Chymosin Concentration Research Articles

Structure and shelf stability of milk protein gels created by pressure-assisted enzymatic gelation

In this work, pressure-assisted enzymatic gelation was applied to milk proteins, with the goal of enhancing the structure and stability of pressure-created milk protein gels. High-pressure processing (HPP) at 600 MPa, 3 min, and 5°C was applied to milk protein concentrate (MPC) samples of 12.5% protein concentration, both in the absence and in the presence of calf chymosin [up to 60 IMCU (international milk-clotting units)/kg of milk] or camel chymosin (up to 45 IMCU/kg of milk). Gel hardness, water-holding capacity, and degree of proteolysis were used to assess network strength and shelf stability. The processing trials and all measurements were conducted in triplicate. Statistical analyses of the data were performed by ANOVA, at a 95% confidence level. After HPP treatment, we observed significant structural changes for all samples. Pressurization of MPC, with or without chymosin addition, led to extensive protein aggregation and network formation. The strength of HPP-created milk protein gels without chymosin addition, as measured by the elastic modulus (G'), had a value of 2,242 Pa. The value of G' increased with increasing chymosin concentration, reaching as high as 4,800 Pa for samples with 45 IMCU/kg of camel chymosin. During 4 wk of refrigerated storage, the HPP and chymosin MPC gels maintained higher gel hardness and better structural stability compared with HPP only (no chymosin) MPC gels. The water-holding capacity of the gels without chymosin remained at 100% during 28 d of refrigerated storage. The HPP and chymosin MPC gels had a lower water-holding capacity (91-94%) than the HPP-only counterparts, but their water-holding capacity did not decrease during storage. Overall, these findings demonstrate that controlled, fast structural modification of high-concentration protein systems can be obtained by HPP-assisted enzymatic treatment, and the created gels have a strong, stable network. This study provides insights into the possibility of using HPP for the development of milk-protein-based products with novel structures and textures and long refrigerated shelf life, along with the built-in safety imparted by the HPP treatment.

The inhibitory roles of native whey protein on the rennet gelation of bovine milk

Rennet gelation is used to produce many types of cheese. The effect of native whey protein on rennet gelation kinetics was investigated. Milks with a wide range of whey protein:casein (WP:CN) ratios (with standardised casein concentrations) were made from powders produced by microfiltration. Measurements of casein macro peptide release showed that native whey protein inhibited the enzymatic action of chymosin, which delayed the onset and reduced the subsequent rate of gelation. Experiments in which increased chymosin concentrations compensated for the inhibition, demonstrated that other factors also contributed to the reduced gelation rate. Neither an increase in viscosity nor a reduction in soluble calcium was responsible, leading to the conclusion that in addition to inhibiting chymosin, native whey proteins present a physical barrier to para-casein aggregation. This study demonstrates and explains how casein-enriched retentates from microfiltration gel faster than regular cheese milk that contains higher amounts of native whey protein.

Changes in volatile composition and sensory properties of Iranian ultrafiltered white cheese as affected by blends of Rhizomucor miehei protease or camel chymosin.

The effect of using various combinations of Rhizomucor miehei protease and camel chymosin (100:0, 75:25, 50:50, 25:75, and 0:100, respectively) on volatile composition and sensory scores in Iranian ultrafiltered white cheese was studied during 90d of ripening. A solid-phase microextraction-gas chromatography-mass spectrometric method was used for determining the volatile compounds of the cheeses. Forty compounds including esters (12), acids (6), ketones (9), alcohols (3), and miscellaneous compounds (10) were identified. The main classes of volatile components in the cheeses are esters, miscellaneous compounds, and ketones. The type and concentration of the coagulants influenced both volatile composition and sensory scores of the cheeses. Principal component analysis separated the cheeses based on the use of 2 coagulants in various combinations and ripening time. The cheeses produced using higher concentrations of R. miehei were separately located on the plot compared with the cheeses produced using higher concentrations of camel chymosin. Sensory evaluation of the cheeses showed that, in general, the cheeses produced using higher concentrations of camel chymosin received higher body and texture and odor and flavor scores than the cheese produced using higher concentrations of R. miehei. In conclusion, 2 combinations of R. miehei and camel chymosin (75:25 and 25:75, respectively) can be successfully used for the production of Iranian ultrafiltered white cheese, considering the results of volatile composition and sensory analysis.

