Original papers
Effects of κ-Casein Dissociation from Casein Micelles on Cheese Curd Formation
2017 Volume 23 Issue 5 Pages 743-748
Details
2017 Volume 23 Issue 5 Pages 743-748
We studied the effects of κ-casein (κ-CN) dissociation from casein micelles upon heating on cheese curd formation. Cheese curd was formed by the addition of chymosin to unheated and heated (80°C, 30 min) defatted milk. In heated defatted milk, cheese curd was not formed; however, the amount of free glycomacropeptides (GMPs) and the degree of formation of para-κ-CN were the same in both the unheated and heated defatted milk. In addition, because calcium ions insolubilize upon heating, the calcium ion concentration in the heated defatted milk was adjusted to its equivalent amount in the unheated defatted milk; however, cheese curd was not formed in this case either. Therefore, chymosin was added to the casein micelles, which dissociated κ-CN. As a result, the amount of free GMP was significantly less (p < 0.05) than that in the native casein micelles. The results suggest that the quantity of formation of para-κ-CN on the micelle surface decreases due to κ-CN dissociation from the casein micelles.
Milk clotting, which is an essential process in the production of cheese, occurs due to the agglutination of casein micelles through the chymosin reaction. Chymosin hydrolyzes the Phe105-Met106 bond in κ-casein (κ-CN), which exists on the surface of casein micelles, and detaches the glycomacropeptide (GMP), thereby converting κ-CN into the highly hydrophobic para-κ-CN. The casein micelles coagulate due to hydrophobic interaction by the produced para-κ-CN and the crosslinking of calcium ions, resulting in milk clotting (Yokoyama and Yamauchi, 1992).
For cheese production, milk with a low degree of heat denaturation, such as raw milk or pasteurized milk, is used, while milk with a high degree of heat denaturation such as high-temperature-sterilized milk, does not undergo milk clotting, and thus, its cheese curd formation properties deteriorate significantly (Yokoyama and Yamauchi, 1992). This phenomenon occurs due to inhibition of the chymosin reaction by the formation of SS-bonds between κ-CN and β-lactoglobulin (β-Lg) (Leaver et al., 1995; Reddy and Kinsella, 1990; Van Hooydonk et al., 1987) and the insolubilization of calcium ions in milk because of heating (Ono, 2005; Tercinier et al., 2014), which prevents calcium crosslinking among para-κ-CN (Choi et al., 2007; Singh, 1988; Yokoyama and Yamauchi, 1992).
Recently, it has been reported that β-Lg forms SS-bonds with κ-CN present in casein micelles upon heating, and part of κ-CN dissociates from the micelles to become solubilized in the form of whey protein/κ-CN complex (Donato and Guyomarc'h, 2009; Jean et al., 2006). It has also been reported that the denatured whey protein and solubilized whey protein/κ-CN complex block the agglutination of casein micelles after the chymosin reaction to prevent milk clotting (Anema et al., 2011; Giroux et al., 2015; Kethireddipalli et al, 2010); thus, high-temperature-sterilized milk does not form cheese curd well. Numerous studies have reported on the relationship between high-temperature sterilization of milk and formation of cheese curd. On the other hand, few studies have focused on the aggregation of casein micelles. Overall, it was inferred that the dissociation of κ-CN affects cheese curd formation because κ-CN participates in micellar stabilization and milk clotting.
Therefore, the objective of this study is to examine the effects of inhibition of the chymosin reaction and insolubilization of calcium ions on milk clotting, and to clarify the effects of dissociation of κ-CN from casein micelles due to heating on the formation of cheese curd.
Preparation of milk material Raw milk was obtained from Holstein cows that were bred at the Tokyo University of Agriculture's Fuji Farm (Fujinomiya, Shizuoka). To eliminate the influence of milk fat, unheated defatted milk was used, which was obtained by removing milk fat at 40°C using a cream separator. Heated defatted milk was prepared by heating the unheated defatted milk at 80°C in a hot-water bath for 30 min.
