Optimization of deamidation of casein by protein-glutaminase and its effect on structural and functional properties

doi:10.21203/rs.3.rs-3963610/v1

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Optimization of deamidation of casein by protein-glutaminase and its effect on structural and functional properties

https://doi.org/10.21203/rs.3.rs-3963610/v1

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published 23 Jun, 2024

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Casein is a commonly used protein in the food industry, and its related products are abundant, such as beverages, desserts, but the further application of casein is limited by its solubility and stability. This study aimed to optimize the deamidation of casein using Protein-glutaminase (PG) and investigate its impact on the structure, solubility and stability of casein. Through center composite experiments, the optimal conditions for PG deamidation were determined to be at pH 6.0, E/S 15 U/g, and a temperature 45°C. The deamidation process alters the secondary structure of casein, resulting in a decrease in α-helix structure and an increase in β-sheet structure. The modification of casein improved emulsifying activity at pH 8.0 and pH 10.0, and significantly enhanced solubility at 5.0 to 6.0. Furthermore, the deamidation of casein leads to an increase in zeta potential and a decrease in particle size, resulting in improved stability of the protein solution due to reduced particle aggregation. The 3% deamidated casein based beverage with carrageenan exhibited reduced precipitation rates compared to the control after sterilization at 121°C for 15 min. In summary, PG deamidation offers a promising method to modify casein, enhancing its functional properties, including solubility, stability and emulsifying activity, thereby expanding the use of casein in the food industry.

Deamidation

Protein-glutaminase

Casein

Structural properties

Functional properties

Drinks.

The optimal conditions for the deamidation of casein by PG were determined.
The secondary structure of deamidated casein decreases in α-helix structure and increases in β-sheet structure.
The deamidation improves emulsifying activity and solubility of casein.
The deamidation improves the zeta potential and stability of the protein solution.

Casein is a crucial protein in bovine milk, constituting around 80% of total protein content and containing essential amino acids (Plank et al., 2008). It exists as casein micelles composed of α_S1-, α_S2-, β- and κ-caseins in proportions 40, 10, 35, and 15% (w/w), respectively (Broyard et al., 2015). These casein micelles are mainly associated with protein-protein interaction, which determine the physicochemical and functional properties of the products containing them. Therefore, casein is not only a recognized source of nutrients like essential amino acids, calcium and phosphorus, but also a widespread techno-functional ingredients in food manufacturing (Lara-Castellanos et al., 2021). In food applications, casein is often used as a protein booster in high protein and low carbohydrate beverages, but the problems of protein flocculation, delamination and precipitation are easy to occur. Frozen desserts such as ice cream and cakes are also heavily fortified with casein, but casein products are unstable and prone to shrinkage and collapse.

Deamidation can make the carboxyl group replace the amide group, so that the protein carries more negative charge. This process leads to protein unfolding, reduced steric hindrance, improved amphiphilicity, and effectively improved functional properties such as solubility, emulsification stability, foaming stability. Therefore, deamidation is a very potential method of modifying proteins. While physical (heating, high-pressure treatment), chemical and enzymatic modification methods can improve the solubility, emulsification, gelling and foaming properties of casein (Broyard et al., 2015), different physicochemical processes can alter the structure and the techno-functionalities of proteins. Of these processes, the charge of casein micelles is a key factor influencing their structure-function relationship, affecting the hydrophilicity/ hydrophobicity balance and techno-functionality of caseins. Chemical modification has the potential to introduce new substances that are difficult to remove, and the operation can be complicated, with the degree of reaction not easy controlled. Enzymatic deamidation of protein has been proposed as an effective approach to enhance the functionality of food proteins and to broaden their potential applications in the food industry (Hamada, 1994). Protein-glutaminase (PG; EC 3.5.1.44) could specifically convert the amide groups of glutamine residues in proteins to carboxyl groups without any side reactions. (Zhang et al., 2021). PG deamidation has been successfully implemented in improving the solubility of soy protein isolate (Jiang et al., 2022), coconut protein (Kunarayakul et al., 2018), wheat gluten (Yong et al., 2006), α-zein (Yong et al., 2004), glutenin and gliadin (Chen et al., 2019), Oat protein (Jiang et al., 2015), and evening primrose protein (Hadidi et al., 2021).

There is limited research regarding casein deamidated by PG. In a study by Miwa et al. (2010), the impact of PG on skim milk proteins was investigated, revealing a dose-dependent increase in their deamidation degree. This treatment led to improved solubility and emulsifying capacity. Miwa et al. (2014) investigated the influence of PG deamidation on the textural and microstructural properties of set yoghurts, demonstrating that PG treatment reduced firmness while increasing adhesiveness. However, to date, there has been a lack of studies directly investigating the impact of PG on casein to assess changes in the structural and functional properties of deamidated casein. Casein is rich in proline peptides that interrupt α-helix and β-strands in its supramolecular structure. Consequently, it is regarded as a rheomorphic protein with minimal secondary or tertiary structure and an exceptionally open and flexible conformation (Holt et al., 1993). These characteristics contribute to its outstanding heat stability (Wusigale et al., 2020), making it highly functional in various food industry applications.

