INFLUENCE OF LIMITED PROTEOLYSIS WITH IMMOBILIZED OR SOLUBLE ENZYMES ON THE WHITENESS OF SKIM MILK

Limited proteolysis with milk clotting enzymes can increase the whiteness of skim milk. Porcine pepsin and chymosin were immobilized onto nonporous ceramic, glass, and controlled pore glass (CPG) beads. The enzymes were coupled to beads either directly or via crosslinker proteins. Pepsin, immobilized onto CPG beads via crosslinker proteins, exhibited the best properties with respect to enzymatic activity, stability, and whitening efficiency. The Hunter L value (whiteness) of this immobilized enzyme‐treated skim milk was 79.5, which approached the whiteness of l% fat milk. Immobilized proteases whitened skim milk more effectively than soluble proteases.     

Influence of solvent extraction on the dietary fibre fractions of alfalfa protein concentrates

“Freezing curd” was prepared from alfalfa juice and extracted with 2-propanol, either untreated or after washing with water. The soluble and insoluble dietary fibre fractions of the two extracted concentrates and the unextracted freezing concentrate were analysed to assess the effect of extraction on fibre content. Compared with unextracted “freezing curd”, direct extraction with 2-propanol without washing increased the insoluble and soluble dietary fibre fractions by approximately 55% and 52% respectively, while washing with water followed by extraction with organic solvent led to an increase of about 79% and 28%, respectively. Thus, the washing with water lowered the soluble fibre by around 16%. Analysis of the ash and proteins in the crude fibre residues showed that while the ash content was similar in the residues for both the soluble and insoluble fibre, the protein content was much higher in the insoluble fibre residue.     

Utilization of immobilized archaeal chaperonin for enzyme stabilization

CpkA and CpkB are thermostable chaperonins from hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. CpkA or CpkB was immobilized onto agarose beads by the carbodiimide coupling method. In order to investigate whether immobilized CpkA or CpkB stabilizes a foreign enzyme, their ability to stabilize enzyme was examined using beta-D-galactosidase from Escherichia coli as a model target. In the absence of chaperonin beads, 38.1% of the total soluble protein was precipitated by heat treatment at 53 degrees C for 30 min. The protein structure of the residual soluble fraction was examined by circular dichroism, indicating that beta-D-galactosidase exists as a mixture of the active folded form and the inactive unfolded form. The specific activity of the residual soluble fraction decreased to 85.1% that of the unheated level (from 149.0 U/mg to 127.0 U/mg), indicating that the active folded form in the heated soluble fraction was 85.2%. In the presence of CpkA- or CpkB-beads, 28.6% or 34.3% of beta-d-galactosidase was precipitated by the same heat treatment. However, the specific activity in the soluble fraction was almost maintained (CpkA, from 151.0 U/mg to 140.3 U/mg; CpkB, from 149.0 U/mg to 140.3 U/mg). These results indicated that CpkA- or CpkB-beads are useful for keeping the enzyme active.     

Stability and Kinetic Properties of a Magnetic Immobilized alpha-Amylase K

The properties of α-amylase K immobilized on hydrous titanium(IV)oxide coated magnetic iron oxide are reported and compared with the previously reported properties of the soluble form of the enzyme. The optimum pH was increased on immobilization but the addition calcium chloride caused a decrease. Compared to the soluble form the immobilized enzyme is less stable at 60 °C in calcium enriched buffers but more stable in calcium free buffers. The temperature-activity profile has a plateau between 40 °C and 60 °C attributable to a comformational change above 60 °C to a more active form. The action pattern in the hydrolysis of soluble starch was found to be unaffected by immobilization. Parameters affecting the amount of bound activity were studied. The magnetic recovery of the immobilized enzyme was complete.     

Quality and ripening changes of Ras cheese made by direct acidification

Trials were carried out to produce Ras cheese of good quality without the use of starter. Cheese was made from pasteurized cow's milk acidified with lactic acid or citric acid to pH 5.8 alone or coupled with mixing the curd with glucono δ lactone (4.5 g/kg curd). Control cheese was made from milk ripened with a starter culture of S. lactis. Resultant cheeses showed poor body and texture, weak flavour intensity and low levels of soluble nitrogen compounds and free volatile fatty acids. Incorporation into the cheese curd of mixtures containing Fromase 100 (fungal protease) and Piccantase B (fungal lipase) or Fromase 100 and Capalase K (animal lipase) enhanced flavour intensity, improved body characteristics and accelerated the formation of both soluble nitrogen compounds and free volatile fatty acids. The organoleptic properties of the experimental cheeses with added enzymes were comparable to those of the control cheese.     