Preparation of cheese curd Cheese curd samples were prepared following the method of Gaygadzhiev et al. (2012). Chymosin (CHY-MAX®, Chr. Hansen, Hoersholm, Danmark), prepared at a concentration of 4.5 IMCU (International Milk-Clotting Unit), was added to the milk material to a final concentration of 0.018 IMCU, and samples of cheese curd were prepared by heating at 32°C in a hot-water bath for 0, 10, 30, 60, 120 and 180 min. In addition, the calcium ion concentrations in both the unheated and heated defatted milk were measured by a calcium ion meter (LAQUAtwin B-751: HORIBA Ltd., Kyoto, Japan). Calcium ion concentrations in both samples were adjusted by supplementing with calcium lactate pentahydrate.
Physical property measurement The strength of the cheese curd was measured using a compression tester (Model 5564; INSTRON, Norwood, MA). Samples were compressed with a plunger of 7 mm diameter to a compression rate of 70%, and the breaking load (mN) was considered the curd strength. Each test was performed in triplicate, and the average value and standard deviation were calculated and analyzed by Tukey-Kramer's multiple comparison tests. Differences were considered significant at p < 0.05.
Electrophoresis analysis The milk material and cheese curd samples were analyzed using SDS-PAGE to determine the amount of dissociated κ-CN induced by heating, and the degree of para-κ-CN formation by the chymosin reaction. As per the method of Jean et al. (2006), the milk material and cheese curd samples were centrifuged (33,000 × g, 20°C, 65 min) and the supernatant and precipitate were separated. Samples (precipitate and supernatant) were respectively dissolved in 0.1 M phosphate buffer (pH 7.0) and treated with sample buffer containing 2-mercaptoethanol for protein reduction. Finally, the samples, in which the protein amount was adjusted to 15 µg, were analyzed by SDS-PAGE. Polyacrylamide gel was prepared at a concentration of 15%, and the protein in the gel was detected by the CBB staining method. Image analysis was performed by Chemi Doc (Bio-Rad Laboratories Inc., Hercules, CA).
Glycomacropeptide (GMP) quantification In order to evaluate the reactivity of chymosin, GMP detached by the dissolution of κ-CN was quantified. Following the method by Gaygadzhiev et al. (2012), 4% trichloroacetic acid (TCA) was added to each sample of cheese curd at a ratio of 1:1 to terminate the enzyme reaction. Samples were then centrifuged (5,000 × g, 20°C, 10 min), and 0.1 M NaOH solution was added to adjust the supernatant pH to 7.0. Then, for the reduction treatment, 50 µL of 0.1 M phosphate buffer (pH 7.0) containing 50 mM EDTA and 500 mM DTT was added to 850 µL of this sample. After the reduction treatment, the SH group was modified by adding 100 µL of 0.1 M phosphate buffer (pH 7.0) containing 500 mM iodine acetamide. Samples were filtered with a 0.45-µm filter, and RP-HPLC was conducted to determine GMP content. A ZORBAX300SB-C8 (Agilent Technologies Co. Ltd., Santa Clara, CA) column was used at an oven temperature of 45°C, and samples were eluted at a flow rate of 0.5 mL/min with a moving bed of linear gradient of A: 0.1% trifluoroacetic acid (TFA) and B: acetonitrile containing 0.1% TFA. Equilibration was obtained at a 30% concentration of B, and elution was performed first with a 37% concentration from 0 – 8 min, maintained until 12 min, and then with 40% from 12 – 18 min. Subsequently, from 18 – 22 min, the concentration was increased to 80% to remove adsorbed substances, and then from 22 – 26 min, the concentration was lowered to 30% to allow the flow of sample for 7 min. The total program duration was 33 min. Protein was detected with a UV detector (SPD10Avp: Shimadzu Corp., Kyoto, Japan) at the wavelength of 214 nm, and the obtained chromatogram was analyzed by Chromato-PRO (RunTime Corp., Tokyo, Japan) to determine the peak area. An excess amount of chymosin was added to the unheated defatted milk and free GMP was calculated as a relative ratio against the maximum amount of free GMP taken as 100%.