This study aims to optimize the deamidation conditions of casein by PG using response surface methodology and assess the resulting changes in protein structure and functional properties, including emulsifying properties, foamability, and solubility, etc. Additionally, circular dichroism was employed to analyze casein’s secondary structure, while zeta potential and particle size measurements were conducted for deamidated casein. Finally, the stability of beverages based on deamidated casein was evaluated. This investigation contributes to a deeper understanding of the impact of deamidation on the functional properties of casein and provide valuable insights into the potential applications of deamidation casein in the food industry.

2.1 Materials

Casein was obtained from Marcus Trading (Shanghai) Co., Ltd. Protein-glutaminase (500 U/g) was provided by Amano Enzyme, Inc. (Japan).

2.2 Optimization of PG deamidation on casein

The optimal condition for PG deamidation on casein was investigated using response surface methodology (RSM). Degree of deamidation (DD) and degree of hydrolysis (DH) were chosen as dependent variables. The response variables for each dependent variable were estimated using a second-order mathematical model (Eq. (1)):

$$\widehat{y}={\beta }_{0}+{\beta }_{1}{x}_{1}+{\beta }_{2}{x}_{2}+{\beta }_{3}{x}_{3}+{\beta }_{12}{x}_{12}+{\beta }_{13}{x}_{13}+{\beta }_{23}{x}_{23}+{\beta }_{11}{{x}_{1}}^{2}+{\beta }_{22}{{x}_{2}}^{2}+{\beta }_{33}{{x}_{3}}^{2}$$

where $\widehat{y}$ is the estimated response value, ${x}_{1}、{x}_{2}、{x}_{3}$ are independent variables, ${\beta }_{0}$ is the constant term, ${{\beta }_{1}{、\beta }_{2}、\beta }_{3}$ are linear terms, ${\beta }_{12}、{\beta }_{13}、{\beta }_{23}$ are interaction terms, ${\beta }_{11}、{\beta }_{22}、{\beta }_{33}$ are quadratic terms of the model. The regression coefficients of the models were predicted using Design-Expert 8.0.

The reaction conditions that were optimized by RSM included a temperature of 45°C, an E/S ratio of 15 U/g, and a pH of 6.0. The enzymatic deamidation reaction was conducted at different times using optimized conditions, and DD and DH were measured at each time point.

2.3 Preparation of samples with different degrees of deamidation

To prepare the samples, an aqueous solution of 1.0% casein was dissolved in 50 mM sodium phosphate buffer at pH 6.0. The casein solutions were then incubated with 15 U of PG per 1 g casein (abbreviated as ’U/g’) at 45°C for 2 min (CDD15, with DD 15.02%), 10 min (CDD27, with DD 26.88%), and 1 h (CDD32, with DD 32.33%). After PG incubation, the samples were water-bath heated at 80°C for 10 min to inactivation the enzyme. The control group of samples was treated under the same conditions without PG, while the untreated group did not undergo any processing during the experiment.

2.4 Determination of degree of deamidation (DD)

The concentration of free ammonia in the deamidated samples was determined using the indophenol method (Ouyang et al., 2021). The DD was calculated as the percentage of ammonia released from deamidated casein by PG in comparison to the amount of ammonia released from a completely hydrolyzed casein sample, obtained by treating casein with 2 N sulfuric acid at 100°C for 4 h.

2.5 Determination of degree of hydrolysis (DH)

The DH was calculated as the percentage ratio of the soluble protein in supernatant after precipitate deamidated of the deamidated casein sample with 0.2N trichloroacetic acid (TCA) and the soluble protein in supernatant of the completed hydrolysis casein sample. The soluble protein in supernatant was quantified using the BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China), and the concentration was determined using standard curve of bovine serum albumin (BSA).

2.6 Determination of molecular mass distribution by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

The samples, prepared as 10 mg/mL solutions in deionized water, were mixed with loading buffer in a 4:1 ratio and boiled for 15 minutes. A total of 30 µl of appropriately diluted marker and samples were added to the wells of gels with a concentration of 12% acrylamide for the separated gel and 5% polyacrylamide for the concentrated gel. The concentrated gel was run at 80 V for 45 min and the separated gel was run at 100V for 120 min. After electrophoresis, gels were stained with SimplyBlue™ SafeStain (Thermo Fisher Scientific, USA), followed by destaining in a solution of isopropanol and acetic acid.

2.7 Determination of protein secondary structure by circular dichroism (CD)

The CD spectra were determined according to modified method of Miwa et al. (2013). Casein was dispersed in phosphate buffer (pH 7.0) at a final concentration of 3 mg/mL and analyzed in the far ultraviolet region (180–260 nm) using a circular dichroism spectrometer (model Applied Photophysics Chiascan, APL, UK). Samples were analyzed using 1 cm diameter rectangular quartz cuvettes with Teflon caps. The molar ovality value was calculated using Eq. (2):

${\left[\theta \right]}_{molar \lambda }\left(deg {cm}^{2}{dmol}^{-1}\right)=100\times {\theta }_{\lambda /m}\times$ d (2)

where ${\theta }_{ \lambda }$ is the observed ellipticity at wavelength λ, m is the molar concentration of the solute, and d is the path length (cm). Secondary structure analysis of CD spectra was performed using CDNN software.

