Influence of Chymosin on Physicochemical and Hydrolysis Characteristics of Casein Micelles and Individual Caseins

The effects of chymosin on the physicochemical and hydrolysis characteristics of casein micelles and individual caseins were investigated. Adding 0.03 units of chymosin/mL led to the casein micelles in skim milk coagulating after a 3 h incubation period at 30 °C. SDS–PAGE investigation showed that β-CN, κ-CN, αs-CN, and a portion of β-lactoglobulin (β-LG) in the milk supernatant fraction (MSF) were precipitated into the milk pellet fraction (MPF). The mean particle size of the MSF with chymosin decreased from 254.4 nm to 179.2 nm after a 3 h incubation period. Mass spectrometry and SDS–PAGE analysis suggested that chymosin hydrolyzed individual β-CN, κ-CN, and αs-CN, but not β-LG. Chymosin hydrolysis led to a decrease in the molecular weights of the hydrolyzed β-CN, κ-CN, and αs-CN. Particle size analysis indicated that there was no difference in the particle size distribution of hydrolyzed β-CN and αs-CN. Moreover, our outcomes demonstrated that the hydrolysis of κ-CN by chymosin occurs before that of β-CN and αs-CN.

Milk is not only an important source of nutrition, but it is also important for industrial and commercial reasons. For example, milk is used in dairy-based products and other processed foods, such as cheese [10]. Cheese is a nutritious food and a significant wellspring of proteins, fatty acids, vitamins, and minerals [11]. Milk coagulation is an important stage in cheddar making, and its success is a significant boundary for obtaining high-quality cheese [12].
Various coagulants are utilized in cheese-making, but the most commonly used coagulant is chymosin [13].
In the process of making cheese, chymosin coagulates milk proteins enzymatically, and casein micelles are enzymatically destabilized during this process. Milk coagulation is a cycle in which liquid milk is turned into a viscoelastic semi-strong coagulum by an explicit chemical called chymosin [14,15].
Traditionally, chymosin (EC 3.4.23.4) is the main enzyme responsible for milk protein coagulation. Chymosin is a protease found in rennet that can precipitate milk proteins and promote the formation of curds during cheese making [16]. The enzyme works on milk κ-CN, somewhat separating these particles. This process causes an adjustment of micelle surface charge, increases their hydrophobicity, and encourages their conglomeration [17]. Furthermore, the natural substrate of chymosin is separated at the peptide connection between amino corrosive deposits as phenylalanine (105) and methionine (106). The chymosin induces the hydrolysis of κ-CN, which causes destabilization and aggregation of casein micelles [18].
In the process of making cheese from milk, when the cheese is aged, the hydrolysis of caseins prompts changes. Proteolysis is frequently considered to be the main biochemical pathway affecting the surface and flavor development [19].
In their 2012 study, Hsieh and Pan (2012) discussed the utilization of proteomic innovation to research the impacts of milk protein coagulation with chymosin [20]. The sample confirmed that the coagulation change of κ-CN is faster than the coagulation change of different caseins, such as β-CN, α s -CN, and a portion of whey protein. However, information about the relative susceptibility and coagulation of β-CN and α s -CN with chymosin treatment, apart from κ-CN, is lacking. Therefore, the purpose of this study is to investigate the effect of chymosin on the physicochemical and hydrolysis characteristics of casein micelles and individual β-CN, κ-CN, α s -CN, and β-LG.

Preparation of Skim Milk, Individual Caseins, and Chymosin
Raw milk was collected from healthy Holstein cows on a dairy farm in Taipei. Skim milk (29.9 mg/mL) was prepared by separating fat from raw milk by centrifugation (5000× g, 20 min), collected, and stored at 4 • C. Individual milk proteins, including β-CN (C6905), κ-CN (C0406), α s -CN (C6780), β-LG (L3908), and chymosin (R4877, 20 units/mg), were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Chymosin was dissolved in a phosphate buffer (pH 6.8, 0.02 mM) prior to use. One unit of coagulation activity was defined as the amount of chymosin that coagulated 10 mL of milk per min at 30 • C.