Differences in cheese curd formation characteristics due to heat Differences between the curd formation characteristics of unheated and heated defatted milk by the chymosin reaction were compared based on curd strength for each time course (Fig. 1). The unheated defatted milk did not form curd until 30 min of reaction and remained as a liquid, showing a compressive strength of around 20 mN. However, after 60 min of reaction, it started forming curd, and at 180 minutes, the curd strength reached 82.3 mN. In contrast, the heated defatted milk did not form curd, even after 180 min of reaction, and showed a compressive strength of around 20 mN.
Temporal change of breaking load (mN) of cheese curd prepared from unheated defatted milk (⋄) and heated (80°C, 30 min) defatted milk (♦) using chymosin (0.018 IMCU). The error bars indicate standard deviation (n = 3).
This confirmed that high-temperature heat sterilization of milk significantly decreases its ability to form cheese curd, and is in agreement with previous reports (Scott, 1977; Ustunol and Brown, 1985; Yokoyama and Yamauchi, 1992).
We thus examined the effects of inhibition of the chymosin reaction on κ-CN caused by SS-bond formation between κ-CN and β-Lg, and that of insolubilized calcium ions on the high-heat-induced phenomenon.
Effects of reactivity of chymosin on κ-CN The effects of inhibition of the chymosin reaction caused by heat-induced SS-bond formation between κ-CN and β-Lg on κ-CN were examined. The amount of free GMP at each time course after addition of chymosin to unheated and heated defatted milk was determined, and the results are shown in Fig. 2. Before the chymosin reaction (0 min), no peaks were detected; however, two peaks appeared after 5 min, whose intensity increased with time. As the peaks were detected at 12 – 14 min, which is earlier than that observed in κ-casein standard (Sigma-Aldrich, St. Louis, MO, USA), the peaks could be attributed to GMP. We calculated the amount of GMP from the total area of the two peaks. In addition, GMP amounts were expressed relative to the maximum amount of the free GMP (100%). In the unheated defatted milk, 35.2% of GMPs were detached after 10 min of reaction time, while after 60 min, when milk clotting was observed, 85.8% of GMPs were detached (Fig. 3). The reaction almost stabilized after this point, and at 180 min, 88.4% of GMPs were detached. On the other hand, in the heated defatted milk, 20.7% of GMPs were detached after 10 min, 78.1% after 60 min, and although the detachment speed of GMP in this case was slower than that in the unheated defatted milk, 82.5% of GMPs were detached after 180 min (Fig. 3). This indicates that the reaction of chymosin was not inhibited, as GMP was significantly isolated in the heated defatted milk.
Chromatograms of GMP produced by a chymosin reaction (32°C for 0 – 180 min) and κ-casein by RP-HPLC. Conditions were determined according to the method of Gaygadzhiev et al. (2012). Column: ZORBAX300SB-C8 (Agilent Technologies); detection: UV at the wavelength of 214 nm; mobile phase: water containing 0.1% TFA/acetonitrile containing 0.1% TFA (refer to the Materials and Methods section for gradient details).
Temporal change in the amount of free GMP by chymosin reaction. Symbols: ⋄, GMP amount of unheated defatted milk; ♦, GMP amount of heated (80°C, 30 min) defatted milk. Amount of free GMP was indicated by the relative ratio against the maximum amount of free GMP, taken as 100%.
Moreover, at 180 min of reaction, the formation of para-κ-CN, which is involved in milk clotting, was examined by SDS-PAGE. In both the unheated and heated defatted milk, the κ-CN bands at around 25 kDa disappeared and para-κ-CN bands at around 14 kDa appeared, showing similar electrophoretic patterns (Fig. 4), and revealing that para-κ-CN was formed in the heated defatted milk as well.
