Preliminary studies on the gelation processes of fermented and GDL-acidified bovine and caprine milk systems

Bovine and caprine milk gels were made by GDL-acidification and yogurt fermentation (using 'ropy' and 'non-ropy' starter cultures). The respective gelation processes were monitored rheologically using dynamic oscillatory testing. The bovine fermented systems produced gel structures with about half the strength of the equivalent chemically acidified gels. The fermented caprine milk systems produced gel structures some eight to 10 times weaker than the equivalent acidified systems. In all cases the caprine systems were weaker than the bovine gels despite having higher protein contents. In all cases the 'ropy' milk systems followed somewhat different gelation patterns and formed weaker gels than the equivalent 'non-ropy' and GDL-acidified systems. These data suggested that the starter culture material (biomass and extracellular polysaccharides) may have interfered with the protein–protein interactions during yogurt fermentation. This produced weaker gel structures, possibly by a modified gelation mechanism .     

Effect of various starter cultures on the viscoelastic properties of bovine and caprine yogurt gels

Bovine and caprine yogurt gels made using 'ropy' (extracellular polysaccharide-producing starters) and 'non-ropy' starter cultures were examined using dynamic (oscillatory) testing. The storage (G¸) and loss (G˝) moduli of the ropy gels were found to be lower than those of the equivalent non-ropy ones. Both moduli of the ropy gels declined on storage (at 4°C). On the other hand, the loss tangents of the gels made using the ropy starters were higher than those of the non-ropy gel systems. Caprine yogurt gels were some eight times weaker than the equivalent bovine systems, the ropy gels again being weaker that the non-ropy systems. These data suggest that the secreted polysaccharide may well interfere with the gel structure and development.     

Starter culture effects on caprine yogurt fermentation

The growth rate of the starter culture and its effects on the gelation process of caprine milk were determined. Caprine milk was fermented using various levels of starter culture inoculum and the acidification, growth and gelation profiles were obtained. The caprine yogurt samples inoculated with lower starter culture levels (1% and 1.5% wlv) produced gel structures about eight to 10 times stronger than that made using a 2% v/v inoculum. The gelation patterns suggested that the formation of the gel structure during the caprine yogurt fermentation was time dependent. This behaviour may be due to the presence of higher cell numbers in the caprine yogurt system.     

Evaluation of tilapia skin gelatin as a mammalian gelatin replacer in acid milk gels and low-fat stirred yogurt

Tilapia skin gelatin (TSG) was studied in a 3-stage process (cooling, annealing, and heating) for pure gelatin gels and in a 4-stage process (acidification, cooling, annealing, and heating) for acid milk gels and cultured yogurt. The aim was to evaluate the use of TSG as a replacement for mammalian gelatin in yogurt. In pure TSG gels, stronger gels with higher melting temperatures were formed with increasing TSG concentrations. Compared with bovine gelatin (BG), which gelled at a concentration of 2.5%, TSG gels had lower gelling (14.1°C) and melting (24°C) temperatures but comparable storage moduli during annealing. In acid milk gels, addition of TSG increased the firmness of the gels with increasing concentration. Gelling and melting points of TSG in milk gels were observed at sufficient concentrations during cooling and heating. Strands and sheets were observed in the electron micrographs of milk gels with 1% TSG and a very dense structure was observed with 2.5% TSG. Yogurt with 0.4% TSG had similar viscosity, consistency, pseudoplasticity, and thixotropy as yogurt containing 0.4% BG; no difference was perceived by sensory panelists according to a triangle test. Addition of 0.4% TSG completely prevented whey separation from the acid milk gel and yogurt. The results suggest that TSG could be a suitable replacement for mammalian gelatin in low-fat stirred yogurt.     

