REDUCTION OF ALLERGENICITY AND INCREASING THE BIOLOGICAL VALUE OF BUFFALO'S MILK PROTEINS BY ENZYMATIC MODIFICATION

Buffalo milk proteins were covalently enriched with methionine (Met) during the enzymatic peptide modification (EPM) process. The maximal Met-enrichment occurred when the α-chymotryptic hydrolysate of buffalo's milk proteins was treated with methionine ethyl ester in the presence of α-chymotrypsin during the EPM. Methionine content of this product was more than three times (5.4%) higher than that of the original buffalo milk protein (1.5%). The electrophoretic patterns of the modified proteins showed that the enzymatic reaction was mainly characterized by transpeptidation. Molecular masses of the modified protein fractions were influenced by the concentration of L-methionine ethyl esters in the reaction mixture during the EPM. EPM products from the buffalo milk protein hydrolysates with α-chymotrypsin and trypsin or with pepsin and trypsin showed low allergenic activities compared with those when buffalo milk proteins were hydrolyzed with α-chymotrypsin or with pepsin alone.     

Charakterisierung pankreatischer Casein-Plasteine

Characterization of pancreatic casein plasteins. In the course of the plastein reaction hydrophobic peptides concentrate mainly in the aggregates (plasteins), whilst hydrophilic peptides remain in solution (supernatant). Liquid chromatographic and sequence analytical studies of pancreatic casein plasteins have shown that the aggregates consist mainly of the free amino acids tyrosine, phenylalanine and tryptophan. Plasteins contain, in addition, short-chain peptides, particularly from the C-terminal of β-casein. Characterization of the functional properties of the plasteins has shown clearly that aggregation of the short-chain peptides and free amino acids is brought by non-covalent, hydrophobic and ionogenic interactions. In the supernatants resulting from the plastein reaction caseinophosphopeptide sequences, in particular from α_s-casein, were determined.     

Functional Properties of Hydrolysates from Proteolysis of Heat-denatured Whey Protein Isolate

Heat-denatured whey protein isolate was hydrolyzed with trypsin, α-chymotrypsin, Alcalase or Neutrase to 2.8, 4.3, 6.0 or 8.0% degree of hydrolysis. Hydrolysates were fractionated by ultrafiltration and freeze-dried. Protein content of retentates showed little variation but permeates differed with enzyme. Surface hydrophobicity increased with hydrolysis but was not linear except for α-chymotrypsin. Ultrafiltration increased solubility and the permeates and retentates had better solubility than hydrolysates. Retentates had higher emulsifying activity index than hydrolysates while permeates did not form stable emulsions. Permeates formed stable foams but hydrolysates and retentates showed poor foaming characteristics. Specificity of the enzyme, and degree of hydrolysis influenced the functional properties of the peptides. Fractions generated by trypsin, at all levels of hydrolysis generally had higher solubility, emulsifying properties and foaming properties. Permeates from Alcalase hydrolysis had the best foam capacity but low foam stability.     

Increase in Curd Tension of Milk Coagulum Prepared with Immobilized Proteases

Curd tension of milk clotted by nine proteases in immobilized or soluble forms was measured by a curd tension meter. In the curd prepared by trypsin, α-chymotrypsin, papain, thermolysin or alkaline-protease in a soluble form, the increasing rate of curd tension was definitely smaller than that of curd formed by rennet. However, curd tension increased linearly when the extent of proteolysis was controlled by employing these proteases in an immobilized form. The amounts of TCA-soluble nitrogenous compounds and sialic acid released at the beginning of clotting were kept constant throughout the incubation time.     

