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.     

The impact of supplementing goats' milk with quinoa extract on some properties of yoghurt

This study investigated the impact of supplementing goats' milk with quinoa extracts, in the range of 5, 10 and 15 g/100 g on the milk fermentation. The properties of yoghurt produced from this milk, which include viscosity, microstructure and sensory acceptability, were assessed. The supplementation of goats' milk with quinoa extracts, particularly permeate extract, reduced the fermentation time and enhanced the viability of lactic acid bacteria. Supplementation of yoghurt with increased levels of quinoa extracts increased the apparent viscosity and changed the yoghurt protein matrix. Panellists highly accepted the yoghurt that contained quinoa permeate extract.     

添加谷氨酰胺转氨酶对凝固型酸奶品质的影响

研究添加谷氨酰胺转氨酶（简称TG酶）的凝固型酸奶与普通凝固型酸奶（未添加TG酶）在发酵过程中理化指标与感官指标的差异性。采用酚酞指示剂法、流变仪、质构仪、离心法和平板计数法分别测定酸奶在发酵过程中酸度、粘度、硬度、持水性及活菌数的变化;用电子眼、电子舌、电子鼻测定色泽、滋味、气味的变化,并进行感官评定。结果表明,每克乳蛋白添加0~5 U的TG酶,发酵结束时酸奶的酸度在71.12~73.64 oT范围内,酸度无显著性差异（P>0.05）;在发酵过程中,粘度、硬度均在3 h后呈显著上升（P<0.05）,发酵结束时,添加TG酶会显著增高凝固型酸奶的粘度和硬度（P<0.05）;随着TG酶添加量的增加,持水性显著增强（P<0.05）;添加不同浓度的TG酶凝固型酸奶中的活菌数无显著差异（P>0.05）。TG酶凝固型酸奶与普通凝固型酸奶的主色号均为4095,发酵结束时,色泽无显著性差异（P>0.05）;经主成分分析,发酵结束后添加不同浓度的TG酶不会对酸奶的滋味、气味造成显著性影响（P>0.05）;经感官评定,TG酶在添加量为2 U/（g蛋白）时感官品质最受喜爱。因此,与未加TG酶的普通凝固型酸奶相比,添加TG酶的凝固型酸奶在酸度、活菌数、色泽、滋味、气味方面均无显著性差异（P>0.05）;在粘度、硬度和持水性方面显著提高（P<0.05）,添加适量TG酶有利于凝固型酸奶品质的提高。     

Microstructure, rheology and storage stability of low-fat yoghurt structured by carrot cell wall particles

Plant cell wall particles derived from fruits and vegetables are natural fibre materials with a low calorie content that can be used as a healthy alternative to gum stabilisers and starches for structuring low-fat yoghurt. In this study we investigated the effect of cell wall particle (CWP) addition on the gelation kinetics, viscoelastic properties, microstructure, texture and whey loss of the set yoghurt gels as a function of CWP concentration, particle size and storage time. Three particle sizes of dried carrot CWP (d_0.5 = 34, 71 and 80 ??m) were produced from an industrial carrot pomace. Rehydrated CWP was added to skim milk prior to acidification. The results showed that the addition of carrot CWP accelerated the rate of pH reduction and induced earlier gelation. The gel viscoelastic properties were enhanced with increased CWP concentration. This was accompanied with progressive reduction in the whey loss. The smallest cell wall particles (d_0.5 = 34 ??m) gave better gel strength and lower whey loss compared to the larger CWP particles, possibly due to higher contact between the CWP and casein particles thus contributing to the stronger gel network. The CLSM images of yoghurt gels containing CWP showed that carrot CWP occupied the void space within casein particle network. The enhanced gel strength and reduced whey loss achieved by the addition of CWP were maintained throughout the 28 day storage period. The reduction of fermentation processing by almost 1 h, yet still achieving good gel properties for the yoghurt type product could be a significant benefit from a manufacturing point of review.     

