Xin Wang,Chenchen Gou,Chunyuan Gao,Yazhen Song,Jinming Zhang,Dandan Zhai,Yawei Zhao,Jihong Huang,Ming Hui

College of Biological Engineering,Henan University of Technology,Zhengzhou 450001,China

ABSTRACT The γ-PGA-based food hydrogel exhibits good biodegradability and biocompatibility,and is non-toxic to humans.In this study,a novel hydrogel(γ-PGA/casein composite hydrogel)was prepared by combining casein with γ-PGA separated from natto using microbial transglutaminase(MTG)as the crosslinker.The effects of the MTG mass concentration,gelation time,crosslinking temperature,pH,and the mass ratio of γ-PGA to casein on the crosslinking ratio of γ-PGA and swelling ratio of the γ-PGA/casein hydrogel were investigated.Furthermore,the synthesis conditions were optimized by response surface methodology to optimize three important factors,i.e.crosslinking temperature,pH and the mass ratio of γ-PGA to casein.The results of single factor experiments showed that the MTG mass concentration and crosslinking time had little impacts on the crosslinking ratio of γ-PGA and the swelling ratio;the preferred MTG mass concentration,crosslinking time,temperature and pH were selected as 0.3%,7 h,40°C and 7.5;and the preferred mass ratio of γ-PGA to casein was 0.1 for the crosslinking ratio of γ-PGA and 0.2 for the swelling ratio.The response surface experiment results showed that the optimum crosslinking temperature,pH and the mass ratio of γ-PGA to casein ratio were 42.4°C,7.33,and 0.18,respectively.Under these optimum conditions,the predicted crosslinking ratio of γ-PGA and swelling ratio reached 90.35% and 112.07%,which were highly consistent with the experimental values(88.00%and 115.87%).The γ-PGA/casein composite hydrogel developed in this study shows promising application potential in food industry,biomedical field,and cosmetic industry.

Keywords:Hydrogel synthesis MTG cross-linking γ-PGA Casein Composite hydrogel

1.Introduction

Gamma-polyglutamic acid(γ-PGA)is a well-known component of the traditional Japanese food,generally called natto,it is the main component of natto mucilage(viscous substances),which is produced by several Bacillus species during food fermentation.γ-PGA hydrogel has a threedimensional(3D)network structure,formed by the crosslinking of γ-PGA and a naturally occurring homopolyamide composed of D-and L-glutamic acid units connected by amide linkages between the α-amino and γ-carboxylic acid groups[1].γ-PGA hydrogel is watersoluble,water-absorptive,biodegradable,edible,and nontoxic to humans and the environment[2-4].Owing to these properties,γ-PGA hydrogel has attracted significant interest as a promising drug carrier,an antifreeze agent,a food thickener,a flocculating agent for environmental protection,and humectants in cosmetics[5,6].

γ-PGA hydrogels are prepared using various methods including the radial method[7-9],chemical method[10,11],and self-assembly method[12].However,the commercialization of the radial method is expensive owing to its complex preparation conditions[7,8].In addition,the chemical agents used in the chemical method are often toxic to humans[13],and the self-assembled hydrogels used in the self-assembly method exhibits poor stability and poor mechanical properties[11].Consequently,the use of crosslinking agents with low toxicity have gained increased attention for the crosslinking of γ-PGA-based materials.

Various materials have been combined to prepare γ-PGA hydrogels with distinctive properties to expand the application scope of γ-PGA hydrogel.For example,chitosan(CS)and alginate(SA)have been combined with γ-PGA to form a CS/γ-PGA/SA composite hydrogel,and the formed hydrogel exhibited significantly improved strength and structural stability[14].In addition,poly(vinyl alcohol),β-tricalcium phosphate,ε-polylysine and others have been incorporated into γ-PGA to form the novel composite hydrogels[15-17].Microbial transglutaminase(MTG)is a common enzyme crosslinker that enables the covalent crosslinking between the lateral chains of the lysine and glutamine residues of proteins such as casein.Caseins are proline-rich openstructured phosphoproteins found in raw milk.More than half of the functional groups in caseins are hydrophilic,including lysine and glutamine residues.Several studies have investigated the crosslinking of caseins by MTG[18-21].