Fat-free yogurt made using a galactose-positive exopolysaccharide-producing recombinant strain of Streptococcus thermophilus

To prevent textural defects in low-fat and fat-free yogurts, fat substitutes are routinely added to milk. In situ production of exopolysaccharides (EPS) by starter cultures is an acknowledged alternative to the addition of biothickeners. With the aim of increasing in situ EPS production, a recombinant galactose-positive EPS⁺Streptococcus thermophilus strain, RD-534-S1, was generated and compared with the parent galactose-negative EPS⁺ strain RD-534. The RD-534-S1 strain produced up to 84 mg/L of EPS during a single-strain milk fermentation process, which represented 1.3 times more than the EPS produced by strain RD-534. Under conditions that mimic industrial yogurt production, the starter culture consisting of RD-534-S1 and (EPS⁻) Lactobacillus bulgaricus L210R strain (RD-534-S1/L210R) led to an EPS production increase of 1.65-fold as compared with RD-534-S1 alone. However, the amount of EPS produced did not differ from that found in yogurts produced using an isogenic starter culture that included the parent S. thermophilus strain RD-534 and Lb. bulgaricus L210R (RD-534/L210R). Moreover, the gel characteristics of set-style yogurt and the rheological properties of stirred-style yogurt produced using RD-534-S1/L210R were similar to the values obtained for yogurts made with RD-534/L210R. In conclusion, it is possible to increase the production of EPS by ropy S. thermophilus strains through genetic engineering of galactose metabolism. However, when used in combination with Lb. bulgaricus for yogurt manufacture, the EPS overproduction of recombinant strain is not significant.     

水牛乳中高产胞外多糖乳酸菌的筛选与鉴定

从生水牛乳中筛选出两株高产胞外多糖（EPS）的乳酸菌株LB2、LB7,经MRS肉汤培养基发酵后,菌液中的胞外多糖（EPS）产量分别达135 mg/L、148 mg/L,通过形态学、生理生化特征、API细菌鉴定系统、16S rRNA序列分析,鉴定出菌株LB2为植物乳杆菌（Lactobacillus plantarum）,LB7为类肠膜魏斯氏菌（Weissella paramesenteroides）。将其应用到水牛乳酸奶的发酵中,结果表明,植物乳杆菌LB2和类肠膜魏斯氏菌LB7均可用来发酵水牛乳酸奶,且能有效增加酸奶的黏度和胞外多糖产量,其黏度和EPS产量分别为4 050 mPa·s和149 mg/L。     

双歧杆菌胞外多糖对酸豆乳品质的影响

为探索益生菌及其胞外多糖对酸豆乳的影响,利用产胞外多糖双歧杆菌及其活性多糖与传统发酵剂共同发酵豆浆,研究其对酸豆乳理化、感官及质构特性的作用。结果表明：添加双歧杆菌（107 CFU/mL）和其活性胞外多糖（质量分数0.3%,下同）均对酸豆乳凝乳速度及后酸化无显著影响（P＞0.05）。添加0.3%胞外多糖能显著提高酸豆乳的黏度、持水力和质构特性（P＜0.05）。添加产胞外多糖双歧杆菌107 CFU/mL,对发酵期间酸豆乳发酵速率及凝胶特性影响不大;在后酵期间产生86 mg/L 胞外多糖,显著提高酸豆乳的黏度和质构特性（P＜0.05）,但对产品持水力的影响并不显著（P＞0.05）。双歧杆菌胞外多糖可有效改善酸豆乳凝胶特性,提高产品品质。     

EVALUATION OF pH CHANGE KINETICS DURING DIFFERENT STAGES OF KASHAR CHEESE PRODUCTION FROM BOVINE, OVINE AND CAPRINE MILK

The objective of this study was to determine the pH change kinetics during Kashar cheese production from bovine, ovine and caprine milk. Kinetics of pH change were determined during milk ripening, cooking/holding and pressing/fermentation phases of Kashar cheese. The pH decreased logarithmically, nonlinearly, with time in the milk ripening period and was reduced linearly with time in the cooking/holding and pressing/fermentation stages. The time of pH decrease in cow's, ewe's and goat's milk was significantly different, except cooking/holding time, for three stages. There was no difference between bovine and caprine milk in cooking/holding time; however, ovine milk was significantly different from the other two kinds of milk. The shortest and the longest overall times were determined for caprine and ovine milk cheeses, respectively. The rate of change in milk pH of the three species was significantly different for three stages. The rate followed the pattern caprine  >  bovine  >  ovine milk.

