Hydrolysis of whey protein isolate with Bacillus licheniformis protease: aggregating capacities of peptide fractions

In a previous study, peptides aggregating at pH 7.0 derived from a whey protein hydrolysate made with Bacillus licheniformis protease were fractionated and identified. The objective of the present work was to investigate the solubility of the fractionated aggregating peptides, as a function of concentration, and their aggregating capacities toward added intact proteins. The amount of aggregated material and the composition of the aggregates obtained were measured by nitrogen concentration and size exclusion chromatography, respectively. The results showed that of the four fractions obtained from the aggregating peptides, two were insoluble, while the other two consisted of 1:1 mixture of low and high solubility peptides. Therefore, insoluble peptides coaggregated, assumedly via hydrophobic interactions, other relatively more soluble peptides. It was also shown that aggregating peptides could aggregate intact protein nonspecifically since the same peptides were involved in the aggregation of whey proteins, beta-casein, and bovine serum albumin. Both insoluble and partly insoluble peptides were required for the aggregation of intact protein. These results are of interest for the applications of protein hydrolysates, as mixtures of intact protein and peptides are often present in these applications.     

Hydrolysis of whey protein isolate with Bacillus licheniformis protease: fractionation and identification of aggregating peptides

The objective of this work was to identify the dominant aggregating peptides from a whey protein hydrolysate (degree of hydrolysis of 6.8%) obtained with Bacillus licheniformis protease. The aggregating peptides were fractionated with preparative reversed-phase chromatography and identified with liquid chromatography-mass spectrometry. The results showed that the dominant aggregating peptide, at pH 7.0, was beta-lg AB [f1-45]. In addition, the peptides beta-lg AB [f90-108]-S-S-alpha-la [f50-113], alpha-la [f12-49]-S-S-alpha-la [f50-113], beta-lg AB [f90-108]-S-S-beta-lg AB [f90-108], beta-lg A [f90-157], and beta-lg AB [f135-157/158] were also identified as main aggregating peptides. The results further showed that aggregation, via hydrophobic interactions, prevented further digestion (at pH 8.0), thereby explaining the large size of the aggregating peptides. It is hypothesized that B. licheniformis protease breaks down hydrophilic segments in the substrate and, therefore, preserves hydrophobic segments that aggregate once exposed to the solvent.     

Heat-induced formation of intermolecular disulfide linkages between thaumatin molecules that do not contain cysteine residues

Thaumatin, a sweet protein that contains no cysteine residues and eight intramolecular disulfide bonds, aggregates upon heating at pH 7.0 above 70 degrees C, and its sweetness thereby disappears. The aggregate can be solubilized by heating in the presence of both thiol reducing reagent and SDS. This molecular aggregation depended on the protein concentration during heating and was suppressed by the addition of N-ethylmaleimide or iodoacetamide, indicating a thiol-catalyzed disulfide interchange reaction between heat-denatured molecules. An amino acid analysis of the aggregates suggested that the cysteine and lysine residues were reduced, and the formation of a cysteine residue and a lysinoalanine residue was confirmed. The reduction and formation of these residues stoichiometrically satisfied the beta-elimination of a cystine residue. The disulfide interchange reaction was catalyzed by cysteine; that is, a free sulfhydryl residue was formed via beta-elimination of a disulfide bond. Intermolecular disulfide bonds were probably formed between thaumatin molecules upon heating at pH 7.0, which led to the aggregation of thaumatin molecules.     