In this study,γ-PGA with a free chemically functionalized carboxyl side group(-COOH)was combined with casein hydrogel via hydrogen bonding using a nontoxic crosslinking agent(MTG)to prepare a novel γ-PGA/casein composite hydrogel.The effects of the MTG concentration,crosslinking time,temperature,pH,and the mass ratio of γ-PGA to casein on the crosslinking ratio of γ-PGA and swelling ratio of the γ-PGA/casein hydrogel were systemically investigated.In addition,a fast and reliable response surface methodology(RSM)was conducted to evaluate the optimum conditions for the preparation of the γ-PGA/casein composite hydrogel.

2.Methods

2.1.Preparation of the MTG-crosslinked γ-PGA/casein hydrogel

Casein powder(Tianjin Guangfu Fine Chemical Research Institute,China)was dispersed in 1 mol/L Tris-HCl buffer(pH 7.5),and the mixture was stirred at 50°C overnight to ensure the complete dissolution of the casein powder.Subsequently,Tris-HCl buffer was added into the mixture until the casein mass concentration reached 10%.The γ-PGA solution was prepared by dissolving the appropriate weight of γ-PGA powder(50-2,000 kD,Hetianlong Biotech Co.,Ltd.,Haining,China)in 1 mol/L Tris-HCl buffer(pH 7.5).Then,equivalent volumes(10 mL)of the casein and γ-PGA solutions were mixed together,and the pH was adjusted to specific values with 1 mol/L HCl and Tris solution,after which specific mass concentration of MTG was added to initiate the gelation reaction.The samples were then incubated at specific temperatures for specific time,and the enzymeinduced crosslinking reactions were terminated by placing the samples in boiling water for 5 min.Subsequently,the samples were aged in a refrigerator at 4°C for 15 h without disturbance to enable the hydrogel formation,after which the supernatant was collected to determine the γ-PGA mass concentration and calculate the crosslinking ratio of γ-PGA.The hydrogel was placed in a freeze-dryer for 48 h for subsequent determination of the swelling ratio.

2.2.Single factor experiments

Crosslinking ratio of γ-PGA and swelling ratio of hydrogel are two key indicators for the crosslinking reaction.We conducted single factor experiments to investigate the effects of four factors on the two indicators.While investigating a certain factor,the other factors were set to a fixed value,7 h for crosslinking time,0.3%for MTG mass concentration,40 °C for crosslinking temperature,0.2 for mass ratio of γ-PGA to casein,and 7.5 for pH.The factors and levels were shown in Table 1.

2.3.Determination of crosslinking ratio of γ-PGA

The mass concentration of γ-PGA in the hydrogel was determined by ultraviolet spectrophotometry.First,50 mg of γ-PGA powder was dissolved in 1 mol/L Tris-HCl buffer(pH 7.5).Subsequently,different volumes of the prepared solution(0.4,0.8,1.2,1.6 and 2.0 mL)were transferred to a 50-mL volumetric flask respectively to obtain the standard solutions,then the standard solutions were diluted with Tris-HCl buffer solution to volume.The absorbance values were measured at 216 nm and the standard curve was plotted(Fig.1).

Table 1 Factors and levels used in One-Factor-at-a-Time optimization.

Fig.1.Standard curve of the γ-PGA.

The mass concentration of non-crosslinked γ-PGA in the hydrogel was determined from the standard curve and the crosslinking ratio of γ-PGA was calculated according to the following equation:

where N0and N1are the mass concentration of the total γ-PGA and the non-crosslinked γ-PGA,respectively.All experiments were conducted in triplicate.

2.4.Determination of swelling ratio

The swelling ratio was determined according to the previous report[22].Briefly,fast-frozen and freeze-dried MTG-crosslinked γ-PGA/casein hydrogels were measured by electronic balance(Sartorius BT 125D and Sartorius BS 124S,Sartorius Co.,Germany).Subsequently,the hydrogels were immersed in 5 mL of distilled water at 25°C for approximately 60 h.A part of the samples were taken out every 5 h from the tenth hour,wiped with filter paper to remove the external solution,and re-weighed.The swelling ratios of the hydrogel samples were calculated by the following equation:

where Wtis the weight of the swollen hydrogel sample and W0is the weight of the freeze-dried hydrogel sample.All experiments were repeated three times.

