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.     

Formation of disulfide bonds in acid-induced gels of preheated whey protein isolate

Cold gelation of whey proteins is a two-step process. First, protein aggregates are prepared by a heat treatment of a solution of native proteins in the absence of salt. Second, after cooling of the solution, gelation is induced by lowering the pH at ambient temperature. To demonstrate the additional formation of disulfide bonds during this second step, gelation of whey protein aggregates with and without a thiol-blocking treatment was studied. Modification of reactive thiols on the surface of the aggregates was carried out after the heat-treatment step. To exclude specific effects of the agent itself, different thiol-blocking agents were used. Dynamic light scattering and SDS-agarose gel electrophoresis were used to show that the size of the aggregates was not changed by this modification. The kinetics of gelation as determined by the development of pH and turbidity within the first 8 h of acidification were not affected by blocking thiol groups. During gelation, formation of large, covalently linked, aggregates occurred only in the case of unblocked WPI aggregates, which demonstrates that additional disulfide bonds were formed. Results of permeability and confocal scanning laser microscope measurements did not reveal any differences in the microstructure of networks prepared from treated or untreated whey protein aggregates. However, gel hardness was decreased 10-fold in gels prepared from blocked aggregates. Mixing different amounts of blocked and unblocked aggregates allowed gel hardness to be controlled. It is proposed that the initial microstructure of the gels is primarily determined by the acid-induced noncovalent interactions. The additional covalent disulfide bonds formed during gelation are involved in stabilizing the network and increase gel strength.     

Aggregation properties of whey protein hydrolysates generated with Bacillus licheniformis proteinase activities

Hydrolysis of whey protein concentrate (WPC) with Alcalase 2.4 L, a Bacillus licheniformis proteinase preparation, induces gelation. The aggregation behavior of WPC hydrolysates generated with Alcalase and Prolyve 1000, a Bacillus licheniformis proteinase that did not induce gelation, were studied by turbidity and particle size analysis. With the use of synthetic peptide substrates, it was shown that Alcalase contains a glutamyl endopeptidase (GE) activity not present in Prolyve. Comparison of the aggregation behavior of WPC hydrolysates generated with Alcalase, Prolyve, and combinations of Prolyve with a GE activity isolated from Alcalase showed that GE was responsible for the observed enzyme-induced peptide aggregation in Alcalase hydrolysates. Hydrolysates generated with Prolyve, having a degree of hydrolysis (DH) of 11.8% and 10.4% of peptide material greater than 10 kDa, could be induced to aggregate by the addition of GE. These results emphasize the contribution of enzyme specificity to the physicochemical and functional characteristics of proteinase hydrolysates of WPC.     

Glucose oxidase-mediated gelation: a simple test to detect glucose in food products

This paper reports a simple, rapid, and sugar-selective method to induce gelation from glucose-containing samples. This method employs glucose oxidase (GOx) to selectively "recognize" and oxidize glucose to generate gluconic acid, which acts to solubilize calcium carbonate and release calcium ions. The release of calcium ions triggers gelation of the calcium-responsive polysaccharide alginate to form a calcium-alginate hydrogel. Rheological measurements confirm that gel formation is triggered by glucose but not fructose or sucrose (consistent with GOx's selectivity). Vial inversion tests demonstrate that gel formation can be readily observed without the need for instrumentation. Proof-of-concept studies demonstrate that this gel-forming method can detect glucose in food/beverage products sweetened with glucose or high-fructose corn syrups. These results indicate that the enzyme-induced gelation of alginate may provide a simple means to test for sweeteners using components that are safe for use on-site or in the home.     

蓝园鲹深度水解过程中蛋白质降解研究

利用风味蛋白酶深度酶解蓝园鲹蛋白，通过比较酶解液的水解度曲线和蛋白质利用率曲线之间的差异、对TCA不溶性氮的变化趋势以及不同酶解时间的凝胶过滤图谱进行分析，探讨了深度酶解过程中蛋白质的降解。酶解6h后大部分蛋白质在酶的作用下降解为水溶性多肽，蛋白质利用率达到83。3％；6h以后蛋白质利用率增长速度降低，这可能是由于可被降解的底物含量降低。此后。风味蛋白酶以水溶性多肽为底物将其进一步降解为小分子肽和氨基酸；21h时水解度达到59．7％。21h以后水解度增长速度降低．这可能是由于亮氨酸氨肽酶难于分解氨基末端上带有甘氨酸和酸性氨基酸的肽。21h以后酶解的主要底物分子量范围在6214到10700的多肽。     