Effect of phenolic acids on the formation and stabilization of soil aggregates

ABSTRACT

To examine the effects of phenolic acids, which are generated by the decomposition of cell walls in plant residues, and other constituents on the stability of soil aggregates, phenolic acids and carbohydrates were mixed into three different types of soil. After a 1-month incubation, the plot containing soil mixed with phenolic acids showed the greatest mean weight diameter of all the soils. In the treated soils, before incubation, the decline of saturated water permeability during continuous water percolation was mitigated in the plot containing soil mixed with phenolic acids compared with that in the other plots. Soil aggregates were synthesized with the addition of phenolic acids and carbohydrates using two methods (mixing and surface brushing) and were incubated for 153 days. The aggregate stability was greatest in the plots surface-brushed with phenolic acids for Andosol and gray lowland soil, whereas the aggregate stability was most stable in the plots mixed with phenolic acids for yellow soil. This difference in the effectiveness of application methods is rationalized by the densities of the active Al and Fe contents, the carbon content, and the specific surface area of the soils. The phenolic acids also affected sandy soil. In a similar experiment using a gray lowland soil, mixing a portion of p-coumaric acid into synthetic aggregates was found to shift the molecular weight distribution of substances to larger molecular weights, as determined by size exclusion chromatography of the liquid extracted from the aggregates, which was likely accompanied by an increase in aggregate stability. The effects of fungi and bacteria on soil long-term stability were not greater than those of phenolic acids. Our findings and previous results show that microorganisms aid in soil-aggregate formation during the early stages, and phenolic acids not only aid in the formation of aggregates but also strongly stabilize them.     

Effect of extended aging of parma dry-cured ham on the content of oligopeptides and free amino acids

The effect of the dry-curing processing time on the release of oligopeptides and amino acids was evaluated with 158 Parma hams subdivided into three groups: (1) traditional processing (450 days); (2) extended processing (570 days); and (3) extended aging (690 days). Most of the oligopeptides and free amino acids detected increased up to the last deadline (690 days); a sharp increase of peptides below 400 Da was the main change in most aged hams. In particular, gamma-glutamyl dipeptides showed a remarkable increase during ham extended aging, acting like permanent taste-active compounds, being unsuitable for further enzymatic breakdown. The pH of fresh hams showed negative relationships (P < 0.001) with most peptides. With regard to free amino acids, the pattern was modified by different processing lengths, together with their taste categories, so that the amino acids having monosodium glutamate-like and bitter tastes were enhanced in more aged hams.     

Factors affecting the formation of alkylpyrazines during roasting treatment in natural and alkalinized cocoa powder

The cocoa roasting process at different temperatures (at 125 and 135 degrees C for 3 min, plus 44 and 52 min, respectively, heating-up times) was evaluated by measuring the initial and final free amino acids distribution, flavor index, formol number, browning measurement, and alkylpyrazines content in 15 cocoa bean samples of different origins. These samples were also analyzed in manufactured cocoa powder. The effect of alkalinization of cocoa was studied. Results indicated that the final concentration and ratio of tetramethylpyrazine/trimethylpyrazine (TMP/TrMP) increased rapidly at higher roasting temperatures. The samples roasted with alkalies (pH between 7.20 and 7.92), such as sodium carbonate, or potassium plus air injected in the roaster during thermal treatment, exhibited a greater degree of brown color formation, but the amount of alkylpyrazines generated was adversely affected. The analysis of alpha-free amino acids at the end of the roasting process demonstrated the importance of the thermal treatment conditions and the pH values on nibs (cocoa bean cotyledons), liquor, or cocoa. Higher pH values led to a lower concentration of aroma and a higher presence of brown compounds.     

Small Scale Heterogeneity of Soil Chemical Properties: Effects on Spring Water Chemistry

Previous soil and spring water analyses in small catchments revealed low pH values in the spring water during high discharge events. This paper analyses the potential which small scale heterogeneity of soil acidity may have to explain decreasing spring water pH as a result of high discharge. Soil aggregates were collected from a C‐horizon of a Spodosol in the Fichtelgebirge. Exchangeable cations and soil solution were examined on both samples from the surfaces and the cores of aggregates which were obtained by a mechanical separation procedure. The Reuss‐Johnson soil chemical equilibrium model was used to predict soil and spring water pH values as a function of acidic input and soil air CO₂ concentration in equilibrium with both aggregate fractions. Ranges of acidic input from 160—570 μeq L⁻¹ and soil air CO₂ concentrations from 0.1 to 3 Vol. % were considered. The model predicted spring water pH values from 5.0 to 5.3 for the acidic aggregate surface samples (base saturation = 12.5%) and from 6.8 to 7.2 for the aggregate core samples (base saturation = 32.1%). The results suggest that small scale acidity gradients may expand the range of predictable spring water pH values. However, very low pH values (<5) still need additional explanation.     