Preparation of Skim Milk and Individual Casein Samples with/without Chymosin
Skim milk samples with/without chymosin (0.03 units/mL) were incubated at 30 • C for 0, 1, 2, and 3 h. Samples were then fractionated into the milk pellet fraction (MPF) and the milk supernatant fraction (MSF) by centrifugation at 5000× g for 20 min after incubation. One milliliter of each MSF sample was collected, and each MPF sample was redissolved in an equal volume (1 mL) of lysis solution containing 7 M urea, 2 M thiourea, and 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate prior to use. To examine the effects of chymosin on the hydrolysis of individual β-CN, κ-CN, α s -CN, and β-LG, 2 mg of each individual casein was dissolved in 0.02 mM phosphate buffer (pH 6.8). Each casein sample with 0.03 units of chymosin/mL was incubated at 30 • C for 0, 1, 2, and 3 h.

SDS-PAGE Analysis
After incubation, skim milk and individual caseins (including β-CN, κ-CN, α s -CN, and β-LG) with/without chymosin were analyzed by SDS-PAGE. In this study, samples were shown by using stacking gel (5%) and separating gel (12.5%). Then, 0.1 mL of each sample was mixed with 0.3 mL sample buffer (pH 6.8, containing SDS (2%), glycerol (10%), β-mercaptoethanol (5%), Tris-HCl (70 mM), and bromophenol blue (0.02%)). Each sample was heated at 95 • C for 5 min. The gel electrophoresis was run at 50 V for 30 min and then at 120 V for 2-3 h at constant current. After electrophoresis, Coomassie Brilliant Blue R-250 solution was used to stain the gels, which were imaged by an EPSON perfection V39 image scanner and then analyzed by Gel-Pro Analyzer software (version 4.0, Media Cybernetics, Inc., Rockville, MD, USA). SDS-PAGE profiles were used to evaluate the effect of chymosin on the hydrolysis of casein micelles and individual caseins.

Mass Spectrometry Analysis
To examine the effects of chymosin on the hydrolysis characteristics of individual caseins, individual β-CN, α s -CN, and β-LG samples with 0.03 units of chymosin/mL were hatched at 30 • C for 0 and 3 h. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to determine the molecular weights of the individual β-CN, α s -CN, and β-LG samples, which were analyzed on a Bruker Autoflex Speed MALDI-TOF-MS (Bruker Daltonics, Billerica, MA, USA) by measuring the massto-charge proportion (m/z) of the ionized analyte and recording the number of ions at each m/z value. The spectrum was acquired in linear positive mode at a laser frequency of 200 Hz. A method optimized for the mass range was established using the following parameters: acquisition range: 5000 to 35,000 Da.

Particle Size Analysis
To investigate the effects of chymosin on the physicochemical characteristics of casein micelles and individual caseins, skim milk, β-CN, and α s -CN with 0.03 units of chymosin/mL were hatched at 30 • C for 0 and 3 h. The particle size analysis was measured using a nanoparticle analyzer (SZ-100Z, HORIBA Instruments, Inc.). The particle size distribution spectra for the nanoparticles were recorded as diameter (nm) versus frequency (%), using the dynamic light scattering technique at a scattering angle of 90 • and a temperature of 25 • C. The experiments on the sample were performed in triplicate.

Statistical Analysis
The results were analyzed using SAS ® version 9.4 (SAS Institute, Cary, NC, USA), and the data are displayed as the mean ± standard deviation. One-way analysis of variance was used to calculate significant differences between treatments. Each treatment was measured 3 times, and the statistical significance level was set to p < 0.05.

