Allergenicity of proteolytic hydrolysates of the soybean 11S globulin

ABSTRACT:  A substantial portion of the human population has immune hypersensitivities to various food materials. Soybean is one of the most common foods involved in such hypersensitivity reactions, especially in younger children. In this study, we investigated the effect of peptic and chymotryptic hydrolysis on the allergenicity of the 11S soybean globulin, which is the primary soybean allergen. The 11S globulin is composed of both acidic and basic polypeptides, and we found that the acidic polypeptide was effectively hydrolyzed, while basic polypeptide was more resistant to hydrolysis. The 11S globulin hydrolysate was size-fractionated by gel filtration, and 9 of the fractions obtained were tested for allergenicity against sera from 6 soybean-allergenic patients. The overall allergenicity of soybean 11S globulin was reduced by peptic and chymotryptic hydrolysis, although a gel filtration fraction with a major peptide of 20 kDa was highly immunoreactive. Hydrolyzed fragments of less than about 20 kDa were not immunoreactive.     

Antilisterial activity of dromedary lactoferrin peptic hydrolysates

The aim of this study was to explore the antibacterial peptides derived from dromedary lactoferrin (LFc). The LFc was purified from colostrum using a batch procedure with a cation exchange chromatography support and was hydrolyzed with pepsin to generate peptic digest. This peptic digest was fractionated by cation exchange chromatography, and the antilisterial activity of LFc, peptic digest, and obtained fractions was investigated using the bioscreen method. The growth of Listeria innocua ATCC 33090 and LRGIA 01 strains was not inhibited by LFc and its hydrolysates. Two fractions of dromedary lactoferrin peptic hydrolysate were active against both strains. A tandem mass spectroscopy analysis revealed that the 2 active fractions comprised at least 227 different peptides. Among these peptides, 9 found in the first fraction had at least 50% similarity with 10 known antimicrobial peptides (following sequence alignments with the antimicrobial peptide database from the University of Nebraska Medical Center, Omaha). Whereas 9 of these peptides presented homology with honeybee, frog, or amphibian peptides, the 10th peptide, F₁₅₂SASCVPCVDGKEYPNLCQLCAGTGENKCACSSQEPYFGY₁₉₂ (specifically found in 1 separated fraction), exibited 54% homology with a synthetic antibacterial peptide (AP00481) derived from human lactoferrin named kaliocin-1. Similarly, the second fraction contained 1 peptide similar to lactoferrampin B, an antibacterial peptide derived from bovine milk. This result suggests that peptic hydrolysis of LFc releases more active antimicrobial peptides than their protein source and thus provides an opportunity for their potential use to improve food safety by inhibiting undesirable and spoilage bacteria.     

Pepsin-induced changes in the size and molecular weight distribution of bovine casein during enzymatic hydrolysis

Bovine casein was digested with pepsin at pH 2.0 in a batch-stirred tank reactor. To investigate the effect of peptic digestion on the aggregate size and molecular weight distribution of bovine casein, the resulting hydrolysates were examined by size-exclusion chromatography coupled with multiangle laser light scattering and dynamic light scattering. Casein was resolved by size-exclusion chromatography into 2 major peaks corresponding to aggregates and monomers, both of which showed a continuous decrease as hydrolysis proceeded. However, the ratio of aggregates to monomers was maintained at almost 1 (2:2.5) during the initial 30-min hydrolysis, indicating that the caseins in solution were in a type of equilibrium between aggregates and monomers. Upon peptic hydrolysis, casein aggregates increased in size and molecular weight, and exhibited a decrease in intermolecular repulsion. This finding was confirmed by dynamic light scattering measurements, which traced the changes in the hydrodynamic radii and light scattering intensities of casein hydrolysates. In addition, the release kinetics of peptide fractions with different molecular weights was also examined. It was concluded that the increase in hydrophobic attraction and the reduction in intermicellar repulsion might promote the growth in aggregate size of bovine casein during the limited hydrolysis.     