蓝园鱼参深度水解过程中蛋白质降解研究(英)

利用风味蛋白酶深度酶解蓝园鱼参蛋白,通过比较酶解液的水解度曲线和蛋白质利用率曲线之间的差异、对TCA不溶性氮的变化趋势以及不同酶解时间的凝胶过滤图谱进行分析,探讨了深度酶解过程中蛋白质的降解。酶解6 h后大部分蛋白质在酶的作用下降解为水溶性多肽,蛋白质利用率达到83.3％;6 h以后蛋白质利用率增长速度降低,这可能是由于可被降解的底物含量降低。此后,风味蛋白酶以水溶性多肽为底物将其进一步降解为小分子肽和氨基酸;21 h时水解度达到59.7％。21 h以后水解度增长速度降低,这可能是由于亮氨酸氨肽酶难于分解氨基末端上带有甘氨酸和酸性氨基酸的肽。21 h以后酶解的主要底物分子量范围在6214到10700的多肽。     

Heat-induced gelation of pea legumin: comparison with soybean glycinin

Gel network formation of pea legumin (8.4% on a protein basis, pH 7.6) was monitored via dynamic rheological measurements. Gelation was performed in the absence and presence of the thiol-blocking reagent N-ethylmaleimide, at different rates of heating and cooling. Overall, it was shown that pea legumin gel formation was not effected by changes in the heating rate, and the two differently heated samples were unaffected by the addition of 20 mM NEM, which indicated that disulfide bonds were not essential within the network strands of these legumin gels. However, slowly cooling the legumin samples caused disulfide bonds to become involved within the network; this was observed by a large increase in gel strength that was then substantially reduced when repeating the sample in the presence of NEM. These experiments were repeated with soybean glycinin in order to determine whether a common model for gel formation of legumin-like proteins could be built, based upon molecular reasoning. The two proteins were affected in the same way by changes in the conditions used, but when applying a procedure of reheating and recooling the gel networks responded differently. Pea legumin gel networks were susceptible to rearrangements that caused the gels to become stronger after reheating/recooling, yet glycinin gel networks were not. It was concluded that the same physical and chemical forces drove the processes of denaturation, aggregation, and network formation. Each process can therefore be readily targeted for modification based upon molecular reasoning. Pea legumin and soybean glycinin gel networks had structurally different building blocks, however. A model of gelation aimed at texture control therefore requires additional information.     

Heat-induced gel formation by soy proteins at neutral pH

Heat-induced gel formation by soy protein isolate at pH 7 is discussed. Different heating and cooling rates, heating times, and heating temperatures were used to elucidate the various processes that occur and to study the relative role of covalent and noncovalent protein interactions therein. Gel formation was followed by dynamic rheological measurements. Heat denaturation was a prerequisite for gel formation. The gelation temperature (84 degrees C) was just above the onset denaturation temperature of glycinin. The stiffness of the gels, measured as the elastic modulus, G', increased with the proportion of denatured protein. An increase in G' was also observed during prolonged heating at 90 degrees C. This increase is explained by the occurrence of rearrangements in the network structure and probably also by further incorporation of protein in the network. The increase in G' upon cooling was thermoreversible indicating that disulfide bond formation and rearrangements do not occur upon cooling.     

Enzymatic hydrolysis as a means of expanding the cold gelation conditions of soy proteins

Acid-induced cold gelation of soy protein hydrolysates was studied. Hydrolysates with degrees of hydrolysis (DH) of up to 10% were prepared by using subtilisin Carlsberg. The enzyme was inhibited to uncouple the hydrolysis from the subsequent gelation; the latter was induced by the addition of glucono-delta-lactone. Visual observations, confocal scanning laser microscopy images, and the elasticity modulus showed that hydrolysates gelled at higher pH values with increasing DH. The nonhydrolyzed soy protein isolate gelled at pH approximately 6.0, whereas a DH = 5% hydrolysate gelled at pH approximately 7.6. Gels made from hydrolysates had a softer texture when manually disrupted and showed syneresis below a pH of 5-5.5. Monitoring of gelation by measuring the development of the storage modulus could be replaced by measuring the pH onset of aggregate formation (pH(Aggr-onset)) using turbidity measurements. The rate of acidification was observed to also influence this pH(Aggr-onset). Changes in ionic strength (0.03, 0.2, and 0.5 M) had only a minor influence on the pH(Aggr-onset), indicating that the aggregation is not simply a balance between repulsive electrostatic and attractive hydrophobic interactions, but is much more complex.     

