Effect of heat and transglutaminase on solubility of goat milk protein‐based films

The effect of heat, transglutaminase and combination of heat and transglutaminase treatments on the solubility of films prepared from goat milk casein, goat milk whey proteins and whole goat milk proteins was investigated. Goat milk casein films were less soluble when treated with transglutaminase and combination of heat with transglutaminase compare with heat‐treated caseins alone. Heat treatment was more effective at decreasing the solubility of whey protein films. SDS‐PAGE patterns demonstrated that goat milk caseins were better cross‐linked by transglutaminase, whereas whey proteins were better cross‐linked by heat. The extent of cross‐linking was further enhanced when a combination of heat and transglutaminase was used.     

Optimization of microbial transglutaminase activity in ice cream using response surface methodology

In order to determine the maximum activity range of transglutaminase (TG) in ice cream, the central composite design was used and the TG concentration along with the reaction time and temperature were evaluated. The rheological behavior of the ice cream mix that provided the best response and the determination of the protein cross-linking by electrophoresis (SDS-PAGE) were evaluated. It was observed that the TG increased the consistency index and favored the pseudoplastic behavior of the ice cream samples. The maximum response (0.69 Pa sⁿ) was obtained using a TG concentration of 4 U g⁻¹ protein at 56.8 °C with a reaction time of 90 min. The protein cross-linking was confirmed by electrophoresis, which showed that κ-casein was more susceptible to attack from TG than whey proteins.     

定性测定羊奶中黄曲霉毒素M1残留高灵敏度检测卡的研制

研制定性测定羊奶中黄曲霉毒素M1残留的高灵敏度检测卡.在硝酸纤维素膜上预包被黄曲霉毒素M1全抗原作为检测线,以及预包被羊抗鼠二抗作为质控线.将抗黄曲霉毒素M1单克隆抗体用胶体金标记作为金标垫.将样品垫、金标垫、预包被黄曲霉毒素M1的硝酸纤维素膜以及吸水垫依次粘贴在PVC板上,切割成试纸条并组装成检测卡.结果表明:该检测卡的检测限为0.5μg/L,对黄曲霉毒素M1的交叉反应率为100％,而与同族的其他物质的交叉反应率小于5％.假阳性率小于5％,假阴性率为0％,检测卡常温储存,保质期18个月.本研究的检测卡,可快速准确地应用于羊奶中黄曲霉毒素M1残留的定性检测,为快速准确地测定羊奶中黄曲霉毒素M1残留提供了依据.     

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％。     

The effects of different incubation temperatures on the acetaldehyde content and viable bacteria counts of bio-yogurt made from ewe's milk

Yogurt and bio-yogurt were manufactured from ewe's milk using a starter culture and a probiotic culture. Incubation was carried out at 37°C and 42°C until pH 4.6 was reached and the yogurts were stored at 4 +1°C for 14 days. Analysis after 1, 7 and 14 days showed that incubation temperature and storage time significantly influenced overall properties of the samples. During the storage, whey separation and pH decreased, but titratable acidity, lactic acid and volatile fatty acid contents increased. Viable bacterial counts in all bio-yogurts were above 10⁷ cfu g⁻¹ at the end of storage.     

Effects of freezing and frozen storage on the microstructure and texture of ewe's milk cheese

Semi-hard ewe's milk cheeses, frozen immediately after manufacture either slowly at –35 °C or rapidly at –80 °C and stored at –20 °C for 4 months were studied for microstructural and textural characteristics during subsequent ripening. Two control groups were used to establish the effect of freezing: the fresh unfrozen cheese and cheese thawed immediately after freezing. Freezing proper did not result in any marked changes in the textural parameters of the cheeses, but considerable changes were found in slowly frozen cheeses after 4 months of frozen storage. Shear strength values were lower in all frozen and stored cheeses, particularly in cheese samples frozen slowly compared to those in the unfrozen control batch. This parameter and firmness values were significantly lower in both slowly and rapidly frozen cheeses at the completion of ripening. Ripening tended to offset differences in elasticity, noticeable in the cheeses during the first 30 days of ripening. Light microscopy and electron microscopy revealed small cracks and ruptures in the cheeses which could not be observed by the naked eye. More extensive damage to the cheese microstructure was found in slowly frozen cheese samples stored frozen for 4 months.     

