Effect of chymotrypsin digestion followed by polysaccharide conjugation or transglutaminase treatment on functional properties of millet proteins

Millet protein was solubilized by chymotrypsin; the soluble protein was conjugated to galactomannan under controlled conditions (60 °C, 76% RH) or polymerised by transglutaminase (TGase). SDS–PAGE patterns showed that the conjugated and polymerised proteins had higher molecular mass bands above the stacking gel. SDS–PAGE patterns also indicated that the digest was conjugated to galactomannan and polymerised by TGase. The free amino groups (OD₃₄₀) of the conjugated and polymerised digest were greatly reduced. Although the chymotrypsin digest was considerably insoluble between pH 2.0 and 5.0, galactomannan conjugate was completely soluble at all levels of pH. TGase polymer was slightly insoluble at pH 4.0. Galactomannan conjugate resisted heat-induced aggregation, even after heating at 90 °C for 20 min, while TGase polymer resisted heat-induced aggregation up to 70 °C, after which its solubility started to decline. The emulsifying properties of the conjugate and the polymerized proteins were greatly improved, compared to the native and chymotrypsin digests.     

Studies on the impact of glycation on the denaturation of whey proteins

It could be shown for technologically relevant whey protein powders that denaturation of β-lactoglobulin (β-Lg) is affected significantly by the extent of covalent modification of lysine residues by lactose. The amount of acid soluble β-Lg as measured via RP-HPLC with UV detection after heating for 10 min at 80 °C increased from 40% (4.6% lysine modification) to 82% (22.4% lysine modification). An increase in glycation leads to a slower denaturation-induced oligomerisation, as shown by SDS-PAGE. Concomitant with an increase in lysine modification, the denaturation temperature increased from 79.5 to 84 °C, as measured by differential scanning calorimetry (DSC). Covalent attachment of lactose to whey proteins during preparation or storage significantly improves the heat stability of whey proteins, which may be of particular importance for the technological use of whey proteins varying in the degree of lysine modification.     

Demonstration of the presence of aminoreductone formed during the Maillard reaction in milk

The Maillard reaction is a common chemical reaction that occurs in food and it generates multiple reaction products. Aminoreductone (AR) is one of the early-stage Maillard reaction products. At present the formation of AR has only been demonstrated in a model system consisting of a monosaccharide or disaccharide and an amino group-containing compound. There is no direct evidence to show the presence of AR in food. In this study, we demonstrated the formation and presence of AR in milk using a combination of 2,4-dinitrophenylhydrazine (DNP) and Cu²⁺. A DNP derivative of AR oxidised by Cu²⁺ was isolated and its detailed structure was identified by NMR analysis. We thus directly demonstrated the formation and presence of AR in milk.     

The use of transglutaminase in dairy products

Modification of proteins by enzymes such as transglutaminase (TG) has recently become of great interest to food scientists. TG (EC 2.3.2.13) catalyses the post-translational modification of proteins by transamidation of available glutamine residues by the formation of covalent cross-links between glutamine and lysine residues in proteins. It is suggested that TG is a useful tool for the production of dairy products. In this review, properties of TG and its possible use in the manufacture of dairy products are discussed. Aspects covered include reactions catalysed by the enzyme and properties of modified proteins of selected milk proteins and products.     

Structural mechanisms leading to improved water retention in acid milk gels by use of transglutaminase

Water retention in transglutaminase (TG)-treated acid milk gels was studied and linked with the gel formation dynamics. Heat-treated skim milk with and without pre-treatment by TG was acidified at 20 °C, 30 °C and 40 °C at constant glucono-δ-lactone (GDL) level to obtain different acidification rates. Formation dynamics and structural properties of acid-induced gels were followed by rheological and near-infrared light backscattering measurements as well as microscopy. TG-treated gels showed decreased tan δ values all through the acidification, which was pronounced around the gelation point. Backscattered light intensity was lowered in TG-treated gels compared to the controls indicating that TG-treated gels were comprised of smaller aggregates. Water holding capacity (WHC) was measured by using centrifugation at selected pH points (pH 5.2, 5.0, 4.8 and 4.6) during acidification. Both acidification temperature and TG treatment had significant effects on the water retention properties of the gels. Spontaneous syneresis observed at high acidification temperatures (≥30 °C) was prevented upon TG-treatment. WHC of TG-treated gels was significantly higher compared to the control gels at all pH points. TG-treated milk gels showed a homogeneous network formed of smaller aggregate and pore sizes at the gelation point and did not show any large-scale re-organisation thereafter. Transglutaminase is likely to act as a fixative of the protein network at an early stage of gelation and thereby limiting network rearrangements that take place in acid milk gels formed at high acidification temperatures leading to contraction and subsequent wheying off.     

Altering functional attributes of proteins through cross linking by transglutaminase – A case study with whey and seed proteins

The effect of cross linking of the major whey protein β-lactoglobulin (β-Lg) and major protein fractions (11S) of soybean (Glycinin) and sesame seed (α-globulin) with the microbial enzyme transglutaminase (EC 2.3.2.13) was studied. The formation of polymerized proteins was followed by poly acrylamide gel electrophoresis, gel filtration high pressure liquid chromatography (HPLC) and evaluation of functional properties. Cross linked proteins were less turbid on heating to higher temperature as compared to untreated samples and the temperature at which the protein turns turbid also increased in the treated samples. In case of β-Lg and α-globulin of sesame seed when the control showed turbidity at 60 °C, the enzyme treated sample indicated at 65 °C and higher. Similar results were obtained in the case of soybean also. The treated samples showed higher emulsifying activity when compared to the control. The control showed an emulsifying activity of 0.55 ± 0.02, and the treated sample showed an emulsifying activity of 0.72 ± 0.02 (optical density at 500 nm is taken as emulsifying activity). Foaming capacity did not improve significantly with the enzyme treatment. The complex formed was investigated by gel filtration chromatography. Nearly 30% of the proteins (11S protein fractions and β-Lg) formed the complex and increase in the concentration of the proteins or the enzyme did not show any increase in the complex formation. The changes in the fluorescence intensity indicated changes in the microenvironment of the chromophores induced by the enzymatic cross linking.     

变性对转谷氨酰胺酶在乳蛋白间交联影响初探

利用SDS-PAGE电泳结合凝胶成像分析技术，比较了在非变性、加入还原剂变性和加热后再加入还原剂变性三种条件下转谷氨酰胺酶对酪蛋白和乳清蛋白之间的交联情况。结果表明：在非变性条件下，酪蛋白质量分数下降96％，乳清蛋白下降15％，酪蛋白和乳清蛋白几乎不能交联。超分子量聚合物是酪蛋白单一聚合物，α-乳白蛋白形成部分低聚体；在加入还原剂时，酪蛋白质量分数下降86％，乳清蛋白下降30％，反应4h后有少量乳清蛋白和酪蛋白中某一组分交联；预热更有助于酪蛋白和乳清蛋白聚合，在第三种条件下，反应24h后乳清蛋白下降60％。