Available lysine. II.—Determination of available lysine in feedingstuffs by dinitrophenylation

A method for determining non-N-terminal lysine by hydrolysis of dinitrophenylated proteins, has been examined critically. Sample concentrations and the presence of carbohydrate at hydrolysis were shown to effect the apparent amount of non-N-terminal lysine found. ?-DNP-lysine can be partly protected from destruction by fluorodinitrobenzene and by dinitrophenol. The method was applied to protein fractions and to feedingstuffs and was compared with that developed by Carpenter; reasons for discrepancies are discussed.     

HOW TO INCREASE β-LACTOGLOBULIN SUSCEPTIBILITY TO PEPTIC HYDROLYSIS

Since β-lactoglobulin is resistant to peptic hydrolysis in physiological conditions, the increase of its digestibility by this enzyme was sought by the destabilization of its folding using methods that do not influence the biological value of protein, such as high pressure, medium polarity changes (alcohol addition), and esterification (ethylation). For example, the rate of hydrolysis of β-lactoglobulin by pepsin (negligible at 0.1 MPa) increased considerably with pressure up to 300 MPa. The susceptibility of all potential β-lactoglobulin proteolytic sites to peptic cleavage remained constant over the pressure range that was studied. The addition of alcohols decreases the bulk dielectric constant of the medium and, according to CD measurements, increases significantly the proportion of helical structure in β-lactoglobulin while increasing susceptibility to peptic hydrolysis. In the presence of alcohols (ethanol, ethylene glycol), β-lactoglobulin hydrolysis by pepsin was initiated when its secondary structure began to change and diversified peptic peptide populations were obtained. The chemical modification of β-lactoglobulin by mild esterification yields a 40%-ethylated β-lactoglobulin derivative that is rapidly hydrolyzed by pepsin. As compared with peptic hydrolysis of β-lactoglobulin in aqueous ethanol, 22 new sites of pepsin cleavage were induced by esterification of the protein.     

大鲵低聚糖肽性质初步研究

采用化学方法对大鲵低聚糖肽的氨基酸组成、糖组成及糖肽连接方式进行了初步研究。结果表明,大鲵低聚糖肽含蛋白量为80.01%、总糖含量为15.15%。其含有的氨基葡萄糖、葡萄糖醛酸、半乳糖醛酸及唾液酸分别为3.39%、0.65%、2.45%和0.60%。其氨基酸组成中,苏氨酸的含量最高,达13.1%,其次是脯氨酸、丙氨酸、亮氨酸、精氨酸、苯丙氨酸等。大鲵低聚糖肽的β-消旋反应和血凝集反应,表明大鲵低聚糖肽中糖肽键以O-连接的形式存在。     

压力烹调对五种食材中淀粉消化性的影响

目的:探讨不同烹调方法对红小豆、莲子、山药片、芡实、薏米5种富含淀粉的保健食材的碳水化合物消化特性的影响,为科学食用传统食材提供依据。方法:体外模拟消化方法测定各时间点的水解率、快消化淀粉、慢消化淀粉和抗性淀粉等指标。结果:压力烹调显著影响样品的水解速率和淀粉类型,水解指数增大,快消化淀粉含量增加,抗性淀粉含量减少。但所有样品的预期血糖负荷均显著低于粳米样品。红小豆和莲子经过压力烹调仍保持低消化速度和低EGL特性。结论:压力烹调对提高难烹煮碳水化合物消化率有利,需要控制血糖的人可以通过选择慢消化食材和烹调参数来控制淀粉类型和EGL的变化。     

Influence of naturally acid-soluble proteins from beans (Phaseolus vulgaris L.) on in vitro digestibility determination

The in vitro digestibility of bean (Phaseolus vulgaris) protein fractions was studied using a pepsin-pancreatin system. Enzymatic hydrolysis was stopped by adding a strong acid and the extent of proteolysis determined by measurement of free amino groups in the soluble fraction. The in vitro digestibility of bean protein fractions was low when in the native state and was differently affected by denaturation. For phaseolin, the main reserve protein, heating caused a significant increase of susceptibility to hydrolysis, whereas heat had no apparent effect on digestibility of glutelins and albumins (II). For the PIL (protease inhibitor-lectin rich) fraction, which was shown to have a composition similar to total albumins, there was a decrease of digestibility, probably associated to disulfide bond formation upon heating. Results of in vitro digestibility were shown to be strongly dependent on the utilization of a sample blank to account for proteins naturally soluble in the acid used to interrupt hydrolysis, which would otherwise be estimated as digested protein. These proteins are characterized by a high carbohydrate content, probably responsible for their high solubility and low digestibility.     