2.8 Determination of emulsifying properties

The emulsifying properties were determined using a slightly modified method from Gao et al. (2021). A 4 g/L protein solution was mixed with olive oil in a ratio of 3:2, homogenized at 18,000 rpm for 2 min, and allowed to stand at room temperature for 1 h, 12 h, 24 h, and 48 h. During this time, a upper milky layer and a lower water layer were formed. The emulsification activity of the time interval (E_t) was calculated using Eq. (3) for the emulsification activity:

$${E}_{t}=\frac{{ℎ}_{e}}{{ℎ}_{t}}\times 100$$

where ${ℎ}_{e}$ is the height of the emulsion layer and ${ℎ}_{t}$ is the total height of the mixture. The experiments were performed in triplicate on three independent occasions.

2.9 Determination of foaming properties

The foaming properties of the samples were determined using a modified method from Siddique et al. (2016), where lyophilized casein samples were dissolved in 0.1 M phosphate buffer (pH 7.0) at a concentration of 0.5% (w/v). A foam was formed by homogenizing 50 mL of protein solution at 18000 rpm for 1 min. Foaming capacity (FC) and Foaming stability (FS) were calculated using equations (4) and (5) respectively:

$$FC\left(\%\right)=\frac{{V}_{FO}-{V}_{L}}{{V}_{L}}\times 100$$

$$FS\left(\%\right)=\frac{{V}_{FT}}{{V}_{FO}}\times 100$$

where ${V}_{FO}$ is the volume of the solution before foaming without whipping, ${V}_{FO}$is the foam volume immediately after homogenization, and ${V}_{FT}$ represents the foaming volume after standing for T min after whipping.

2.10 Determination of water- and oil-holding capacity

Water-holding capacity (WHC) and oil-holding capacity (OHC) were determined using a slightly modified method based on Jiang et al. (2022). Freeze-dried samples of 0.1 g (W) were added to a 10 mL centrifuge tube (the weight of the tube is W_A), and dissolved in 1mL of deionized water or dispersed in 1 mL of olive oil, followed by vortexed for 30 s. After standing for 30 min, the mixtures were centrifuge at 10,000 rpm for 10 min, the supernatant was discarded, and the tube weight was measured (W_B). WHC or OHC was expressed as grams of water or oil retained per gram of protein, as shown in Eq. (6):

WHC/OHC=(W_B - W_A)/W (6)

2.11 Determination of solubility

The solubility of casein was determined using a modified method from Yong et al. (2004). Firstly, 2 mg of casein was dissolved in 2 mL of buffers with different pH values (2, 3, 3.5, 4, 4.2, 4.4, 4.6, 4.8, 5, 6, 7). The samples were oscillated in a shaker at 25℃ 220 rpm for 1 h, followed by centrifugation at 8000 rpm for 2 min at 10°C. The soluble protein in supernatant was quantified using the BCA Protein Assay Kit (Leagen Biotechnology, Beijing, China), and the concentration was determined using standard curve of bovine serum albumin (BSA). The total protein content was determined by Kjeldahl method. The solubility calculation is shown in Eq. (7):

solubility(%)=$\frac{\text{p}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n} \text{i}\text{n} \text{s}\text{u}\text{p}\text{e}\text{r}\text{n}\text{a}\text{t}\text{a}\text{n}\text{t}\left(\text{m}\text{g}/\text{m}\text{L}\right)}{\text{i}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l} \text{p}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n}\left(\text{m}\text{g}/\text{m}\text{L}\right)}\times 100$ (7)

2.12 Determination of zeta potential and particle size

Zeta potential and particle size were measured using a Nanoparticle Size and Zeta Potential Analyzer (Model NANO ZS3600) following the method of Dan et al. (2020). The proteins were dissolved in deionized water to prepare a 2 mg/mL solution and measurements were taken at room temperature.

2.13 The stability of casein based beverages

First, modified casein was dissolved in water to prepared a 3% protein solution at 50℃ with stirring. Then, 0.01%, 0.03% and 0.05% of carrageenan were added as stabilizer, respectively. Subsequently, the solutions were homogenized by an ATS homogenizer (AH Basic, ATS Engineer Inc., China) at 20MPa and sterilized at 121 ℃ for 15 min. The stability of the casein based beverages was determined by calculating the centrifugal precipitation rate (Peng, 2020). A 10 mL sample of the protein drink was centrifuged at 1000 rpm for 2 min, and the centrifugal precipitation rate was calculated using Eq. (8):

$$\text{C}\text{e}\text{n}\text{t}\text{r}\text{i}\text{f}\text{u}\text{g}\text{a}\text{l} \text{p}\text{r}\text{e}\text{c}\text{i}\text{p}\text{i}\text{t}\text{a}\text{t}\text{i}\text{o}\text{n} \text{r}\text{a}\text{t}\text{e}（\text{%}）=\frac{\text{W}\text{e}\text{i}\text{g}\text{h}\text{t} \text{o}\text{f} \text{p}\text{r}\text{e}\text{c}\text{i}\text{p}\text{i}\text{t}\text{a}\text{t}\text{e}（\text{g}）}{\text{W}\text{e}\text{i}\text{g}\text{h}\text{t} \text{o}\text{f} \text{m}\text{i}\text{l}\text{k}（\text{g}）}\times 100$$

2.14 Statistical analysis

All measurements were performed in triplicate unless otherwise specified. The results are presented as means ± SD. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, Dan Diego, California, United States). Significance was considered at p < 0.05.

