Factors that affect the aggregation of rennet-altered casein micelles at low temperatures

The effects of temperature, CaCl₂ concentration, pH and ionic strength of milk on the aggregation of casein micelles in milk renneted at 15ºC were studied using particle size analysis determined by laser-light scattering. The rate of aggregation of rennet-altered casein micelles became significantly slower on reducing the temperature of renneting from 30 to 10ºC. At 15ºC, the rate of aggregation of rennet-altered casein micelles increased significantly on adding CaCl₂, on reducing the pH of renneted milk or on adding NaCl (up to 50 m m ). These results indicate that particle size analysis can be used successfully to study the aggregation of rennet-altered casein micelles.     

Growth kinetics and fractal dimensions of casein particles during acidification

Small angle static light scattering was used to study the effect of milk dilution in permeate on the mechanism of acid-induced aggregation of casein particles. Growth kinetics of casein aggregates during acidification was characterized by the succession of four populations of particles. The first one corresponded to casein particles ranging from 0.1 to 1 microm, with a mean value of 0.3 microm. The second population, from 1 to 10 microm, was quickly replaced by a third population, from 10 to 100 microm, which gave rise to the last population measurable, from 100 to 1000 microm. The angular dependence of static light scattering from about 0.01 to 50 degrees was used to determine the fractal dimension (D) of pH-induced casein aggregates. With the formation of about 10-microm aggregates, fractal structures appeared. The D values, determined from double logarithmic plots of intensity versus scatteringvector resulted in values between 1.85 and 2.03.     

A method for the large-scale isolation of β-casein

A method for the large-scale isolation of β-casein from renneted skim milk was developed. The curd from renneted skim milk was dispersed in hot (?70 °C) water to inactivate residual chymosin. The heated curd was subsequently recovered by centrifugation, resuspended in water and incubated at 5 °C, during which β-casein dissociated from the curd; the suspension was centrifuged and the aqueous phase lyophilised. The isolated protein consisted mainly of β-casein, containing a minor amount of γ-caseins and traces of other caseins. Unless chymosin was fully inactivated by heating, some β-casein was hydrolysed at the Leu₁₉₂–Tyr₁₉₃ bond. The yield of β-casein increased with incubation time, up to ∼20% of the β-casein present in the milk after 24 h at 5 °C. Reducing milk pH to 5.5 or 6.0, prior to renneting, caused a high level of contamination with α_s-caseins. This isolation procedure can be easily scaled-up to an industrial process and the β-casein-depleted curd may be used for the manufacture of rennet casein or processed cheese.     

Sodium caseinate-stabilized fat globules inhibition of the rennet-induced gelation of casein micelles studied by Diffusing Wave Spectroscopy

Sodium caseinate (NaCas)-stabilized oil-in-water emulsions were added to skim milk and the rennet-induced aggregation was observed in situ using light scattering and dynamic oscillatory rheology. The gelation of the recombined milk was greatly inhibited by the addition of the oil droplets, at volume fractions >0.025. The development of the turbidity parameter, 1/l*, and the apparent hydrodynamic radius during renneting were determined using diffusing wave spectroscopy. Although the recombined milk samples contained two scattering particles, namely, casein micelles and fat globules, the latter overwhelmingly contributed to the overall light-scattering signal. This made possible to follow the behaviour of NaCas-stabilized fat globules during the gelation process. The enzymatic reaction associated with the hydrolysis of micellar κ-casein was not significantly affected by the presence of the NaCas-stabilized fat globules. However, the emulsion droplets impeded the aggregation of rennet-altered casein micelles preventing the formation of a gel network. The inability of renneted casein micelles to develop a gel network can be attributed in part to an altered equilibrium between soluble and micellar calcium phosphate, caused by the association of soluble Ca²⁺ with casein molecules, but mostly can be attributed to the effect of non-adsorbed caseins on the surface of the casein micelles.     

Effect of Heat-Stable Proteases on the Kinetic Parameters of Milk Clotting by Chymosin

The milk clotting per unit casein hydrolytic activities of proteases from 14 psychrotrophic pseudomonad isolates of raw milk ranged from 0.77 to 9.97 at 30°C. The milk clotting activities of chymodn and T16 protease were not completely additive, especially at high chymosin concentration when clotting time was relatively fast. The T16 protease was not effective in catalyzing the enzymic step of milk clbtting at O°C in the time expected on the basis of its milk clotting at 30°C. Milk incubated with the T25 motease for 8 days at 4°C and then clotted with chymosin at 30°C exhibited weak curd consistency.     

