INFLUENCE OF TRANSGLUTAMINASE-INDUCED CROSS-LINKING ON IN VITRO DIGESTIBILITY OF SOY PROTEIN ISOLATE

The influence of covalent cross‐linking by microbial transglutaminase (MTGase) on the sequential in vitro pepsin and trypsin digestion process and the digestibility of soy protein isolate (SPI), was investigated by sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and nitrogen release analyses. Various subunits of β‐conglycinin and acidic subunits of glycinin were cross‐linked by MTGase to form high molecular weight (MW) biopolymers, while basic subunits of glycinin were unaffected. SDS‐PAGE analysis indicated that the cross‐linking mainly affected in vitro pepsin digestion pattern of various subunits of β‐conglycinin, while the trypsin digestion pattern of native SPI was nearly unaffected. Nitrogen release analysis showed that the in vitro pepsin or/and trypsin digestibility of native SPI (at the end of pepsin or trypsin ingestion) was significantly decreased (P ≤ 0.01) by the MTGase treatment (for more than 2 h). The cross‐linking by MTGase also significantly decreased the in vitro digestibility of preheated SPI. These results suggest that the cross‐linking by means of transglutaminase may negatively affect the nutritional properties of food proteins.     

Studies on the remedial effect of transglutaminase on protease anti‐felting treated wool

Oxidative protease treatment can improve anti‐felting of wool and also result in its loss of strength. In this study, one kind of microbial transglutaminase (MTG) was applied in the wet process of wool fabrics. The results indicated that transglutaminase could remediate wool damage following hydrogen peroxide and protease anti‐felting finishing, resulting in an increase in wool fabric strength and a decrease in alkali solubility. In addition, the evidence was provided that some cross‐linking through transglutaminase had occurred in the wool protein. The SEM showed that the scale structure became smoother after MTG treating. Analyzing of the thermal behavior revealed that the thermal stability of the MTG‐treated wool was enhanced. UV‐spectrum analysis suggested a decrease in protein extracted from the intercellular cements of the treated wool with MTG.     

Effect of Cross‐linking with Epichlorohydrin on the Properties of Cassava (Manihot esculenta Crantz) Starch

Cassava starch was cross‐linked with epichlorohydrin (EPI) at 45°C for 2 h in three different media which include water, water in the presence of a phase transfer catalyst (PTC) and N,N‐dimethylformamide (DMF). The products were characterized by determining their physicochemical, thermal and retrogradation properties. In aqueous medium, the use of a PTC, tetrabutylammonium bromide (TBAB) produced derivatives with higher degree of cross‐linking than those prepared without the use of the catalyst. The degree of cross‐linking was found to be higher using the same concentration of EPI when the reaction was carried out in DMF. At low levels of cross‐linking, the peak viscosity of the cross‐linked starches increased in comparison to that of the native starch. With increasing degree of cross‐linking, the peak viscosity showed a significant reduction. The swelling volume, solubility and light transmittance of the starch pastes were lower for the modified starches. The cross‐linked starches showed slightly reduced values for the gelatinization temperatures, T_onset, T_peak and T_end. The enthalpy of gelatinization of the modified starches increased with increase in the degree of cross‐linking. The modified starches exhibited higher water‐binding capacities (WBC) than the native starch; but with increase in the degree of cross‐linking, there was a gradual decrease in WBC. The in vitro alpha amylase digestibility of the modified starches decreased gradually with increase in the level of cross‐linking.     

超高压对大豆球蛋白抗原性及结构的影响

以脱脂大豆粉为原料,利用碱溶酸沉法分离提取大豆球蛋白,采用间接竞争酶联免疫吸附法测定不同压 力、加压时间及不同质量浓度下大豆球蛋白的抗原性变化,并对超高压后产物的免疫原性及结构特性进行分析。结 果表明：超高压能显著影响大豆球蛋白的抗原性;免疫印迹结果显示超高压处理后大豆球蛋白的免疫原性有一定 程度的降低,但不能完全消除;傅里叶变换红外光谱结果表明超高压处理之后样品蛋白中α-螺旋和β-折叠的含量减 少,β-转角和无规卷曲含量增加;非还原性电泳与荧光光谱结果表明,大豆球蛋白的空间结构解聚,疏水性氨基酸 残基暴露在蛋白质表面,蛋白质的三、四级空间结构被破坏;蛋白质空间结构的改变可能会引起抗原表位的掩盖, 从而使其抗原性降低。     