3.1 Optimization of PG deamidation on casein

To optimize the deamidation of casein by PG, the center composite experiment was conducted, using enzyme-to-substrate ratio (E/S), temperature, and pH as independent variables, and degree of deamidation (DD) and degree of hydrolysis (DH) as response values. Table 1 displays the DD and DH of 20 experimental combinations. The DH values ranged from 1.03–3.45% across all experimental groups indicating minimal hydrolysis of PG to casein under the experimental conditions, and little impact on the deamidated casein’s quality. Therefore, the following analysis primarily focuses on the DD value.

Table 1

Experimental design (coded and actual values) for the degree of deamidation (DD, %) and degree of hydrolysis (DH, %) for the enzymatic deamidation of casein using protein-glutaminase
design point	independent variables
	coded			actual			DD(%)	DH(%)
	X₁	X₂	X₃	pH	E/S	T
1	1	1	-1	8	15	45	25.62	1.03
2	0	0	0	7	10	50	24.10	2.94
3	0	0	0	7	10	50	24.64	3.15
4	1	-1	-1	8	5	45	16.73	3.45
5	0	0	0	7	10	50	24.48	2.99
6	0	0	0	7	10	50	24.23	2.74
7	-1	-1	1	6	5	55	28.99	1.23
8	1.68	0	0	8.7	10	50	23.21	2.27
9	-1	1	1	6	15	55	28.54	3.02
10	0	0	0	7	10	50	24.40	2.95
11	-1	-1	-1	6	5	45	25.40	1.06
12	0	0	1.68	7	10	58.4	26.29	2.54
13	0	0	0	7	10	50	26.94	2.75
14	0	1.68	0	7	18.4	50	25.97	2.44
15	-1.68	0	0	5.3	10	50	33.87	1.33
16	1	-1	1	8	5	55	21.25	2.84
17	0	-1.68	0	7	1.6	50	11.68	2.29
18	-1	1	-1	6	15	45	32.71	1.17
19	1	1	1	8	15	55	25.53	2.36
20	0	0	-1.68	7	10	41.5	22.80	1.65

The model was fitted with second-order polynomial equations, using actual values to explain DD, as shown in Eq. (9).

DD (%) = 24.74 − 3.25 X₁ + 3.23 X₂ + 0.71 X₃ + 0.79 X₁X₂ + 0.63 X₁X₃ − 1.55 X₂X₃ + 1.68 X₁² − 1.76 X₂² + 0.26 X₃² (9)

DD is the degree of deamidation (%), X₁ is pH, X₂ is E/S (U/g protein), and X₃ is the temperature (°C). The R² value of DD is 0.9389, indicating a significant model. The lack of fit is not significant, and the model is credible.

The Design-Expert software was used to determine the optimal conditions for DD, which were found to be a pH of 6.0, an E/S of 15 U/g, and a temperature of 45°C. Using these conditions, the software calculated a maximum DD of 32.08%.

Figure 1 illustrates that the DD was significantly influenced by pH and E/S, with temperature held constant at 50°C. Increasing the E/S at this temperature led to a rise in DD, but this increase was more pronounced at lower pH values. Yamaguchi et al. (2001) found that the enzymatic activity of PG is significantly reduced at pH values above 6. Therefore, at pH values above 6, increasing E/S does not lead to a considerable increase in DD.

3.2 Degree of deamidation and degree of hydrolysis of Casein

The time-dependent increases in DD and DH of casein induced by PG under the optimized conditions are shown in Fig. 2. The DD was 23.36% at a reaction time of 0.5 h, which increased to 30.23% at 1 h. The DD continued to increase gradually until it reached a peak of 35.83% at 3 h, and then remained relatively stable. The degree of hydrolysis remain stable around 5.5% from 0 to 24h. Deamidated casein samples were obtained after deamidation reactions for 2 min, 10 min and 1 h of under the optimized conditions. The resulting deamidation degrees were 15.02%, 26.88%, and 32.33%, respectively, while the corresponding hydrolysis degree was 3.49%, 3.57%, and 3.80%. The samples were designated as CCD15, CCD27 and CCD32.

Hadidi et al. (2021) used the response surface method to measure the optimal conditions for the PG deamidation of evening primrose seed cake. Under the optimized extraction conditions, DD was 39.40%. Jiang et al. (2015) determined the optimal extraction conditions of PG for oat protein deamidation, and DD was up to 59%. Yongle et al. (2011) deamidation of rice glutelin by PG, and DD was 52.29% under optimal conditions. The maximum DD of casein was similar to that of evening primrose seed cake, but slightly lower than oat protein and rice glutelin.