Effects of mineral salts and calcium chelating agents on the gelation of renneted skim milk

The effects of adding CaCl2, orthophosphate, citrate, EDTA, or a mixture of these, to reconstituted skim milk (90 g of solids/kg solution) on the gelation of renneted milk were mediated by changes in Ca2+ activity and the casein micelle. At pH 6.65, the addition of citrate or EDTA, which removed more than 33% of the original colloidal calcium phosphate with the accompanying release of 20% casein from the micelle, completely inhibited gelation. Reformation of the depleted colloidal calcium phosphate and casein in the micelle, by the addition of CaCl2, removed this inhibition. When the minimum requirements for colloidal calcium phosphate and casein in the micelle were met, the coagulation time decreased with increasing Ca2+ activity, leveling off at high Ca2+ activity. The storage modulus of renneted gels, measured at 3 h, increased with increasing colloidal calcium phosphate content of micelles up to a level at which it was approximately 130% of the original colloidal calcium phosphate in the micelles. Further increases in colloidal calcium phosphate by the addition of CaCl2, orthophosphate, or mixtures of these, which did not change the proportion of casein in the micelle, decreased the storage modulus. The gelation of the renneted milk was influenced by Ca2+ activity, the amounts of colloidal calcium phosphate, and casein within the micelle, with the effects of colloidal calcium phosphate and casein within the micelle clearly dominating the storage modulus. These results are consistent with the model of Horne (Int. Dairy J. 8:171-177, 1998) which postulates that, following cleavage of the stabilizing K-casein hairs by rennet, the properties of the rennet gel are determined by the balance between the electrostatic and hydrophobic forces between casein micelles.     

Rheological methods to analyse the thermal aggregation of calcium enriched milks

The colloidal state of calcium enriched milks during heating and its microstructures were analysed through interpretation of rheometric data with the Brownian aggregation theory, which can be considered as complementary to the methods currently used. Viscosity was measured at 100 s⁻¹ through temperature and time sweeps. It was observed that from 25 to 60 °C, viscosity slowly decreased as temperature increased; above 60 °C, viscosity sharply increased at different temperatures depending on the amount of CaCl₂. From time sweeps, the aggregation of casein micelles was described through the Smoluchowski theory. The addition of CaCl₂ at concentrations of 20–30 mmol kg⁻¹ decreased the stability factor 5 to 6 orders of magnitude compared with non-enriched milk. Values of the fractal dimension indicated that aggregation yields disordered aggregates occluding high amounts of solvent. The methodology described may help to analyse colloidal stability and structures when different calcium salts are used for enrichment.     

混料设计优化牦牛“曲拉”凝乳酶干酪素的工艺及产品的性质分析

为提高牦牛产业的附加值,以牦牛乳提取酥油后进行凝固沉淀,再经自然发酵、风干而成的蛋白质含量丰富的产品“曲拉”为原料,采用胃蛋白酶、木瓜凝乳酶和酵母凝乳酶复配成混合凝乳酶,通过单因素实验和混料设计对凝乳干酪素的制备工艺进行研究,并对干酪素的理化性质、红外光谱特性、热力学性质进行分析。结果表明,混合酶质量分数1%(其中胃蛋白酶∶木瓜凝乳酶∶酵母凝乳酶的质量比为0.60∶0.18∶0.22),在pH 6.3、45℃、添加质量分数CaCl 21%条件下,凝乳30 min,出品率为80.35%。混合酶法制备干酪素的理化性质、红外光谱特性和热力学性质与小牛皱胃酶干酪素差异不显著,而且符合GB31638—2016要求。     

Evaluation of a vat wall-mounted image capture system using image processing techniques to monitor curd moisture during syneresis with temperature treatments

The objective of this study was to evaluate a vat wall-mounted image capture system with a range of image processing techniques (threshold, first order and second order grey level statistics and fractal dimension) to monitor curd moisture content during syneresis with a range of temperature treatments. Milk was renneted using three temperature treatments (32 °C throughout, a cooking step from 32 to 38 °C and 38 °C throughout). Prediction models were evaluated in term of fit to reference measurements on samples of curd at 10 min intervals. The best fitting model was based on the threshold technique giving a standard error of prediction (SEP) = 1.06 g/100 g and correlation coefficient (R) = 0.90. These results demonstrated that the threshold image processing technique was the most useful in this study and showed adequate potential to monitor syneresis and predict curd moisture which influences the final texture of cheese.     