酪蛋白类蛋白物的溶剂分级和酶水解对ACE抑制活性的影响

利用响应面法优化类蛋白反应条件修饰酪蛋白水解物制备酪蛋白类蛋白物.酪蛋白类蛋白物的ACE抑制活性高于酪蛋白水解物,IC50值从52.6 mg/L降低到14.9 mg/L.利用乙醇-水混合溶剂对酪蛋白水解物和类蛋白物进行分级,结果表明,极性最低的溶剂得到的上清液部分活性较高,而沉淀部分活性较低.4种蛋白酶水解酪蛋白类蛋白物的分级产物,导致活性下降,除碱性蛋白酶外,木瓜蛋白酶、胃蛋白酶和胰蛋白酶的水解产物ACE抑制活性为31.5％～46.8％,但是仍然高于酪蛋白水解物的ACE抑制活性(27.8％),表明类蛋白反应提高了酪蛋白水解物对一些蛋白酶的体外抵抗能力.     

The effect of enzymatic modification on lysinoalanine formation in field-bean protein isolate and beta-casein

Lysinoalanine (LAL) was determined in alkali-treated partial hydrolysates (PH) of casein, peptides isolated from these PH and in PH of field-bean protein to clarify whether intermolecular or intramolecular LAL bridges are preferentially formed. Furthermore, the formation of LAS in plasteins was studied as a contribution to plastein research. The formation of LAL in the peptide mixtures of beta-casein and the decrease of the LAL content in the PH (as compared to intact proteins) indicates that the formation of LAL favours the intramolecular cross-linking of polypeptide chains. The LAL content decreases as the degree of hydrolysis of the PH of the field-bean protein isolate increases, and depends upon the protease used in the production of the hydrolysates. The LAL contents of the alkali-treated plasteins are less than those of the initial hydrolysates. The decrease of the LAL content is directly proportional to the hydrolysis proceeding during the plastein reaction.     

Plasteins. Their preparation, properties, and use in nutrition

The theoretical and practical aspects of the plastein reaction, which consist in the formation of a gel following the addition of an endopeptidase to a concentrated solution of a partial protein hydrolysate, are examined and the properties and possibilities of using plasteins in nutrition are discussed. It is shown that valuable protein food products can be obtained with the aid of the plastein reaction from proteins with an unbalanced amino acid composition and from chemically synthesized amino acids. Other applications of plasteins in nutrition are discussed and the studies carried out hitherto on the mechanism and driving forces of plastein formation are considered.     

Effect of animal (lamb) diet and meat storage on myofibrillar protein oxidation and in vitro digestibility

Effect of pasture- or concentrate-diet on myofibrillar protein oxidation and in vitro digestibility was measured in lamb meat (M. longissimus dorsi) during a refrigerated storage of 7days under gas permeable film. Protein oxidation was measured by the carbonyl content determined chemically using 2,4-dinitrophenylhydrazine (DNPH) and specific targets of oxidation were identified by immunoblotting. Carbonyl content significantly increased during storage and diet affected protein oxidation where animals fed concentrate showed higher carbonyl group levels than animals fed pasture. To evaluate effect of diet and storage time on protein digestibility, myofibrillar proteins were exposed to proteases of the digestive tract (pepsin, and a mixture of trypsin and α-chymotrypsin) in conditions of pH and temperature which mimic digestive process. The myofibrillar protein digestibility was not influenced by the diet. Storage time had no significant effect on myofibrillar protein susceptibility to pepsin while an important increase in digestibility by trypsin and α-chymotrypsin was detected during storage.     

Reduction of major peanut allergens Ara h 1 and Ara h 2, in roasted peanuts by ultrasound assisted enzymatic treatment

This study investigated the effects of ultrasound, enzyme concentration and enzyme treatment time on soluble protein and major allergenic proteins (Ara h 1 and Ara h 2) of roasted peanut kernels. A 3-factor, five-level orthogonal experimental design was implemented with various ultrasonication times, concentrations of trypsin or α-chymotrypsin and treatment times. The total soluble proteins were determined by the Bicinchoninic acid (BCA) method, Ara h 1 and Ara h 2 were evaluated by SDS–PAGE and sandwich ELISA. The IgE-binding of peanut extracts was analysed by a competitive inhibition ELISA. Results indicate that ultrasound treatment, followed by protease digestion of peanuts, significantly increased the solubility of peanut protein and decreased the concentrations of Ara h 1 and Ara h 2. The sequential treatment of peanuts by ultrasonication–trypsin–alpha chymotrypsin, resulted in maximum reductions of Ara h 1/Ara h 2, and lowest IgE-binding. This study provides an approach to significantly reduce allergenic proteins in peanut product.     