PRACTICAL APPLICATIONS

Optimum acid development is essential to creating the desired cheese mass. For each major cheese type, lactic acid must develop in the appropriate time, usually not too rapidly or too slowly, and in a specific concentration. The progress of acidification is monitored by pH change in the industrial Kashar cheese production. The final pH of cheese is determined not only by the amount of lactic acid but also by the buffering capacity (BC) of the milk and curd. However, the BC is different between milk species. This study evaluates and compares the pH change kinetics during various stages of Kashar cheese making using cow's, ewe's and goat's milk. This work may help to compare milk and cheese curds made from bovine, ovine and caprine milk.     

Effect of epigallocatechin gallate on the fermentative and physicochemical properties of fermented milk

Yogurt, a traditional fermented dairy product, is made with a starter that contains Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. The aim of this study was to investigate the effects of different concentrations of epigallocatechin gallate (EGCG; 0, 0.5, 1.0, 3.0, and 5.0 mg/mL) on the growth, metabolism, and acid production of lactic acid bacteria, as well as the texture, stability, and antioxidant activity of fermented milk (yogurt). The results showed that a low concentration of EGCG had no significant effect on the acid production capacity of the starter or on the water-holding capacity of the yogurt but did increase its viscosity. A high concentration (5.0 mg/mL) of EGCG delayed the acid production rate of the starter and decreased the water-holding capacity, but significantly increased the antioxidant activity of yogurt. The addition of EGCG significantly increased the hardness of yogurt. Therefore, EGCG can improve the texture of fermented milk and enhance its antioxidant activity and stability, thus improving the overall quality of yogurt.     

Isolation and identification of exopolysaccharide producer lactic acid bacteria from Turkish yogurt

Lactic acid bacteria (LAB) were isolated from traditional yogurt samples and genotypic characterization of these isolates revealed the presence of 21 distinct LAB strains belonging to Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Leuconostoc mesenteroides, and Lactobacillus plantarum as new LAB strains. Determination of the exopolysaccharide (EPS) production characteristics of the selected strains of each species revealed that all strains possessed at least one gene required for both homopolymeric‐ and heteropolymeric‐type EPS production. Structural analysis of the EPSs showed that L. delbrueckii subsp. bulgaricus Y39 and S. thermophilus Y102 produced heteropolymeric EPS containing glucose and galactose, whereas Leuc. mesenteroides Y35 and L. plantarum Y36 produced homopolymeric glucan‐type EPS. The level of EPS production in these strains was found to be in a similar range. These strains with EPS production characteristics are good candidates for future studies as new LAB for yogurt production.

Practical applications

Recent trends in yogurt production technology have led to an increased use of ropy starter cultures in yogurt production due to the technological roles of exopolysacharides (EPS) produced by these cultures. The main role of EPS in yogurt production is to improve the textural properties of yogurt as an in situ produced natural polymer. In addition to the yogurt starter cultures, use of adjunct cultures during production of yogurt is also of special interest to enhance the technological and nutritional characteristics of yogurt. Therefore, in this study, potential yogurt starter and adjunct cultures from traditional yogurt samples with EPS production characteristics were isolated. From these isolates, Lactobacillus delbrueckii subsp. bulgaricus Y39 and Streptococcus thermophilus Y102 produced heteropolymeric EPS containing glucose and galactose, whereas Leuconostoc mesenteroides Y35 and Lactobacillus plantarum Y36 produced homopolymeric glucan.     