Partial renneting of pasteurised bovine milk: Casein micelle size, heat and storage stability

The effects of partial renneting at low temperature on the casein micelle (CM) size and the storage stability of milk were investigated. Low chymosin concentrations (≤0.03IMCUmL−1) was applied to pasteurised skim milk at 4°C and enzyme activity was terminated by thermal application at 60°C/3min and 85°C/30min, referred to as low heat (LHT) and high heat (HHT) treatment milk, respectively. The addition of rennet with concentrations of 0.01, 0.02 and 0.03 IMCU mL−1 for 15min resulted in κ-casein hydrolysis of 10, 20 and 25%, respectively. Moreover, mean CM size of milk was reduced by up to 10nm. For LHT milk, the renneted micelles appeared to be stable for up to 17days, especially in response to the application of 0.01IMCUmL−1 and at a storage temperature of 4°C. Severe heating at 85°C/30min to inactivate the enzyme caused an increase in CM size.

Factors influencing the gelation and rennetability of camel milk using camel chymosin

The effects of temperature, pH, concentration of camel chymosin and addition of CaCl2 on the hydrolysis of κ-casein (κ−CN) and the coagulation kinetics of camel milk were investigated. The rate of κ−CN hydrolysis was higher at 40 °C than at 30 °C and with increasing addition of chymosin and decreasing pH. For all samples gelation was initiated at levels of camel milk κ−CN hydrolysis >95%. The gelation time (Tg) of camel milk was significantly reduced (from 717 to 526 s) at 30 °C when the concentration of chymosin was increased, but was independent of chymosin concentration at 40 °C. Reducing pH also reduced Tg. The gel firmness increased at 40 °C (58 Pa) compared with 30 °C (44 Pa) and effect of CaCl2 addition on the gelation properties of camel milk was found to be dependent on pH; a significant improvement was only found at pH 6.3.

Effect of various blends of camel chymosin and microbial rennet (Rhizomucor miehei) on microstructure and rheological properties of Iranian UF White cheese

Abstract Various blends of milk coagulants Rhizomucor miehei protease and camel chymosin were mixed at levels of 100:0, 75:25, 50:50, 25:75 and 0:100, respectively and used in the manufacture of Iranian ultrafiltered (UF) White cheese. Effect of different blends of these coagulants on microstructure and rheological properties of Iranian UF White cheese were studied during 90 days of ripening. The results showed that both microstructure and rheology of the cheeses were influenced by type and concentration of these coagulant blends due to their different proteolytic activities. Use of high concentrations of camel chymosin resulted in a more compact protein network and firmer structure in the cheeses. Increasing levels of camel chymosin in the coagulant blends increased the storage ( G′ ) and loss ( G″ ) moduli and decreased loss tangent (δ) in the cheeses. In conclusion, increasing levels of camel chymosin provided less protein breakdown and more solid-like viscoelastic structure in UF cheeses.

Detection of glycomacropeptide content in breast milk and comparison with varieties of newborn infant formula

Objective To detect the glycomacropeptide(GMP) content of human breast milk, and take it as reference of newborn infant formula milk powder to optimize the nutritional content of infant formulas. Methods Thirty primiparas who fit the following conditions were selected: healthy, no special diet habits, living stably, having adequate milk, aged between 25 to 39 years old, and term delivery, were selected.Then they were divided into 2 groups, the colostrum group and mature milk group, and each group had 15 cases.Each case had been collected of breast milk 5 mL(front milk). The gathering time of colostrum group was the 2nd day postpartum and mature milk group was the 42nd day postpartum.Hydrolyzing the breast milk by chymosin at 37 ℃, then the sialic acid(SA) content was detected by a sialic acid detection kit, and GMP content was represented by SA content.In addition, 6 brands of formula milk powder were detected in the same way as breast milk after being made into standard liquid milk.The differences between groups were compared by analysis of variance(ANOVA). Results Best conditions for enzymolysis were: chymosin concentration 0.25 g/L, hydrolysis time 120 min.SA content of colostrum group was (3486.98±406.70) mg/L, while mature milk group was(2687.95±375.85) mg/L, as the former was significantly higher than the latter(P<0.01), but the diffe-rences between individuals within each group were small(CVcolostrum=0.12, CVmature milk=0.14). The average level of SA content of various infant formulas was (1196.93±608.40)mg/L, which was significantly lower than colostrum and mature milk(all P<0.01). SA contents of various brands of formulas were various, and the difference among these brands was relatively big(CV=0.63). Conclusions The content of GMP in human colostrum was higher than that of the mature milk.The contents of the GMP in different brands of infant formula milk powder were uneven, and the measured values were quite different with human milk.However, because of the difference of the molecular weight of GMP and the number of amino acid residues between human milk and bovine milk, the amount of GMP required to achieve the same physiological effects may be different in breast milk and bovine milk.In order to optimize the nutritional content of infant formula, and make it even closer to human milk, it is necessary to further explore the best GMP content relative to human milk. Key words: Glycomacropeptide; Sialic acid; Breast milk; Formula

The effect of concentration of chymosin on the yield and sensory properties of camel cheese and on its microbiological quality

The transformation of camel milk into soft cheese by using chymosin and yoghurt starter culture (Streptococcus thermophilus and Lactobacillus bulgaricus) was investigated. The cheese yield and sensory properties were related to the concentration of chymosin. A yield of 16.74 g/100 mL of milk was obtained with a chymosin concentration of 1.7 mL/L of milk. The cheeses obtained with concentrations ranging between 1.0 mL and 2.9 mL of chymosin/L of milk scored highly regarding their sensory properties and had an acceptable microbiological quality. This study demonstrated that cheesemaking from camel milk can be made successfully providing that the appropriate chymosin concentration is used; and that 1.7 mL of chymosin/L of milk was optimal.

