Microwave-assisted cross-linking of milk proteins induced by microbial transglutaminase

We investigated the combined effects of microbial transglutaminase (MTGase, 7.0 units/mL) and microwave irradiation (MI) on the polymerization of milk proteins at 30 °C for 3 h. The addition of MTGase caused the milk proteins to become polymerized, which resulted in the formation of components with a higher molecular-weight (>130 kDa). SDS-PAGE analysis revealed reductions in the protein content of β-lactoglobulin (β-LG), αS-casein (αS-CN), κ-casein (κ-CN) and β-casein (β-CN) to 50.4 ± 2.9, 33.5 ± 3.0, 4.2 ± 0.5 and 1.2 ± 0.1%, respectively. The use of MTGase in conjunction MI with led to a 3-fold increase in the rate of milk protein polymerization, compared to a sample that contained MTGase but did not undergo MI. Results of two-dimensional gel electrophoresis (2-DE) indicated that κ-CN, β-CN, a fraction of serum albumin (SA), β-LG, α-lactalbumin (α-LA), αs1-casein (αs1-CN), and αs2-casein (αs2-CN) were polymerized in the milk, following incubation with MTGase and MI at 30 °C for 1 h. Based on this result, the combined use of MTGase and MI appears to be a better way to polymerize milk proteins.


Results and Discussion
Effects of MI and MTGase on the polymerization of milk proteins. We first investigated the effects of MI and MTGase (7.0 units/mL) on the polymerization of milk proteins (Fig. 1). Milk samples with/without MI were treated at 30 °C for 0, 1, 2, or 3 h prior to analysis by SDS-PAGE. No signs of polymerization were observed during this period, whether or not they underwent MI. Tyler-Cross and Schirch 17 reported that the naturally occurring deamidation of protein is ubiquitous, and that it can affect both the function and structure of proteins. The rate of deamidation is affected by a number of factors, including the primary sequences of the proteins, temperature, pH, and the constituents of solutions. MI induces molecular rotation in the direction of dipole alignment by creating an external oscillating electric field. It is therefore reasonable to expect that MI can accelerate the deamidation of proteins. Liu, Moulton, Auclair, and Zhou 18 developed a novel method for the study of protein deamidation using mass spectrometry, and it is very likely that their approach is also suitable for the study of milk proteins. In this study, milk samples incubated with MTGase at 30 °C for 3 h presented a reduction in the quantity of α S -CN, κ -CN, and β -CN (Fig. 1C). In agreement with our previous research, we observed that MTGase caused these proteins to be polymerized into higher-molecular-weight proteins (> 130 kDa) 10 . Specifically, components with higher molecular weights were produced by casein (α S -CN, β -CN, and κ -CN) crosslinking. Smiddy, Martin, Kelly, de Kruif, and Huppertz 19 reported that MTGase is an acyltransferase capable of inducing intra-and intermolecular crosslinking reactions involving milk proteins. Thus, the disappearance of caseins suggests that MTGase-induced polymerization occurred. Liu et al. 20,21 developed a method that uses 18 O-labeling and mass spectrometry to identify protein cross-linking. Their strategy, which is based on XChem-Finder, can be used to identify cross-linking in individual caseins.
Molecules with high dipole moments, such as enzymes and proteins, are particularly susceptible to the effects of microwaves 22,23 . Indeed, MI has been shown to accelerate MTGase-polymerized reactions in milk samples; therefore, we propose that MI could also be used to induce enzymatic reactions involving MTGase. In this study, the use of MTGase in conjunction with MI for 1 h led to a 3-fold increase in the rate of milk protein polymerization, compared to a sample that contained MTGase but did not undergo MI (Fig. 1D). These results corroborate findings from previous works. For example, Huang et al. 11 used MI to increase the rates of reactions involved in the esterification between n-caprylic acid and pentanol isomers catalyzed via lipase. Wan, Sun, Hu, and Xia 24 suggested that MI has nonthermal effects on enzymatic reactions, which is primarily due to the polarities of the substrates and solvents. Young, Nichols, Kelly, and Deiters 25 reported that MI is an effective tool for increasing the reaction rates in the activation of enzymatic catalysis. Figure 2 illustrates the SDS-PAGE analysis of caseins and whey proteins incubated with MTGase under MI at 30 °C. As shown in Fig. 2A, no polymerization occurred after 1 h of incubation; however after 3 h, the protein contents of β -LG, α S -CN, κ -CN and β -CN were reduced to 50.4 ± 2.9, 33.5 ± 3.0, 4.2 ± 0.5 and 1.2 ± 0.1%, respectively. These results indicate that the MTGase-induced polymerization of κ -CN, β -CN, and α S -CN occurred earlier than that of β -LG. De Jong and Koppelman 26 previously reported that β -LG is a poor substrate for MTGase. In this study, after being incubated at 30 °C for 1 h, the protein content of β -LG, β -CN, κ -CN, and α S -CN was noticeably lower in samples where MTGase was used in conjunction with MI than in samples which contained MTGase but did not undergo MI (Fig. 2B). After incubation at 30 °C for 1 h, the protein content of β -LG, α S -CN, κ -CN, and β -CN decreased to 30.6 ± 1.3, 2.1 ± 0.7, 1.9 ± 0.2, and 0.3 ± 0.1%, respectively. Bornaghi and Poulsen 27 reported the use of MI to accelerate enzymatic processes. Our results indicate that MI treatment can be used to accelerate the MTGase-polymerized reactions of caseins and whey proteins. showed signs of depletion after incubation for 3 h in the MTGase-containing milk. This indicates that most of the casein micelles and a portion of the whey proteins were polymerized by MTGase. We also observed that the casein polymerization of α s1 -CN and α s2 -CN was slower than that of β -CN and κ -CN. Smiddy, Martin, Kelly, de