枯草芽孢杆菌克隆与表达微小毛霉凝乳酶基因

从自制的酒酿利用酪蛋白培养基分离纯化到了一株产凝乳酶的微小毛霉菌株（ZZMZ-19）,从ZZM-19菌株的cDNA文库筛选到了两个凝乳酶基因chl和ch2并实现了凝乳酶基因ch1和ch2在枯草芽孢杆菌菌株（ZZMZ-01）中的的克隆与表达。chl和曲2阳性克隆菌株在酪蛋白培养基r1]发酵30h左右的时间凝乳酶酶活达到最人,分别为48．27SU／mL和41．02SU／mL,相比出发菌株发酵时间缩短了15h。用交联葡聚糖凝胶柱G100分离纯化其发酵卜清液后,用十二烷基硫酸钠聚丙烯酰胺凝胶电泳方法测得CHII和cHIII分子草分别为32ku和30ku。     

Effect of cycling temperatures on the production of deoxynivalenol and zearalenone by Fusarium graminearum NRRL 5883

The effects of three regimens of cycling incubation temperatures and incubation at constant 25 degrees C on the growth of Fusarium graminearum NRRL 5883 and production of deoxynivalenol (DON) and zearalenone (ZEN) on rice were compared. The effects of low-temperature stress were also studied by incubating rice cultures at a constant 15 degrees C for 4 weeks following incubation at constant 25 degrees C for 2 weeks. Both incubation temperature and time significantly (P < or = 0.05) affected growth of F. graminearum NRRL 5883 and production of DON and ZEN. The highest amount of free ergosterol (640 microg/g culture material) that was used as a measure of fungal growth was found in cultures incubated at temperatures cycling between 15 and 30 degrees C during a 6-week period. The highest amounts of DON (1,679 microg/g culture material) and ZEN (603 microg/g culture material) were produced in cultures incubated at a constant 25 degrees C for 2 weeks prior to incubation at a constant 15 degrees C for an additional 4 weeks. Under cycling incubation temperatures, maximum amounts of DON (850 microg/g culture material) and ZEN (98 microg/g culture material) were produced in cultures incubated at temperatures cycling between 15 and 30 degrees C for 6 weeks. Overall, there was no correlation between mold growth and production of either DON or ZEN. However, DON production and ZEN production were correlated.     

Growth and structure of aggregates of heat-denatured β-Lactoglobulin

Summary The aggregation of the globular protein β-lactoglobulin after heat-denaturation was studied in aqueous solution at pH 7 using static and dynamic light scattering. The structure of the aggregates is self-similar with fractal dimension 2.0. Size exclusion chromatography and dynamic light scattering measurements show that the aggregates have a broad size distribution. Initially clusters of about 85 proteins and 15 nm radius are formed which are the elementary units of the larger fractal aggregates. At low ionic strength the formation of the larger aggregates is impeded by electrostatic interactions.
The structure of the aggregates is independent of the concentration and the temperature. The rate of aggregation has an Arrhenius temperature dependence with an activation energy of about 350 kJ/mol weakly dependent on the concentration. The apparent reaction order of the aggregation is 1.5. In the mixture both variants A and B have the same aggregation rate. The gel time increases with decreasing concentration and diverges at about 0.7g L⁻¹. At lower concentration the aggregate growth stagnates when all protein has aggregated.     

Effect of pH at heat treatment on the hydrolysis of κ-casein and the gelation of skim milk by chymosin

Skim milk was adjusted to pH values between 6.5 and 7.1 and heated at 90 °C for times from 0 to 30 min. After heat treatment, the samples were re-adjusted to the natural pH (pH 6.67) and allowed to re-equilibrate. High levels of denatured whey proteins associated with the casein micelles during heating at pH 6.5 (about 70-80% of the total after 30 min of heating). This level decreased as the pH at heating was increased, so that about 30%, 20% and 10% of the denatured whey protein was associated with the casein micelles after 30 min of heating at pH 6.7, 6.9 and 7.1, respectively. Increasing levels of κ-casein were transferred to the serum as the pH at heating was increased. The loss of κ-casein and the formation of para-κ-casein with time as a consequence of the chymosin treatment of the milk samples were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The loss of κ-casein and the formation of para-κ-casein were similar for the unheated and heated samples, regardless of the pH at heating or the heat treatment applied. Monitoring the gelation properties with time for the chymosin-treated milk samples indicated that the heat treatment of the milk markedly increased the gelation time and decreased the firmness (G^′) of the gels formed, regardless of whether the denatured whey proteins were associated with the casein micelles or in the milk serum. There was no effect of pH at heat treatment. These results suggest that the heat treatment of milk has only a small effect on the primary stage of the chymosin reaction (enzymatic phase). However, heat treatment has a marked effect on the secondary stage of this reaction (aggregation phase), and the effect is similar regardless of whether the denatured whey proteins are associated with the casein micelles or in the milk serum as nonsedimentable aggregates.     