Contribution of Sourdough Lactobacilli,Yeast, and Cereal Enzymes to the Generation of Amino Acids in Dough Relevant for Bread Flavor

The amino acid release was determined in wheat doughs supplied with salt, acid, dithiothreitol, or starter cultures to evaluate the relevance of the amino acid concentration on bread flavor. Wheat flour proteinases almost linearly released amino acids and the highest activity of wheat flour proteinases was found in acidified and reduced doughs. The effects of starter cultures on amino acid concentrations depended on their composition. Yeasts exhibited a high demand for amino acids, however, the total amino acid concentrations were not markedly affected by lactic acid bacteria. The individual amino acid contents were determined by the pH during fermentation and microbial metabolism. The formation of proline was favored by values higher than pH 5.5, whereas release of phenylalanine, leucine and cysteine mainly occurred at lower pH. Ornithine was found only in doughs fermented with Lactobacillus pontis. To determine effects of the amino acid concentration on bread aroma, fermented doughs were evaluated in baking experiments. An increased intensity of bread flavor was obtained by preferments prepared with lactic acid bacteria. The roasty note of wheat bread crust could be markedly enhanced by L. pontis. This results support the assumption that flavor of wheat bread is enhanced by increasing the concentration of free amino acids and especially ornithine in dough.     

Identification of free amino acids in peat by gas chromatography and mass spectrometry

The qualitative and quantitative composition of free amino acids in a typical Finnish peat bog at various depths down to 5.3 m below the surface was studied using capillary gas chromatography and mass spectrometry. Sixteen amino acids were identified at each depth: α-alanine, β-alanine, glycine, valine, leucine, proline, isoleucine, serine, threonine, glutamic acid, aspartic acid, phenylalanine, tyrosine, γ-aminobutyric acid, ornithine and lysine. Their amounts decreased markedly at a depth of 40–100 cm. The total amount of amino acids varied between 0.6 and 5.6 g kg^?1 dry matter (i.e. 0.06–0.56%) depending on the depth. The proportion of neutral amino acids was greatest at all depths studied, except at the surface layer where it ranged between 41 and 72% by mass. The acidic amino acids decreased with depth from 56 to 23% of the total. The proportion of aromatic amino acids was very small, 3.2–5.5% by mass. In samples from aerobic conditions, where the microbial production of free amino acids was the greatest, α-ala, gly, glu and asp were most abundant. In peat from anaerobic conditions, where the microbiological activity was low, the proportion of the most chemically stable amino acid was exceptionally high. This may have been because glycine was a degradation product of other amino acids or peptides. Peat type and degree of decomposition had a strong influence on the total amount of free amino acids and their qualitative composition.     

Enzyme-induced gelation of extensively hydrolyzed whey proteins by Alcalase: peptide identification and determination of enzyme specificity

Extensive hydrolysis of whey protein isolate by Alcalase was shown to induce gelation mainly via hydrophobic interactions. The aim of this work was to characterize the peptides released in order to better understand this phenomenon. The apparent molecular mass distribution indicated that aggregates were formed by small molecular mass peptides (<2000 Da). One hundred and thirty peptides with various lengths were identified by reversed-phase high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. Alcalase was observed to have a high specificity for aromatic (Phe, Trp, and Tyr), acidic (Glu), sulfur-containing (Met), aliphatic (Leu and Ala), hydroxyl (Ser), and basic (Lys) residues. Most peptides had an average hydrophobicity of 1-1.5 kcal/residue and a net charge of 0 at the pH at which gelation occurred (6.0). Therefore, an intermolecular attractive force such as hydrophobic interaction suggests the formation of aggregates that further leads to the formation of a gel.     