3.3 SDS-PAGE

The untreated samples, the control samples, and CDD15, CDD27, CDD32 samples were analyzed by SDS-PAGE, and the electrophoresis patterns are shown in Fig. 3.

Casein is composed of three main monomers, α-, β-, and κ-casein, with molecular weights of about 27.1 kDa, 24.9 kDa, and 19.0 kDa, respectively (Bobe et al., 1998). The positions of these monomers in the untreated casein are indicated in Fig. 3. The positions of the three monomers in the deamidated casein samples (Fig. 3) were consistent with those of the control casein, indicating that the deamidation treatment only modified the protein side chain and did not significantly alter the molecular weight of casein. This finding is in agreement with the study by Miwa et al. (2010), who observed that PG deamidation of skim milk did not result in changes in molecular weight.

The band density reflects the relative protein content. As can be observed from the density of each band in the figure, the content of the α-casein monomer was the highest, while the content of the κ-casein monomer was lower, indicating differences in the content of each monomer in casein. SDS-PAGE also showed that the density of the α- and β-monomer bands in the different casein samples increased with the degree of deamidation. This may be attributed to the increase in soluble protein resulting from PG deamidation (Temthawee et al., 2020). The band density of the control casein was higher than that of untreated casein because it was heated at 50°C for 1 h, which increased its solubility and thus the band density.

3.4 Circular Dichroism (CD)

The far-ultraviolet CD spectroscopy (180–260 nm) was used to analyze the secondary structure changes induced by deamidation. The secondary structure composition of the samples is presented in Table 2. When comparing control and deamidated casein, deamidation treatment reduced the α-helix proportion while increasing the β-sheet proportion. However, the ratio of random coils and β-turns was minimally affected by deamidation. Yong et al. (2004) reported a decrease in a α-helix structure and an increase in β-sheet structure after PG deamidation of α-zein, which is consistent with the results of this experiment. The control casein exhibited reduced α-helices and increased β-sheets compared to untreated casein. This shift in secondary structure resulted from a known heat-induced conversion of α-helices to β-sheets, as reported by Drummy et al., (2005). Deamidation of glutamines into negatively charged glutamic residues through the PG reaction increases electrostatic repulsion, altering the packing arrangement of the α-helix domain and consequently destabilizing α-helix formation.

Table 2

Determination of secondary structure of casein by circular dichroism
casein samples	secondary structure(%)
casein samples	α-helix	β-sheet	β-turn	random coil
untreated	44.3	34.4	21.2	0.1
control	32.4	47.8	19.7	0.1
CDD15	17.8	58.7	23.4	0.1
CDD27	27.2	51.1	21.5	0.2
CDD32	18.0	58.3	23.5	0.2

3.5 Determination of emulsifying properties

In assessing casein’s emulsifying properties, five types of vegetable oils (coconut oil, linoleic acid, olive oil, sunflower oil, and corn oil) were individually mixed with deamidated casein CDD32 at a ratio of 3:2. As shown in Fig. 4, these emulsions remained relatively stable after 1 h. Notably, the coconut oil emulsion exhibited poor stability, becoming unstable after 12 h, with emulsifying activity declining rapidly over time. In contrast, when linoleic acid, olive oil, sunflower oil, or corn oil was used as the oil phase, the emulsion system remained relatively stable for 48 h. The emulsifying activity of olive oil emulsion did not significantly decrease even after 72 h of standing, indicating good emulsifying stability. Consequently, olive oil was selected as the emulsified oil phase for subsequent experiments.

Emulsifying activity of a 4 mg/mL deamidated casein solution, mixed at a 3:2 ratio with olive oil and homogenized to form an emulsion, was evaluated under different pH levels and various standing times (Table 3). At pH 2.0, the emulsifying activity of deamidated casein was comparable to the control. However, the emulsion became highly unstable at pH 4.0, likely due to the proximity of pH 4.0 to the isoelectric point of casein. At this point, proteins tend to be poorly soluble, leading to precipitation and aggregation. This condition may also hinder effective encapsulate of oil molecules, resulting in reduced emulsifying activity (Cornacchia et al., 2014; Raak et al., 2017). Importantly, at H 8.0 and pH 10.0, deamidation significantly enhanced the emulsifying activities of casein, with CCD15, CDD27 and CDD32 displaying higher emulsifying activities than control casein.