豆渣腐乳酿制的食用胶筛选及后发酵中营养品质的变化

为解决豆制品企业鲜豆渣大量堆积腐败变质和低值化利用问题，以纯豆渣为原料、毛霉为发酵菌种进行前发酵，利用食用胶将前发酵后的基料成型再后发酵酿制豆渣腐乳。先通过跟踪检测豆渣腐乳的质构，从瓜尔豆胶、魔芋胶、可得然胶、沙蒿子胶、海藻酸钠、聚丙烯酸钠6种食用胶中筛选适宜的食用胶种类及添加量，再通过跟踪考察在40 ℃、28 ℃、室温条件（25~35 ℃）下后发酵的豆渣腐乳中粗蛋白、可溶性蛋白、氨基酸态氮和总酸含量随发酵时间的变化规律以及挥发性风味物质含量，确定后发酵适宜的温度和时间。结果表明，添加0.1%魔芋胶使豆渣腐乳具有较优质构；3种温度下发酵的豆渣腐乳中粗蛋白、可溶性蛋白、总酸和氨基酸态氮含量均随发酵时间的增加呈现先增大后逐渐趋于平稳的变化趋势，3种温度下酿制的豆渣腐乳的粗蛋白含量相差不大，而40 ℃下发酵的豆渣腐乳中可溶性蛋白和氨基酸态氮的含量最高，分别为5.294和0.63075 g/100 g，总酸变化稳定且含量最低，为8.772 g/100 g，产品所需要的成熟时间最短，为35 d，另外，40 ℃下的酯类有12种，含量为55.10%，种类最多含量最高，产品风味品质更优。     

Characterization of Cheese Curd Ripened with Penicillium caseicolum for Producing a Flavor Concentrate

Camembert cheese was prepared from homogenized milk containing veal oral lipase and ripened in a loose curd form to speed up the ripening process. Traditional hooped cheese was a control. Free fatty acid titer increased from 58 μmole in the fresh cheese to 359 μmole in control cheese and 2,289 μmole/g of cheese fat in loose curd cheese. After 3 wk of ripening, the cheese had 70% of its total protein as water soluble protein as compared to 39% in the hooped cheese. Polyacrylamide gel electrophoresis showed 86% of α_S1-caseins and 35% of β-casein had been degraded in the loose curd cheese. Sensory analysis demonstrated that the loose curd process rapidly produced cheese with intense flavor.     

The localization of phytate in tofu curd formation and effects of phytate on tofu texture

ABSTRACT:  The localization of phytate on tofu making and its effects on tofu texture were investigated. Thirty-eight percent and 3% of phytate in soymilk were bound to soluble protein and particulate protein, respectively, and the others were in free form. In the early stage of curd formation, phytate bound to particulate proteins and then a large part of phytate was taken into the tofu curd. Increase of phytate contents in soymilk resulted in the increase of coagulant requirement to make firm tofu. Optimal coagulant concentration (OCC) for making tofu was influenced by phytate contents. The increase of phytate in tofu caused decrease of hardness at OCC and resulted in the increase of brittleness and viscosity of tofu. We concluded that the phytate content is one important factor that contributes to tofu texture.     

Effect of fat content on sensory and physico‐chemical properties of laboratory‐pasteurised calcium‐ and vitamin D‐fortified mixture of cow and buffalo milk

The influence of milk fat on physico‐chemical properties of calcium and vitamin D‐fortified milk was investigated. Sensory scores, curd tension, viscosity, rennet coagulation time and TBA value increased with the increase in fat content. Calcium and vitamin D fortification had no effect on sensory scores, whereas a significant increase was observed in curd tension and viscosity. The TBA value of fortified milk was significantly lower than that of the unfortified milk. The rennet coagulation time of milk increased significantly with addition of calcium phosphate, whereas calcium citrate fortification had no significant effect. All milk samples were stable to alcohol.     