Methionine Enrichment of Milk Protein by Enzymatic Peptide Modification

Methionine-enriched protein was produced from an enzymatically pre-hydrolyzed milk protein using an enzymatic peptide modification (EPM) method with α-chymotrypsin as catalyst. Methionine of the product was twice as high as that of the substrate protein. The incorporated methionine formed a covalent bond with the peptide chain in the product protein. The change in the number of peptide bonds was monitored by the degree of hydrolysis (DH). The slight change of the DH values revealed that a portion of the free amino acids was bound to the peptide chains during the reaction and that transpeptidation was the main process during the EPM treatment. The location of the newly incorporated amino acids was determined by identification of the terminal amino acids. The covalently bound methionine was located in C- and N-terminal positions in a ratio of 3:1.     

Partial purification of horse-specific soluble muscle proteins by immunoadsorption chromatography

Immunoadsorption chromatography has been used to isolate horse-specific soluble muscle proteins from crude horse protein extracts. Horse-specific polyclonal antibodies against soluble horse muscle proteins immobilised on a Protein A-Sepharose CL-4B matrix were used to adsorb the corresponding antigens. Analysis of the crude soluble horse muscle proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed the existence of 20 protein subunits in the gel. SDS-PAGE of the proteins recovered by affinity chromatography showed nine protein subunits, three of which were enriched after the immunopurification step. These affinity-recovered horse-specific soluble muscle proteins may be used as antigens in the development of monoclonal antibodies to detect and quantify horse meat in raw, unheated meat mixtures.     

Physicochemical changes of myofibrillar proteins during processing of Cantonese sausage in relation to their aggregation behaviour and in vitro digestibility

The physicochemical changes of myofibrillar proteins, especially oxidation behaviour, were measured to determine their mechanism of action on in vitro protein digestibility during Cantonese sausage processing. The results indicated that the carbonyl level significantly increased (p < 0.05) during the process. The SH group level decreased, while S–S group level increased gradually. Protein aggregation was induced by oxidation and heat treatment. Result from Fourier transform infrared (FTIR) spectroscopy confirmed protein aggregation occurred. The analysis of in vitro digestibility showed a highly significant (p < 0.05) correlation between pepsin activity and carbonyl group formation, S–S group level, protein surface hydrophobicity, D_4,3. A negative and highly significant correlation between trypsin, α-chymotrypsin activity and carbonyl group formation was measured, while no significant correlation with S–S groups, protein surface hydrophobicity, D_4,3 was observed. It indicated that not only protein oxidation and aggregation but also degradation by pepsin would influence proteolysis with trypsin and α-chymotrypsin.     

嗜酸乳杆菌产类细菌素Lactobacillin NX2-6的初步研究

从内蒙古传统乳制品酸马奶中筛选出1株产生具有广谱抑菌活性物质的嗜酸乳杆菌NX2-6。在排除酸性产物和过氧化氢的干扰后,菌株NX2-6所产生的类细菌素对金黄色葡萄球菌等革兰氏阳性菌和大肠杆菌等革兰氏阴性菌及米曲霉等真菌具有较强的抑制作用;经胰蛋白酶、胃蛋白酶、蛋白酶K处理后发酵液抑菌活性明显下降,但仍具有较强的抑菌活性,对α-胰凝乳蛋白酶抗菌活性没有显著变化,因此表明该抗菌活性物质为蛋白类物质或非单一物质。在酸性条件下它具有很好的热稳定性,pH2.0时活性最强,中性或碱性条件下不具有抗菌活性,抑菌活性pH值范围在2.0-5.0。此抗菌物质不同于一般细菌素,属于类细菌素,命名为Lactobacillin NX2-6。菌株NX2-6产物在生防、食品具有潜在的应用价值。     