TG酶和漆酶对双歧杆菌益生菌酸奶品质的影响

为了比较谷氨酰胺转氨酶(TG酶)和漆酶的添加对双歧杆菌益生菌酸奶感官、蛋白质交联及组织质构变化的影响,测定和分析了两种酶交联益生菌酸奶的游离氨基变化率、感官、质构、表观黏度、蛋白条带及微观结构,并添加阿魏酸改善漆酶酸奶的品质。结果表明:当TG酶用量增加时,TG酶交联酸奶的游离氨基变化率、感官得分和硬度显著增大(p<0.05),胶黏性、粘聚性及表观黏度先增大后减小(p<0.05),内聚性变化不显著(p>0.05),TG酶最佳用量为1.8 U/g;随着漆酶用量的增加,漆酶酸奶游离氨基变化率、感官得分、硬度、内聚性、胶黏性、粘聚性及表观黏度均先增大后减小(p<0.05),漆酶最佳用量为0.3 U/g;添加4.5 mmol/L的阿魏酸明显改善了漆酶交联酸奶的感官、质构及表观黏度;所有待测酸奶中均缺少β-lg条带,TG酶交联酸奶的κ-CN和β-CN条带消失,聚集成了新的蛋白质,而漆酶交联酸奶和阿魏酸+漆酶酸奶与对照及TG酶酸奶相比,分子量在14 kDa的蛋白条带明显变宽;经两种酶交联的酸奶,三维网络结构变得致密,TG酶交联酸奶的胶粒分布更均匀,网络结构更致密,且在漆酶交联酸奶中添加了阿魏酸后,相较于漆酶交联酸奶,蛋白胶束聚集的更紧密,从而得出,不同类型酶对牛奶乳蛋白质交联的催化作用不同,但均可改善酸奶的感官及食用品质。     

Cross-linking with microbial transglutaminase: Relationship between polymerisation degree and stiffness of acid casein gels

Acid casein in phosphate buffer was used as a simplified substrate for cross-linking experiments with microbial transglutaminase. The importance of the intensity of enzyme treatment on the rheology of acid gels was evaluated by preparing mixtures of casein that were cross-linked to a different extent and a native reference to obtain samples with a similar polymerisation degree. The stiffness of acid gels from caseins incubated longer then 3 h was always lower at identical polymerisation degree larger than approximately 30%. The quantification of isopeptides formed by the enzyme reveals a substantial increase of bonds at a stage of cross-linking where only 10% of monomers are available. This indicates that cross-linking must occur between and/or within already existing aggregates, which become larger and less flexible, and supports the hypothesis that, starting with a critical aggregate size the network formation is somewhat impaired by the size and reduced flexibility of the proteins.     

Improvement of enzymatic cross-linking of casein micelles with transglutaminase by glutathione addition

The impact of glutathione (GSH) on cross-linking of micellar casein and sodium caseinate by microbial transglutaminase (TG) was investigated. Micellar casein was obtained by removing whey proteins from skim milk by means of membrane separation techniques. The addition of GSH (0.05–0.1 mm) was found to enhance the cross-linking of micellar casein suspended in milk serum. Cross-linking of sodium caseinate remained unaffected by GSH addition. Mixing TG and milk serum before addition of substrate proteins, however, resulted in an almost complete inhibition of the enzyme. When GSH and TG were present in the system before substrate addition, the reactivity of TG can be maintained. Hence, it was concluded that the addition of GSH mainly affected interactions between TG and an indigenous TG inhibitor present in milk serum. The addition of GSH allows cross-linking in milk products by TG without requiring a prior heat treatment of the milk beyond pasteurisation conditions.     

Effect of Trichoderma reesei tyrosinase on rheology and microstructure of acidified milk gels

The aim of this work was to study the potential of tyrosinase enzymes in structural engineering of acid-induced milk protein gels. Fat free raw milk, heated milk or a sodium caseinate solution were treated with tyrosinases from Trichoderma reesei (TrTyr) and Agaricus bisporus (AbTyr) and the reference enzyme transglutaminase (TG) prior to acid-induced gelation. TrTyr treatment increased the firmness of raw milk and sodium caseinate gels, but not that of heated milk gels, even though protein cross-linking was detected in heated milk. AbTyr did not cross-link proteins in any of the studied milk protein systems. TG was superior to TrTyr in gels prepared of heated milk. In acidified heated milk and sodium caseinate, TrTyr and TG treatment resulted in a decrease of the pore size. Scanning electron microscopy revealed more extensive particle interactions in the heated milk gels with TG than with TrTyr.     