糖接枝处理改善大豆蛋白纤维聚集体泡沫稳定性

为了探究糖接枝对大豆蛋白纤维聚集行为和泡沫性质的影响,明确蛋白质结构与功能的关系,该研究以大豆蛋白(soy protein isolation,SPI)和乳糖(lactose)为原料,通过干热法制备糖接枝大豆蛋白(SPI-lactose conjugate,SPI-Lac),以及在酸性条件下加热诱导其形成纤维聚集体(p H值2.0),制备了一种糖接枝大豆蛋白纤维聚集体(SPI-lactose conjugate fibillar aggregates),并考察了糖接枝对大豆蛋白的纤维聚集行为及泡沫性质的影响。研究结果表明:大豆蛋白在酸性条件下(p H值2.0)经加热后会发生水解,同时水解产物不断聚集形成大分子的纤维聚集体。糖接枝导致大豆蛋白的水解速度下降,但荧光光强和粒径的结果表明糖接枝能增强纤维聚集能力。SPI-Lac在中性条件下的溶解度(p H值5.0—7.0)显著高于SPI(P0.05),且不同时间处理的SPI-Lac纤维聚集体均能改善SPI在酸性条件下的溶解度(p H值2.0—5.0)。此外,不同时间处理的SPI-Lac纤维聚集体在酸性条件下的起泡能力均高于SPI纤维聚集体。SPI和SPI-Lac纤维聚集体的形成会导致SPI起泡能力的下降,但是短时间酸热处理形成的纤维聚集体泡沫稳定性得到显著改善。因此,糖接枝结合短时间酸热处理制备的糖接枝大豆蛋白纤维聚集体在中性条件下的泡沫稳定性显著提高(P0.05),是合理有效的蛋白质改性方法。     

适宜含水率保持油茶籽贮藏品质

为了确定油茶籽贮藏适宜的含水率,研究了在4℃,不同含水率(7%、10%、13%、16%、20%)油茶籽贮藏期间的品质变化。结果表明,较低的含水率能较好保持油茶籽的贮藏特性及营养品质。其中,含水率为7%的油茶籽贮藏效果较好,但与10%处理效果差异不明显(P>0.05)。在整个贮藏期,含水率为7%时油茶籽可溶性蛋白下降了13.05 mg/g,油酸含量下降了2.38%,酸值、过氧化值等品质指标上升速率较慢,同时能较好保持β-谷甾醇和角鲨烯等生物活性成分;其次是10%的含水率处理。而含水率为20%的油茶籽贮藏期间可溶性蛋白下降较快,贮藏结束时为25.47 mg/g,油茶籽劣变严重,所提取的油样品质变差,营养物质含量较少,因此含水率20%的油茶籽不适宜长期贮藏。综合考虑油茶籽品质因素和处理成本,认为控制含水率在10%以下能较好保持油茶籽的贮藏品质。该研究可为科学合理地贮藏油茶籽提供参考。     

Protein-peptide interactions in mixtures of whey peptides and whey proteins

The effects of several conditions on the amounts and compositions of aggregates formed in mixtures of whey protein hydrolysate, made with Bacillus licheniformis protease, and whey protein isolate were investigated using response surface methodology. Next, the peptides present in the aggregates were separated from the intact protein and identified with liquid chromatography-mass spectrometry. Increasing both temperature and ionic strength increased the amounts of both intact protein and peptides in the aggregates. There was an optimal amount of added intact WPI that could aggregate with peptides, yielding a maximal amount of aggregated material in which the peptide/protein molar ratio was around 6. Under all conditions applied, the same peptides were observed in the protein-peptide aggregates formed. The dominant peptides were beta-lg AB [f1-45], beta-lg AB [f90-108], and alpha-la [f50-113]. It was hypothesized that peptides could form a kind of glue network that can include beta-lactoglobulin via hydrophobic interactions at the hydrophobic binding sites at the surface of the protein.     