Reactions of Chlorogenic Acid with Lysozyme: Physicochemical Characterization and Proteolytic Digestion of the Derivatives

ABSTRACT: The lysozyme-chlorogenic acid-derivatives formed at different pH were characterized in terms of their physicochemical and digestion properties, whereby pH 10 led to the highest derivatization. The results showed reduction of lysine residues and a distinctive decrease of the tryptophan fluorescence with increasing lysozyme derivatization. The solubility decreased over a broad pH range with a parallel increase in hydrophobicity of the derivatives. The isoelectric points were shifted to lower pH values and high molecular fractions were formed. The influence of the protein derivatization on the in vitro digestibility was also demonstrated. The peptic digestion of the derivatized lysozyme was adversely affected, whereas the tryptic and chymotryptic hydrolysis seemed to be favored.     

Reactions of phenolic substances with lysozyme — physicochemical characterisation and proteolytic digestion of the derivatives

Lysozyme was modified by covalent attachment of selected phenolic compounds (m-, o-, p-dihydroxybenzenes, ferulic and gallic acid) at pH 9. The derivatives formed were characterized in terms of their physicochemical and digestion properties. The derivatization was accompanied by a reduction of lysine and tryptophan residues. Moreover, the solubility of the derivatives decreased over a broad pH range and the derivatization increased the hydrophobicity. The isoelectric points were shifted to lower pH values and formation of high molecular weight fractions occurred. In vitro experiments showed that, the peptic digestion of the derivatized lysozyme was adversely affected, whereas the tryptic, chymotryptic and pancreatic hydrolysis seemed to be favoured. The lytic activity of all the resulting lysozyme derivatives was reduced.     

Optimization of peptic hydrolysis parameters for the production of angiotensin I-converting enzyme inhibitory hydrolysate from Acetes chinensis through Plackett-Burman and response surface methodological approaches

BACKGROUND: Angiotensin I‐converting enzyme (ACE) plays an important physiological role in regulating blood pressure. The elevation of blood pressure could be suppressed by inhibiting ACE. ACE inhibitory peptides derived from food proteins could exert antihypertensive effects without side effects. Acetes chinensis is a marine shrimp suitable for the production of ACE inhibitory peptides. The principal objective of this study was to screen for the significant variables, and further to optimize the levels of the selected variables, for the enzymatic production of ACE inhibitory peptides from Acetes chinensis. RESULTS: Plackett–Burman design and response surface methodology were employed to optimize the peptic hydrolysis parameters of Acetes chinensis to obtain a hydrolysate with potent ACE inhibitory activity. The peptic hydrolysis variables were subject to a Plackett–Burman design for screening the main factors. The selected significant parameters such as pH, hydrolysis temperature and enzyme/substrate (E/S) ratio were further optimized using a central composite design. The optimized conditions were: pH 2.5, hydrolysis temperature 45 °C, E/S ratio 17 800 U kg^?1 shrimp and substrate concentration 200 g L^?1. The results showed that 3–5 h hydrolysis could result in a hydrolysate with ACE inhibition IC₅₀ of 1.17 mg mL^?1 and a high DH of 25–27%. CONCLUSION: Plackett–Burman design and RSM performed well in the optimization of peptic hydrolysis parameters of Acetes chinensis to produce hydrolysate with ACE inhibitory activity. A hydrolysate with potent ACE inhibitory activity and high degree of hydrolysis was obtained, so that the yield of ACE inhibitory peptides in it was high. Copyright © 2011 Society of Chemical Industry     

Antigenicity of Ovomucoid Remaining in Boiled Shell Eggs

Antigenicity of egg white proteins which remained in heated shell eggs was analyzed quantitatively by using rabbit antibodies specific to egg white proteins and human antibody from patients allergic to egg. The major antigenic component in heated (100°C for 20 min) and coagulated egg white was ovomucoid. A considerable amount of ovomucoid remained immunoreactive even after heating at 100°C for 15 min, though ovomucoid in stored eggs was a little less stable to heating than that of fresh eggs. Even long-time heating (100°C for 45 min) could not completely eliminate the ovomucoid immunoreactivity to human IgE antibody.     