Structural properties of stirred yoghurt as influenced by whey proteins

The effect of whey protein addition on structural properties of stirred yoghurt systems at different protein and fat content was studied using laser diffraction spectroscopy, rheology and confocal laser scanning microscopy (CLSM). The composition of heated milk systems affected micro- and macroscopic properties of yoghurt gels. Particle size increased as a function of increasing whey protein content and decreased as a function of increased fat level. Firmness (elastic modulus) and apparent viscosity of manufactured yoghurt samples increased as a function of increased interparticle interactions, mainly caused by self-aggregation of whey proteins or aggregated whey protein-coated fat globules, respectively. The resistance towards shear-induced disruption of yoghurt gels increased with an increasing proportion of casein protein in the protein mixture, whereas products with high whey protein level revealed lower resistance behaviour towards shear-forces. CLSM images illustrated that the presence of large whey protein aggregates and lower number of fat globules lead to the formation of an interrupted and coarse gel microstructure characterised by large interstitial spaces. The higher the casein fraction and/or the fat level, the less interspaced voids in the network were observed. However, it is evident that the addition of whey proteins reinforces firmness properties of low-fat yoghurts comparable to characteristics of full-fat yoghurt.     

Excessive cross-linking of caseins by microbial transglutaminase and its impact on physical properties of acidified milk gels

By varying cross-linking intensity, the effect of microbial transglutaminase on acid gels made from casein solution and raw milk was studied. To avoid any impact of heating, N-ethylmaleimide was used for enzyme inactivation after appropriately checking its efficiency. Up to a specific degree of oligomerisation gel stiffness and firmness increased and tan δ, time at gelation onset and syneresis decreased. Above approximately 70% and 25% of cross-linked protein in casein solution and raw milk, respectively, these parameters showed an opposite behaviour, and weak gels with high syneresis were obtained. Substrate differences, such as preferred cross-linking of adjoining κ-caseins on the surface of the micelle enhanced the effect of steric hindrance in raw milk and impaired proper rearrangements upon acidification at a much lower level of oligomerised protein. It is mainly dimeric and trimeric casein that successfully contributed to the enhanced properties of milk protein gels.     

Acid-induced gelation of whey protein polymers: effects of pH and calcium concentration during polymerization

Heating whey protein dispersions (90°C for 15 min) at low ionic strength and pH values far from isoelectric point (pH>6.5) induced the formation of soluble polymers. The effect of mineral environment during heating on the hydrodynamic characteristics and acid-induced gelation properties of polymers was studied. Whey protein dispersions (80 g/l) were denatured at different pH (6.5–8.5) and calcium concentrations (0–4 mm) according to a factorial design. At pH 6.5, the hydrodynamic radius of protein polymers increased with increasing calcium concentration, while the opposite trend was observed at pH 8.5. Intrinsic viscosity results suggested that heating conditions altered the shape of protein polymers. Whey protein polymers were acidified to pH 4.6 with glucono-δ-lactone and formed opaque particulate gels. The storage modulus and firmness of gels were both affected by conditions used to prepare protein polymers. As a general trend, polymers with high intrinsic viscosity produced stronger gels, suggesting a relationship between polymer shape and gel strength.Acid gelation properties of whey protein polymers makes them suitable ingredients for yoghurt applications. Using whey protein polymers to standardize protein content increased yoghurt viscosity to 813 Pa.s while using skim milk powder at same protein concentration increased yoghurt viscosity to 393 Pa.s. Water holding capacity of protein polymers in yoghurt was 19.8 ml/g compared to 7.2 ml/g for skim milk powder protein. Acid gelation properties of whey protein polymers are modulated by calcium concentration and heating pH and offers new alternatives to control the texture of fermented dairy products.     

Post-processing of fermented milk to stirred products: Reviewing the effects on gel structure

BackgroundThe structure of stirred fermented milk products such as yoghurt, Greek-style yoghurt and fresh cheese is an essential criterion for consumer acceptance. It is determined by various factors ranging from the milk base composition to starter culture to mechanical forces occurring during filling. The effect of upstream processing parameters and fermentation conditions on the structure of stirred fermented milk gels have been extensively studied and reviewed by different authors.Scope and approachThis review examines the effect of mechanical stresses in different unit operations downstream of fermentation, or post-processing, on the milk coagulum, often referred to as a microgel suspension. The process steps can be indispensable, e.g. pumping, cooling and filling, and concentration for concentrated milk products, or optional, e.g. mechanical or thermal treatment. They are evaluated with regards to their impact on the structure of microgel suspensions. In-situ laboratory scale experiments in rheometers provide an insight into structural changes of milk gels due to shearing.Key findings and conclusionsThe structural properties, including rheological parameters, of the microgel suspension depend on the cumulative effect of the type, magnitude and duration of the mechanical stresses occurring in the different post-processing unit operations. The latter can be optimised to achieve the desired gel properties and save raw material, mainly protein and stabilisers. It can also be adjusted for low viscosity at high protein contents. Thus, the efficiency of the process can be improved by considering the design of the process downstream of fermentation.     