SDS-PAGE Analysis of Chymosin's Effect on the Coagulation of Milk Proteins
Skim milk samples with/without chymosin (0.03 units/mL) were incubated at 30 • C for 3 h, and the resulting MPF and MSF samples were analyzed by SDS-PAGE ( Figure 1). The analysis results revealed that β-CN, κ-CN, α s -CN, and β-LG were the major milk proteins in skim milk. As shown in Figure 1A, the protein bands of β-CN, κ-CN, α s -CN, and a part of β-LG in chymosin-containing MSF disappeared after incubation for 3 h. However, these milk proteins were observed in the chymosin-containing MPF ( Figure 1B). Furthermore, there were no significant changes in the protein bands of β-CN, κ-CN, α s -CN, and β-LG in the MSF and MPF without chymosin after the 3 h incubation period. These results indicate that some whey proteins and casein micelles were coagulated by chymosin into the MPF from the MSF. Horne (2006) reported that β-CN, κ-CN, and α s -CN in milk are consolidated into large colloidal structures called casein micelles [21]. These β-CN and α s -CN are connected with colloidal calcium phosphate, and κ-CN are assembled at the surface. Zhao and Corredig (2020) also reported that chymosin can precipitate casein micelles and promote the formation of curds during the cheese-making process [16]. Chymosin can cleave to κ-CN, resulting in a decrease in steric repulsion and net negative charge. It rapidly and specifically hydrolyzes κ-CN's Phe105-Met106 bond, resulting in instability of casein micelles [22,23]. Hsieh and Pan (2012) suggested that these unstable casein micelles (β-CN, κ-CN, and α s -CN) and a part of whey protein such as β-LG are thought to be trapped in cheese curds [20]. Therefore, the above results demonstrate that chymosin induces the hydrolysis of κ-CN, which causes aggregation and destabilization of casein micelles. This result corresponded with that of Corredig and Salvatore [24].

MALDI-TOF-MS Analysis of Chymosin's Effect on the Hydrolysis of Individual Milk Proteins
κ-CN is known to be a good substrate for chymosin, and κ-CN is hydrolyzed by chymosin to frame two peptide-like response items, which were the result of the specific cleavage of the Phe105-Met106 bond of κ-CN [27]. According to the results above, chymosin also hydrolyzed β-CN and α s -CN. Horneffer et al. (2007) indicated that MALDI-TOF-MS can be used to determine the molecular weights of proteins and is suitable for food research [28]. Therefore, individual β-CN, α s -CN, and β-LG samples with 0.03 units of chymosin/mL were incubated at 30 • C for 0 and 3 h, and the molecular weights of the resulting samples were analyzed by MALDI-TOF-MS. As shown in Figure 4A,C,E, the molecular weights of β-CN, α s -CN, and β-LG were 23858.5751 Da, 23497.3545 Da, and 18265.4257 Da, respectively. Haginaka (2000) reported that the molecular weights of β-CN, α s -CN, and β-LG were 24.0 kDa, 23.6 kDa, and 18.3 kDa, respectively [29]. However, the molecular weights of hydrolyzed β-CN and α s -CN induced by chymosin after 3 h incubation were decreased to 21998.9416 Da and 20751.5721 Da, respectively ( Figure 4B,D). Kim et al. (2004) reported that chymosin hydrolyzed the Leu192-Tyr193 and Phe23-Phe24 bonds in β-CN and α s -CN, respectively [26]. These processes resulted in a decrease in the molecular weights of the hydrolyzed β-CN and α s -CN. However, there were no significant changes in the molecular weights of β-LG (18258.5634 Da) after a 3 h incubation with chymosin ( Figure 4F). This result demonstrated that β-LG was not hydrolyzed by chymosin after 3 h of incubation.

