Evaluation of antigenicity and nutritional properties of enzymatically hydrolyzed cow milk

While enzymatic hydrolysis is an effective method for lowering the antigenicity of cow milk (CM), research regarding the antigenicity and nutritional traits of CM hydrolysate is limited. Here, we evaluated the protein content, amino acid composition, sensory traits, color, flow behavior, and antigenicity of CM following enzymatic hydrolysis. The results showed that enzymatic hydrolysis increased the degree of hydrolysis, destroyed allergenic proteins, including casein, β-lactoglobulin, and ɑ-lactalbumin, and significantly increased the content of free amino acids and nutritional quality. In particular, the antigenicity of CM was significantly reduced from 44.05 to 86.55% (P < 0.5). Simultaneously, the taste, color, and flow behavior of CM were altered, the sweetness and richness intensity decreased significantly (P < 0.5), and astringency and bitterness were produced. A slightly darker and more yellow color was observed in CM hydrolysate. In addition, apparent viscosity decreased and shear stress significantly increased with increasing shear rate intensity. The results will provide a solid theoretical foundation for the development of high-quality hypoallergenic dairy products.

Enzymatic hydrolysis. Alcalase, Protamex, and Flavourzyme were diluted to 100 mg/mL with distilled water. The skim CM was preheated for 20 min at the optimal temperature (Alcalase: 55 ± 5 °C; Protamex; 50 ± 5 °C; Flavourzyme: 50 ± 5 °C). Then, followed by the addition of enzyme solutions according to the enzyme activity to substrate ratio (Alcalase, Protamex, and Flavourzyme was 10,000 U/g, 10,000 U/g, and 6,000 U/g, respectively). The mixture solutions were continuously stirred and the reaction was terminated by heating the solution at 90-100 °C for 10 min and cooling on ice water. The solutions were centrifuged at 5000 × g for 10 min and stored at − 80 °C until use.
Determination of the degree of hydrolysis. The degree of hydrolysis (DH) of the hydrolysates was evaluated using the OPA method, according to the method of 23 . In brief, 400 μL of sample was added to 3 mL of OPA solution, the mixture was combined by inversion and incubated for 2 min at room temperature in the dark. A serine solution (100 μg/mL) was used as a standard control. The absorbance was measured at 340 nm using a spectrophotometer.
Trisine SDS-PAGE. SDS-PAGE was performed according to the procedure described by 24 . Stacking and separating gels were prepared using 3% and 15% acrylamide concentrations, respectively. The samples were added to the loading buffer, and the mixtures were heated in boiling water for 10 min. The protein (15 μg) was transferred to each well, and the gels were stained with Coomassie Brilliant Blue G-250 after electrophoresis. Image analysis was performed using a gel scanner (Amersham Pharmacia Biotech, Uppsala, Sweden).
Enzyme-linked immunosorbent assay (ELISA). IgG-binding capacity was determined by ELISA, according to the method described by 25  Determination of total protein content. The total protein content was measured using the Quick Start Bradford Assay kit (BioRad), in accordance with the manufacturer's instructions 26 . BSA was used as the standard.
Determination of AA composition. AA composition analysis was performed using Agilent 1200 HPLC (Agilent Technologies, Madrid, Spain), according to the method of 27 . Chromatographic separation ( Fig. 1) was performed using a Zorbax Eclipse XDB C18 column (5 mm) and an Agilent guard cartridge C18 (5 mm). Sample (50 µL) was added to the column and eluted at a flow rate of 0.9 mL min −1 , according to the linear gradient used by 28 .
Evaluation of sensory characteristics. The sensory properties of the samples were evaluated using an electronic tongue (ISENSO SuperTongue, USA), according to the method described by 29 . The sensory attributes of samples, including sourness, sweetness, bitterness, saltiness, umami, astringency, and richness were evaluated. Untreated CM samples were used as controls.
Measurement of color. The color of the samples was determined with a chromameter (Minolta CM-3600d, Japan), which was calibrated using a standard white and black plate; L* represents lightness, ranging from black to white (0.00-100.00). In addition, a positive value of a* indicates red, and a negative number indicates green. A positive value of b* indicates yellow, and a negative value represents blue.