The association of chymosin with artificial casein micelles

The influence of casein micelle composition on the extent of chymosin association is described. The association of chymosin with artificial micelles, made with combinations of αs-or β-casein with κ-casein, in a compositional range of αs or β:κ of 12 : 1–1 : 5, and at various pH values has been studied. Isotherms for chymosin association with micelles were constructed. Those composed of β- and κ-casein retained more chymosin than micelles composed of αs- and κ-casein. The chymosin association increased with higher κ-casein content and with dilution of the system. Furthermore the association did depend on contact time, on casein and chymosin concentration, on temperature and on pH. It is hypothesized that chymosin associates mainly with para-κ-casein but that αs- and β-casein and para-κ-casein itself also associate with para-κ-casein; competition between these caseins and chymosin then determines the extent of association of the latter with the para-casein micelles. A simplified model for this competitive association is presented.The model can explain several observations on rennet retention in curd.

Effect of Salt and Chymosin on the Physico-Chemical Properties of Feta Cheese During Ripening

The physico-chemical properties during ripening of Feta cheese made from bovine milk were monitored at 7-d intervals for 63 d. Samples were analyzed for moisture, fat, protein and ash contents, salt uptake, pH, and hardness. Three percentages of salt in brine (8, 15, and 18%) and three concentrations of double-strength chymosin (0.5, 1, and 2.5 ml/10 L of milk) were investigated. Salt had a significant effect on moisture, uptake of salt, pH, and hardness. Results indicated that higher percentages of salt in brine developed a harder cheese with higher salt content and higher pH values but lower moisture content. High rennet concentration had a significant effect on the uptake of salt. Increasing the concentration of rennet gave a softer cheese with higher salt content. Rennet level had no significant effect on the moisture or protein contents or pH of the cheese.

Gel firming rate of rennet curd as a function of rennet concentration

The rate of gel firming, expressed as dG∗/dt passes through a maximum when plotted as a function of measured gel firmness. This maximum rate is a non-linear function of chymosin concentration, indicating that it is not solely determined by the rate of κ-casein proteolysis.

Sensitive fluorometric assay for the activity of chymosin.

Fluorogenic substrates for chymosin [Dns-Leu-Ser-Phe-Trp-Ala-Leu-OCH2Py (I), Dns-Leu-Ser-Phe-Met-Trp-Leu-OCH2Py (II), Dns-Leu-Ser-Leu-Trp-Ala-Leu-OCH2Py (III), Dns-Leu-Ala-Phe-Trp-Ala-Leu-OCH2Py (IV), Dns-Leu-Ser-Phe-Leu-Ala-Leu-OCH2Py (V) and Dns-Leu-Ser-Phe-Phe-Ala-Leu-OCH2Py (VI)] were synthesized by a solution method. The obtained substrates I-VI were cleaved specifically (between the Phe and Trp residues for substrates I and IV, the Phe and Met residues for substrate II, the Leu and Trp residues for substrate III, the Phe and Leu residues for substrate V, and the Phe and Phe residues for substrate VI) by chymosin. The fluorescence of substrates I-IV (345 nm) increased with their hydrolysis, and hydrolysis rates were obtained by measuring the increase in fluorescence. The minimum detectable chymosin concentrations for substrates I and IV were about 1 nM; those for substrates II and III were about 4 and 2 nM. This assay method is very sensitive, and it is possible to determine the chymosin activity rapidly and easily. Substrates I and IV-VI were hydrolyzed by chymosin two times faster than substrates II and III. The effect of the amino-acid residues of the substrates on the hydrolysis rate is discussed.

Detection of residual chymosin in cheese by an enzyme-linked immunosorbent assay

SummaryA simple and rapid competitive enzyme-linked immunosorbent assay method has been developed to detect the residual activity of chymosin in various types of cheese without extraction. In a cheese of the Saint-Paulin type, concentrations of chymosin detected at the end of cheesemaking in curd and whey represented 22·5 and 73·5% respectively of the total amount added to milk, so most of the chymosin was detected using this method. In a cooked pressed cheese of the Comté type, only 14% of the chymosin added to milk was detected in curd and all the chymosin in whey was inactivated. It was thus possible to demonstrate the presence of active residual chymosin in cooked pressed cheese. When measurements were carried out in 12 different cheeses, chymosin concentrations ranged between 384 ng/g in a Camembert cheese and 86 ng/g for an 8-month old Comté cheese. Chymosin appeared to be highly stable during cheese ripening.