2.5.Box–Behnken design and RSM

The Box-Behnken Design(BBD)was used to further investigate the optimum conditions of the key factors in the preparation of the γ-PGA/casein hydrogel.The coded values and real values of the variables were listed in Table 2.The obtained results were analyzed by RSM.The experimental variables were coded using the following equation:

where Xiis the coded value of an independent variable,xiis the real value of an independent variable,x0is the real value of an independent variable at the center point,and Δxiis the value of the step change.The behavior of the system was described using the following quadratic equation:

where Y is the predicted response(herein it is the crosslinking ratio of γ-PGA or swelling ratio of thehydrogel),β0is the offset term,βiis the linear effect,βiiis the squared effect,βijis the interaction effect,Xiand Xjare the coded independent variables.The second-order polynomial coefficients were calculated and analyzed using the“Design Expert”software package(Version 12.0.3.0,Stat-Ease Inc.,Minneapolis,USA).Statistical analysis of the model was performed to evaluate the analysis of variance(ANOVA).

Table 2 Factors and levels and results of BBD.

3.Results and discussions

3.1.Preparation of the γ-PGA/casein hydrogel

We could see from Fig.2 that both the casein solution and the γ-PGA solution were liquid,but they are quite different in viscosity(Fig.2c and d).At the beginning of the crosslinking reaction,casein and γ-PGA formed a viscous solution in the absence of MTG,but no hydrogel was formed(Fig.2a).With the addition of MTG,the γ-PGA/casein solution formed a stable hydrogel(Fig.2b),indicating that the MTG contributed to the crosslinking between γ-PGA and casein.A casein-MTG hydrogel was also formed in the mixture of casein solution and MTG(Fig.2f),which was consistent with the previous report[18].However,the casein-MTG hydrogel was different from the γ-PGA/casein hydrogel we prepared in appearance.

Fig.2.Photographs of γ-PGA/casein precursor solution and the hydrogel formed with MTG.

3.2.Results of single factor experiments

3.2.1.Effects of the MTG mass concentration on the crosslinking reaction

As shown in Fig.3,with the increase of the MTG mass concentration,both the crosslinking ratio of γ-PGA and the swelling ratio increased and stabilized from the MTG mass concentration of 0.3%.These results indicated that the addition of MTG was beneficial to the crosslinking of γ-PGA and casein.The lateral chains of the lysine and glutamine residues present on the casein could be crosslinked with MTG covalently,then γ-PGA could be reacted with casein through hydrogen bonds to form novel γ-PGA/casein hydrogels.Therefore,a certain amount of MTG was necessary to initiate hydrogel formation,but higher amount of MTG showed no positive influence on the swelling ratio of hydrogels.In general,the MTG mass concentration had little effect on the crosslinking ratio of γ-PGA and swelling ratio of the hydrogel,and the optimum mass concentration of MTG was 0.3%.

Fig.3.Effect of MTG mass concentration on the crosslinking ratio of γ-PGA(a)and swelling ratio of the γ-PGA/casein hydrogel(b).

3.2.2.Effects of reaction time on the crosslinking reaction

As shown in Fig.4,both the crosslinking ratio of γ-PGA and swelling ratio of hydrogel increased to the highest values(79% at 6 h,and 93.18% at 7 h),then decreased slightly with the reaction time increasing.The longer reaction time may inhibit the enzyme activity so as to result in the declines of crosslinking ratio and the swelling ratio.Comprehensively considering the two indicators,the optimum reaction time was selected as 7 h for subsequent study.

Fig.4.Effect of the reaction time on the crosslinking ratio of γ-PGA(a)and swelling ratio of the γ-PGA/casein hydrogel(b).

3.2.3.Effects of temperature on the crosslinking reaction

As shown in Fig.5,the temperature had obvious effects on the crosslinking ratio of γ-PGA and the swelling ratio of the hydrogel,and two indicators had the same tendency,both increasing sharply from 30°C to 40°C then dropping dramatically from 40 °C to 50 °C.Typically,the optimum MTG crosslinking temperature was in the range of 37-45 °C[23].Therefore,the optimum temperature was selected as 40°C for subsequent experiments.

Fig.5.Effect of temperature on the crosslinking ratio of γ-PGA(a)and swelling ratio of the γ-PGA/casein hydrogel(b).