葛根发酵乳发酵条件优化

以从酸奶中筛选的高产胞外多糖（EPS）的乳酸菌为出发菌株,用葛根和脱脂乳进行乳酸菌的发酵,通过单因素试验及响应面试验对葛根发酵乳发酵条件进行优化。结果表明,最优的发酵条件为发酵温度39 ℃,葡萄糖添加量1.4%,发酵时间20 h。在此优化条件下,葛根发酵乳总黄酮含量为1.91 mg/mL,EPS含量为0.155 mg/mL。     

EFFECT OF HIGH HYDROSTATIC PRESSURE PROCESSING ON RHEOLOGICAL AND TEXTURAL PROPERTIES OF PROBIOTIC LOW-FAT YOGURT FERMENTED BY DIFFERENT STARTER CULTURES

The effect of milk processing on rheological and textural properties of probiotic low‐fat yogurt (fermented by two different starter cultures) was studied. Skim milk fortified with skim milk powder was subjected to three treatments: (1) thermal treatment at 85C for 30 min; (2) high hydrostatic pressure (HHP) at 676 MPa for 5 min; and (3) combined treatments of HHP (676 MPa for 5 min) and heat (85C for 30 min). The processed milk was fermented using two different starter cultures containing Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus and Bifidobacterium longum at inoculation rates of 0.1 and 0.2%. Rheological parameters were determined and a texture profile analysis was carried out. Yogurts presented different rheological behaviors according to the treatment used, which could be attributed to structural phenomena. The combined HHP and heat treatment of milks resulted in yogurt gels with higher consistency index values than gels obtained from thermally treated milk. The type of starter culture and inoculation rate, providing different fermentation pathways, also affected the consistency index and textural properties significantly. The combined HHP and heat treatment of milks before fermentation, and an inoculation rate of 0.1% (for both cultures), led to desirable rheological and textural properties in yogurt, which presented a creamy and thick consistency that does not require the addition of stabilizers.     

Effect of mono or co-culture of EPS-producing Streptococcus thermophilus strains on the formation of acid milk gel and the appearance of texture defects

One acidifying (ST1) and two texturing strains (ST2 and ST3) of Streptococcus thermophilus were used as pure or co-cultures to identify and understand their effects on the structuring of acid milk gels and on the appearance of texture defects, i.e., syneresis and graininess. Symbiosis between specific texturing and acidifying strains reduced acidification time and increased exocellular polysaccharide (EPS) content. The texturing strain could simultaneously produce low and high molar mass EPS and their distribution in mass and/or in number were influenced by the proportion of acidifying to texturing strain used. The results of this study suggest that the high molar mass EPS contributes to acid gel firmness, but less so compared with the acidification rate. The ability of strain ST3 to prevent texture defects, specifically graininess, did not depend on the acidification kinetics or final EPS content, but rather on the structural properties of EPS and/or the bacterial chain morphology.     

Power ultrasound as a tool to improve the processability of protein-enriched fermented milk gels for Greek yogurt manufacture

One approach to avoid production of acid whey during the manufacture of high-protein yogurt and related products is to concentrate the milk before fermentation. However, the resultant gels are firm so that stirring in the tank and further processing are difficult on an industrial scale. We hypothesize that power ultrasound (US) during fermentation softens the gel because sound waves cause cavitation and strong shear forces in the fluid. Skim milk was standardized to different protein contents up to 12%, heated (85°C, 30 min), and acidified with thermophilic or mesophilic starter cultures. An excessive increase in gel firmness as a function of protein content was detected. In the next series of experiments, US was applied during fermentation. Milks (10% protein) were acidified at 43.5°C and sonicated from pH 5.8 to 5.1 with a sonotrode (20 kHz, 20 W). Immediately after fermentation, gels were agitated using a rheometer with a vane geometry. The maximum torque required to break the gel was reduced by 75% following US, and gel firmness was reduced by 80%. Gels were then processed into stirred yogurt and analyzed. Sonicated samples were smoother with fewer large aggregates. Confocal laser scanning microscopy images suggested a less cohesive structure and more compact microgel particles, resulting in reduced viscosity. We concluded that US is a promising tool to weaken the gel and facilitate further processing. This enables new approaches for the manufacture of Greek yogurt, particularly in regard to avoiding production of acid whey and developing products with novel textures.     