Hypochlorous and peracetic acid induced oxidation of dairy proteins

Hypochlorous and peracetic acids, both known disinfectants in the food industry, were compared for their oxidative capacity toward dairy proteins. Whey proteins and caseins were oxidized under well controlled conditions at pH 8 as a function of the sanitizing concentration. Different markers for protein oxidation were monitored. The results established that the protein carbonyl content was a rather unspecific marker for protein oxidation, which did not allow one to differentiate the oxidant used especially at the lower concentrations. Cysteine, tryptophan, and methionine were proven to be the most vulnerable amino acids for degradation upon hypochlorous and peracetic acid treatment, while tyrosine was only prone to degradation in the presence of hypochlorous acid. Hypochlorous acid induced oxidation gave rise to protein aggregation, while during peracetic acid induced oxidation, no high molecular weight aggregates were observed. Protein aggregation upon hypochlorous acid oxidation could primarily be linked to tryptophan and tyrosine degradation.     

The effect of successive wet/dry cycles on aggregate size distribution in a clay texture soil

Aggregates of a fine textured (61% clay, 33% silt) soil with a high coefficient of linear expansion (20%) were exposed to successive wet/dry cycles in their natural state and after mechanical reworking. In both cases aggregate size decreased; the reworked sample initially consisted of larger aggregates, but their size fell more rapidly, and after four cycles both the reworked and natural aggregates had a similar size distribution. A model was developed which successfully predicts the proportion of aggregates in each size grade at the n th cycle from that present at the outset. The rate of decrease in aggregates size declined with aggregate size and on both reworked and natural samples the dispersion ratio was low (≤5%).     

Mozzarella干酪成熟中蛋白水解与功能特性的变化

为控制干酪的质量,对Mozzarella干酪成熟过程中蛋白质的水解(测定SDS凝胶电泳和可溶性氮)和未融化干酪的质构变化以及融化干酪功能特性变化进行了研究,干酪成熟过程中由于凝乳酶和乳酸菌酶的作用使蛋白水解,从而使pH 4.6可溶性氮(SN)和12% TCA SN逐渐增加;凝乳酶主要影响酪蛋白的水解范围,乳酸菌及其酶,不但影响酪蛋白的水解范围,而且主要影响酪蛋白的水解深度。干酪中的残留凝乳酶和乳酸菌酶使酪蛋白水解为大分子量的肽段,而乳酸菌酶还可将大分子量的肽段进一步降解为小分子量的肽段和游离氨基酸。由于酪蛋白的水解,使干酪的硬度和弹性下降,融化性和油脂析出性增加,随着小分子量肽和游离氨基酸的增加,干酪的褐变性提高。     

Physical and chemical interactions in cold gelation of food proteins

pH-Induced cold gelation of whey proteins is a two-step process. After protein aggregates have been prepared by heat treatment, gelation is established at ambient temperature by gradually lowering the pH. To demonstrate the importance of electrostatic interactions between aggregates during this latter process, beta-lactoglobulin aggregates with a decreased iso-electric point were prepared via succinylation of primary amino groups. The kinetics of pH-induced gelation was affected significantly, with the pH gelation curves shifting to lower pH after succinylation. With increasing modification, the pH of gelation decreased to about 2.5. In contrast, unmodified aggregates gel around pH 5. Increasing the iso-electric point of beta-lactoglobulin via methylation of carboxylic acid groups resulted in gelation at more alkaline pH values. Comparable results were obtained with whey protein isolate. At low pH disulfide cross-links between modified aggregates were not formed after gelation and the gels displayed both syneresis and spontaneous gel fracture, in this way resembling the morphology of previously characterized thiol-blocked whey protein isolate gels (Alting, et al., J. Agric. Food Chem. 2000, 48, 5001-5007). Our results clearly demonstrate the importance of the net electric charge of the aggregates during pH-induced gelation. In addition, the absence of disulfide bond formation between aggregates during low-pH gelation was demonstrated with the modified aggregates.     