Changes in the apparent viscosity profiles of casein suspensions as affected by plant enzymes

The aim of this work was to study the degree of hydrolysis and changes in the apparent viscosity of casein suspensions as a result of various enzymes addition. Suspensions with 3, 12 and 15 g/100 mL of casein, at pH 5.2, 6.0 and 6.5 were prepared in buffer solutions. Previous standardization; plant (papain and bromelain) and animal (chymosin) enzymes were added to hydrolize the casein suspensions. A control with no enzyme addition was used. The rheological behaviour was determined using a rotational rheometer (Haake RV20), with a cone and plate geometry. The Casson and the power law equations were applied to the data. The degree of hydrolysis was a function of the enzyme, pH and casein concentration, presenting chymosin the highest values. All enzymes showed the highest activity at acidic pH. Also, some substrate inhibition was observed. All samples behaved as non-Newtonian, shear-thinning systems with a yield stress value. In all cases, a significant increase in the viscosity was observed when shifting from 3 to 12 g/100 mL. Further increase in concentration caused an opposite effect. Changes in pH of the casein suspensions affected the viscosity, presenting maximum values at pH 6.0. The Casson equation fitted the results better than the power law model.     

Influence of pressure release rate and protein concentration on the formation of pressure-induced casein structures

The formation of pressure-induced casein structures (600 MPa for 30 min at 30 degrees C) was investigated for different pressure release rates (20 to 600 MPa min-1) and casein contents (1 to 15 g/100 ml). Structures from liquid (sol) to solid (gel) were observed. The higher the protein content and the pressure release rate, the higher was the dynamic viscosity. A firm gel was built up at a casein content of 7 g/100 ml for a pressure release rate of 600 MPa min-1, while lower release rates resulted in less firm gels (200 MPa min-1) or liquid structures (20 MPa min-1). In a 5 g/100 ml casein solution and at a pressure release rate of 600 MPa min-1, casein aggregates were generated which were built from smaller casein particles with a larger hydrodynamic diameter and higher voluminosity than in the untreated solution. After a slow release rate casein micelles had a smaller hydrodynamic diameter and a lower voluminosity, but were similar in shape and diameter as compared with the micelles in solution before high pressure treatment.     

Addition of sodium caseinate to skim milk increases nonsedimentable casein and causes significant changes in rennet-induced gelation,heat stability,and ethanol stability

The protein content of skim milk was increased from 3.3 to 4.1% (wt/wt) by the addition of a blend of skim milk powder and sodium caseinate (NaCas), in which the weight ratio of skim milk powder to NaCas was varied from 0.8:0.0 to 0.0:0.8. Addition of NaCas increased the levels of nonsedimentable casein (from ～6 to 18% of total casein) and calcium (from ～36 to 43% of total calcium) and reduced the turbidity of the fortified milk, to a degree depending on level of NaCas added. Rennet gelation was adversely affected by the addition of NaCas at 0.2% (wt/wt) and completely inhibited at NaCas ≥0.4% (wt/wt). Rennet-induced hydrolysis was not affected by added NaCas. The proportion of total casein that was nonsedimentable on centrifugation (3,000 × g, 1 h, 25°C) of the rennet-treated milk after incubation for 1 h at 31°C increased significantly on addition of NaCas at ≥0.4% (wt/wt). Heat stability in the pH range 6.7 to 7.2 and ethanol stability at pH 6.4 were enhanced by the addition of NaCas. It is suggested that the negative effect of NaCas on rennet gelation is due to the increase in nonsedimentable casein, which upon hydrolysis by chymosin forms into small nonsedimentable particles that physically come between, and impede the aggregation of, rennet-altered para-casein micelles, and thereby inhibit the development of a gel network.     

Effect of heat treatment of rennet skim milk induced coagulation on the rheological properties and molecular structure determined by synchronous fluorescence spectroscopy and turbiscan

Heat treatment applied to milk induces denaturation of whey proteins, leading to a complex mixture of whey protein and whey protein coated casein micelles. The present paper investigates the effects of heat treatment (60 and 80°C during 20min) and rennet-induced coagulation temperature (30 and 40°C) determined by rheology, synchronous fluorescence spectroscopy (SFS) and turbiscan measurements. The gelation times determined by rheology and SFS increased with the increase of heat treatment applied to milk. The rise in temperature induced a decrease in the maximum curd firming rate and an increase in the viscosity of the investigated milk samples. The principal component analysis (PCA) applied, separately, to the SF and turbiscan spectra showed a clear discrimination between: (i) raw milks and heated milks; and (ii) milks renneted at 30°C from those renneted at 40°C. The results showed the ability of SFS as a rapid and non-destructive technique for the: (i) monitoring network structure and molecular interaction during the coagulation process; and (ii) determination of gelation time of rennet-induced coagulation of studied milk samples.     

