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Abstract In this study， with chestnut as a raw material， the effects of enzymolysis time， enzymolysis temperature and enzyme amount on hydrolysis process were investigated. The optimal reaction conditions were obtained by single factor and response surface experiment as follows： enzymolysis time of 75 min， enzymolysis temperature at 58 ℃ and enzyme amount of 7 U/g. Under these conditions， the browning degree of chestnut serosity was 1.228.
Key words Chestnut； Enzymatic hydrolysate； Browning
Chinese chestnut is called as "the King of Nuts"， also known as Lizi and Maoli， belonging to Castanea of Fagaceae family in Dicotyledoneae of Pteridophyta[1]. Chinese chestnut has the starch content of about 40%， total sugar content of 9%-13%， protein content of 4%-10%， and the Vc content 10 times of apple， and also contains folic acid， fat， tannin， Fe， Cu， Mg， Ca and Zn[2]. Among the amino acids essential for human body in Chinese chestnut， glutamic acid， asparaginic acid， leucine and lysine have higher contents[3]. Furthermore， Chinese chestnut also has very high medicinal value， and could be prepared with other foods or Chinese herbal medicine into tonic diet and functional foods， which could be used for treating trachitis， diarrhea， stomachache， and dyspepsia[4-5].
At present， Chinese chestnut is mainly processed into sugar frying chestnut and chestnut cans. Sugar frying chestnut has simple treatment method， and the product is golden yellow with fragrant and sweet aroma. However， after being stored for several days， the sugar frying chestnut is prone to aging and retrogradation， and its color become dark[6-8]. Chestnut cans are the main product of Chinese chestnut deep processing. Though chestnut cans solve the problem of storage of Chinese chestnut， they have the disadvantages of high production cost， easy browning， poor quality and unsatisfactory economic benefit[9]. The existing of these problems has certain effect on the industrialization of Chinese chestnut processing.
Chinese chestnut cloudy beverage is clear， fragrant and sweet， with better flavor and taste， and less nutritional components are lost during processing[10]. During the processing of Chinese chestnut cloudy beverage， the enzymolysis of Chinese chestnut serosity could rapidly reduce the viscosity of gelatinized starch， which turns to liquefied starch， and the stability of the cloudy beverage is thus improved[11]. Due to the effects of enzymatic browning and non-enzymatic browning， phenolic substances of Chinese chestnut serosity could be oxidized easily， thereby exhibited changed color， and browning would be observed during heating and storage of Chinese chestnut[12-14]. Therefore， the understanding of the effect of enzymolysis on the color of chestnut serosity is of certain significance to the reduction of browning of Chinese chestnut serosity. In this study， with the browning degree of chestnut as a basis， the enzymolysis process of Chinese chestnut serosity was optimized， and the effect of the enzymolysis process on browning of Chinese chestnut was analyzed. This study lays a foundation for research and development of Chinese chestnut cloudy beverage.
Materials and Methods
Materials and Reagents
Luotian chestnut： commercially available； α-amylase： Xingtai Wanda Bioengineering Co.， Ltd.； other common regions， which were all analytically pure， and purchased from Sinopharm Chemical Reagent Co.， Ltd.
Instruments and equipment
IKA T18 digital dispersion machine （IKA Works Guangzhou）； JJ-2 tissue blender （Jintan Xicheng Xinrui Instruments Factory）； PB2002-10 electronic scale （Mettler-Toledo）； H/18MM desk type high-speed centrifuge （Hunanherexin Instrument & Equipment Co.， Ltd.）； cutting machine （Linyi Huole Machine Manufacturing Co.， Ltd.）.
Methods
Enzymolysis process of Chinese chestnut
The enzymolysis process included selection of Chinese chestnut， cutting， cooking， peeling， beating， homogenization and enzymolysis， and finally gave Chinese chestnut serosity.
At first， full seeds without worm sting free of mildewing were selected， and cut， giving an opening. The Chinese chestnut was then cooked in boiling water for 30 min， and the peels were removed when hot. Chinese chestnut and water were added into a blender at a ratio of 1∶4， and pulverized for 3 min. Homogenization was performed at 7 000 r/min for 5 min. Enzymolysis was performed with the addition of α-amylase.