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.     

Monovalent salt-induced gelation of enzymatically deesterified pectin

Pectin gels were induced by monovalent salts (0.2 M) concurrently with deesterification of high methoxy pectin using a salt-independent orange pectin methylesterase (PME). Constant pH was maintained during deesterification and gelation. If salt or PME was absent, the pectin did not form a gel. The gel strength was influenced by both pH and species of monovalent cation. At pH 5.0, the pectin gel induced by KCl was significantly stronger than the NaCl-induced gel. In contrast, a much stronger gel was produced in the presence of NaCl as compared to KCl at pH 7.0. LiCl did not induce pectin gelation at either pH. Molecular weights of pectins increased from 1.38 x 10(5) to 2.26 x 10(5) during NaCl-induced gelation at pH 7. One proposal to explain these pectin molecular weight changes is a hypothetical PME transacylation mechanism. However, these pectin molecular weight changes can also be explained by metastable aggregation of the enzymatically deesterified low methoxy pectin. We postulate that gelation was induced by a slow deesterification of pectin under conditions that would normally salt out (precipitate) low methoxy pectin in the absence of PME.     

Influence of the extent of hemoglobin hydrolysis on the digestive absorption of heme iron. An in vitro study

This study was designed to assess the interactions of heme with peptides produced by enzyme hydrolysis of hemoglobin, and their relationship with heme iron absorption. Bovine hemoglobin was hydrolyzed by pepsin or by subtilisin, which differ in their hydrolysis processes. The hydrolysis rate ranged from 0 (native hemoglobin) to 15%. Heme solubility and heme-peptides interactions were compared to iron absorption by the Ussing chamber model, at intestinal pH (7.5). Increasing hemoglobin hydrolysis enhanced iron absorption; the highest value was reached between 8 and 11% hydrolysis, whatever the enzyme used. Comparing the products of hydrolysis of the two enzymes showed that heme iron absorption depends not only on its solubility, but relies mainly on the balance between the strength of heme-peptides and the polymerization rate of heme.     

马面鱼皮胶原抗氧化肽的分离制备及稳定性研究

为促进对马面鱼皮资源的综合利用,开发高附加值产品,本试验以DPPH自由基清除率和水解度(DH)为评价指标,探讨马面鱼皮胶原蛋白的最佳酶解工艺,并采用超滤和凝胶柱层析法分离制备抗氧化肽,通过超高效液相色谱-质谱联用(UPLC-MS)法对其进行结构解析。此外,还探讨了pH值、温度及体外模拟消化对多肽抗氧化活性的影响。结果表明,利用双酶分步酶解法可制备高活性抗氧化多肽,即在底物浓度3%,加酶量3 600 U·g^-1以及温度50℃的条件下先用Proteasea A ‘Amano’2G酶解3 h,再用酸性蛋白酶酶解2 h,清除DPPH自由基的IC₅₀值为13.03 mg·mL ^-1。经超滤及柱层析分离后,可得到抗氧化活性较高的A₁组分,其清除DPPH自由基的 IC₅₀值为1.80 mg·mL^-1。稳定性研究结果表明,所制备的胶原蛋白抗氧化肽热稳定性好,在偏酸性条件下能保持较高的活性,经体外模拟胃肠消化后仍能保持较高的抗氧化活性。根据UPLC-MS分析推测A₁的氨基酸序列可能为Gly-Glu-Gly-Ala-Cys-Asn或Asn-Glu-Gly-Ala-Cys-Gly。本研究结果为马面鱼皮的高值化利用及高活性抗氧化肽的筛选提供了一定的理论依据。     

Effects of sugars on whey protein isolate gelation

Whey protein isolate (WPI) gels were prepared from solutions containing ribose or lactose at pH values ranging from 6 to 9. The gels with added lactose had no color development, whereas the gels with added ribose were orange/brown. Lactose stabilized the WPI to denaturation, which increased the time and temperature required for gelation, thus decreasing the fracture modulus of the gel compared to the gels with added ribose and the gels with no sugar added. Ribose, however, favored the Maillard reaction and covalent cross-linking of proteins, which increased gel fracture modulus. The decreased pH caused by the Maillard reaction in the gels containing ribose occurred after protein denaturation and gelation, thus having little if any effect on the gelation process.     