Improvement of low fat mozzarella cheese properties using denatured whey protein

Mozzarella cheese was made from buffalo milk (6% fat) or from partially skimmed buffalo milk (2 and 4% fat) with 0.5 and 1% denatured whey protein. Adding whey protein to buffalo milk decreased rennet coagulation time and curd tension whereas increased curd synaeresis. Addition of whey protein to cheese milk increased the acidity, total solids, ash, salt, salt in moisture, also some nitrogen fractions. The meltability and oiling‐off values increased but the calcium values of mozzarella cheese decreased. The sensory properties of low fat mozzarella cheese were improved by addition of whey protein to the cheese milk.     

Mineral equilibrium in commercial curd and predictive ability of near-infrared spectroscopy

Curd samples (n = 83) from 3 European dairy companies were analyzed for micellar and soluble mineral fractions content using inductively coupled plasma optical emission spectrometry as a gold standard method. The same curd samples were analyzed through 3 different near-infrared (NIR) instruments, and NIR spectra were merged with reference data. Prediction equations were developed using modified partial least squares analysis, and the accuracy of prediction was evaluated through leave-one-out cross validation. Overall, NIR spectroscopy was capable of predicting micellar and soluble mineral fractions in curd, but with differences among instruments. Fitting statistics showed that the visible NIR instrument in reflectance mode outperformed the NIR instrument in transmittance mode as well as the portable NIR instrument in reflectance mode. Prediction accuracies for most of the analyzed mineral fractions can be used for curd quality control in dairy companies and to aid in decision-making during the cheesemaking process.     

Comparison of the milk-clotting properties of three plant extracts

Several proteases from plant sources have been proposed as milk coagulants, however, limited research has been done on their milk-clotting properties. The effect of temperature on the milk-clotting activity of kiwi fruit, melon and ginger extracts was evaluated, as well as the effects of the different extracts on curd properties. Melon extracts showed high milk-clotting activity over a broad temperature range (45–75 °C) while kiwi fruit and ginger extracts showed high activity over a narrower temperature range, with a maximum at 40 and 63 °C, respectively. Curds produced using kiwi extracts had textural properties comparable with those obtained using commercial rennet, while melon extracts produced a fragile gel and low curd yield. The milk-clotting behavior of the three plant extracts was related to the protease specificity present in these extracts. The kiwi proteases displayed chymosin-like properties and thus hold the best potential for use as a milk coagulant in cheese production.     

Milk Curdling by Rennet under High Pressure

The effect of pressure on milk curd formation initiated by rennet was studied under high pressures. The milk used was a 20% solution of skim milk powder in 0.02M CaCl₂. After holding the milk with rennet at 35°C for given periods under high pressure, up to 1300 kglcm², the extent of proteolytic digestion and the curd tension were measured under atmospheric pressure. Rennet and milk protein were not denatured by pressure under the experimental conditions used. The primary phase reaction of milk curdling by rennet (hydrolysis of casein) was not affected by compression. The secondary phase reaction (core formation of casein micelle aggregates) was delayed under high pressure and the tertiary phase reaction (milk curd formation) was accelerated.     

Enzymic differentiation between curds of heated and raw milk II. Milk peroxidase

Unlike phosphatase, peroxidase, once destroyed by heating of milk, is not regenerated by the acidity or microbial growth of curd formation or by reasonable ageing. This reveals that curd-forming micro-organisms do not have or form peroxidase. The test was repeated in various heating conditions of milk. Peroxidase is destroyed even when the temperature of milk is gradually raised to 80 °C (in 3 min). Less-heated milk, e.g. pasteurised milk and its corresponding curd, retain this enzyme. A positive test in the curd of raw milk is not affected by reasonable ageing or increased acidity but by excessive fermentation (secondary fermentation or putrefaction) which again can be halted by addition of formalin. The peroxidase test, therefore, appears to be a test for differentiating curds of heated and unheated milk.     