Rheological and textural properties of goat and cow milk set type yoghurts

Yoghurts were made from pasteurised and homogenised goat milk (2.5% protein), goat milk fortified with a milk protein isolate (5% protein) and cow milk (3% protein), by acidification with a starter culture at 43 °C until a pH of 4.6 was reached. The rheological and textural properties of gels and yoghurts were analysed using dynamic low amplitude oscillatory rheology and back extrusion texture analysis. Gelation and fermentation times of goat milk were longer, while gelation pH, storage moduli (G′) and yield stress values were lower, compared with those of cow milk. Textural properties of goat milk yoghurts such as firmness, consistency, cohesiveness and viscosity index were very poor. Consequently, the products could not be classified as set type yoghurts. Fortification of goat milk with a milk protein isolate contributed to a significant improvement of the rheological and textural properties of yoghurt.     

The quality of set‐style yoghurt responsible to partial lactose hydrolysis followed by protein cross‐linking of the skimmed milk

Skimmed milk used for set‐style yoghurt production was treated with lactase at 0.1 g/kg for 30 min to give partial lactose hydrolysis and then treated with horseradish peroxidase and glucose oxidase at 200 and 6 kU/kg protein to result in protein cross‐linking. Two treatments conferred higher apparent viscosity on the milk, but led to the yoghurt prepared from it with insignificantly different chemical compositions to the counterparts (P > 0.05). The prepared yoghurt also showed decreased syneresis (about 17.7%), higher apparent viscosity and viscoelastic modulus, firmer texture and finer microstructure. This ternary enzyme system is a potential approach to improving the quality of set‐style yoghurt.     

Influence of heat treatment of goat milk on casein micelle size,rheological and textural properties of acid gels and set type yoghurts

Acid gels and yoghurts were made from goat milk that was heated at 72°C/30 s, 85°C/5 min, and 95°C/5 min, followed by acidification with starter culture at 43C until pH 4.6. The rheological and textural properties of acid gels and yoghurts were analyzed using dynamic low amplitude oscillatory rheology and back extrusion texture analysis, respectively. The effect of goat milk heat treatment on the mean casein micelle diameter and protein profile was also determined by dynamic light scattering and SDS PAGE electrophoresis, respectively. The shortest gelation and fermentation time was recorded for yoghurt prepared from milk heated at 85°C/5 min. Also, the pH of gelation, the storage moduli (G′) and yield stress were higher for this yoghurt, compared with the other two. Textural properties of goat milk yoghurts such as firmness and consistency were strongly affected by milk heat treatment, and the highest values were recorded for yoghurt produced from milk preheated at 85°C/5 min, as well. The largest casein micelles were measured after 85°C/5 min treatment and their size decreased at higher temperature, despite higher denaturation of whey proteins at the most intense heat regime, indicating the structure changes that influence on the acid gelation.     

Effect of transglutaminase on rheological properties and microstructure of chemically acidified sodium caseinate gels

The mechanical properties and microstructure of 2.7% and 4.5% sodium caseinate gels chemically acidified by glucono-δ-lactone (GDL) and cross-linked by microbial transglutaminase (TG) were studied. The acidification was performed at different temperatures. According to SDS–PAGE TG clearly caused polymerisation of caseinate irrespective of the treatment temperature (4–50 °C), The cross-linking of the proteins was more extensive at temperatures 22–50 °C. Low amplitude viscoelastic measurements showed that 4.5% caseinate gels acidified at 50 °C were formed much faster than gels acidified at 22 °C. TG only slightly increased the time of gelling. Control gels prepared without TG at temperatures of 4, 22, 37 and 50 °C were mechanically weak. Examination of the control gels with a confocal laser scanning microscope showed that gels formed at 37 and 50 °C were coarse and porous with large cavities between particle aggregates, whereas those formed at 22 °C were much more homogeneous. The TG-treated and acidified sodium caseinate dispersions formed firm gels, indicating cross-linking of casein proteins. Interestingly, the strongest gels were formed at 22 and 37 °C. TG treatment improved the homogeneity of the gel structure at temperatures of 37 and 50 °C. The hardness of TG-treated gels acidified at 4 °C increased during 1 week of storage.     