Table 3

Emulsifying activity of casein samples mixed with olive oil under different pH
casein samples	E_t	pH
casein samples	E_t	2.0	4.0	6.0	8.0	10.0
untreated	E₁	61.50 ± 0.50	50.40 ± 0.40	63.10 ± 1.00	71.00 ± 1.00	80.60 ± 0.60
	E₁₂	59.00 ± 1.00	0.00 ± 0.00	61.00 ± 1.00	65.56 ± 1.00	75.50 ± 0.50
	E₂₄	58.90 ± 1.10	0.00 ± 0.00	60.10 ± 0.10	64.45 ± 0.45	75.40 ± 0.20
	E₄₈	58.80 ± 1.20	0.00 ± 0.00	60.05 ± 0.05	64.05 ± 0.05	75.20 ± 0.00
control	E₁	60.70 ± 0.70	42.20 ± 0.20	61.80 ± 0.20	74.40 ± 0.40	79.90 ± 0.30
	E₁₂	60.00 ± 0.00	0.00 ± 0.00	61.20 ± 0.10	63.50 ± 0.50	64.50 ± 0.50
	E₂₄	60.00 ± 0.00	0.00 ± 0.00	61.08 ± 0.10	63.50 ± 0.10	64.10 ± 0.10
	E₄₈	58.10 ± 0.90	0.00 ± 0.00	60.50 ± 0.50	62.20 ± 0.20	63.50 ± 0.50
CDD15	E₁	62.20 ± 0.20	51.00 ± 1.00	69.60 ± 0.60	80.50 ± 0.50	85.80 ± 0.20
	E₁₂	59.80 ± 0.20	0.00 ± 0.00	60.10 ± 0.10	70.50 ± 0.50	70.90 ± 0.10
	E₂₄	59.40 ± 0.00	0.00 ± 0.00	60.10 ± 0.30	69.90 ± 0.10	70.20 ± 0.20
	E₄₈	59.10 ± 0.10	0.00 ± 0.00	60.10 ± 0.10	69.30 ± 0.30	69.60 ± 0.00
CDD27	E₁	62.80 ± 0.80	45.40 ± 0.20	80.10 ± 0.10	91.00 ± 1.00	96.60 ± 0.60
	E₁₂	60.60 ± 0.20	0.00 ± 0.00	62.20 ± 0.20	77.40 ± 0.40	82.20 ± 0.20
	E₂₄	59.70 ± 0.10	0.00 ± 0.00	61.50 ± 0.10	76.60 ± 0.20	80.10 ± 0.10
	E₄₈	59.17 ± 0.17	0.00 ± 0.00	60.20 ± 0.20	76.50 ± 0.10	79.90 ± 0.50
CDD32	E₁	69.50 ± 0.50	45.30 ± 0.10	80.20 ± 0.20	94.00 ± 0.20	96.60 ± 0.20
	E₁₂	60.10 ± 0.10	0.00 ± 0.00	61.80 ± 0.20	81.80 ± 0.20	80.30 ± 0.10
	E₂₄	59.90 ± 1.10	0.00 ± 0.00	61.05 ± 0.05	81.70 ± 0.10	80.20 ± 0.20
	E₄₈	59.20 ± 0.20	0.00 ± 0.00	60.60 ± 0.40	80.20 ± 0.20	80.10 ± 0.10

E₁, E₁₂, E₂₄ and E₄₈ mean emulsification index after 1, 12, 24 and 48 h, respectively.

At pH 8.0, E₄₈ of deamidated casein samples was higher than that of untreated and control samples, indicating an improvement in protein emulsification duo to deamidation. This finding is consistent with the observations made by Miwa et al. (2010), who reported that the emulsifying capacity of deamidated skim milk increased with higher deamidation degree. While interactions between the protein backbone and side chains can promote the formation of aggregates (Zaitsu et al., 2015), deamidation, by converting glutamine to glutamate, weakens this aggregation effect by altering the side chain. Moreover, deamidation reduces NH₂ groups, disrupting intramolecular and intermolecular hydrogen bonds and enhancing electrostatic repulsion (Chen et al., 2021). These changes may reduce protein aggregation, enhance dispersion, and improve the emulsification of the oil phase. Deamidation also causes protein unfolding, exposing previously hidden hydrophobic regions (Miwa et al., 2013), and improving amphiphilicity. This results in the complete encapsulation of oil molecules by soluble casein in the solution and strong emulsifying activity.

3.6 Determination of foaming properties

The foaming ability of deamidated casein was significantly higher than that of the untreated and control groups, and it increased with the degree of deamidation increased (Table 4). Specifically, the foaming ability of sample CDD32 was 2.25 times greater than that of the untreated casein and 2.34 times greater than that of the control casein. Hadidi et al. (2021) also observed an improvement in the foaming ability of evening primrose protein following deamidation by PG. This enhance foaming ability may be attributed to increased protein solubility (Townsend et al., 1983), where the increment in foam ability could be attributed to the presence of solubilized proteins at the air-water interface. The deamidation treatment could enhance the net electrostatic charge of the protein, resulting in an improved propensity for foam formation.

The foaming stability of casein decreased with increasing deamidation degree (Fig. 5A), significantly within 40 min. CDD15’s foaming stability dropped to around 75%, and CDD32’s to about 40%. Notably, control casein and CDD15 had better foaming stability than untreated, suggesting that heat treatment and mild deamidation treatment may enhance it. However, while deamidation enhances foaming ability, it also increases protein charge, potentially destabilizing the cohesive protein membrane and affecting foam retention (Suppavorasatit et al., 2011).