Gelation properties of salt-extracted pea protein isolate catalyzed by microbial transglutaminase cross-linking

Gelation is a fundamental functional characteristic of plant proteins. In this paper, a salt-extracted pea protein isolate (PPI) was mixed with microbial transglutaminase (MTG) to produce gels and the gelation properties were studied. When the MTG level increased, the magnitude of both the G′ and G″ moduli also increased, which means the gel strength increased. A second order polynomial equation was used to describe the relationships between the G′, G″ modulus and TG level. It was found that with increased heating and cooling rate at the same MTG level, G′ and G″ tended to decrease, resulting in a weaker gel. This was attributed to the rearrangement time of pea protein molecules; slower heating and cooling rates enabled protein molecules to have more time to rearrange and therefore form a stronger gel. At the same MTG level, higher pea protein concentration resulted in higher G′ and G″ values and a power law relationship was found between G′ and pea protein concentration or G″ and pea protein concentration. Frequency sweep data of PPI show that the MTG treatment resulted in higher G′ values and lower tan delta values, indicative of a stronger more elastic gel. The minimum gelation concentration was found to be 3% (w/v) with 10 U MTG treatment, lower than 5.5% required when no MTG was present. When compared to PPI and soy protein isolate (SPI) with and without 10 U MTG treatment, the gel strength of PPI with MTG was stronger than that of SPI with MTG treatment, whereas the opposite was true without the MTG treatment. SDS-PAGE showed that at the same pea protein concentration, higher MTG level induced more cross-linking as fainter bands were seen on the gel and there was a shift in the relative intensities of the bands in the molecular weight range of 35–100 kDa.     

谷氨酰胺转氨酶交联对牦牛乳曲拉酪蛋白功能性质的影响

以曲拉和荷斯坦牛乳酪蛋白为原料,利用安全、高效的谷氨酰胺转氨酶对其进行交联修饰,研究谷氨酰胺转氨酶对酪蛋白功能性质的影响,测定交联前后样品的持水性、持油性、热稳定性、黏度、起泡性。结果表明,牦牛乳曲拉酪蛋白和荷斯坦牛乳酪蛋白经过谷氨酰胺转氨酶交联后,两种酪蛋白的交联度分别可达到20.04%(p<0.05)和25.15%(p<0.05),曲拉酪蛋白的持水性、热稳定性、黏度和泡沫稳定性显著增加(p<0.05),荷斯坦酪蛋白的黏度和热稳定性显著增加(p<0.05),其余性质均无显著差异。研究结果能为酶法改性酪蛋白的工业化生产提供理论依据。      

Optimization of ingredient levels for manufacturing malted milk beverage using response surface methodology

The malted milk beverage was prepared with varying levels of milk fat, wort and stabilizer–emulsifier mix using response surface methodology. Milk fat had a significant effect on all sensory attributes at the linear, quadratic or interactive levels. Wort level affected the flavour of the beverage at the linear ( P <  0.01) term. The addition of stabilizer–emulsifier mix enhanced the overall acceptability of the product by significantly affecting the flavour ( P <  0.01) and mouthfeel ( P <  0.01). The optimum levels of ingredients to manufacture acceptable quality malted milk beverages were 1.93 g/100 g milk fat, 62.96 g/100 g wort and 0.09 g/100 g stabilizer–emulsifier mix.     

Dependence of microbial transglutaminase on meat type in myofibrillar proteins cross-linking

The objectives of this study were to determine the factors that cause differences in the improvements of gel strength and ε(γ-glutamyl)lysine (G-L) content in chicken and beef (Japanese black cattle) myofibrillar proteins after adding microbial transglutaminase (MTG). As the amount of MTG added increased, the breaking strength increased progressively (p < 0.01) in chicken and beef samples, with the exception of chicken samples treated at 40 °C. The values of elasticity in the chicken samples were lower than those of the beef samples (p < 0.01). Surprisingly, the elasticity level, ε(γ-glutamyl)lysine contents and myosin heavy chain (MHC) band sizes of chicken and beef at all levels of MTG were significantly different (p < 0.01). The results of this study suggest that MTG activity was affected by MTG inhibitors; that MTG develops the texture of myofibrils differently in different species. However, the activity is limited and inconstant among meat proteins, as suggested by the data collected from the chicken samples. As a result, when the transferable amino acid residues are depleted (cross-linked) by MTG activity, the function of MTG will be insignificant. The correlation between MTG and different sources of meat protein is quite unstable but it is strong, which was observed when chicken and beef responded differently to MTG because their chemical and physiological properties were different. The remarkable rate of formation of cross-linked proteins and the discrepancy between the expected and observed amount of dipeptide raises the possibility that there are enzymes capable of reversing the reaction induced by transglutaminase in chicken and beef myofibrils. In summary, our results suggest that access of MTG to chicken and beef myofibrils is different because it depends on physiological (muscles and their fibre types), biological (substrates) and biochemical (inhibitors and amino acids) variables.     