3.2.4.Effects of pH on the crosslinking reaction

The effects of pH on the crosslinking ratio of γ-PGA and swelling ratio of hydrogel were shown in Fig.6.With the increase of pH from 5.5 to 9.5,the crosslinking ratio and the swelling ratio increased firstly and then dropped sharply,indicating that the MTG preferred a pH-neutral environment(Fig.6a)[24-26].Furthermore,the swelling ratio increased significantly after soaking for 25 h,and the maximum value was observed at pH 7.5(Fig.6b).Similar to the temperature,pH was also a key parameter for the preparation of γ-PGA/casein hydrogel,and was chosen for the RSM experiments.

Fig.6.Effect of pH on the crosslinking ratio of γ-PGA(a)and swelling ratio of the γ-PGA/casein hydrogel(b).

3.2.5.Effects of the mass ratio of γ-PGA to casein on the crosslinking reaction

We could seen from Fig.7 that the crosslinking ratio decreased significantly from the mass ratio of 0.1 to 0.5(Fig.7a),while the swelling ratio rose to the highest value at the mass ratio of 0.2 and then decreased(Fig.7b).Combined with the crosslinking ratio of γ-PGA and swelling ratio results,the mass ratio of γ-PGA to casein at 0.2 should be a better choice for subsequent experiments.

Fig.7.Effects of the mass ratio of γ-PGA to casein on the crosslinking ratio of γ-PGA(a)and swelling ratio of the γ-PGA/casein hydrogels(b).

3.3.Results of BBD design and RSM

RSM has been extensively used to optimize γ-PGA production by the microorganisms[4,27-30],however,little attention has been paid to the optimization of the properties of γ-PGA-based composite hydrogels using the RSM approach.In this study,results of the single factor experiments have confirmed that the crosslinking temperature,pH,and the mass ratio of γ-PGA to casein had significant effects on the crosslinking ratio of γ-PGA and swelling ratio of the hydrogel.Thus,we employed the BBD to investigate the interaction effects of the three factors on the crosslinking ratio of γ-PGA and 50-hour swelling ratio of the γ-PGA/casein hydrogel.Seventeen experiments were conducted with three independent variables at three levels(-1,0,1).The factors and levels and results of BBD were shown in Table 2.

3.3.1.Optimizaiton results of the crosslinking ratio of γ-PGA

The ANOVA results for the crosslinking ratio of γ-PGA were shown in Table 3.The ANOVA results indicated that the model parameters,X1,X2,X3,X1X2,X1X3,X2X3,,,andhad significant influences on the crosslinking ratio(P＜0.01)(Table 3).The model F-value was 636.90,and the F-value for lack of fit was 3.69,indicated that the model had a goodness of fit.The regression equation coefficients were calculated and the data were fitted to a secondorder polynomial equation.The prediction model of crosslinking ratio of γ-PGA(Y1)of the hydrogel can be expressed in terms of the following regression equation:

where Y1is the predicted value of crosslinking ratio,X1is pH,X2is the mass ratio of γ-PGA to casein,X3is temperature.

The results of ANOVA analysis showed that the P-value of the regression model was statistically significant(P＜0.0001,Table 3).In addition,the coefficient of determination of the model was 0.9988,indicating that the regression model was highly reliable.

To further understand the relationships between the variables and responses,a series of 3D response surface plots from the prediction model were examined using the Design-Expert software.Fig.8a showed the interaction between the mass ratio of γ-PGA to casein and pH at the optimum temperature(44.2 °C).As shown in Fig.8a,the crosslinking ratio reached the highest value 93.22%at the mass ratio of γ-PGA to casein of 0.14 and pH of 7.35.As shown in Fig.8b,with an increase in the crosslinking temperature and pH,the crosslinking ratio of γ-PGA increased to the optimum level at the mass ratio of γ-PGA to casein of 0.14.As shown in Fig.8c,with an increase in the crosslinking temperature and mass ratio of γ-PGA to casein ratio,the crosslinking ratio of γ-PGA increased and peaked at 93.22%.

Fig.8.Response surface and contour plots of the crosslinking ratio of γ-PGA.