Effect of transglutaminase on the functional properties of GDL (glucono‐delta‐lactone) cold‐set soybean glycinin gel

Cold‐set soybean glycinin gel was prepared by cross‐linking of soybean glycinin (non‐preheated and preheated) at pH 8.0 and 50 °C using transglutaminase (TG), followed by cold‐set acidification using glucono‐δ‐lactone (GDL). Dynamic oscillatory rheological measurements (both under non‐preheat and preheat conditions) showed that there was a substantial increase in the elastic (G′) and viscous (G″) moduli of enzyme treated gels. The moduli also showed similar frequency dependence. The yield strains of enzyme treated gels were clearly higher than that of the gels without enzyme treatment. Texture profile analysis indicated that preheat and enzyme treatments have beneficial effect on hardness, fracturability and gumminess of gels. Thermal and enzyme treatments have synergetic effect to improve the strength of gels, which is also consistent with the results of rheological measurements. These results demonstrated that heat treatment followed by TG treatment and GDL acidification improved the functional properties of soybean glycinin cold‐set gel.     

Physicochemical Properties of Non‐thermally Cross‐linked Corn Starch with Phosphorus Oxychloride using Ultra High Pressure (UHP)

Corn starch (20%, w/w) was non‐thermally and conventionally cross‐linked with phosphorus oxychloride (POCl₃; 0.01, 0.05, or 0.1%, based on dry weight of starch) at 400 MPa for 5, 15 and 30 min and at 45°C for 2 h, respectively. Swelling power and solubility of both non‐thermally and conventionally cross‐linked corn starches were relatively lower than those of native corn starch. The pressure holding time did not affect the solubility and swelling power of non‐thermally cross‐linked corn starches. X‐ray diffraction patterns and relative crystallinity were not significantly altered by both conventional and non‐thermal cross‐linking. DSC thermal characteristics of both non‐thermally and conventionally cross‐linked corn starches were not significantly changed indicating that the double helical structure of amylopectin was not influenced by both conventional and non‐thermal cross‐linking reactions. Both non‐thermal and conventional cross‐linking greatly affected the Rapid Visco Analyser (RVA) pasting properties, such as increase in pasting temperature and decrease in peak viscosity compared to native starch. This result suggests that in case of cross‐linking using POCl₃, both non‐thermal and conventional methods result in similar physicochemical properties and non‐thermal cross‐linking with POCl₃ can reduce the reaction time from 2 h to 15 min. This work shows the potential and possibility of non‐thermal starch modification and provides the basic and scientific information on the physicochemical properties of non‐thermally cross‐linked corn starches with phosphorus oxychloride using UHP.     

Optimisation of cross‐linking β‐cyclodextrin and its recycling efficiency for cholesterol removal in milk and cream

This study was carried out to investigate the optimum conditions of cross‐linking β‐cyclodextrin (β‐CD) and recycling for cholesterol removal in milk and cream. The cross‐linked β‐CD was prepared with a 15% adipic acid solution, and the water solubility of the β‐CD was measured for the optimum conditions based on mixing temperature, mixing time, cross‐linking temperature, cross‐linking reaction time and cooling time. In the results of this study, optimum conditions were 80 °C mixing temperature, 2 h mixing time, 60 °C cross‐linking temperature, 24 h cross‐linking reaction time and 48 h cooling time. After determining the optimum conditions, the recyclable yields of the cross‐linked β‐CD ranged from 90.01% to 55.17% in six recyclings and the percentage of cholesterol removal by 15% cross‐linked β‐CD was over 90% until eighth recycling. On the basis of the results, this study suggests that 15% adipic acid‐added cross‐linked β‐CD maximised recyclable yield and that cholesterol removal was improved during recycling.     

Casein gelation under simultaneous action of transglutaminase and glucono‐δ‐lactone

Casein solutions (5% w/v) were treated with microbial transglutaminase (MTG) and glucono‐δ‐lactone (GDL) under varying conditions in order to obtain gels. Storage modulus (G ′) and gelation time of the gels were measured by oscillation rheometry, while protein cross‐linking was determined by gel permeation chromatography. The addition of only GDL to milk resulted in very weak gels, while MTG on its own was not able to create gel networks. Simultaneous action of both ingredients led to gels, the firmness of which was linearly related to the added amount of MTG, but passed through a maximum with rising GDL concentrations. Using chromatographical analysis, increasing G ′ values were interrelated with the formation of MTG‐induced oligomers. The gelation time was directly proportional to the GDL concentration but not influenced by the addition of MTG within the studied range of concentration.     