Soil humic acid aggregation by dynamic light scattering and laser Doppler electrophoresis

Humic acids (HAs), similar to other fractions of humic substances (HSs), have a large number of reactive functional groups enabling them to aggregate in solutions. Regardless of the origin of humic acid (aqueous or soil), this aggregation process is dependent on environmental conditions and strongly influences the mobility of soluble ionic and molecular pollutants. The aim of this work was to monitor the aggregation process of two humic acids isolated from different mineral soils (IHSS Elliot soil HA standard and Rendzic Leptosol HA) in the 2–11 pH range. Changes in aggregate size in HA sols were followed up using dynamic light scattering (DLS), while zeta potential (ZP) measurements in the same pH range were performed applying laser Doppler electrophoresis (LDE) technique. The effect of HA sol concentration and soil source on aggregation was examined as well. Besides, HA samples were characterized using Fourier transform infrared (FT‐IR) spectroscopy. By inspecting HA‐particle‐size dependence on pH, it can be concluded that both HAs in corresponding sols behave as molecular aggregates or supramolecular structures, formed from small individual moieties (sizes < 10 nm) at higher pH values. The ZP vs. pH curve for both HAs revealed the ZP minimum in the 5–7 pH range, caused most likely by dissociation of acidic functional groups prevailing at lower pH values and deaggregation predominating over dissociation at higher pH values.     

湿润速度和累积降雨对土壤表面结皮发育的影响

土壤表面强度和微结构显微照片是研究表土结皮的重要指标和直接表征。以两种典型的土壤(垆土和黑土)为研究对象,采用2mmh-1(慢速)和50mmh-1(快速)两种速度湿润后进行雨强为60mmh-1的降雨,研究不同湿润速度和累积降雨对结皮发育的影响。结果表明:湿润速度对垆土结皮发育过程的影响不明显,土壤表面强度主要由累积降雨打击夯实引起;快速湿润对黑土结皮发育有显著的影响,慢速湿润后黑土在60min降雨过程中没有明显的结皮,累积降雨的打击起次要作用;湿润速度和累积降雨的对结皮发育的影响取决于土壤团聚体稳定性。     

Physical properties of homoionic montmorillonite and kaolinite complexed with amino acids and peptides

X-ray diffractometry was used to investigate whether various amino acids and peptides expanded montmorillonite (M) homoionic to different cations, and the electrophoretic mobility (EPM) was measured to determine whether the binding of these amino acids and peptides influenced the net surface charge of M and kaolinite (K). Neither aspartic acid, adsorbed at equilibrium at concentrations of 20–250 nM mg^?1 M homoionic to Ca or Zn, nor cysteine, at 50–830 nM mg^?1 M homoionic to Zn or Al, caused expansion, whereas proline, at 90–870 nM mg^?1 clay, caused expansion of M homoionic to Ca or Zn, and both proline and arginine, at 60–1150 nM mg^?1 clay, caused expansion of M homoionic to H or Al. The extent of expansion caused by proline and arginine was related to the concentration of the amino acids and the exchangeable cation on the homoionic M. Only M-A1 complexed with arginine remained expanded after extensive washing of the clay-amino acid complexes. The expansion of M was apparently dependent on both the type of exchangeable cation on the clay and the characteristics of the amino acid (i.e. the functional groups, dielectric constant increment, dipole moment and concentration). M homoionic to H, Na, Zn, Al or La was not expanded by a series of di- to tetrapeptides bound on these clays and none of the homoionic K-amino acid or peptide complexes was expanded. The EPM of the homoionic M and K and of the corresponding clay-amino acid or peptide complexes was identical, indicating that the concentration of the amino acids or peptides bound was not sufficient to cover enough of the surface of the clays to alter significantly their net charge. The lack of differences in the bulk pH of the clays after binding with amino acids or peptides (with the exception of complexes with arginine) also indicated limited coverage of the clays by these organics.