Interaction Between Ovomucoid and Lysozyme

The interaction between ovomucoid and lysozyme was investigated by precipitation experiments, polyacrylamide gel electrophoresis and inactivation experiments on ovomucoid. The ovomucoid-lysozyme mixture produced turbidity at low salt concentrations over the pH range 6–11. Although both ovomucoid and lysozyme solutions (0.2%, pH 8.5) yielded no precipitation when heated separately at 80°C for 10 mm, about 40% of the proteins in the 0.2% solution of the ovomucoid-lysozyme mixture (2:3, in weight; 1:3, in mole) was precipitated by the same treatment. Ovomucoid was found to form complexes with lysozyme, some of which were precipitated by centrifugation (2000 ×g, 20 mm), and it was presumed that ovomucoid and lysozyme molecules were brought close together by the electrostatic attractive force, unfolded by heating, and then aggregated through intermolecular forces such as hydrophobic forces, hydrogen bonds and disulfide bonds.     

ENZYME AND ACID HYDROLYSIS OF MALTED MILLET (Pennisetum typhoidees) AND SORGHUM (Sorghum bicolor)

Malted millet and sorghum were hydrolysed by the enzymes produced during malting. Malted and unmalted millet and sorghum were acid hydrolysed. The enzyme hydrolysed carbohydrate was mostly glucose, with only traces of disaccharides, in the case of malted millet. The yield of enzyme hydrolysed carbohydrate was however low in both millet and sorghum. Acid hydrolysis of malted millet or sorghum showed that glucose of malted grains was destroyed during acid hydrolysis more than pentoses in the malted grains.     

Thermal modifications of structure and co-denaturation of alpha-lactalbumin and beta-lactoglobulin induce changes of solubility and susceptibility to proteases

Study of heat denaturation of major whey proteins (beta-lactoglobulin or alpha-lactalbumin) either in separated purified forms, or in forms present in fresh industrial whey or in recomposed mixture respecting whey proportions, indicated significant differences in their denaturation depending on pH, temperature of heating, presence or absence of other codenaturation partner, and of existence of a previous thermal pretreatment (industrial whey). alpha-Lactalbumin, usually resistant to tryptic hydrolysis, aggregated after heating at > or = 85 degrees C. After its denaturation, alpha-lactalbumin was susceptible to tryptic hydrolysis probably because of exposure of its previously hidden tryptic cleavage sites (Lys-X and Arg-X bonds). Heating over 85 degrees C of beta-lactoglobulin increased its aggregation and exposure of its peptic cleavage sites. The co-denaturation of alpha-lactalbumin with beta-lactoglobulin increased their aggregation and resulted in complete exposure of beta-lactoglobulin peptic cleavage sites and partial unveiling of alpha-lactalbumin tryptic cleavage sites. The exposure of alpha-lactalbumin tryptic cleavage sites was slightly enhanced when the alpha-lactalbumin/beta-lactoglobulin mixture was heated at pH 7.5. Co-denaturation of fresh whey by heating at 95 degrees C and pH 4.5 and above produced aggregates stabilized mostly by covalent disulfide bonds easily reduced by beta-mercaptoethanol. The aggregates stabilized by covalent bonds other than disulfide arose from a same thermal treatment but performed at pH 3.5. Thermal treatment of whey at pH 7.5 considerably enhanced tryptic and peptic hydrolysis of both major proteins.     

Sediments in coffee extracts: Composition and control by enzymatic hydrolysis

The water-insolubility of some coffee extract components is one of the major limitations in the production of instant coffee. In this work, fractions from coffee extracts and sediments were prepared, and their chemical composition determined. Based on the carbohydrate analysis, galactomannan was found to be the main polysaccharide component of the insoluble fractions and probably responsible for sediment formation. The suitability of twelve commercial enzymes for the hydrolysis of the insoluble fractions was investigated. Pectinase 444L was the most effective enzyme in releasing sugars, mainly mannose and galactose, from these substrates. Biopectinase CCM, Rohapect B1L, Pectinase 444L and Galactomannanase ACH were found to be the most effective enzymes for reducing the sediment of coffee extracts. The highest sediment reduction was obtained using Rohapect B1L and Galactomannanase ACH, at enzyme concentrations of 0.3 and 0.1 mg protein/g substrate, respectively.     