SDS-PAGE (T = 15%, Me+) of cheese curd prepared from unheated defatted milk (U) and heated defatted milk (H) by chymosin reaction for 180 min. N: Non-treated defatted milk, M: Molecular weight marker.
We have previously reported that κ-CN forms SS-bonds with β-Lg when heated at 80°C for 30 min (Kikuchi, 2012). Thus, we inferred that inhibition of the chymosin reaction caused by the association of β-Lg with κ-CN does not hinder curd formation.
Relationship between calcium ion concentration and cheese curd formation Next, the effects of insolubilization of calcium ions upon heating on cheese curd formation in high-temperature-sterilized milk were examined. The calcium ion concentration in unheated defatted milk was 90 ppm, which reduced by 33% to 60 ppm in heated defatted milk. As it was assumed that this difference (approximately 30 ppm) was affecting cheese curd formation, the difference was compensated by supplementing with soluble calcium lactate to adjust the calcium ion concentration to 90 ppm, followed by preparation of the cheese curd. Figure 5 shows the strength of the curd in each time course after chymosin was added to the milk material. The heated defatted milk with its calcium ion concentration adjusted to 90 ppm did not show milk clotting. Furthermore, the heated defatted milk with 120 ppm calcium ion concentration did not show milk clotting either. However, a significantly hard curd was formed in the unheated defatted milk with 120 ppm calcium ion concentration. This result is in agreement with other reported results (Choi et al., 2007; Singh, 1988; Yokoyama and Yamauchi, 1992) and suggests that the calcium ions participate in milk clotting after the chymosin reaction. However, it is inferred that factors other than calcium ions are responsible for the suppression of cheese curd formation in heated defatted milk.
Temporal change of breaking load (mN) of cheese curd prepared from unheated defatted milk (○⋄) and heated (80°C, 30 min) defatted milk (●♦) with calcium ions adjusted to 90 ppm (○●), 120 ppm (⋄♦). The error bars indicate standard deviation (n = 3).
State of dissociated κ-CN in cheese curd It has been reported that in heated defatted milk, part of κ-CN is dissociated from micelles (Guyomarc'h et al., 2003; Guyomarc'h et al., 2007; Vasbinder et al., 2003). In the present work, the state of the dissociated κ-CN in cheese curd was examined. After centrifuging the cheese curd samples obtained from both unheated and heated defatted milk, the supernatant and the precipitate were analyzed by SDS-PAGE. The results are shown in Fig. 6. Only whey protein was found in the supernatant of unheated defatted milk. In contrast, both whey protein and para-κ-CN were detected in the supernatant of heated defatted milk, which revealed that dissociated κ-CN was not involved in the milk clotting of casein micelles, even when para-κ-CN was formed by the chymosin reaction. In the precipitate, the band of κ-CN in heated defatted milk was found to be weaker than that in unheated defatted milk.
SDS-PAGE (T = 15%, Me+) of the supernatant (sup.) and the precipitation (ppt.) of cheese curd fractionated by centrifugation (33,000×g). M: Molecular weight marker.
This suggests that, in the casein micelles of heated defatted milk, κ-CN is partly dissociated from the casein micelle surface due to heating, which decreases the amount of para-κ-CN. It can be inferred that the decrease in para-κ-CN weakens the formation of curd.