A study on the preparation and some functional properties of a cross‐linked casein–gelatin composite by a microbial transglutaminase

A microbial transglutaminase (TGase) was used in this work as biocatalyst to prepare a cross‐linked casein–gelatin composite, a modified protein product with 4‐hydroxyproline about 41 mg g^?1 peptides. Some cross‐linking conditions such as total protein content, the ratio of caseinate to gelatin and original pH were fixed at 5% (w/v), 4:1 (w/w) and 7.5, respectively. Other suitable conditions selected by single factor trials were TGase addition of 20 U g^?1 peptides, reaction temperature of 45 °C and reaction time of 4 h. Peptide profiles from SDS‐PAGE analysis showed the composite was peptide polymers. Compared to that of the original caseinate or the cross‐linked caseinate, the dispersion of the composite exhibited a markedly enhanced apparent viscosity, storage modulus and viscous modulus. Meanwhile, the composite also showed a better water holding capacity, unchanged oil binding capacity and lower enzymatic digestibility in vitro, conferring its applicability as a new protein ingredient.     

Improved microstructure and properties of the gelatin film produced through prior cross‐linking induced by horseradish peroxidase,glucose oxidase and glucose

The effects of prior enzymatic cross‐linking of bovine gelatin via horseradish peroxidase, glucose oxidase and glucose on microstructure and properties of the target film (cross‐linked gelatin film) were assessed. The cross‐linked gelatin film exhibited similar film thickness and moisture content, lower water solubility and higher opacity than the gelatin film directly prepared with bovine gelatin. The cross‐linked gelatin film also demonstrated improved thermal stability and mechanical properties, characterised by higher melting point and glass transition temperature, enhanced tensile strength and elongation at break and greater storage modulus. Prior gelatin cross‐linking resulted in 30.2% and 68.6% reductions in water vapour and CO₂ permeability of the cross‐linked gelatin film, respectively, but did not affect oil permeability. Furthermore, the cross‐linked gelatin film possessed smaller cross‐sectional voids (diameter 100?360 nm vs. 200?595 nm) than the bovine gelatin film. This study shows that cross‐linking can efficiently improve film microstructure and properties of the gelatin‐like products.     

Development of a probiotic delivery system from agrowastes, soy protein isolate, and microbial transglutaminase

Abstract: Probiotic delivery system was developed via the use of microbial transglutaminase (MTG) cross‐linked soy protein isolate (SPI) incorporated with agrowastes such as banana peel (BE), banana pulp (BU), and pomelo rind (PR). Inoculums of Lactobacillus bulgaricus FTDC 1511 were added to the cross‐linked protein matrix. The incorporation of agrowastes had significantly (P < 0.05) reduced the strength, pH value, and the lightness of the SPI gel carriers, while sodium dodecyl sulfate‐polyacrylamide gel electrophoresis profiles revealed that the occurring cross‐links within the SPI gel carriers were attributed to the addition of MTG. Scanning electron microscope micrographs illustrated that SPI carriers containing agrowastes have exhibited a less‐dense protein matrix. All the SPI carriers possessed maximum swelling ratio at 4 to 4.5 within 15 min in simulated gastric fluid (SGF), whereas the maximum swelling ratios of SPI/BE, SPI/BU, and SPI/PR were higher compared to that of control in simulated intestinal fluid (SIF). Additionally, SPI carriers in SGF medium did not show degradation of structure, whereas a major collapse of network was observed in SIF medium, indicating controlled‐release in the intestines. The addition of agrowastes into SPI carriers led to a significantly (P < 0.0001) lower release of L. bulgaricus FTDC 1511 in SGF medium and a higher release in SIF medium, compared to that of the control. SPI carriers containing agrowastes may be useful transports for living probiotic cells through the stomach prior to delivery in the lower intestines. Practical Application: Agrowastes could be utilized as a new probiotic carrier for enhanced gastrointestinal transit and during storage. This also reduces the amount of agrowastes accumulated.     