Quantitative determination of chymosin activity by thrombelastography

An accurate and objective thrombelastographic procedure for the quantitative determination of chymosin in solution and in cheese whey is proposed. Linear relationships between coagulation time and the inverse of chymosin concentration in solution showed determination coefficients (r 2) in the range 0·989–0·997. A satisfactory reproducibility was achieved, with 0·88% as mean coefficient of variation. Determination coefficients for linear relationships between coagulation time and the inverse of chymosin present in whey ranged from 0·995 to 0·998. Reproducibility of chymosin quantification in whey was high, with 0·98% as mean coefficient of variation.

An Enzyme-Linked Immunosorbent Assay for Detection of Chymosin in Dairy Products

An enzyme-linked immunosorbent assay has been developed to detect very low concentrations of chymosin. The method was evaluated by determination of chymosin in fermented milk. A four-layer sandwich ELISA was used, which allows a sensitivity between .5 to 10ng chymosin/ml fermented milk. Samples of fermented milk, with and without addition of calf rennet, were tested. The blind samples were clearly distinguished from the samples containing chymosin. However, some adsorption of the chymosin to the caseins and a high standard deviation within the samples disturb a reliable estimation of the true chymosin concentration, indicating that some unknown components interfere with the analytical procedure.

Actual Milk Coagulation Time and Inverse of Chymosin Activity

Coagulation of Berridge substrate by several different chymosin concentrations was followed with a spectrophotometer to obtain actual coagulation times. Actual coagulation times plotted as functions of inverse enzyme concentrations produced an intercept within experimental error of zero. Deviations of this intercept from zero are artifacts of methods used to measure coagulation time.

Identification of human milk kappa-casein on polyacrylamide gels by differential staining with Ethyl-Stains-all and chymosin sensitivity.

Ethyl-Stains-all (ESA), a cationic carbocyanine dye that stains phosphorylated, sialylated, and unmodified proteins differentially, was used to stain a human casein fraction enriched for its kappa-casein-like characteristics. The staining properties and chymosin sensitivity of this fraction were compared with those of human milk and bovine casein proteins. Phosphorylated human and bovine beta caseins stained blue with ESA. The sialic acid-containing bovine kappa-casein stained blue-green. The human kappa-like fraction was enriched for a protein that stained blue-green with ESA. Both bovine kappa-casein and the human blue-green-staining protein were susceptible to chymosin digestion at lower concentrations of chymosin than that required for digestion of beta-caseins. In each case, following chymosin digestion, a green-staining peptide of lower molecular weight replaced the original protein and para-kappa-casein was formed. Identification of human kappa-casein on SDS-polyacrylamide gels was based on its differential staining with ESA and chymosin sensitivity with respect to beta-casein.

Interactions of Calcium, pH, Temperature, and Chymosin During Milk Coagulation1,2

Holstein milk samples with good and poor chymosin-coagulation characteristics were coagulated in the Formagraph using different combinations of five levels of chymosin, three pH, and three temperatures in the presence and absence of .02% added calcium chloride.All the main factors significantly altered both coagulation time and curd firmness. Multiple comparisons of mean coagulation times showed that lower concentrations of chymosin (.01, .02, and .03 rennin units/ml milk) were significantly different in altering coagulation time and were different from higher concentrations (.04 and .05 rennin units/ml milk). The three pH produced significantly different mean coagulation times. Addition of more than .02 rennin units/ml milk was not necessary for adequate curd firmness in 30min after chymosin addition where the pH of the milk was 6.4 or lower. Addition of .02% calcium chloride to milk was not necessary for adequate curd firmness 30min after chymosin addition if other milk coagulation factors were adequately adjusted (pH≤6.4; chymosin concentration=.02 rennin unit/ml milk; temperature=37°C).

Effects of pH, Calcium Chloride, and Chymosin Concentration on Coagulation Properties of Abnormal and Normal Milk

Individual Holstein cow milk samples were selected for good and poor chymosin-coagulation characteristics. The effect of pH adjustment, addition of .02% calcium chloride, and variation in chymosin concentration on coagulation properties of good and poor coagulating samples was evaluated. Pooling 50% good and 50% poor coagulating samples did not improve the average coagulation properties of the poor samples. Reducing milk pH to 6.3 caused a significant decrease in coagulation time but a less marked increase in curd firmness. The greatest increase in curd firmness was obtained by a combination of reducing milk pH, addition of .02% calcium chloride, and reducing chymosin concentration. High-chymosin concentration at reduced pH decreased coagulation time without substantially increasing curd firmness. Curd disintegration was more apparent at high-chymosin concentration in the poor coagulating samples.

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.