first stage, which involves a "loosening" of the native structure, is influenced by the environmental conditions, such as pressure, pH, and added solutes. In the present study, four potential beta-LG ligands [palmitate, sodium dodecyl sulfate (SDS), 8-anilino-1-naphthalenesulfonate (ANS), and all-trans-retinol (retinol)] were added to beta-LG solutions prior to heat treatment for 12 min at temperatures between 40 and 93 degrees C. The extent of the changes in secondary and tertiary structures, unfolding, and aggregation at 20 degrees C were determined by circular dichroism, fluorescence, and alkaline- and SDS-polyacrylamide gel electrophoresis (PAGE). Both palmitate and SDS stabilized the native structure of beta-LG against heat-induced structural flexibility, subsequent unfolding, and denaturation. Retinol was less effective, probably because of its lower affinity for the calyx-binding site, and ANS did not stabilize beta-LG, suggesting that ANS did not bind strongly in the calyx. It was also noted that holding a beta-LG solution with added SDS or ANS promoted the formation of a hydrophobically associated non-native dimer.     

Determination of exposed sulfhydryl groups in heated beta-lactoglobulin A using IAEDANS and mass spectrometry

This paper takes a new approach to determining which sulfhydryl groups are exposed during the heat denaturation of bovine beta-lactoglobulin A. The sulfhydryl groups exposed after heating were blocked with 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS). The results show that IAEDANS is a suitable blocking agent, and its absorbance at 336 nm enabled the quantification of exposed sulfhydryl groups in a mixture of protein species by gel permeation chromatography. Combined with the specific fragmentation of bound IAEDANS by matrix-assisted laser desorption ionization (MALDI) MS/MS in negative ionization mode, this facilitated the identification of peptides that contained blocked cysteines after enzymatic digestion of the protein. During MALDI MS/MS of the peptides, in positive ionization mode, the IAEDANS molecule remained bound to the cysteines, making it possible to identify exactly which cysteine had been exposed after heating. In beta-lactoglobulin A it was found that cysteine 66 and cysteine 160 were predominantly exposed regardless of the length of exposure to heat.     

稻瘟菌细胞壁热解物诱导水稻抗稻瘟病的研究

以水稻近等基因系和广东省稻瘟病菌优势小种ZC13的细胞壁热解物(HDS)为材料进行诱导抗病性的研究。HDS通过细胞壁121°C 20 m in热解获得。HDS中葡萄糖质量浓度为176.83 m g/mL。HDS在体外对稻瘟病菌的孢子萌发和菌丝生长均无抑制作用,但处理水稻后可以显著提高水稻对稻瘟病菌的抗病性。在亲和性品系中,诱导抗病效果最高可达82.47%,且浓度与活性间存在正相关趋势;在非亲和品系中,热解物处理引起HR反应。HDS处理后,水稻过氧化物酶(POD)和苯丙氨酸解氨酶(PAL)活力明显升高。     

Land‐use change in subalpine grassland soils: Effect on particulate organic carbon fractions and aggregation

Abandonment of mountain grassland often changes vegetation composition and litter quantity and quality, but related effects on labile soil organic matter (SOM) are largely unknown. The aim of this study was to investigate the impacts of grassland management and abandonment on soil carbon distribution in light (< 1.6 g cm^–3) particulate organic matter (POM) and aggregation along a gradient of management intensity including hay meadows, pastures, and abandoned grasslands. The reduction of management intensity is an interregional phenomenon throughout the European Alps. We therefore selected sites from two typical climate regions, namely at Stubai Valley, Austria (MAT: 3°C, MAP: 1097 mm) and Matsch Valley, Italy (MAT: 6.6°C, MAP: 527 mm), to evaluate effects of land‐use change in relation to climate. Free water‐floatable and free POM (wPOM, fPOM), and an occluded POM fraction (oPOM), were isolated from three water‐stable aggregate size classes (2–6.3 mm, 0.25–2 mm, < 0.25 mm) using density fractionation. Aggregate mean weight diameter slightly decreased with decreasing management intensity. In contrast to absolute POM‐C, fPOM‐C increased in aggregates at both sites with abandonment. Because the oPOM‐C was less affected by abandonment, the ratio of oPOM‐C : fPOM‐C shifted from > 1 to < 1 from meadow to abandoned grassland in aggregates at both sites and thus independent of climate. This suggests that in differently managed mountain grasslands free and occluded POM are functionally different SOM fractions. In bulk soil, the oPOM‐C : fPOM‐C ratio is better suited as an indicator for the response of SOM to management reduction in subalpine grasslands than the total soil C, absolute or relative POM‐C content.     

地源热泵—地板散热系统在温室冬季供暖中的应用

针对浅层地能低品位的特点,在中国农业科学院Venlo型试验温室内设计建造了一套地源热泵与地板散热方式相结合的供暖系统,并对加热效果进行了试验,结果表明:温室内垂直方向的气温随着高度的增加而下降,水平方向气温分布均匀,温度分布有利于植物的生长。整套地源热泵系统的实际制热系数COP值达到3.14,与燃煤锅炉相比节能36.3%,节能效果明显。     

A continuum approach to soil classification by modified fuzzy k-means with extragrades

The need for a more continuous approach to soil classification is discussed, and methods based on the mathematical constructs known as fuzzy sets are considered most appropriate for this. A centroidal grouping method, fuzzy k-means with extragrades, which quantifies the intragrading and extragrading of soil individuals is described. An example of the application of this technique to an area of 4800 ha at Wesepe in The Netherlands is presented. The results show that the technique could create a classification that reflects the main pedological features of the area in a continuous way. Although there may be problems in selecting the optimal number of groups and degree of fuzziness, we conclude that the method is most promising and worthy of consideration when any type of quantitative soil classification is required.     

陆生植物对全球环境变化的适应

全球环境变化对陆地生态系统产生了深刻影响,而陆生植物对环境变化具有一定的适应机制。在生理过程中,信号物质能够对植物的生理过程进行调节,植物会主动积累或合成小分子物质以减轻逆境的伤害。在个体水平上,植物通过调节同化物在不同器官中的分配来适应环境条件的变化;植物对水氮资源的权衡利用可以使其适应不同的资源环境条件;由于对非结构碳水化合物的奢侈利用,植物生长不会受碳供应的限制。植物生理生态过程中的适应机制会影响陆地生态系统的碳收支。只有在综合分析的基础上建立整合植物适应机理的数学模型,才能准确预测环境变化对陆地生态系统的影响。