3.3.2.Optimization results for the swelling ratio of hydrogels

The ANOVA results indicated that the model parameters,X1,X2,X3,X1X2,,,andhad significant influences on the swelling ratio(P＜0.05)(Table 4).The model F-value was 42.14,and the F-value for lack of fit was 2.52,indicating that the model was had a goodness of fit.The regression equation coefficients were calculated and the data were fitted to a second-order polynomial equation.The prediction model of swelling ratio(Y2)of the hydrogel can be expressed in terms of the following regression equation:

Table 4 Analysis of variance(ANOVA)for the swelling ratio of the BBD.

The results of the ANOVA revealed that the R2was 0.9819,indicating that the model was capable of explaining 98.19%of the variation in response.The adjusted R2was 0.9586,indicating that the model was reliable.To determine the optimal levels of each variable to achieve the maximum swelling ratio,3D response surface plots were constructed by plotting the response values on the Z-axis against any two independent variables,while maintaining other variables at their optimal levels.As shown in Fig.9,the three response surface curves exhibited a convex shape and outlined the position of the optimum conditions.The optimal levels for each factor as calculated by the regression formula were 7.32 for pH,0.21 for the mass ratio of γ-PGA to casein,and 41.8°C for temperature.The maximum swelling ratio under these optimal conditions was 113.51%.

Fig.9.Response surface and contour plots of the swelling ratio of the γ-PGA/casein hydrogel.

3.4.Verification test

Under the optimum predicted conditions,pH of 7.33,the mass ratio of γ-PGA to casein of 0.18,and temperature of 42.4°C,the predicted values of crosslinking ratio of γ-PGA and swelling ratio were 90.35%and 112.07%,respectively.To verify the predicted results and model accuracy,three validation experiments were performed under the optimum conditions.The average values of the crosslinking ratio of γ-PGA and the swelling ratio of the γ-PGA/casein hydrogel were 88.00%and 115.87%respectively,which were consistent with the predicted value,indicating that the proposed model was valid for predicting the optimum preparation conditions of the γ-PGA/casein hydrogel.

4.Conclusion

In this study,an enzyme-crosslinked γ-PGA/casein hydrogel was prepared by crosslinking casein solution with γ-PGA using MTG as the crosslinker,and the minimum mass concentration of MTG was 0.3%.Since γ-PGA is derived from microorganism,both the MTG and casein are biocompatible,the γ-PGA/casein hydrogel is non-toxic to cells.The effects of the reaction time,temperature,pH,and the mass ratio of γ-PGA to casein on the crosslinking ratio of γ-PGA and swelling ratio of the γ-PGA/casein hydrogel were also systematically investigated.The results of the single factor experiments revealed that the optimum reaction time,temperature,pH,and the mass ratio of γ-PGA to casein were 7 h,40 °C,7.5,and 0.2,respectively.RSM was conducted to optimize the conditions to obtain the highest crosslinking ratio of γ-PGA and the highest swelling ratio of the γ-PGA/casein hydrogel.The predicted optimal factors were 7.33 for pH,0.18 for the mass ratio of γ-PGA to casein,and 42.4°C for temperature,and the predicted values of the crosslinking ratio of γ-PGA and swelling ratio were 90.35%and 112.07%respectively,highly consistent with the measured values(88.00%and 115.87%),indicating that the predication model is reliable.

Owing to its high water absorption capacity,the γ-PGA/casein hydrogel could be used as a superior drug carrier system in biomedical field.The hydrophilic salicylic acid and hydrophobic docetaxel drugs'release profiles based on the casein hydrogel system had been studied extensively,but there are no reports of the new developed γ-PGA/casein hydrogel.Due to the large number of hydrophilic carboxyl groups present on the PGA molecular,we believed the γ-PGA/casein hydrogel had high potential in the drug delivery,and this will be studied in the future.

Author Contributions

Xin Wang:Investigation,writing-original draft,writing-review&editing,funding acquisition;Chenchen Gou:Investigation,writing-original draft;Chunyuan Gao:Investigation,writing-original draft;Yazhen Song:Formal analysis;Jinming Zhang:Formal analysis;Dandan Zhai:Writing-review&editing,visualization;Yawei Zhao:Writing-review&editing;Jihong Huang:Project administration;Ming Hui:Supervision.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by the Doctoral Scientific Research Start-up Foundation from Henan University of Technology(No:2018BS072).