Gelation properties of partially renneted milk

Acid- and rennet-induced gelation properties of milk with modified casein micelles, produced by partial renneting at 4^oC for 15 min, followed by inactivation of enzymes by heat at 60^oC/3 min (referred as low heat treatment milk) and 85^oC/30 min (high heat treatment milk), were investigated to provide a mechanistic understanding of gel formation from partially renneted milk. Acidification of low heat treatment milk gave firmer gel quality, this was reflected by its high elastic modulus (G′) and hardness. In addition, the high heating condition for enzyme inactivation of high heat treatment milk alone increased the elastic modulus of both the control and renneted milk samples. Gel development of the two milk types (low heat treatment and high heat treatment milks) was different. In contrast with acid gelation, rennet-induced gelation of partially pre-rennet treated milk had no impact on the elastic modulus of low heat treatment milk and the rennet gels were very weak. Similarly, the addition of rennet to pre-rennet treated high heat treatment milk did not produce “true gels,” most likely due to the effect of the heat treatment on impairing the rennet coagulation. The findings in this study confirmed that pre-rennet treated milk had positive effects on the end-product acid gels of low heat treatment and high heat treatment milk.     

The effect of nisin on growth kinetics from activated Bacillus cereus spores in cooked rice and in milk

The growth kinetics of germinated cells from activated spores of Bacillus cereus in cooked white rice and in milk were evaluated at different temperatures for control samples and for samples with 25 microg of nisin per ml added. Nisin was applied in the form of Nisaplin (10(6) IU/g), which contained 25,000 microg of nisin per g. The length of the lag phase for cooked white rice controls was 120 h at 10 degrees C, 8 h at 25 degrees C, and 2.5 h at 33 degrees C. The generation times for cooked rice were 327.7 min at 10 degrees C, 59.0 min at 25 degrees C, and 42.3 min at 33 degrees C; those for milk without nisin were 297.0 min at 20 degrees C, 31.2 min at 30 degrees C, 28.6 min at 35 degrees C, and 33.7 min at 40 degrees C; and those for milk with nisin added were 277.2 min at 20 degrees C, 66.9 min at 30 degrees C, and 66.4 min at 35 degrees C. No development of B. cereus was observed for milk with nisin added at 40 degrees C for 12 h, in which germinated cells decreased by a decimal reduction time (D) of 4.7 h. A temperature of 45 degrees C was shown to be harmful to B. cereus, decreasing the germinated cells in both formulations with D-values of 4.3 to 4.6 h. Similar inhibition of cell growth at 40 degrees C was not observed with lower nisin concentrations.     

Fumonisin B1 production by Fusarium moniliforme and Fusarium proliferatum as affected by cycling temperatures

The effects of temperatures cycling between 5 and 20 degrees C, 10 and 25 degrees C, and 15 and 30 degrees C on the production of fumonisin B1 (FB1) and ergosterol by Fusarium moniliforme and Fusarium proliferatum on rice was studied. Temperatures were cycled at 12-h intervals by manually moving cultures from one temperature to another. Constant temperature incubation at 25 degrees C and a low temperature stress were compared with the cycling temperature incubations. Low temperature stress was achieved by incubating rice cultures at 25 degrees C for 2 weeks followed by 15 degrees C for 4 weeks. The maximum yields of FB1 were found to be 247 microg/g by F. moniliforme at temperatures that cycled between 10 and 25 degrees C after 2 weeks and 284 microg/g by F. proliferatum when the temperatures cycled between 5 and 20 degrees C after 6 weeks. Ergosterol content of the rice cultures was also monitored. Overall, the two Fusarium species showed differences in production of FB1 and ergosterol under the various temperature treatments. The most notable differences were that the temperature treatments that stimulated greatest FB1 production were different for each species: cycling temperatures between 10 and 25 degrees C for F. moniliforme and cycling temperatures between 5 and 25 degrees C for F. proliferatum. At most temperatures, F. proliferatum produced more ergosterol than F. moniliforme. Maximum production of ergosterol by F. proliferatum occurred at 6 weeks, with temperatures that cycled between 10 and 25 degrees C, whereas F. moniliforme produced maximum amounts of ergosterol at 6 weeks, with temperatures that cycled between 15 and 30 degrees C.