Impact of microbial transglutaminase on the viscoelastic profile of formulated bread doughs

The effects of microbial transglutaminase (TGM) on the viscoelastic profile of wheat flour doughs when added singly and in combination with hydrocolloids—hydroxypropylmethylcellulose (HPMC) and a high ester pectin (GENU pectin type BIG, or BIG)—, amylolytic (-amylase) and non amylolytic enzymes (xylanase) and an emulsifier [diacetyl tartaric acid ester of mono-diglycerides (DATEM)] have been investigated by applying response surface analysis to a Draper-Lin small composite design of formulated dough samples. In spite of the major effects on mixing, textural, extensional and viscometric parameters, which were provided by hydrocolloid/surfactant combinations, incorporation of TGM into dough formulas improved some viscoelastic properties. TGM, when added to DATEM and/or HPMC-containing doughs, induced synergistic effects on mixing parameters, resulting in increased water absorption, development time and stability. Highly cohesive doughs with improved water holding capacity and gluten strength during mixing and fermentation, and suitable pasting behaviour during cooking were achieved using TGM/BIG/DATEM mixtures, mainly associated with suitable interactions of the pairs TGM/DATEM and DATEM/BIG.     

Effect of formaldehyde on protein cross-linking and gel forming ability of surimi from lizardfish induced by microbial transglutaminase

Impact of formaldehyde (FA) at various levels (0–9 μmol/g surimi) on gel properties of surimi from lizardfish added with microbial transglutaminase (MTGase) was studied. During iced storage of 10 days, total and free FA in lizardfish flesh increased continuously (P < 0.05). In the presence of FA, breaking force of gels slightly increased, whilst the deformation decreased (P < 0.05). The addition of MTGase (0.4 units/g surimi) was able to increase gel strength and water holding capacity of resulting gel. Nevertheless, gel strengthening effect of MTGase was lowered when FA at higher level was present. Myosin heavy chain (MHC) dominantly underwent polymerisation to a higher extent when either MTGase or FA was added. The higher reduction in ε-amino group content was observed in natural actomyosin (NAM) when FA at higher levels (0–30 μmol/g protein) was incorporated. Acyl transfer reaction mediated by MTGase was impeded in NAM containing FA, especially at higher levels. Generally, FA had an adverse effect on cross-linking ability towards surimi proteins induced by MTGase. Therefore, cross-linking and gel-forming ability of lizardfish surimi could be maximised by MTGase when surimi contained no FA.     

The effect of protein profile and preheating on denaturation of whey proteins and development of viscosity in milk protein beverages during heat treatment

The effect of preheat temperature (63 or 77 °C for 30 s; final heat 120 °C for 30 s) and casein to whey protein ratio on the physical characteristics of 3.3%, w/w, dairy protein beverages was investigated. Dispersions preheated at 77 °C had lower viscosity than dispersions preheated at 63 °C. Casein‐containing dispersions had significantly lower levels of α‐lactalbumin denaturation than whey protein‐only dispersions. A higher proportion of casein improved the thermal stability of protein dispersions. Overall, alteration of preheat temperature and casein to whey protein ratio can influence dairy beverage quality, with increasing levels of casein reducing physical changes due to heat treatment.     

Effect of enzymatic cross-linking of milk proteins on functional properties of set-style yoghurt

This paper presents research on the effect of enzymatic cross-linking of milk proteins on the properties of yoghurt. Whole milk was incubated with transglutaminase (TG) prior to fermentation (2 h, 40°C, E/S ratio 1/2000). Enzyme action was stopped by heating (1 min, 80°C). Skim-milk was treated by simultaneous use of TG and thermophilic yoghurt starter culture without inactivation of the enzyme. A TG treatment of milk prior to fermentation led to prolonged fermentation, while the concomitant use of TG and culture had no influence on fermentation time. Post acidification of yoghurt during storage was lower for products from enzyme-treated milk. This applies both for products cross-linked prior to fermentation with enzyme inactivation, and for simultaneous use of culture and TG without inactivation of the enzyme. Scanning electron microscopic studies revealed that a TG treatment of milk led to reduced mesh sizes of the protein network, and a more regular distribution of the proteins in the yoghurt gel. As a result, yoghurt products from enzyme-treated milk showed increased gel strength and less syneresis, especially when the enzyme was not inactivated. Sensory studies revealed that odour and consistency properties of products from TG-treated milk were assessed as less 'yoghurt specific'. On the other hand, products from enzyme-treated milk were described as being more creamy, indicating that a TG treatment may simulate fat in fermented milk products.     