Amino-N compounds found in soil organic matter hydrolysates of a loamy sand using an immobilized protease reactor column

Summary Soil organic matter (OM) from seven different fertility plots of a loamy sand was extracted and fractionated into high- and low-molecular-weight (HMW, LMW) fractions using gel filtration. The fractions were acid-hydrolyzed to determine the amino sugar and amino acid contents. The same fractions were hydrolyzed with an immobilized protease reactor column. Reverse-phase high-performance liquid chromatography (HPLC) was used to identify the soil amino-N compounds. With the HMW fraction as substrate, the enzyme released less than 1% of 11 amino-N compounds determined by acid hydrolysis. Phenylalanine and leucine, however, were recovered in quantities of 2% and 4%, respectively. Immobilized protease hydrolysis of the LMW fraction recovered considerably more amino-N compounds compared with acid hydrolysis of the same fractions. Each system of hydrolysis produced some amino-N compounds not found in the other. We conclude that an immobilized enzyme reactor column will allow a researcher to perform time-course hydrolysis, so that hydrolysis intermediates, e.g. peptides, can be separated and identified.     

Gelation of iota-carrageenan and micellar casein mixtures under high hydrostatic pressure

Effects of high-pressure treatment (HPT) on the rheological parameters and gelation of iota-carrageenan (iota-Car) and mixtures of micellar casein (MC) and iota-Car have been investigated under neutral pH conditions. It was established that HPT showed no significant effect, in the presence or absence of ionic calcium, with or without initial thermal processing, on the rheology or gelation/melting temperatures of the pure iota-Car solution. However, in mixed systems containing varying concentrations of iota-Car (up to 1 wt %) and MC (up to 8 wt %), considerable changes were detected. At the higher molar ratios of MC to iota-Car, and especially at the higher pressures, the dispersions were not thermoreversible in gelation, presumably due to the strong interactions of disrupted casein micelles with iota-Car molecules, as well as due to the formation of a dominant proteinaceous network at higher concentrations of MC. The associative protein-polysaccharide interactions in these systems are highly dependent on the ionic calcium content.     

Influence of aroma compounds on the mechanical properties of pectin gels

A detailed study of the rheological behavior of pectin gels, in the presence of aroma compounds in food concentration is reported. In high methoxylated pectin (HMP) gels, it has not yet been shown if aroma compounds can be responsible for modifications in rheology. Two rheological techniques were used to measure the impact of aroma substances on rheological properties of HMP-based systems. Maximum strain to fracture (sigma F) was compared between flavored and unflavored gels on stress-displacement curve, which was obtained with uniaxial compression until fracture. An oscillatory rheometer was applied to determine the gelation time (Tgel). It appeared that all the aroma compounds studied increased significantly sigma F. It is generally acknowledged that hydrophobic interactions are the main interactions leading to HMP gelation, and the more hydrophobic interactions there are, the higher the sigma F. It is assumed that esters might increase sigma F through the increase of hydrophobic interactions in HMP network. For solvents (ethanol, propylene glycol), a cosolute effect could explain the increase in firmness for HMP-based gels.     

Functional and structural properties of 2S soy protein in relation to other molecular protein fractions

The purpose of this investigation is to develop a better understanding of the structure-function relationship of the 2S fraction of soy protein that has not been considered in earnest by the research community. Defatted soy flour was used to extract the three major fractions of the protein (2S, 7S, and 11S). It was found that 2S exhibits better foaming and emulsification properties than the other two molecular fractions. Work was extended to structural properties, which were monitored using spectrophotometry, atomic force microscopy, scanning electron microscopy, small-deformation dynamic oscillation on shear, and large-deformation compression testing. An experimental protocol utilizing glucono-delta-lactone (GDL), GDL with N-ethylmaleimide, or GDL with urea was capable of identifying the nature of molecular interactions responsible for gelation. Surprisingly, it was found that in the initial stages of structure formation, 2S fared better than 7S, with 11S exhibiting the highest rates of aggregation. Given time, however, 7S produced a firmer network with a better water-holding capacity than that of 2S. Non-covalent interactions, as opposed to disulfide bridging, were found to be largely responsible for the changing functionality of the molecular fractions throughout the experimentation from the formation of a vestigial structure to that of a mature gel.     