Isolation,purification and identification of intracellular protein targets of ochratoxin A: mitochondrial aldehyde dehydrogenase class 2 is a high-affinity ochratoxin A-binding protein

An affinity chromatography technique was utilised to isolate and purify the soluble intracellular protein targets of ochratoxin A (OA). OA was covalently immobilised on a beaded agarose matrix. Several proteins (approximately nine) in the soluble fraction of crude protein extracts of different bovine tissues were specifically bound to the immobilised OA. The binding of these proteins to the modified agarose could not be dissociated with mild buffer (buffer plus 0.5 M NaCl and 1% Tween 20), but the proteins were competitively eluted with free 25 mM OA in mild buffer. Only one major protein (55 kDa) remained tightly bound to the immobilised OA when the column was incubated with bovine liver extract and then washed extensively with higher concentrations of salt and detergent (2 M buffer plus NaCl and 5% Tween 20). The bound protein was eluted competitively with 25 mM OA and further purified using SDS-PAGE. The N-terminal sequence of 24 residues of the high-affinity OA-binding protein was shown to be AATQAVPTPNQQPEVLYNQIFINN. This sequence is identical to the 24 residues of the N-terminal sequence of bovine mitochondrial aldehyde dehydrogenase class 2 (ALDH2, EC 1.2.1.3). The molecular weight and tissue distribution of ALDH2 are the same as those of the high-affinity protein isolated in this study. Immobilised OA can serve as an excellent affinity matrix for the identification of target proteins of OA and for the purification of ALDH2 and possibly other proteins. © 1999 Society of Chemical Industry     

Characterisation of trypsin and α-chymotrypsin inhibitors in Australian wattle seed (Acacia victoriae Bentham)

Crude water extracts from Australian wattle seed (Acacia victoriae Bentham) and their salt (ammonium sulphate)-precipitated fractions were analysed for trypsin and α-chymotrypsin (chymotrypsin) inhibitor activity, using gel electrophoresis and spectrophotometric methods. Three different bands with molecular weight 30.20, 38.03 and 39.81 kDa were active, with the 50% salt-precipitated fraction exhibiting highest activity and number of active bands. The same proteins also appeared to be responsible for both trypsin and chymotrypsin inhibitor activity. To establish conditions for the inactivation of these inhibitors, whole seed and uncoated (dehulled) cotyledon were subjected to different heat treatments. Moist heat treatment at 100 °C for 30 s was sufficient to inactivate both protease inhibitors although the trypsin inhibitor was found to be more heat-resistant than was the chymotrypsin inhibitor. Soaking overnight, before thermal treatment, improved the trypsin inhibitor activity but enhanced the efficiency of thermal inactivation in both inhibitors.     

Effects of Enzyme Modified Soy Protein on Rennet-Induced Reconstituted Nonfat Dry Milk Coagulum Properties

Soy protein was hydrolyzed by trypsin, pepsin and α-chymotrypsin. Effects of adding hydrolyzed soy protein on rennet-induced nonfat milk coagulum texture and syneresis were studied. Pepsin-modified soy protein increased coagulum firmness and syneresis greater than untreated soy protein. Soy protein hydrolyzed with trypsin for 150 min and added to nonfat milk increased coagulum firmness greater than nonfat milk with no soy protein added. Chymotrypsin-modified soy protein acted similarly to the trypsin modified soy protein except the coagulum firmness was less than nonfat milk alone. Soy protein hydrolyzed by trypsin followed by pepsin increased coagulum firmness more than hydrolysis by a single enzyme and was close to nonfat milk without soy protein.     