Comparison of micellar casein isolate and nonfat dry milk for use in the production of high-protein cultured milk products

High protein levels in yogurt, as well as the presence of denatured whey proteins in the milk, lead to the development of firm gels that can make it difficult to formulate a fluid beverage. We wanted to prepare high-protein yogurts and explore the effects of using micellar casein isolate (MCI), which was significantly depleted in whey protein by microfiltration. Little is known about the use of whey protein-depleted milk protein powders for high-protein yogurt products. Microfiltration also depletes soluble ions, in addition to whey proteins, and so alterations to the ionic strength of rehydrated MCI dispersions were also explored, to understand their effects on a high-protein yogurt gel system. Yogurts were prepared at 8% protein (wt/wt) from MCI or nonfat dry milk (NDM). The NDM was dispersed in water, and MCI powders were dispersed in water (with either low levels of added lactose to allow fermentation to achieve the target pH, or a high level to match the lactose content of the NDM sample) or in ultrafiltered (UF) milk permeate to align its ionic strength with that of the NDM dispersion. Dispersions were then heated at 85°C for 30 min while stirring, cooled to 40°C in an ice bath, and fermented with yogurt cultures to a final pH of 4.3. The stiffness of set-style yogurt gels, as determined by the storage modulus, was lowest in whey protein-depleted milk (i.e., MCI) prepared with a high ionic strength (UF permeate). Confocal laser scanning microscopy and permeability measurements revealed no large differences in the gel microstructure of MCI samples prepared in various dispersants. Stirred yogurt made from MCI that was prepared with low ionic strength showed slow rates of elastic bond reformation after stirring, as well as slower increases in cluster particle size throughout the ambient storage period. Both the presence of denatured whey proteins and the ionic strength of milk dispersions significantly affected the properties of set and stirred-style yogurt gels. Results from this study showed that the ionic strength of the heated milk dispersion before fermentation had a large influence on the gelation pH and strength of acid milk gels, but only when prepared at high (8%) protein levels. Results also showed that depleting milk of whey proteins before fermentation led to the development of weak yogurt gels, which were slow to rebody and may be better suited for preparing cultured milk beverages where low viscosities are desirable.     

Effect of fermentation temperature on the properties of exopolysaccharides and the acid gelation behavior for milk fermented by Streptococcus thermophilus strains DGCC7785 and St-143

We investigated the effect of fermenting milk with 2 strains (DGCC7785 and St-143) of Streptococcus thermophilus, which are known to produce different types of exopolysaccharide (EPS) structures. The yields and physical properties of these ropy EPS were monitored during the fermentation of milk at different temperatures. We wanted to understand how these types of EPS properties affected yogurt gelation. Reconstituted skim milk was fermented at 33, 39, or 45°C until pH values reached 5.2, 4.9, 4.7, and 4.5. Molar mass of ropy EPS samples was determined using size exclusion chromatography coupled with multiangle laser light scattering. Rheological properties of fermented milk gels were analyzed using small-strain dynamic oscillatory measurements. In both strains, concentrations of ropy EPS increased during fermentation and at all temperatures. Fermentation times, by both strains, were shortest at 45°C and longest at 33°C. For both strains, molar mass of ropy EPS ranged from 2 to 4 × 10⁶ g/mol during fermentation. A major proteinaceous contaminant that was co-isolated with the ropy EPS fraction by our isolation method was identified as a milk-derived phosphoglycoprotein PP3. Increase in fermentation temperature from 33 to 45°C significantly decreased the storage modulus values (from 170 to 41 Pa) for milk gelled by strain DGCC7785, whereas the gels made with St-143 had very low storage modulus values (11–17 Pa) regardless of fermentation temperatures. For both strains, the values of maximum loss tangent in the milk gels increased with fermentation temperature; the maximum loss tangent occurred at higher pH values when milk was fermented by strain DGCC7785. The specific type of EPS produced appeared to be responsible for the differences in yogurt texture rather than the concentration or molar mass of the EPS.     