Enzymatic production of infant milk fat analogs containing palmitic acid: optimization of reactions by response surface methodology

Infant milk fat analogs resembling human milk fat were synthesized by an enzymatic interesterification between tripalmitin, coconut oil, safflower oil, and soybean oil in hexane. A commercially immobilized 1,3-specific lipase, Lipozyme RM IM, obtained from Rhizomucor miehei was used as a biocatalyst. The effects of substrate molar ratio, reaction time, and incubation temperature on the incorporation of palmitic acid at the sn-2 position of the triacylglycerols were investigated. A central composite design with 5 levels and 3 factors consisting of substrate ratio, reaction temperature, and incubation time was used to model and optimize the reaction conditions using response surface methodology. A quadratic model using multiple regressions was then obtained for the incorporation of palmitic acid at the sn-2 positions of glycerols as the response. The coefficient of determination (R²) value for the model was 0.845. The incorporation of palmitic acid appeared to increase with the decrease in substrate molar ratio and increase in reaction temperature, and optimum incubation time occurred at 18 h. The optimal conditions generated from the model for the targeted 40% palmitic acid incorporation at the sn-2 position were 3 mol/mol, 14.4 h, and 55°C; and 2.8 mol/mol, 19.6 h, and 55°C for substrate ratio (moles of total fatty acid/moles of tripalmitin), time, and temperature, respectively. Infant milk fat containing fatty acid composition and sn-2 fatty acid profile similar to human milk fat was successfully produced. The fat analogs produced under optimal conditions had total and sn-2 positional palmitic acid levels comparable to that of human milk fat.     

Optimization of process parameters for the development of a cheese analogue from pigeon pea (Cajanus cajan) and soy milk using response surface methodology

Response surface methodology was used to optimize starter culture concentration (3–7%), pigeon pea–soy solids ratio (1 : 0.3–1 : 1.1), pigeon pea–water ratio (1 : 12–1 : 9), incubation temperature (37–45C) and coagulation temperature (75–95C) in the development of a cheese analogue from pigeon pea and soy milk. Pigeon pea milk and soy milk were prepared by a hot water grinding method, followed by filtration and inoculation. Cheese analogue was obtained through coagulation of the fermented milk by boiling for 15 min, followed by filtration using double-layered muslin cloth and pressing at 1 kg/cm ² pressure for 1 h. The results showed that the total solids recovery (TSR), protein content, fat content and pH varied from 51.09–62.61%, 40.04–55.93%, 9.35–15.36% and 4.48–4.9, respectively, depending on the experimental conditions. The optimum conditions of process parameters for maximum total solids recovery were 6.0% starter culture concentration, 1 : 0.4 pigeon pea–soy solids ratio, 1 : 11 pigeon pea–water ratio, 40.5C incubation temperature and 88.5C coagulation temperature.     

Optimization of Enzymatic Hydrolysis Pretreatment Conditions for Enhanced Juice Recovery from Guava Fruit Using Response Surface Methodology

The effect of enzyme concentration (0.16–0.84 mg/100 g guava pulp), incubation temperature (36.6–53.4 °C), and incubation time (0.95–11 h) on juice yield was studied. A central composite rotatable design was used to establish the optimum conditions for enzymatic hydrolysis of guava to obtain maximum juice yield. Significant regression model describing the changes of juice yield with respect to hydrolysis parameters were established with the coefficient of determination, R ² = 0.85. Enzyme concentration was the most significant variable affecting the juice yield. The recommended enzymatic treatment condition from the study was at the enzyme concentration 0.70 mg/100 g guava pulp, incubation time 7.27 h, and incubation temperature 43.3 °C.     

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.     

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.     

Comparison between structural changes of heat-treated and transglutaminase cross-linked beta-lactoglobulin and their effects on foaming properties

The main whey protein, β-lactoglobulin, was enzymatically modified by transglutaminase and analyzed for structural and conformational changes and their impact in protein foaming properties: foamability and foam stability. Solutions containing 25 mg mL⁻¹ of β-lactoglobulin, 0.07 M cysteine in 20 mM sodium phosphate buffer pH 8.0 were incubated with transglutaminase, at a level of 1 U g⁻¹ substrate, for different periods of time: 30, 60, 120 and 180 min. Protein structural characterization was discussed based on electrophoresis, fluorescence and viscosity studies. Comparison between the effects on foaming properties of this enzymatic treatment with those produced by heating, assayed in a previous work, was made. While 3 min was pointed as the critical time in heating treatment, 60 min was identified as the corresponding crucial time for transglutaminase treatment. The most significant conformational change, the greatest amount of dimers and trimers, and approximately the same proportion among protein species were verified at these times. Foamabilities were similar regardless of the treatment, but foam stability for heated β-lactoglobulin, measured through the change in foam volume with time is ∼250% higher than the same property for the protein enzymatically treated. Heating produces a higher degree of unfolding and index of surface hydrophobicity; less compact and more asymmetrical structures, with higher flexibility, which implies a greater capacity of rearrangement in the interface, producing a stiffer viscoelastic film, which slows down disproportionation through mechanisms that involve resisting compression and reducing gas transport. This improved film can be responsible for the higher foam volume stability.