Effect of angiotensin I converting enzyme inhibitory peptide purified from skate skin hydrolysate

Our objective was to evaluate the angiotensin I converting enzyme (ACE) inhibitory activity of skate skin protein hydrolysates and its corresponding fractions. The skate skin hydrolysates were obtained by enzymatic hydrolysis using alcalase, α-chymotrypsin, neutrase, pepsin, papain, and trypsin. Amongst the six hydrolysates, the α-chymotrypsin hydrolysate had the highest ACE inhibitory activity compared to other hydrolysates. The amino acid sequences of the purified peptides were identified to be Pro–Gly–Pro–Leu–Gly–Leu–Thr–Gly–Pro (975.38 Da), and Gln–Leu–Gly–Phe–Leu–Gly–Pro–Arg (874.45 Da). The purified peptides from skate skin had an IC₅₀ value of 95 μM and 148 μM, respectively, and the Lineweaver–Burk plots suggest that they act as a non-competitive inhibitor against ACE. Our study suggested that novel ACE inhibitory peptides derived from skate skin protein may be beneficial as anti-hypertension compounds in functional foods.     

Preparation of Alcalase-catalyzed casein plasteins in the presence of proline addition and the ACE-inhibitory activity of the plasteins in vitro

Casein hydrolysates were prepared by hydrolysis of casein with alkaline protease Alcalase for 6 h and showed the highest ACE-inhibitory activity in vitro with an IC₅₀ value of 47.1 μg mL⁻¹. Casein hydrolysates prepared were subjected to Alcalase-catalyzed plastein reaction in the presence or absence of proline addition to prepare casein plasteins. Some optimal reaction conditions of plastein reaction in the presence of proline addition were studied using response surface methodology with the decrease in free amino groups in the casein plasteins as response. When the concentration of casein hydrolysates was fixed at 35% (w w⁻¹) and reaction time at 6 h, the optimal conditions were reaction temperature 48 °C, addition level of proline 0.54 mol/mol free amino groups of casein hydrolysates and addition level of Alcalase 9.5 kU g⁻¹ proteins. With these conditions, the maximal decrease in free amino groups in casein plasteins was 195.7 μmol g⁻¹ proteins. The ACE-inhibitory activities of twelve casein plasteins in vitro, prepared in the presence or absence of proline addition with different reaction extents, were evaluated and compared. The results showed that the ACE-inhibitory activity of the casein plasteins prepared in the presence of proline addition changed irregularly, different to that of the casein plasteins prepared in the absence of proline addition, and might relate to the different linking of proline to the peptides in casein hydrolysates during plastein reaction. When the casein plasteins prepared in the presence of proline addition had a decrease in free amino groups 195.7 μmol g⁻¹ proteins, the IC₅₀ value of the casein plasteins was lowered to 0.2 μg mL⁻¹.     

Immobilisation of α-Amylase on Polyaromatic and Titanium Compounds Incorporating a Magnetic Material

Three methods of preparing immobilised α-amylase on supports which incorporate magnetic properties have been developed. The use of diazotized 1,3-diaminobenzene coated on magnetic iron oxide, which was itself either uncoated or coated with hydrous titanium (IV) oxide, was found to result in higher bound activities of the immobilised enzyme. The immobilised enzyme prepared from diazotized 1,3-diaminobenzene coated magnetic iron oxide support showed excellent stability towards freeze drying. A preparation of immobilised enzyme using magnetic iron coated with hydrous titanium (IV) oxide resulted in a greater thermostability. This support can be prepared in situ, overcoming the problem of loss of activity of the freeze dried immobilised enzyme.     

A preliminary note on the detection and partial characterization of chicken muscle soluble proteins by immunoelectrophoresis in agarose gels

Immunoelectrophoresis in agarose gels has been used to detect and partially characterize specific protein precipitin bands of chicken muscle soluble proteins (CHSP), free of crossreactions with muscle soluble proteins of cow (CSP), pig (PSP) and horse (HSP). Six precipitin bands were obtained by reacting CHSP against an anti-CHSP antiserum produced by a rabbit. These precipitin bands did not appear when the protein extracts from cow, pig and horse were analyzed against the same anti-CHSP antiserum. The six precipitin bands were specific of the chicken muscle soluble proteins. This technique may have the potential to detect the presence of chicken meat in unheated ground meat products.