Effects of dissociation of κ-CN on cheese curd formation The properties of casein micelles with partial κ-CN dissociation were examined. First, to obtain such casein micelles, the heated defatted milk was centrifuged using the casein micelle fractionation method of Jean et al. (2006). Then, the precipitate and the supernatant were analyzed by SDS-PAGE to verify the casein micelle fraction. In the heated defatted milk, κ-CN that was not detected in the supernatant of the unheated defatted milk was observed (Fig. 7). Moreover, the weaker band intensity of κ-CN in the precipitate as compared to that of unheated defatted milk indicates that the casein micelles with partial κ-CN dissociation were fractionated. The obtained casein micelles were redispersed in deionized water, and calcium lactate pentahydrate was added to obtain 100 ppm calcium ions, which had caused milk clotting in the preparatory examination, to prepare cheese curd. The results showed that the native casein micelles obtained from the unheated defatted milk formed cheese curd, and the curd strength at 120 min of reaction time was 65.7 mN (Table 1). In contrast, casein micelles with κ-CN dissociation did not form cheese curd, and the sample exhibited a strength of 19.0 mN. It has been reported that, in milk clotting after the chymosin reaction, denatured whey and solubilized whey protein/κ-CN complex inhibit the agglutination of casein micelles (Anema et al., 2011; Giroux et al., 2015; Kethireddipalli et al., 2010). However, this result suggests that even when there is no substance that inhibits the agglutination of casein micelles, κ-CN-dissociated casein micelles do not cause milk clotting. In addition, the amount of free GMP was found to be 69.5% in the cheese curd prepared from native casein micelles, while it was significantly lower (47.9%) in κ-CN-dissociated casein micelles (Table 1). This suggests that the amount of free GMP greatly decreased because the amount of κ-CN on the micellar surface decreases owing to heating. That is, in heated defatted milk, the amount of para-κ-CN on the micellar surface formed by the chymosin reaction is smaller than that in unheated defatted milk, and it would weaken the cheese curd formation.
SDS-PAGE (T = 15%, Me+) of the supernatant (sup.) and the precipitation (ppt.) of the unheated defatted milk and heated defatted milk fractionated by centrifugation (33,000×g). N: Non-treated defatted milk, M: molecular weight marker.
| Breaking load (mN) | GMP (%) | |
|---|---|---|
| Native casein micelle (Unheated) | 65.7±7.4 a | 69.5±1.7 a |
| κ-casein dissociation casein micelle (Heated) | 19.0±1.0 b | 47.9±1.5 b |
κ-CN dissociation casein micelle was obtained by centrifuging heated defatted milk. The GMP amount was determined according to the method of Gaygadzhiev et al. (2012). The amount of free GMP was indicated using the relative ratio against the maximum amount of free GMP, taken as 100%. Data are expressed as mean ± standard deviation (n = 3). Values with different superscripts are significantly different (Tukey-Kramer, p < 0.05).
The results of this study clearly indicate that the weakening of the formation of cheese curd in high-temperature-sterilized milk is not inhibition of the chymosin reaction by SS-bonds between κ-CN and β-Lg or insolubilization of calcium ions, but the dissociation of κ-CN from casein micelles.
This study aimed to clarify the factors that weaken cheese curd formation in high-temperature-sterilized milk. Heated defatted milk did not form cheese curd in contrast to unheated defatted milk. However, no significant differences were observed in the amount of free GMP and the formation behavior of para-κ-CN owing to the chymosin reaction. The results suggest that the main factor weakening cheese curd formation in high-temperature-sterilized milk is inhibition of the chymosin reaction by SS-bonds between β-Lg and κ-CN. Even if the calcium ions decreased by heating were adjusted to equal levels as those in unheated defatted milk, the heated defatted milk did not form cheese curd. This observation suggests that insolubilization of calcium ions was not responsible for the weakening of cheese curd formation. When chymosin was added to casein micelles with partial κ-CN dissociation, milk clotting did not occur and the amount of free GMP by the chymosin reaction decreased significantly in comparison to that observed with native casein micelles.
These results revealed that amount of para-κ-CN necessary for calcium bridging decreased because κ-CN on casein micelles, which is a substrate of chymosin, decreased by heating, which is the main factor that weakened cheese curd formation in high-temperature sterilized milk.
