Protein cross‐linking and the Maillard reaction decrease the solubility of milk protein concentrates

Abstract Milk protein concentrate (MPC) is a widely used material in the food industry. However, despite its widespread use, the mechanism underlying the decreased solubility of MPC that occurs during storage has not yet been clarified. In this study, the solubility changes, protein cross‐linking, and Maillard reaction and the relationships between them were investigated in modified MPC powders (MMPC) containing different concentrations of protein and/or lactose stored at 50°C for 15–45 days. The results demonstrated that both the protein and lactose contents affected solubility. The proteins interacted through hydrogen bonding, disulfide bonding, hydrophobic interactions, and nondisulphide covalent bonding, which led to cross‐linking. The Maillard reaction promoted protein cross‐linking and was in turn influenced by protein cross‐linking. The Maillard reaction was slower when the degree of protein cross‐linking was greater. These results improve our understanding of the mechanism leading to poor solubility of MPC powders during storage.


| INTRODUC TI ON
Milk protein concentrate (MPC) is a powder manufactured from skim milk through membrane separation and spray drying. The protein content of MPC powders ranges from 40 to 90%, based on total solid content (Kelly, 2011;Sikand, Tong, Roy, Rodriguez-Saona, & Murray, 2011). MPCs are named according to their protein content.
For example, MPC85 contains approximately 85% protein content. MPC powders are widely applied in cheese, yogurt, beverage, and confection manufacturing due to their desirable functional properties and nutritional qualities (Farkye & Yim, 2003;Francolino, Locci, Ghiglietti, Iezzi, & Mucchetti, 2010). The solubility of specific MPC powders is closely related to their various functional properties, such as emulsification, gelation, and foaming (Dybowska, 2008;Sandra & Corredig, 2013;Ye, 2011). Despite their widespread use, the solubility of MPC powders gradually decreases during storage, which limits their application (De Castro-Morel & Harper, 2002;(Anema, Pinder, Hunter, & Hemar, 2006;Havea, 2006). For example, it was reported that the solubility of MPC85 was 53% and 32% after 2 days and 24 months of storage at 20°C, respectively (Havea, 2006). Thus, various approaches have been used to improve the solubility of MPC powders, such as raising the water temperature and extending hydration time (Fang, Selomulya, Ainsworth, Palmer, & Chen, 2011;Kuo & Harper, 2003). However, after extended storage, the solubility was still low, even at a high dissolution water temperature (McKenna, 2000). Generally, it is believed that the loss of solubility is linked to processing. Some studies have suggested that ultrasonication and the addition of salt could increase the solubility (Mao, Tong, Gualco, & Vink, 2012;McCarthy, Kelly, Maher, & Fenelon, 2014;Sikand, Tong, & Walker, 2013). For example, the solubility of stored MPC80 was increased from 63 to 100% by the addition of 50-150 mM NaCl during the diafiltration step of manufacturing. However, these studies did not fundamentally solve the low solubility of stored MPC. Thus, determination of the mechanism underlying the decreased solubility of MPC powders during storage is very important for improving its functionality and expanding its applications in the food industry.
Protein cross-linking and the Maillard reaction are the two main factors that contribute to the decreased solubility of MPC powders. The objective of this study was to investigate the changes in the chemical and physical properties of modified commercial MPC powders after storage for a certain time period at 50°C. The solubility, protein cross-linking, and Maillard reaction were examined in stored MMPC powders to clarify the mechanism underlying the solubility decrease.

| Solubility assay
The rehydration procedure was carried out as described previously, with a minor modification (Arnaud Mimouni, Deeth, Whittaker, Gidley, & Bhandari, 2009). MMPC solutions (4%) were reconstituted by stirring the powders in distilled water at 1500 rpm for 10 min at 30°C. The rehydrated solutions were then centrifuged at 2400 × g for 10 min at 10°C. The centrifugation sediments and rehydrated solutions were subjected to solid determination by oven-drying (at 105°C) to a constant weight. The solubility of MMPC powders was calculated using the equation below.

| Changes in color
The surface color properties of the MMPC powders were measured using a Color Meter ZE6000 (Nippon Denshoku Industries, Tokyo, Japan), and the results were expressed as L*, a*, and b* values (Morales & Van Boekel, 1998). The color values reported are the means of three measurements.

| Statistical analysis
Data were analyzed by analysis of variance and the F test using the Statistical Product and Service Solutions (SPSS) 11.0 software package (SPSS Inc., Chicago, IL, USA). p values <0.05 were considered statistically significant.

Solubility (%) =
Solids in the solution − Solids in the sediment Solids in the solution × 100% The final protein contents of MPC85-NS and MPC70-NS were determined; All values are the mean ± SD (n = 3).

| Effects of protein content on solubility
The solubility of MMPC-P samples stored at 50°C for up to 45 days is shown in Figure 1. MMPC40, MMPC55, and MMPC70 initially showed relatively good solubility. Then, the solubility decreased rapidly during storage. The MMPC85 sample showed lower solubility than others, even at the beginning of storage (day 0). As shown in Table 2

| Effects of lactose content on solubility
The solubility changes of MMPC-L samples stored at 50°C for various time points are shown in Figure 2. Compared with the

| SDS-PAGE analysis
The SDS-PAGE of MMPC-P samples after 0, 15, 30, and 45 days of storage is shown in Figure 3. The changes in the MMPC-P protein pattern increased with storage time. The molecular weights of bands observed in the SDS-PAGE are summarized in Table 4.
There were reductions in the densities of the α-, β-, and κ-casein bands after 15, 30, and 45 days when compared to the densities of those of protein bands on day 0. Cross-linking between casein and others proteins could account for the insolubility of MPC powders with higher protein content. MMPC85 showed that most obvious changes among the MMPC-P samples, which was consistent with the changes in the solubility of the MMPC-P samples.
As the hydrophobic bonds and hydrogen bonding were dis-

| Changes in color during storage
The L*, a*, and b* values were used to assess the changes in color of MMPC-L samples during storage. These color values, L*, a*, and b*, represent lightness (or whiteness), redness (or greenness), and yellowness (or blueness), respectively. Figure  Moreover, advanced glycation end products can lead to browning and the formation of high molecular weight protein complexes (Mottram, Wedzicha, & Dodson, 2002;Singh, 1991).
Compared with MMPC75 (14%), MMPC77 (7%) showed more remarkable changes in color. Thus, the occurrence of the Maillard reaction in MPC was not linearly proportional to the lactose content. The protein and lactose contents in MMPC77, 77% protein and 7% lactose (a mass ratio of 11:1), facilitated the Maillard reaction.

| CON CLUS IONS
In summary, the solubility of all modified MPC powders decreased during storage, and protein cross-linking was the major reason for the solubility decrease in MPC powders during storage. The protein molecules interacted through hydrogen bonding, disulfide bonding, hydrophobic interactions, and nondisulphide covalent bonding. In addition, the Maillard reaction also decreased solubility during storage. A small amount of lactose in the MPC reacted with proteins or amino acids with free amino groups; this process formed products that promoted protein cross-linking, and in turn, the Maillard reaction influenced protein cross-linking.

ACK N OWLED G M ENTS
This study was financially supported by the National Natural

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interests.

O RCI D
Ming Du http://orcid.org/0000-0001-5872-8529 F I G U R E 5 Changes in the color of MMPC-L samples during storage. L* represents lightness, and a* and b* represent redness and yellowness, respectively