Combined effect of autoclaving‐cooling and cross‐linking treatments of normal corn starch on the resistant starch formation and physicochemical properties

Normal corn starch (NCS) was treated by both autoclaving‐cooling and cross‐linking to produce resistant starch (RS). The RS yield, crystalline structure, as well as other physicochemical properties of the modified starch were investigated. The yield of RS was 12.2% by four autoclaving‐cooling cycles, and it could be further increased up to 31.0% by the subsequent cross‐linking using sodium trimetaphosphate/sodium tripolyphosphate. The scanning electron microscopy images clearly illustrated that the granular structure of native starch was disrupted and a continuous network with irregular shape was formed after autoclaving‐cooling cycles. The subsequent chemical cross‐linking appeared to make the network structure more compact and dense. X‐ray diffraction patterns showed that B‐ and V‐types coexisted in all the modified corn starches, and all these modified starches exhibited very low viscosity which remained almost constant regardless of temperature changes.     

Impacts of glucosamine/oligochitosan glycation and cross‐linking by transglutaminase on the structure and in vitro antigenicity of whey proteins

With the fixed and selected conditions, whey protein concentrate (WPC) was glucosamine‐/oligochitosan‐glycated and cross‐linked by transglutaminase, resulting in glucosamine conjugation of 4.18–5.88 g/kg. Electrophoretic analysis showed cross‐linking and glycation of whey proteins, while circular dichroism analysis indicated that the two reactions contributed less ordered secondary structure to the two products. Enzyme‐linked immunosorbent assay results showed that the two products lost 64–95% antigenic responses of WPC, and oligochitosan was more powerful than glucosamine to reduce the responses. Rocket immuno‐electrophoretic analysis also evidenced antigenicity loss. This applied treatment is efficient to modify the structure and to decrease the allergenicity of whey proteins.     

Physicochemical and structural characteristics of cross‐linked banana starch using three cross‐linking reagents

Banana starch was cross‐linked using different cross‐linking reagents, phosphoryl chloride (POCl₃), sodium trimetaphosphate (STMP), and epichlorohydrin (ECH), under alkaline conditions. The reaction conditions were selected to produce similar pasting profiles. The effects of the different cross‐linking reagents on the physicochemical and structural characteristics of cross‐linked starches were evaluated. The microscopy study did not show difference on the surface of the granules. Slight decrease in the peak temperature and enthalpy were found in the cross‐linked banana starch. The chemical groups introduced in the starch molecules by the diverse reagents promoted the re‐association of starch chains during storage. The rheological analysis of all starch dispersion at 10% (flow curves) showed a non‐Newtonian shear‐thinning; pastes obtained were time‐independent, suggesting an important contribution of the continuous phase. Structural study showed that the cross‐linked STMP‐starch had the lowest level of amylose and the ratio short/long amylopectin chains. The three reagents used for cross‐linking presented different action mode on starch granule and its components.     

Effect of a microbial calcium‐independent transglutaminase on functional properties of a partially purified cowpea (vigna unguiculata) globulin

A partially purified globulin from cowpea seeds was polymerised by calcium‐independent microbial transglutaminase. The level of free amino groups in the globulin decreased with increase in enzyme concentration, suggesting that the cross‐linking reaction involved participation of the amino residues. At transglutaminase concentrations of 6–18 μg ml⁻¹ emulsifying activity decreased with increasing enzyme concentration and degree of cross‐linking, while a similar trend was observed for the foaming property at 6–12 μg ml⁻¹ transglutaminase concentrations. The resultant emulsions and foams formed by the cross‐linked proteins were more stable than those formed by the untreated protein. Sodium dodecyl sulphate polyacrylamide gel electrophoresis showed that of the two major polypeptides, the 55 kDa protein was more susceptible to transglutaminase action than the 50 kDa protein. Scanning electron microscopy revealed an amorphous structure for the control protein gel while a more defined and cross‐linked gel structure was observed for the protein treated with transglutaminase. The results suggest that protein products differing in functionalities can be obtained by controlling the degree of enzymatic protein cross‐linking. © 1999 Society of Chemical Industry     

Gel‐enhancing effect and protein cross‐ linking ability of tilapia sarcoplasmic proteins