Single factor tests of enzymolysis conditions
（1） Enzymolysis time： A certain amount of Chinese chestnut was cut and cooked for 30 min， and peels were removed quickly， followed by homogenization. During enzymolysis， the material-to-liquid ratio was 1∶4， the addition amount of enzyme was 7 U/g， the temperature of water bath was set at 65 ℃， and enzymolysis was performed at 30， 50， 70， 90 and 110 min， respectively. The values of browning degree were determined and averaged.
（2） Enzymolysis temperature： A certain amount of Chinese chestnut was cut and cooked for 30 min， and peels were removed quickly， followed by homogenization. During enzymolysis， the material-to-liquid ratio was 1∶4， the addition amount of enzyme was 7 U/g， the enzymolysis time was 90 min， and the temperatures of water baths were set at 50， 55， 60， 65 and 70 ℃， respectively. The values of browning degree were determined and averaged. （3） Amount of enzyme： A certain amount of Chinese chestnut was cut and cooked for 30 min， and peels were removed quickly， followed by homogenization. During enzymolysis， the material-to-liquid ratio was 1∶4， the temperature of water bath was set at 65 ℃， the enzymolysis time was 90 min， and the enzyme was added according to 5， 6， 7， 8 and 9 U/g， respectively. The values of browning degree were determined and averaged.
Optimization of enzymatic process
According to the results of single factor tests， enzymolysis time （min）， enzymolysis temperature （℃） and amount of enzyme （U/g） were selected as evaluation factors， and with the browning degree of Chinese chestnut as response value， three-factor three-level response surface method analysis were designed with Design-Expert. The test results were analyzed， and the factors and levels are shown in Table 1.
Determination of browning degree
Chinese chestnut sample was added with 20 ml of 10% trichloroacetic acid solution and subjected to 10 min of vortex oscillation. The sample was stood at 4 ℃ in a refrigerator for 24 h， and centrifuged at 5 000 r/min for 20 min， obtaining supernatant， which was determined with an infrared spectrophotometer for absorbance at 420 nm， and distilled water was also determined as control.
Results and Analysis
Effects of enzymolysis conditions on browning degree of Chinese chestnut serosity
Effect of enzymolysis time on browning degree of Chinese chestnut serosity
It could be seen from Fig. 1 that over time， the browning degree of the enzymolysis liquid became higher and higher， after exceeding 70 min， the increasing trend of the liquid was no longer remarkable， and when the enzymolysis time reached 90 min， the browning degree of the enzymolysis liquid became lower. Therefore， the enzymolysis time was selected as 90 min.
As shown in Fig. 2， the browning degree of Chinese chestnut serosity was lowered with the increase of temperature， and over 55 ℃， the decreasing trend was not remarkable. Due to the effect of high temperature on enzyme activity， taking economic benefit into consideration， the enzymolysis temperature was selected at 55 ℃.
It could be seen from Fig. 3 that the browning degree of chestnut liquid increased with the amount of enzyme increasing， and when the amount of enzyme increased from 7 to 8 U/g， the browning degree of Chinese chestnut serosity decreased remarkably. At the enzyme amount of 8 U/g， the browning degree of Chinese chestnut serosity reached its maximum value， and thus， the amount of enzyme was selected at 8 U/g. Optimization of enzymolysis conditions
According to the results of single factor tests， three factors （enzymolysis time， enzymolysis temperature and amount of enzyme） were selected as evaluation factors， and with the browning degree of chestnut as response value， three-factor three-level response surface method （RSM） analysis were designed with Design-Expert. The test results were analyzed， and the results are shown in Table 2.
Significance testing was performed with modes and regression equations in Design Expert. The effects of the three factors on the browning degree of Chinese chestnut serosity ranked as enzymolysis time>enzyme amount>enzymolysis temperature. The regression equation between the browning degree of Chinese chestnut serosity and enzymolysis was obtained：
R1=1.25+8.500E-003A+0.039B+0.022C+5.000E-004AB+0.022AC-7.250E-003BC+0.049A2+0.018B2-0.013C2
It could be known from the equation， the enzymolysis conditions giving the enzymolysis liquid with the best stability were as follows： enzymolysis time of 75.27 min， enzymolysis temperature at 57.34 ℃， and enzyme amount of 7.02 U/g， under which the browning degree of Chinese chestnut serosity was 1.195.