Particle Size Analysis of Milk Proteins and Individual Milk Proteins Treated with Chymosin
Skim milk and individual milk protein samples with 0.03 units of chymosin/mL were incubated at 30 • C for 0 and 3 h, respectively. The resulting MSF and individual milk protein samples were analyzed by a nanoparticle analyzer ( Figure 5). The particle size of MSF with chymosin was 180-460 nm after 0 h incubation, while the mean particle size of MSF with chymosin was 254.4 nm after 0 h of incubation ( Figure 5A). De Kruif and Holt (2003) reported that the normal particle size of casein micelles is approximately 50-600 nm, and the mean particle size is approximately 200 nm [30]. Huppertz et al. (2018) reported that casein micelles consisting of β-CN, κ-CN, and α s -CN are highly hydrated particles. κ-CN exists on the surface of casein micelles, and β-CN and α s -CN are predominantly found in the interior [4]. In solution, the milk protein spreads to form a polyelectrolyte layer, such as a hairy layer, which brings sterics and charges repulsion between protein particles, providing stability. When the casein micelles are linked with chymosin, the polyelectrolyte layer of the casein micelles is hydrolyzed while causing the casein micelles to become unstable and precipitate out of solution. The particle size of the MSF with chymosin was 92-316 nm after 3 h of incubation, while the mean particle size of the MSF with chymosin was 179.2 nm after a 3 h incubation period ( Figure 5B). This is because only whey proteins such as β-LG and some casein micelles remained in the MSF, and it decreased the mean particle size of the MSF. The outcome revealed that β-CN, κ-CN, α s -CN, and some β-LG were trapped in the curds, and the MSF mostly consisted of whey protein and some casein micelles.
The effects of chymosin (0.03 units/mL) on the hydrolysis of individual β-CN and α s -CN samples at 30 • C for 0 and 3 h were also investigated, however, no significant changes were observed in either. The particle sizes of α s -CN with chymosin were 4.2-17.8 nm and 7.0-14.9 nm after 0 h and 3 h incubation, respectively ( Figure 5C,D). The particle sizes of β-CN with chymosin were 8.8-18.2 nm and 11.1-28.4 nm after 0 h and 3 h incubation, respectively ( Figure 5E,F). We observed that the molecular weights of β-CN and α s -CN decreased after chymosin hydrolysis; however, there was no difference in particle size distribution. These results suggest that chymosin induced the hydrolysis of β-CN and α s -CN, but it did not cause aggregation of β-CN and α s -CN.

Hydrolysis Reaction Scheme on Casein Micelles and Individual Caseins Induced by Chymosin
Chymosin hydrolyzes the specific peptide bond of κ-CN, leading to destabilization and aggregation of casein micelles. It has a specific function during the bioactive reaction in cheese-making. According to our results, a hydrolysis reaction scheme on the casein micelles and individual caseins induced by chymosin in the cheese-making process is shown in Figure 6. As shown in Figure 6A, the coagulation reaction on casein micelles induced by chymosin consists of two steps. κ-CN was surrounded by the external area of casein micelles. First, chymosin hydrolyzes κ-CN to form caseinomacropeptide and para-κ-CN in the external area of casein micelles [18]. The Phe105-Met106 bond of κ-CN was hydrolyzed by chymosin, resulting in the instability of the casein micelles. Then, the aggregation of unstable casein micelles was induced by chymosin. Hydrophobic interactions are an important impetus for coagulation and are indirectly enhanced by calcium ions in milk. These unstable casein micelles and a part of whey protein, such as β-LG, are then thought to be trapped to form cheese curds after 3 h of incubation. During the coagulation process, the mean particle size of MSF with chymosin decreased from 254.4 nm to 179.2 nm. The hydrolysis reaction scheme on the individual caseins induced by chymosin in the cheese-making process is shown in Figure 6B. The peptide bonds in β-CN, κ-CN, and α s -CN were hydrolyzed by chymosin, and this protease hydrolyzed the Leu192-Tyr193, Phe105-Met106, and Phe23-Phe24 bonds in α s -CN and β-CN, respectively [26]. Chymosin hydrolyzed most κ-CN after 1 h of incubation, while some β-CN and α s -CN were hydrolyzed by chymosin after 1 h of incubation. However, β-LG was not hydrolyzed by chymosin after 3 h of incubation. These hydrolyzed β-CN, κ-CN, α s -CN, and β- LG were entrapped into the cheese curd.

Conclusions
We analyzed the effect of chymosin on the physicochemical and hydrolysis characteristics of casein micelles and individual caseins. SDS-PAGE analysis showed that milk proteins, including β-CN, κ-CN, α s -CN, and some β-LG, were coagulated by chymosin. In the catalysis process, chymosin hydrolyzed β-CN, κ-CN, and α s -CN but not β-LG. The hydrolysis of κ-CN by rennet occurred earlier than the hydrolysis of β-CN and α s -CN. MALDI-TOF-MS analysis indicated a decrease in the molecular weights of the hydrolyzed β-CN and α s -CN. Particle size analysis suggested that there was no difference in particle size distribution between β-CN and α s -CN after hydrolysis. Moreover, β-LG and hydrolyzed β-CN, κ-CN, and α s -CN were entrapped in the cheese curd.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.