鸡卵类黏蛋白结构与性质研究进展

鸡卵类黏蛋白是鸡蛋中最主要也是过敏原性最强的过敏原蛋白。本文总结该蛋白的结构,包括氨基酸序列、糖基组成、二硫键位置、二级结构以及组成该蛋白的3 个结构域,并描述其理化性质,最后着重分析讨论其过敏原性,特别是其分子结构中二硫键、糖基和结构域等因素与过敏原性之间的内在联系。     

Effect of Acetylation on Composition of Phenolic Acids and Proteolysis of Rapeseed Flour

Rapeseed flour was treated with various levels of acetic anhydride to produce flours with 35, 70, and 90% of the ε-amino groups of lysine acetylated. With the extent of acetylation, free phenolic acids ranged from 86 to 35 mg/100g, phenolic acids from hydrolyzed esters ranged from 1.51 to 340 mg/100g, and no insoluble-bound phenolic acid was found. With the extent of acetylation, an increase in rapeseed flour was less susceptible to tryptic and peptic hydrolysis and an increament of water- soluble protein fragments corresponding to molecular weights of 3700–4500 and 1600–2100 daltons was observed. However, these peptides were devoid of phenolic constituents. Acetylated rapeseed flours were less susceptible to tryptic and peptic hydrolysis.     

The allergenicity of ovomucoid and the effect of its elimination from hen's egg white

In order to remove the ovomucoid from hen's egg white, chitin and hydrazide polystyrene beads were used as affinity ligands with 8.9 and 7.1 mg trypsin g^?1 ligand respectively. Ovomucoid was successfully depleted using the trypsin affinity column without hydrolysation of the other egg white constituents. The components of the egg white were purified by high‐performance liquid chromatography, and then the allergenicity of each of these components was compared with that of pooled human serum derived from patients who are allergic to hen's eggs. The importance of using pure protein for studies of the allergenicity of egg white is highlighted, and it was determined (using an enzyme‐immunosorbent assay) that ovomucoid and ovalbumin are major allergenic proteins in egg white. The ovomucoid‐eliminated egg white preparation exhibited significantly less IgE‐binding activity than normal egg white. The ovomucoid‐specific IgE antibodies may have important implications with regard to the egg‐allergic reaction in humans. © 2001 Society of Chemical Industry     

Thermal modifications of structure and codenaturation of α‐lactalbumin and β‐lactoglobulin induce changes of solubility and susceptibility to proteases

Study of heat denaturation of major whey proteins (β‐lactoglobulin or α‐lactalbumin) either in separated purified forms, or in forms present in fresh industrial whey or in recomposed mixture respecting whey proportions, indicated significant differences in their denaturation depending on pH, temperature of heating, presence or absence of other co‐denaturation partner, and of existence of a previous thermal pretreatment (industrial whey). α‐Lactalbumin, usually resistant to tryptic hydrolysis, aggregated after heating at ⪈85°C. After its denaturation, α‐lactalbumin was susceptible to tryptic hydrolysis probably because of exposure of its previously hidden tryptic cleavage sites (Lys‐X and Arg‐X bonds). Heating over 85°C of β‐lactoglobulin increased its aggregation and exposure of its peptic cleavage sites. The co‐denaturation of α‐lactalbumin with β‐lactoglobulin increased their aggregation and resulted in complete exposure of β‐lactoglobulin peptic cleavage sites and partial unveiling of α‐lactalbumin tryptic cleavage sites. The exposure of α‐lactalbumin tryptic cleavage sites was slightly enhanced when the α‐lactalbumin/β‐lactoglobulin mixture was heated at pH 7.5. Co‐denaturation of fresh whey by heating at 95°C and pH 4.5 and above produced aggregates stabilized mostly by covalent disulfide bonds easily reduced by β‐mercaptoethanol. The aggregates stabilized by covalent bonds other than disulfide arose from a same thermal treatment but performed at pH 3.5. Thermal treatment of whey at pH 7.5 considerably enhanced tryptic and peptic hydrolysis of both major proteins.