Robust PCR‐based method for quantification of bovine milk in cheeses made from caprine and ovine milk

To prevent fraud and enhance quality assurance, credible analysis of dairy products is crucial. A common problem is the addition of cheaper bovine milk to caprine and/or ovine dairy products and when not declared addition of bovine milk constitutes fraud. The aim was to develop a rapid, robust and sensitive method for the identification of adulteration of caprine and/or ovine cheeses with bovine milk. New quantitative real‐time polymerase (qPCR) assays were designed for the specific determination of bovine DNA (Cow1) and bovine, caprine and ovine DNA (BoCaOv). These were applied to 17 samples of caprine cheese and 24 of ovine cheese. Results showed that 17% (7/41) of these cheeses contained >5% bovine milk. As bovine milk was not declared as an ingredient in any of the samples, this represents adulteration. Other cheeses that contained detectable bovine milk at ≤5% (22%; 5/41) might pose a health risk to people allergic to bovine milk.     

Qualitative and quantitative analysis of bovine milk adulteration in caprine and ovine milks using native-PAGE

Native-PAGE (polyacrylamide gel electrophoresis) was used for the simultaneous qualitative and quantitative analysis of bovine milk adulteration in caprine and ovine milk using whole milk samples as well as their whey protein fraction. Quantification was based on measuring band intensity of bovine β-lactoglobulins in all milk mixtures and bovine α-lactalbumin in caprine/bovine milk blends. Linear relationships were established between the band intensity of bovine β-lactoglobulins and α-lactalbumin vs. volume percentage of added bovine milk in all milk analysed, with the correlation coefficient from 0.9950 to 0.9998. These correlations enabling the quantification of bovine milk percentage within the wide range from 3% or 5% to 90% in caprine/bovine and ovine/bovine milk blends, respectively. The differences between the actual percentages of bovine milk present in the adulterated milk samples and those calculated using the regression lines were less than or equal to 5% for all samples. This method offers a rapid determination combined with unequivocal identification of the bovine whey proteins in almost every caprine/bovine or ovine/bovine milk mixtures.     

Effect of Tetrasodium Pyrophosphate on the Physicochemical Properties of Yogurt Gels

The effect of tetrasodium pyrophosphate (TSPP) on the properties of yogurt gels was investigated. Various concentrations (0.05 to 0.2%) of TSPP were added to preheated (85°C for 30 min) reconstituted skim milk, which was readjusted to pH 6.50. Milk was inoculated with 2% starter culture and incubated at 42°C until the pH reached 4.6. Acid-base buffering profiles of milk and total and soluble calcium levels were measured. Turbidity measurements were used to indicate changes in casein dispersion. Storage modulus (G′) and loss tangent (LT) values of yogurts were monitored during fermentation using dynamic oscillatory rheology. Large deformation properties of gels were also measured. Microstructural properties of yogurt were observed using fluorescence microscopy. The addition of TSPP resulted in the disappearance of the buffering peak during acid titration at pH ∼5.1 that is due to the solubilization of colloidal calcium phosphate (CCP), and a new peak was observed at lower pH values (pH 4.0-4.5). The buffering peak at pH 6.0 during base titration virtually disappeared with addition of TSPP and a new peak appeared at pH ∼4.8. The addition of TSPP reduced the soluble Ca content of milk and increased casein-bound Ca values. The addition of up to 0.125% TSPP resulted in a reduction in turbidity because of micelle dispersion but at 0.15%, turbidity increased and these samples exhibited a time-dependent increase in turbidity because of aggregation of casein particles. Gels made with 0.20% TSPP were very weak and had a very high gelation pH (6.35), probably due to complete dispersion of the micelle structure in this sample. The LT value of gels at pH 5.1 decreased with an increase in TSPP concentration, probably due to the loss of CCP with the addition of TSPP. The G′ values at pH 4.6 of gels made with ≤0.10% TSPP were not significantly different but the addition of ≥0.125% TSPP significantly decreased G′ values. The addition of 0.05 to 0.125% TSPP to milk resulted in a reduction in the yield stress values of yogurt compared with yogurt made without TSPP. Greater TSPP levels (>0.125%) markedly reduced the yield stress values of yogurt. Lowest whey separation levels were observed in yogurts made with 0.10% TSPP. High TSPP levels (>0.10%) greatly increased the apparent pore size of gels. Addition of very low levels of TSPP to milk for yogurt manufacture may be useful in reducing the whey separation defect, but at TSPP concentrations ≥0.125% very weak gels were formed.