Effects of incubation condition on MTGase activity in buffer and milk samples.
MTGase-containing buffer (0.1 M Tris-acetate buffer, pH 6.0) was incubated in a dry bath at 30 °C for 3 h. Residual MTGase activity in the buffer decreased slightly as incubation time increased (Fig. 7). After 3 h of incubation, the residual MTGase activity had decreased to 72.3 ± 1.4% of the original level. Similar trends were observed for residual MTGase activity in the MTGase-containing buffer when MI was performed at 30 °C for 3 h. Specifically, residual MTGase activity decreased to 74.5 ± 1.5% after 3 h of incubation. Jiang, Hsieh, and Tsai 31 also reported a reduction in residual MTGase activity (to 42.1 ± 2.9%) in MTGase-containing buffer (50 mM sodium phosphate buffer, pH 7.0) when samples were incubated at 45 °C for 2 h. Ho, Leu, Hsieh, and Jiang 32 reported that the rate constant (K D ) for the thermal inactivation of MTGase at 45 °C was 6.2 × 10 −5 /min. In addition, the residual MTGase activity in MTGase-containing milk was 99.8 ± 0.8% when the sample was incubated in a dry bath incubator at 30 °C for 0 h. These results suggest that the components of milk can not affect the activity of MTGase. Nonetheless, the residual MTGase activity in milk decreased significantly to 25.2 ± 2.7% after 3 h of incubation. Therefore, the residual MTGase activity in milk samples incubated at 30 °C for 3 h was lower than that in buffer samples (P < 0.05). As previously mentioned, we observed that caseins and whey proteins were polymerized into   higher-molecular-weight proteins by MTGase, which means that MTGase was probably entrapped within the high-molecular-weight polymers, thereby leading to a reduction in residual MTGase activity.
MTGase-containing milk samples were also incubated in a microwave reactor at 30 °C for 3 h, which resulted in a significant decrease in residual MTGase activity to just 42.4 ± 2.5% (P < 0.05). The residual MTGase activity in all milk samples incubated in the microwave reactor exceeded that of the samples incubated in the dry bath incubator. This indicates that MTGase is more stable under MI, and these findings are supported by Yu et al. 33 who reported that the lipase Novozym 435 is more stable under MI than under conventional heating. Réjasse, Lamare, Legoy, and Besson 34 proposed that MI enhances interactions between chemical bonds involved in maintaining the enzymatic structure, thereby protecting enzymes from thermal denaturation.
Catalytic procedure for the microwave-assisted polymerization of milk proteins induced by MTGase. Figure 8 presents the microwave-assisted catalytic procedure applied to the polymerization of milk proteins by MTGase. The MTGase-induced polymerization of milk proteins was observed after 3 h of incubation (Fig. 8A). MTGase catalyzes the cross-linking of κ -CN and β -CN before catalyzing SA, α -LA, α s1 -CN, α s2 -CN, and β -LG. The degree of β -CN and κ -CN polymerization exceeded that of SA, α -LA, α s1 -CN, α s2 -CN, and β -LG. De Kruif and Holt 35 reported that MTGase-induced polymerization occurs more readily for β -CN and κ -CN than for other milk proteins, which may be an effect of the specific location of MTGase within the casein micelle. Sharma, Lorenzen, and Qvist 36 reported that the porous nature of casein micelles allows MTGase to diffuse into the interior. This means that the flexible conformation of β -CN in casein micelles also allows for catalysis by MTGase. In the current study, MI treatment was found to accelerate the MTGase-polymerized reactions in milk samples, wherein the MTGase-induced polymerization of milk proteins was observed after just 1 h of incubation (Fig. 8B). Within 1 h, most of the κ -CN and β -CN as well as fractions of SA, α -LA, β -LG, α s2 -CN, and α s1 -CN had been polymerized by MTGase into higher-molecular-weight proteins. Thus, the polymerized milk proteins contained κ -CN, β -CN, SA, β -LG, α -LA, α s2 -CN, and α s1 -CN fractions.
In summary, we examined the effects of MTGase and MI on the polymerization of individual proteins in milk. After 3 h of incubation, the MTGase had polymerized κ -CN, β -CN and a portion of SA, α s1 -CN, α s2 -CN, α -LA, and β -LG fractions to form high-molecular-weight polymers. Furthermore, the polymerization of SA, β -LG, α -LA, α s2 -CN, and α s1 -CN was slower and occurred earlier than the polymerization of β -CN and κ -CN. The use of MTGase in conjunction with MI for 1 h substantially improved the polymerization of caseins and whey proteins, indicating that MI accelerates MTGase-polymerized reactions in milk.