BACKGROUND: Tilapia (Oreochromis niloticus) sarcoplasmic proteins contain substantial transglutaminase (TGase) activity. The enzyme catalyzes the protein cross‐linking reaction, resulting in a more elastic gel. The objective was to investigate the gel‐enhancing effect of sarcoplasmic proteins from tilapia as related to TGase activity. RESULTS: Total TGase activity of sarcoplasmic proteins concentrate (SpC) increased about 3.6‐fold after ultrafiltration using 30 kDa membrane, but specific activity remained unchanged, indicating minimal TGase purification by ultrafiltration. Addition of 1 mg mL^?1 SpC containing 40 units TGase activity induced cross‐linking of tilapia actomyosin, and the extent of cross‐linking increased with added level of SpC. Myosin heavy chain (MHC) and troponin were preferably cross‐linked by tilapia SpC, while actin and tropomyosin were not affected. Higher retention of MHC was observed concomitantly with greater content of cross‐linked protein when SpC was added to lizardfish surimi. Lizardfish surimi with 10 g kg^?1 SpC added and pre‐incubated at 37 °C for 1 h exhibited 91.6% and 26.7% increase in breaking force and deformation, respectively, when compared to the control. CONCLUSIONS: Residual TGase activity in SpC played an important role in catalyzing the protein cross‐linking and enhancing actomyosin gelation. SpC could be a potential ingredient for improving textural properties of fish protein gel. Copyright © 2007 Society of Chemical Industry     

土壤分离转谷氨酰胺酶生产菌株

根据转谷氨酰胺酶催化反应结果的特性 ,设计了一个利用廉价的酶作用底物实施的蛋白质交联凝絮 -沉淀性能测定方法 ,并将其作为初筛手段 ,用于从土壤中分离生产转谷氨酰胺酶微生物菌种。从 13 9株放线菌中经初筛和摇瓶发酵测定酶活的复筛试验筛选得到 10株产酶菌株 ,其中一株酶活达 0 2 4U/mL ,并对其进行分类鉴定实验 ,确定为链霉菌属 ,编号为Streptomycessp .WZFF .W 12。     

The preparation of an oligochitosan‐glycosylated and cross‐linked caseinate obtained by a microbial transglutaminase and its functional properties

A glycosylated and cross‐linked caseinate (GCC) with glucosamine amount of 4.74 g/kg protein was generated from caseinate and oligochitosan by a microbial transglutaminase. The applied temperature, pH and molar ratio of acyl donor/acceptor were 37 °C, 7.5 and 1:3, respectively; while caseinate concentration, transglutaminase addition and reaction time selected from single‐factor trials were 50 g/L, 10 kU/kg protein and 3 h, respectively. Electrophoretic analysis revealed the cross‐linking and glycosylation of caseinate. Compared with caseinate, GCC showed improved solubility in pH 4–11, higher digestibility in vitro and water binding capacity, about 3‐fold, but lower surface hydrophobicity and oil binding capacity (34%).     

热处理对大豆11S球蛋白溶解性和二级结构的影响

采用差示扫描量热仪、Lowry法以及傅里叶变换红外光谱法分别对大豆11S球蛋白的热性质、溶解性和二级结构进行测定与分析,研究热处理对11S球蛋白溶解性和二级结构的影响。热处理能够使大豆11S球蛋白Td和ΔH降低,导致蛋白质发生部分或完全变性。80?℃热处理使大豆11S球蛋白的溶解性降低,这可能由于热处理改变了蛋白质的空间结构和表面电荷所致,此时蛋白质的α-螺旋结构逐渐转变为β-折叠和无规卷曲结构。而90?℃和100?℃热处理一定时间后,蛋白质溶解性又稍有升高,这可能是形成可溶性聚集体造成的。此时蛋白质的α-螺旋和β-折叠结构向β-转角和无规卷曲结构转变,表明β-转角和无规卷曲结构对热聚集体的形成具有重要作用。此外,蛋白质变性和氢键断裂是导致二级结构相互转变的重要因素。     

Synthesis and evaluation of physicochemical properties of cross‐linked Phaseolus aconitifolius starch

Highly substituted moth bean starch (MBS) phosphate ester was synthesized using POCl₃ as cross‐linking reagent. Titrimetric and FT‐IR spectral analysis was used to characterize the substitution. The physicochemical properties of cross‐linked moth bean starch (CLMBS) were done by using X‐ray powder diffractometer (XRD), thermogravimetric analysis (TGA) and swelling study at various temperatures. The change in starch morphology was done using scanning electron micrograph (SEM), and found that it lost its crystallinity after cross‐linking. The results revealed that crystalline nature of native MBS was transformed into amorphous after cross‐linking. TGA report exhibited higher thermal stability, which make it suitable for various industrial applications. Swelling behavior showed high swelling at low temperatures (30 and 60°C) as compared to at high temperature (90°C).