Table 4

Effects of PG deamidation on foaming ability, water-/oil- holding capacity, zeta potential and particle size of casein
samples	FC(%)	WHC (g water/g protein)	OHC (g oil/g protein)	zeta potential (mV)	Z-Average (d.nm)
untreated	72.00^d ± 2.00	3.66^d ± 0.16	4.13^d ± 0.11	-11.4^c ± 0.5	6440.0^d ± 999.0
control	69.00^d ± 1.00	3.94^c ± 0.08	5.15^c ± 0.26	-19.0^b ± 0.6	1066.0^c ± 49.0
CDD15	86.00^c ± 4.00	4.49^b ± 0.02	8.35^b ± 0.02	-20.7^b ± 1.7	435.5^b ± 82.1
CDD27	120.00^b ± 4.00	4.66^a ± 0.09	9.42^a ± 0.08	-23.8^a ± 0.2	401.0^b ± 29.3
CDD32	162.00^a ± 2.00	4.78^a ± 0.02	9.31^a ± 0.04	-24.2^a ± 1.5	195.4^a ± 21.7

Values are mean ± standard deviation. Different lowercase letters (a-d) in each column indicate significant differences within the same group (p < 0.05).

3.7 Determination of water- and oil-holding capacity

Deamidated casein exhibited higher WHC and OHC compared to untreated and control casein (Table 4). Specifically, CDD15, CDD27 and CDD32 had OHC value 1.62, 1.83 and 1.81 times greater than that of control casein. Ye et al. (2018) showed that the flexibility of protein conformation significantly influenced water-oil adsorption capacity. Deamidation by PG converted amide groups into carboxyl groups, increasing negative charge and strengthening protein binding with water/oil molecules. Deamidation also altered the secondary structure of the protein (Table 2), reduced steric hindrance, and increased conformational flexibility (Santos et al., 1999), ultimately enhancing the ability of the protein to adsorb water or oil molecules.

3.8 Determination of Solubility

The solubility curves of casein samples reveal a U-shaped pattern with the highest and lowest solubility at the pH extremes (Fig. 5B), which is consistent with the findings of Post et al. (2012). The pH range of 3.0 to 4.8, near the isoelectric point of casein, results in low solubility for all casein samples. The solubility rapidly increased below pH 3.0 and above pH 5.0. Notably, at pH 5.0 to 6.0, deamidated casein exhibits higher solubility than untreated and control casein. PG-induced deamidation converts the amide group of the protein side chain into a more stable carboxyl group in the ionic form, increasing the net charge of the protein and enhancing the interaction between the protein and water, leading to increased solubility. Within the pH range of 6.0 to 7.0, the differences in solubility among samples decreased, and the solubility of all casein samples remained at around 55%, and remained relatively constant.

Untreated and control casein samples were least soluble at pH 4.2, whereas deamidated samples exhibited their lowest solubility at pH 4.0. As pH approached the isoelectric point, solubility decreased for all samples. Figure 5C illustrates the appearance of the dissolved casein samples at pH 4.0, where the flocculation of the casein markedly increased with increasing deamidation, indicating that pH 4.0 was close to the isoelectric point of modified casein. Hadidi et al. (2021) also observed that the isoelectric point of evening primrose protein shifted to the left after PG deamidation. The conversion of amide groups to carboxyl groups via deamidation increased the negative charge and improved the polyelectrolyte properties of the protein, leading to a change in the isoelectric point.

3.9 Measurement of zeta potential and particle size

Untreated casein had a zeta potential of -11.4 mV, while control casein showed an absolute zeta potential increase to -19.0 mV, potentially attributed to heat treatment. The absolute zeta potential value rose with higher degree of deamidation. Specifically, CDD 15, CDD27 and CCD32 had zeta potentials of -20.7 mV, -23.8 mV and − 24.2 mV, respectively. Previous studies have also reported an increase in zeta potential with increasing deamidation degree. The zeta potential of deamidation deamidated wheat gluten (Liao et al. 2016) and rice glutenin (Yongle et al. 2011) both exhibited an increase with a higher degree of deamidation. The increase in absolute zeta potential could be attributed to reduced intramolecular and intermolecular hydrogen bonds caused by the deamidation treatment, which generated strong electrostatic repulsion and enhanced solution stability (Dan et al., 2020).

Particle size is a critical factor in maintaining the stability and cohesion of casein solution (Bouzid et al., 2008). The untreated casein exhibited a larger particle size (Z-average) of 6440 nm, indicating a higher degree of casein micelles aggregation (Table 4). Heat treatment in the control casein disrupted the casein micelles, leading to a significantly reduced in particle size to 1066 nm. Deamidation further decreased the casein particle size. As the degree of deamidation increased, the particle size of casein gradually decreased to 195.4 nm for CCD32. Deamidated pea protein isolate also displayed a reduced particle size (Fang et al. 2020). Smaller particle sizes intensify Brownian motion, which enhances system stability (Cui et al., 2014). Miwa et al. (2010) reported that transmission electron microscopy (TEM) analysis revealed the image of PG-deamidated skim milk samples, showing the complete disintegration of casein micelles into smaller particles. This suggests that PG induces the disruption of casein micelles into smaller, sub-micellar particles. Therefore, PG treatment reduced casein particle size and improved casein solution stability.