Results
Degree of hydrolysis. The DH of enzymatic hydrolysis-treated CM (HM) produced by Alcalase, Protamex, and Flavourzyme was quantified using the OPA method (Fig. 2). It can be seen that the DH of Flavourzymetreated CM (FT) and Protamex-treated CM (PT) gradually increased with the time of enzymatic hydrolysis. Flavourzyme displayed the ability to hydrolyze CM, and the DH ranged from 13.87% to 31.36%. The hydrolysis capacity of Protamex to CM was lower, with the DH ranging from 2.99% to 6.86%. In addition, with the increase in enzymatic hydrolysis time, the DH of Alcalase-treated CM (AT) showed an increasing trend, and reached a maximum value (13.81%) after 60 min, but as the enzymatic hydrolysis time reached 90-120 min, the DH decreased gradually (90 min: 8.81%; 120 min: 11.43%). Figure 3 shows the electrophoretic patterns of CM and HM obtained with Alcalase, Protamex, and Flavourzyme. The electrophoretic patterns of CM showed protein bands with apparent molecular weights (MWs) that ranged from to 4.0 to 66.1 kDa. The electrophoretic pattern exhibited three higher intensity bands with apparent MWs of approximately 19.0-25.0, 18.0, and 14.0 kDa, which most likely correspond to CNs, BLG, and ALA, respectively, and also showed a lower density protein band that might correspond to BSA  IgG-binding capacity. The IgG-binding capacity of HM was evaluated by icELISA using anti-CM rabbit polyclonal antibodies (Fig. 4). The IgG reactivity reduction of HM ranged from 44.05% to 86.55%. With increasing time of enzymatic hydrolysis, the reduction in IgG reactivity of AT and PT did not decrease significantly (P > 0.05). Interestingly, the enzymatic hydrolysis time reached 15-60 min, the reduction in IgG reactivity of FT was 82.27%, 69.46%, and 72.94%, but when the enzymatic hydrolysis time reached 90 min, the IgG reactivity reduction of FT significantly increased (44.05%).

SDS-PAGE profile.
Total protein content. Figure 5 shows that with the increase in enzymatic hydrolysis time, the total protein content was significantly reduced (P < 0.05). Among these, the content of FT was significantly lower than that of AT and PT (P < 0.05).
AA composition. The AA composition was analyzed using HPLC. The total free amino acid (TFAA) of HM significantly increased (P < 0.05) in comparison with CM (Fig. 6). The content of essential amino acids (EAAs) and non-essential amino acids (non-EAAs) significantly increased with time. Furthermore, the content of TFAAs in FT was higher than those of AT and PT. With respect to the AT, when the enzymatic hydrolysis time    PCA of EAAs and non-EAAs showed that two principal components, the cumulative explained variance, was 40.6%, indicating that these two principal components could not be distinguished between CM and HM (Fig. 7). PC1 mainly contained Met, Val, Leu, Phe, Thr, His, Gln, Arg, and Tyr. PC2 principally included Ile, Leu, Lys, Gly, Asp, Glu, Pro, and Cys. Figure 8  In order to compare the expression patterns of AAs from CM and HM, hierarchical cluster analysis was used to analyze the significant, differentially expressed AAs (Fig. 9).
Taste. The sensory characteristics of CM and HM were determined using an electronic tongue (e-tongue).
High intensity positive tastes, such as sweetness, richness, and umami, were found in CM (Fig. 10). Negative  Figure 10D shows that AT, PT, FT, and CM can be clearly distinguished in the PCA score plot. FT showed saltiness and bitterness, and PT had a slight sourness, and AT had heavy astringency and an aftertaste of astringency (aftertaste-A). Additionally, Fig. 10E shows a significant correlation between the sensory characteristics of AT, PT, FT, and CM. Among these correlations, 15 were positive and the remaining 21  Flow behavior. The relationship between shear rate and shear stress or apparent viscosity of CM, AT, PT, and FT were assessed (Fig. 12). The trend of the relationship between shear rate and apparent viscosity was similar among CM and AT, PT, or FT. Generally, apparent viscosity decreased with the increasing intensity of shear rate, and the apparent viscosity gradually stabilized when the shear rate reached a high level (0.10-10.00 S −1 ), which exhibited pseudoplastic fluid behavior. At a lower shear rate (0.01-0.10 S −1 ) the apparent viscosity decreased sharply and the shear thinning effects were observed. www.nature.com/scientificreports/ Furthermore, the shear stress significantly increased with the intensity reinforcement of the shear rate (Fig. 12A,C,E). Notably, when CM was incubated with Alcalase, Protamex, and Flavourzyme, and enzymatic hydrolysis time was 90 min, 60 min, and 15 min, respectively, and the apparent viscosity and shear stress reached high levels.