Improving the quality of Ras cheese made without starter

Trials were carried out to produce Ras cheese of good quality without the use of starter. Cheese was made from cows' milk, pasteurized and acidified with lactic acid or citric acid to pH 5·8; cheese was also made after adding 4·5 g glucono-δ-lactone per kg to the acidified curd. Control cheese was made by the traditional method.The cheese had poor body and texture, weak flavour intensity, and low levels of soluble nitrogen compounds and free volatile fatty acids.Incorporation into the cheese curd of mixtures containing Fromase 100 (fungal protease) and Piccantase B (fungal lipase) or Fromase 100 and Capalase K (animal lipase) enhanced flavour intensity and body characteristics and accelerated the formation of both soluble nitrogen compounds and free volatile fatty acids. The organoleptic properties of the experimental cheese with added enzymes were comparable to the control cheese.     

姜汁凝乳影响因素的研究

生姜经清洗、打浆榨汁后可制得姜汁,将姜汁以2%的比例添加到经杀菌和冷却处理的鲜乳中可制成具有凝乳状态的姜汁牛乳。为了探讨姜汁凝乳的影响因素,文中研究了温度、pH值、牛乳蛋白质和盐类等对姜汁凝乳的影响。结果表明,温度和pH值是影响姜汁凝乳的关键外部因素。牛乳蛋白质、CaCl2、NaCl是凝乳促进物。     

固定化碱性蛋白酶制备麦胚降血压肽研究

采用中空壳聚糖作为载体,通过双功能试剂戊二醛作偶联剂,将碱性蛋白酶连接到中空壳聚糖,制备固定化碱性蛋白酶。研究固定化碱性蛋白酶的部分性质及水解麦胚蛋白质条件。实验结果显示,1.5% 戊二醛溶液活化的壳聚糖偶联1mg 碱性蛋白酶,酶催化相对活性可达90%;固定化酶最适pH 值为9.5,最适反应温度约65℃,催化水解麦胚蛋白质的水解率达16.5% 约需10min;固定化酶与麦胚蛋白质的比达到1:5 时,一次性获取含降血压肽水解物质的得率为15.6%。     

Chemical interactions among caseins during rennet coagulation of milk

Rennet milk curds were prepared under 4 different temperature and acidity conditions. The development of different types of inter-protein chemical bonds (disulfide, hydrophobic, electrostatic, hydrogen, and calcium bridges) was monitored for 60 min after curd cutting. Hydrophobic inter-protein interactions originally present in casein micelles in milk were substituted by electrostatic, hydrogen, and calcium bonds throughout the curd curing period. Disulfide bonds were not disturbed by the experimental conditions employed in the study, remaining at a constant level in all studied treatments. Acidification of curds increased the availability of soluble ionic calcium, increasing the relative proportion of calcium bridges at the expense of electrostatic-hydrogen bonds. Although pH defined the nature of the interactions established among proteins in curd, temperature modified the rate at which such bonds were formed.     

Study of Two Different Immobilized Acid Proteases for Wine Application

This study compares the hydrolytic efficiency in model wine (pH 3.2 tartaric acid buffer with 12% v/v ethanol) of two immobilized enzymes. Food-grade acid proteases (from Aspergillus saitoi and stem bromelain) were covalently immobilized on two solid supports, aminopropyl controlled pore glass beads and Eupergit®C. The effects of pore diameter, immobilization procedure, and protein properties on the activity of biocatalysts were evaluated to determine the feasibility of immobilized proteases in wine. The results expressed in terms of yield of immobilized enzyme, enzyme activity, and kinetic parameters (V_max and K_m) demonstrated that the best performance of both proteases was when immobilized in Eupergit®C.     

Technical note: Development and validation of a new method for the quantification of soluble and micellar calcium,magnesium, and potassium in milk

Milk mineral content is a key trait for its role in dairy processes such as cheese-making, its use as source of minerals for newborns, and for all traits involving salt-protein interactions. This study investigated a new method for measuring mineral partition between soluble and micellar fractions in bovine milk after rennet coagulation. A new whey dilution step was added to correct the quantification bias due to whey trapped in curd and excluded volume. Moreover, the proposed method allowed the quantification of the diffusible volume after milk coagulation. Milk mineral content and concentration in whey, and diluted whey were quantified by acid digestion and inductively coupled plasma optical emission spectrometry. The repeatability of the method for micellar Ca, Mg, and K was between 2.07 and 8.96%, whereas reproducibility ranged from 4.01 to 9.44%. Recovery of total milk minerals over 3 spiking levels ranged from 92 to 97%. The proposed method provided an accurate estimation of micellar and soluble minerals in milk, and curd diffusible volume.