It could be seen from Table 3 that the F value of regression model was 33.21， P<0.0001， indicating a very significant difference； the F value of the lack of fit was 0.73. P=0.585 1， indicating a non-significant difference； and the interaction term BC and quadratic terms A2， B2 and C2 exhibited significant effects. Therefore， the fitting effect of the model was good.
By fixing B and C in the model at 0 level， respectively， the response curve of the effect of the interaction between other two factors on the browning degree of the chestnut beverage was obtained， as shown in Fig. 4. According to the analysis of response surface analysis figure， the best parameters could be found out， and interaction between various parameters could be analyzed.
In order to check the reliability of the results obtained by the response surface method， an enzymolysis test should be carried out according to above optimized conditions. According to practical operating conditions， the enzymolysis conditions were corrected to be enzymolysis time of 75 min， enzymolysis temperature at 58 ℃， and enzyme amount of 7 U/g. Under these conditions， three parallel tests were carried out， obtaining the browning degree of 1.228. The relative error between the actual value and the theoretical value was 2.76%， so the model could well reflect the effects of various factors on the stability of Chinese chestnut serosity， demonstrating that the method is feasible for the study on enzymolysis conditions on the stability of Chinese chestnut serosity. Discussion
In this study， single factor tests of enzymolysis conditions were carried out， and combined with response surface method， the optimized conditions were obtained as follows： enzymolysis time of 75 min， enzymolysis temperature at 58 ℃， and enzyme amount of 7 U/g. Under these conditions， the browning degree of Chinese chestnut serosity was 1.228.
References
[1] HAN JC， ZHANG XZ， LIU QX， et al. The progress of the molecular biological studies on Castanea （Tourn.） L.[J]. Molecular Plant Breeding， 2006（1）： 117-124.
[2] WANG XH， SANG JX， ZHANG ZD， et al. Quality analysis of different chestnut[J]. Food Science and Technology， 2004（3）： 95-97.
[3] PENG HZ， CHEN SW， JIANG MD， et al. Difference of amino acid in Castanea mollissima between varieties and yield areas and analysis on its nutrient value[J]. Journal of Zhejiang Forestry Science and Technology， 2000（5）： 30-35.
[4] QI M， YUE CF， LI YM. Medicinal value and development and application of chestnut[J]. Forest By-product and Speciality in China， 1997（03）： 51-52.
[5] MARLENA DUDEK-MAKUCH， EL BIETA STUDZI SKA-SROKA. Horse chestnut-efficacy and safety in chronic venous insufficiency： an overview[J]. Revista Brasileira de Farmacognosia， 2015， 25（5）.
[6] LIANG JL， LIU H， LIU XF， et al. Effect of different processing methods on chestnut fragrance[J]. Food Science and Technology， 2013， 38（07）： 84-88.
[7] SU X， LIU PF， SUN Y， et al. Research of processing characteristics of different species of sugar frying chestnut[J]. Science and Technology of Food Industry， 2012， 33（12）： 128-131.
[8] ZHOU JH， CHANG H， XIE M. Research of processing characteristics of different species of sugar frying chestnut[J]. Food Industry， 2012， 33（10）： 60-62
[9] SU X， GUO LJ， SUN Y， et al. Processing characteristics of different species of chestnut cans[J]. Food and Fermentation Industries， 2012， 38（05）： 129-133.
[10] WU CP. Research of chestnut beverage processing technology[D]. Hefei： Hefei University of Technology， 2015.
[11] PRC Hygiene Ministry. GB/T 24401-2009 α-amylase preparation standards.[S]. Beijing： Standards Press of China， 2004.
[12] WU YH， QI JH， HUANG MQ， et al. Analysis of Substrates and Products of Non-enzymatic Browning in Chinese Chestnut[J]. Chinese Agricultural Science Bulletin， 2012， 28 （30）： 267-271.