Gelation of edible blue-green algae protein isolate (Spirulina platensis Strain Pacifica): thermal transitions, rheological properties, and molecular forces involved

Proteins isolated from blue-green algae Spirulina platensis strain Pacifica were characterized by visible absorption, differential scanning calorimetry (DSC), viscometry, and dynamic oscillatory rheological measurements. Unique thermal unfolding, denaturation, aggregation, and gelation of the algal protein isolate are presented. DSC analysis showed that thermal transitions occur at about 67 and 109 degrees C at neutral pH. Calcium chloride stabilized the quaternary structure against denaturation and shifted the transitions at higher temperatures. Viscometric studies of Spirulina protein isolate as a function of temperature showed that the onset of the viscosity increase is closely related to the dissociation-denaturation process. Lower viscosities were observed for the protein solutions dissolved at pH 9 due to an increased protein solubility. Solutions of Spirulina protein isolate form elastic gels during heating to 90 degrees C. Subsequent cooling at ambient temperatures caused a further pronounced increase in the elastic moduli and network elasticity. Spirulina protein isolate has good gelling properties with fairly low minimum critical gelling concentrations of about 1.5 and 2.5 wt % in 0.1 M Tris buffer, pH 7, and with 0.02 M CaCl(2) in the same buffer, respectively. It is suggested that mainly the interactions of exposed hydrophobic regions generate the molecular association, initial aggregation, and gelation of the protein isolate during the thermal treatment. Hydrogen bonds reinforce the network rigidity of the protein on cooling and further stabilize the structure of Spirulina protein gels but alone are not sufficient to form a network structure. Intermolecular sulfhydryl and disulfide bonds were found to play a minor role for the network strength of Spirulina protein gels but affect the elasticity of the structures formed. Both time and temperature at isothermal heat-induced gelation within 40-80 degrees C affect substantially the network formation and the development of elastic modulus of Spirulina protein gels. This is also attributed to the strong temperature dependence of hydrophobic interactions. The aggregation, denaturation, and gelation properties of Spirulina algal protein isolate are likely to be controlled from protein-protein complexes rather than individual protein molecules.     

不同热处理大豆分离蛋白凝胶冻藏特性

为探究冻藏过程中不同加热温度处理大豆分离蛋白(soybean isolate protein,SPI)凝胶特性变化及评估不同热处理对SPI凝胶冻藏特性的影响。该文以65、90和135℃3个不同温度处理所得SPI为研究对象(分别记为65SPI、90SPI和USPI),采用离心法、质构分析法、可溶蛋白含量测定和电泳等方法对其冻藏过程中的凝胶持水性、凝胶硬度、凝胶弹性、可溶蛋白含量及亚基组成和凝胶作用力进行了分析研究。结果表明:随冻藏时间延长,不同温度处理SPI凝胶持水性、凝胶弹性和凝胶可溶蛋白含量呈下降趋势,而凝胶硬度呈增大趋势。凝胶持水性、弹性的下降和凝胶硬度的升高标志着凝胶品质的劣变。不同温度处理对SPI凝胶的冻前凝胶特性和冻藏特性有较大影响,65和90℃的温度处理降低了冻前SPI凝胶的持水性,增强了冻前SPI凝胶硬度,有更多的β和B亚基参与了凝胶形成,冻藏前后的亚基组成没有变化;超高温瞬时加热(ultra high temperature,UHT)处理则降低了冻前SPI凝胶硬度,冻藏过程中可溶蛋白含量大幅下降且可溶蛋白中β和B亚基含量下降。3种温度处理SPI的凝胶劣变程度均高于未处理SPI。加热处理会造成SPI发生部分或完全变性,变性后疏水基团的暴露会加快蛋白凝胶形成过程中聚集速率,进而增大粗糙凝胶结构形成的几率,而粗糙凝胶网络在冻藏过程中其劣变程度更甚于未加热SPI。由此可知,加热处理尽管在一定程度上增大了凝胶硬度,但会加速其凝胶品质冻藏劣变。