Materials and Methods
Preparation of MTGase and milk samples. Milk was first centrifuged at 5,000 × g for 20 min, and a layer of fat was subsequently removed from the top of the milk using a spatula. The resulting skimmed milk was then stored at 4 °C. A Bio-Rad protein assay kit was used in accordance with manufacturer's instructions to determine the protein concentrations in the skimmed milk, using SA as a standard. MTGase from Streptoverticillium mobaraense (1.0 units/mg) was purchased from Ajinomoto Co. Inc. (Activa TG-B, solution form; Ajinomoto Co. Inc., Tokyo, Japan). All experiments on the microwave-assisted polymerization of milk proteins were performed using 1 mL milk samples contained within a closed vessel, where they did not undergo any stirring or shaking. To prepare milk with/without MTGase (7.0 units/mL), we first incubated the milk samples in a dry bath incubator at 30 °C for 0, 1, 2 and 3 h, respectively. The resulting samples were then heated to 80 °C for 3 min to deactivate the MTGase. The microwave-assisted polymerization of milk proteins induced by MTGase was investigated using a microwave reactor (CEM ® Discover system, CEM Corporation, Matthews, NC, USA). Milk samples with/without MTGase (7.0 units/mL) were incubated in the microwave reactor at 30 °C at a power of 30W for 0, 1, 2, or 3 h. Samples were then heated to 80 °C for 3 min to deactivate the MTGase. Experiments were conducted under one of four conditions: (1) milk without MTGase or MI; (2) milk with MTGase; (3) milk with MI; and (4) milk with MTGase and MI.

SDS-PAGE and 2-DE analysis.
Milk samples were analyzed using SDS-PAGE and 2-DE, in accordance with protocol outlined by Hsieh and Pan 10 . For SDS-PAGE analysis, milk samples were analyzed using 12.5% separating gels. Milk samples were mixed with loading buffer before being denatured at 100 °C for 5 min. Five μ L of the milk samples with protein markers (10-200 kDa) were loaded onto the SDS-PAGE gel, and separation was performed at 90 V in the separating gel. Following electrophoresis, Coomassie Brilliant Blue R-250 staining was used for the detection of protein bands. Gel images were then scanned and analyzed using Gel-Pro Analyzer software. For 2-DE analysis, milk proteins (100 μ g) were solubilized in rehydration buffer to a final sample volume of 350 μ L. The IPG gel strips were then focused on the soluble proteins. For this, we used 12.5% separating gels in the second dimension. Each 2-DE gel was stained and scanned using the Typhoon 9200 imaging system. Finally, we used Progenesis SameSpots software to analyze gel images.
Determination of MTGase activity. MTGase (7.0 units/mL) was respectively added to buffer (0.1 M Tris-acetate buffer, pH 6.0) and milk samples. The MTGase-containing buffer and milk samples were incubated in a dry bath incubator or microwave reactor (CEM ® Discover system, power of 30 W) at 30 °C for 0, 1, 2, or 3 h. This experiment was conducted under one of four conditions: (1) MTGase-containing buffer without MI; (2) MTGase-containing buffer with MI; (3) MTGase-containing milk without MI; and (4) MTGase-containing milk with MI. The resulting samples were then analyzed using MTGase assay to assess residual MTGase activity in accordance with the method proposed by Ho, Leu, Hsieh, and Jiang 32 .
Statistical analysis. SPSS statistical software was used for statistical analysis. The results are presented as mean ± standard deviation. Duncan's multiple range tests and one-way ANOVA were used to calculate significant differences between treatments. Three determinations were performed for each treatment, and the level of statistical significance was set at P < 0.05.