Discussion
Many strategies have been widely used to improve the functional properties of food, including chemical and enzymatic modifications, but enzymatic hydrolysis might be the preferable method due to milder process conditions, higher specificity, and minimal by-product generation during the reaction 30 . Moreover, Wróblewska et al. suggested that enzymatic hydrolysis is the most effective method to alter the immunoreactivity of food proteins 31 . In addition, Yang et al. demonstrated that enzymatic hydrolysis is a safe and mild process method, which can break peptide and disulfide bonds and result in the collapse of conformational and linear epitopes, destroying the allergenic proteins. Most importantly, it does not reduce the nutritional quality or destroy the nutritional composition 32 . In the present study, we found that CM proteins were more sensitive to Flavourzyme and exhibited a higher DH. While Alcalase and Protamex showed a lower DH than Flavourzyme, the DH did not increase with time. Similarly, some studies have indicated that limited hydrolysis, ranging from 1.0 to 15.0%, was needed, which was beneficial for improving the functional properties of food 33 .
Enzymatic hydrolysis is the process of using certain digestive enzymes to break down the peptide bonds of proteins and convert the whole protein into smaller peptide fragments. However, the species of enzymes are different, and the degradation abilities of these enzymes also vary 34 . Our findings showed that the electrophoretic patterns of HM became less visible with increasing time, especially the bands of PT, which vanished completely with increasing enzymatic hydrolysis time. Some studies have reported that the protein bands of AT became less visible, compared to WPC, and a large amount of high MW proteins decreased, with many peptide bands below 14.0 kDa 35 . Previous studies have demonstrated that WPC obtained from Alcalase (0.6 L) generates lower MW peptides, ranging from 10.0-20.0 kDa 36 .
During the enzymatic hydrolysis reaction, the structure of food allergens is altered, which interferes with the antigen-antibody complex-forming ability. In the present study, we found that the IgG-binding capacity of HM was significantly reduced. Thus, we speculated that this might be due to the splitting of epitope sequences during enzymatic hydrolysis. Similar research carried out by Jordan et al. suggested that enzymatic hydrolysis could damage or destroy antigenic epitopes of allergenic proteins, resulting in the reduction of IgG-binding capacity and immunoreactivity 37 . In addition, Villas-Boas et al. indicated that enzymatic hydrolysis reduced the amount of allergenic epitopes, and the IgE or IgG-binding ability of β-LG could be significantly decreased 38 . Jing et al. showed that reduction of the IgE-binding ability of ALA and BLG in CM was reduced, ranging from 15.0 to 90.0% 39 . These findings are in line with the DH of CM obtained with Alcalase, Protamex, and Flavourzyme, indicating that enzymatic hydrolysis is an effective method to reduce the immunoreactivity of CM. Taken together, enzymatic hydrolysis could effectively reduce the immunoreactivity of CM, but the effect of enzymatic hydrolysis on the nutritional properties of natural CM should be further studied.
The contents of the total protein and AA composition were systematically analyzed in our study. Our findings showed that the total protein content was significantly lower than that of CM, and the content of FT was significantly lower than that of AT and PT. These results are consistent with the DH of AT, PT, and FT. The reason  AAs are important nutrients in the body, and most people in developed countries get enough AAs from proteins in the diet 42 . The composition of EAAs and non-EAAs were evaluated in this study, and it was found that the TFAAs of HM significantly increased, and the content of TFAAs in FT was higher than those of AT and PT. Previous studies have suggested that the AAs of hydrolysates depend on the manufacturing technology, such as the type of enzymes, enzymatic conditions, and the extent of proteolysis 43 . Yvon & Rijnen evaluated the hydrolysates of cheese, and found that the content of EAAs and non-EAAs varied significantly according to the extent of proteolysis 44 . In particular, we found that the Thr, Leu, Ile, Trp, His, Glu, and Tyr in HM were significantly higher than those in CM. Liang et al. reported that FAAs could be directly absorbed by the human body, which helps the body regulate the immune system and provide energy for the body and brain 45 . Baldeón et al. observed that the content, composition, and ratio of FAAs could reflect the nutritional value of food products 46 . In addition, recent studies have indicated that Trp can promote the growth and development of infants and children, enhance intelligence, and even play a significant role in the treatment of diseases such as cancer 47 . Zhang et al. suggested that His is very important for infant growth and development, which can promote the early development of immune system function of infants, strengthen physiological metabolism, stabilize the utilization rhythm of proteins in the body, and promote body development in infants 48 . Therefore, our findings provide a theoretical foundation for the development of functional and hypoallergenic dairy products. www.nature.com/scientificreports/ Taste is an important characteristic of CM products, with a good taste being a key factor in gaining the favor of consumers. Thus, the effect of Alcalase, Protamex, and Flavourzyme treatment on the taste of CM was evaluated in this study. Our results showed that sourness, aftertaste-B, and astringency were observed in HM, and the sweetness significantly decreased, which indicated that enzymatic hydrolysis with Alcalase, Protamex, and Flavourzyme significantly affected the taste profile of CM. This may be due to the break-down of proteins, which results in the release of a large amount of FAAs and small-molecule peptides with different taste characteristics 49 . Similarly, Phat et al. reported that the different taste of peanut meal hydrolysates might be due to different compositions of FAAs and polypeptides 50 .
Here, color parameters indicated that the color of HM is significantly different to that of CM (lower L*, and higher b* and a* values). This phenomenon occurred because the protein micelles changed into smaller particles during enzymatic hydrolysis. Furthermore, the change in color could be beneficial for incorporation into certain foods, such as cookies and extruded snacks 51 . A similar result was obtained by 52 , who indicated that a significant decrease in L* values were observed in camel milk treated with ultra-high pressure, which is in line with our findings.
Flow behavior can be used to describe and measure the texture of products; it can not only accurately reflect the essential properties of food, but also guide the development of CM products. The findings of the present study showed that the relationship between the shear rate and apparent viscosity of HM is similar to that of CM, and with the increase in shear rate intensity, the apparent viscosity decreased, while shear stress significantly increased. Falcone et al. suggested that the change in apparent viscosity and shear stress is closely related to the shear rate, and the change in apparent viscosity and shear stress might be caused by changes in the shape of CN