Some properties of yoghurt produced by adding mulberry pekmez (concentrated juice)

Fruit-flavoured yoghurt was made by adding 2.5, 5.0, 7.5 and 10.0% mulberry pekmez (MP) into milk. The effects of the MP on the quality and fermentation process of the yoghurt were determined. The titratable acidity, pH, viscosity, whey separation and lactic acid bacteria (LAB) counts were determined at weekly intervals for 28 days. The pH range of the MP yoghurts was 4.65–5.57 and the pH of the plain yoghurt was 4.47 ( P  < 0.05). The addition of MP led to an increase in the fermentation time and a decrease in the viscosity of the yoghurts. Statistically significant differences were found between the plain and MP yoghurts in terms of pH (4.01 and 4.35), viscosity (5429 and 3175 cP) and number of LAB (7.07 and 6.48 log cfu). During storage, the titratable acidity, viscosity and LAB counts of MP yoghurts were lower and the whey separations higher than those of controls.     

Influence of cofermentation by amylolytic Lactobacillus strains and probiotic bacteria on the fermentation process,viscosity and microstructure of gruels made of rice,soy milk and passion fruit fiber

Gruels tailored to school-age children and made of soy milk and rice flour with or without total dietary fiber from passion fruit by-product were fermented by amylolytic lactic acid bacteria strains (Lactobacillus fermentum Ogi E1 and Lactobacillus plantarum A6), by commercial probiotic bacteria strains (Lactobacillus acidophilus L10, Lactobacillus casei L26 and Bifidobacterium animalis subsp. lactis B94) and by co-cultures made of one amylolytic and one probiotic strain. The influence of ingredient composition and bacterial cultures on kinetics of acidification, α-amylase activity of the bacteria, apparent viscosity and microstructure of the fermented products was investigated. During fermentation of the gruels, α-amylase activity was determined through the Ceralpha method and apparent viscosity, flux behavior and thixotropy were determined in a rotational viscometer. Rheological data were fitted to Power Law model. The combination of amylolytic and probiotic bacteria strains reduced the fermentation time of the gruels as well as increased the α-amylase activity. The addition of passion fruit fiber exerted less influence on the apparent viscosity of the fermented products than the composition of the bacterial cultures. Scanning electron microscopy provided evidence of exopolysaccharide production by amylolytic bacteria strains in the food matrices tested. The co-cultures made of amylolytic and probiotic bacteria strains are suitable to reduce the fermentation time of a soy milk/rice matrix and to obtain a final product with pH and viscosity similar to yoghurt.     

金黄色葡萄球菌在酸奶中的生长状况与产毒规律研究

将酸奶发酵剂和不同菌数的金黄色葡萄球菌同时接种到原料乳中,检测酸奶发酵与冷藏过程中酸奶的pH值、乳酸含量、金黄色葡萄球菌菌数以及成品酸奶中金黄色葡萄球菌耐热核酸酶的活性。结果表明,当原料乳中金黄色葡萄球菌初始接种量分别为1.8×10、1.8×102cfu/ml时,在酸奶发酵与冷藏过程中均未检测出金黄色葡萄球菌,成品酸奶中金黄色葡萄球菌耐热核酸酶活性呈阴性;当原料乳中金黄色葡萄球菌的接种量分别为8.5×103、2.7×104、2.7×105、1.8×106cfu/ml时,在酸奶发酵过程中(42℃、4～5h),金黄色葡萄球菌菌数呈明显上升趋势;在冷藏(4℃)过程中,金黄色葡萄球菌菌数呈逐渐下降趋势;对于原料乳中金黄色葡萄球菌初始接种量为8.5×103cfu/ml的样品,在冷藏的第3d就检测不出金黄色葡萄球菌,成品酸奶中耐热核酸酶活性呈阳性。在酸奶发酵与冷藏过程中同时伴随着乳酸含量的升高和pH值的降低。     

The formation and structure of milk protein gels

The formation of gels from milk proteins is irreversible. Most are not suitable for optical or rheological approaches.Various gels from milk products are discussed: those made by the action of rennet on whole milk, yoghurt, those from heated milk and by the storage of heated milk and gels made from whey protein of very high nutritional quality.