3.10 Stability of beverages based on deamidated casein

The food industry utilizes casein for various purposes, including protein beverage production and enhancing protein content. Figure 5B demonstrates that deamidated casein considerably improve solubility. Carrageenan, a common stabilizer in milk-base beverages, form a helical aggregation state with casein to stable the solution (Toshifumi et al. 2023).

The effects of carrageenan addition on the precipitation rate of 3% of deamidated casein based beverages (pH 7.0) sterilized at 121°C for 15 minutes is presented in Fig. 6. Deamidated casein based beverages exhibited lower precipitation rates than untreated and control casein based beverages at different carrageenan concentrations. CDD32 beverage had the lowest precipitation rate, with value of 0.34%, 0.27% and 0.25% with the addition of 0.01%, 0.03% and 0.05% carrageenan, respectively.

Table 5 analyzes the appearance of 3% deamidated casein based beverages with varying carrageenan concentration. Untreated and control casein based beverages demonstrated significantly precipitation after 1 d. Deamidated casein based beverages with 0.01% and 0.03% carrageenan exhibited precipitation after 1 d as well. For samples with 0.05% carrageenan, CDD15 and CDD27 based beverages displayed precipitation after 4 d or 8 d, respectively. However, CDD32 based beverages remained stable without precipitation for 15 d. This result suggested that the casein based beverages stability increases with greater casein deamidation.

Table 5

Appearance analysis of 3% deamidated casein based beverages with different carrageenan concentration
Carrageenan concentration(%)
days	untreated			control			CDD15			CDD27			CDD32
	0.01	0.03	0.05	0.01	0.03	0.05	0.01	0.03	0.05	0.01	0.03	0.05	0.01	0.03	0.05
	1	+	+	+	+	+	+	+	+	−	+	+	−	+	+	−
4	+	+	+	+	+	+	+	+	+	+	+	−	+	+	−
8	+	+	+	+	+	+	+	+	+	+	+	+	+	+	−
15	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+

+ means precipitation occurs, − means on precipitation occur.

This study optimized the PG deamidation process of casein using a center composite experiment, achieving high deamidation rates while minimizing hydrolysis. Deamidation altered the casein structure, improving emulsifying activity at pH 8.0 and 10.0 and solubility at pH 5.0–6.0. The modified casein demonstrated better foaming, water retention, and oil-holding capabilities, as well as improved stability due to reduced particle aggregation. Deamidated casein based beverages with carrageenan also showed lower precipitation rates, indicating potential for creating stable protein beverages. These findings suggest that deamidated casein has a wide range application in the food industry, particularly in beverages, and can address some prevailing issues, such as unstable foaming, and poor solubility under weakly acidic environment, to meet the demands of the market.

CRediT author statement

Deming Jiang: Methodology, Investigation, Data curation; Wei Ouyang: Investigation, Data curation, Writing – original draft; Lingling Huang: Investigation, Data curation, Writing – original draft; Jinjin Niu: Data curation, Writing – review & editing; Zheng Zhang: Data curation; Congli Jin: Data curation; Siyi Gu, Data curation; Mengmeng Liu, Data curation; Zhongyi Chang: Conceptualization, Data curation; Yanning Niu: Methodology; Chunjing Zou: Data curation; Jing Huang: Data curation; Caifeng Jia: Data curation; Lihua Tang: Methodology, Data curation, Supervision; Hongliang Gao: Conceptualization, Methodology, Data curation, Writing – review & editing, Funding acquisition.

Conflict of interest

The authors declare that they have no conflicts of interest in this work.

Funding

The authors appreciate the support provided by the Natural Science Foundation of Shanghai (22ZR141200), and we thank the Instruments Sharing Platform of School of Life Sciences, East China Normal University for technical service.

Data availability statement

The data that support the findings of this study area available from the corresponding author upon reasonable request.

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Optimization of deamidation of casein by protein-glutaminase and its effect on structural and functional properties

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Abstract

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Highlights

1 Introduction

2 Materials and methods

2.1 Materials

2.2 Optimization of PG deamidation on casein

2.3 Preparation of samples with different degrees of deamidation

2.4 Determination of degree of deamidation (DD)

2.5 Determination of degree of hydrolysis (DH)

2.6 Determination of molecular mass distribution by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

2.7 Determination of protein secondary structure by circular dichroism (CD)

2.8 Determination of emulsifying properties

2.9 Determination of foaming properties

2.10 Determination of water- and oil-holding capacity

2.11 Determination of solubility

2.12 Determination of zeta potential and particle size

2.13 The stability of casein based beverages

2.14 Statistical analysis

3 Results and discussion

3.1 Optimization of PG deamidation on casein

3.2 Degree of deamidation and degree of hydrolysis of Casein

3.3 SDS-PAGE

3.4 Circular Dichroism (CD)

3.5 Determination of emulsifying properties

3.6 Determination of foaming properties

3.7 Determination of water- and oil-holding capacity

3.8 Determination of Solubility

3.9 Measurement of zeta potential and particle size

3.10 Stability of beverages based on deamidated casein

4 Conclusion

Declarations

References

Additional Declarations

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