[13] ZHOU D， LI YJ， WANG JZ， et al. Changes in activities of polyphenol oxidase and peroxidase activity during enzymatic browning of chestnut[J]. Food Science and Technology， 2014， 39（6）： 47-50.
[14] ZHOU JW， SHAO LY， LIU DH， et al. The kinetic analysis of non-enzymatic browning in blume can during the storage[J]. J Chin Inst Food Sci Tech ， 2015， 15（7）： 18-25.
Key words Chestnut； Enzymatic hydrolysate； Browning
Chinese chestnut is called as "the King of Nuts"， also known as Lizi and Maoli， belonging to Castanea of Fagaceae family in Dicotyledoneae of Pteridophyta[1]. Chinese chestnut has the starch content of about 40%， total sugar content of 9%-13%， protein content of 4%-10%， and the Vc content 10 times of apple， and also contains folic acid， fat， tannin， Fe， Cu， Mg， Ca and Zn[2]. Among the amino acids essential for human body in Chinese chestnut， glutamic acid， asparaginic acid， leucine and lysine have higher contents[3]. Furthermore， Chinese chestnut also has very high medicinal value， and could be prepared with other foods or Chinese herbal medicine into tonic diet and functional foods， which could be used for treating trachitis， diarrhea， stomachache， and dyspepsia[4-5].
At present， Chinese chestnut is mainly processed into sugar frying chestnut and chestnut cans. Sugar frying chestnut has simple treatment method， and the product is golden yellow with fragrant and sweet aroma. However， after being stored for several days， the sugar frying chestnut is prone to aging and retrogradation， and its color become dark[6-8]. Chestnut cans are the main product of Chinese chestnut deep processing. Though chestnut cans solve the problem of storage of Chinese chestnut， they have the disadvantages of high production cost， easy browning， poor quality and unsatisfactory economic benefit[9]. The existing of these problems has certain effect on the industrialization of Chinese chestnut processing.
Chinese chestnut cloudy beverage is clear， fragrant and sweet， with better flavor and taste， and less nutritional components are lost during processing[10]. During the processing of Chinese chestnut cloudy beverage， the enzymolysis of Chinese chestnut serosity could rapidly reduce the viscosity of gelatinized starch， which turns to liquefied starch， and the stability of the cloudy beverage is thus improved[11]. Due to the effects of enzymatic browning and non-enzymatic browning， phenolic substances of Chinese chestnut serosity could be oxidized easily， thereby exhibited changed color， and browning would be observed during heating and storage of Chinese chestnut[12-14]. Therefore， the understanding of the effect of enzymolysis on the color of chestnut serosity is of certain significance to the reduction of browning of Chinese chestnut serosity. In this study， with the browning degree of chestnut as a basis， the enzymolysis process of Chinese chestnut serosity was optimized， and the effect of the enzymolysis process on browning of Chinese chestnut was analyzed. This study lays a foundation for research and development of Chinese chestnut cloudy beverage.
Materials and Methods
Materials and Reagents
Luotian chestnut： commercially available； α-amylase： Xingtai Wanda Bioengineering Co.， Ltd.； other common regions， which were all analytically pure， and purchased from Sinopharm Chemical Reagent Co.， Ltd.
Instruments and equipment
IKA T18 digital dispersion machine （IKA Works Guangzhou）； JJ-2 tissue blender （Jintan Xicheng Xinrui Instruments Factory）； PB2002-10 electronic scale （Mettler-Toledo）； H/18MM desk type high-speed centrifuge （Hunanherexin Instrument & Equipment Co.， Ltd.）； cutting machine （Linyi Huole Machine Manufacturing Co.， Ltd.）.
Methods
Enzymolysis process of Chinese chestnut
The enzymolysis process included selection of Chinese chestnut， cutting， cooking， peeling， beating， homogenization and enzymolysis， and finally gave Chinese chestnut serosity.
At first， full seeds without worm sting free of mildewing were selected， and cut， giving an opening. The Chinese chestnut was then cooked in boiling water for 30 min， and the peels were removed when hot. Chinese chestnut and water were added into a blender at a ratio of 1∶4， and pulverized for 3 min. Homogenization was performed at 7 000 r/min for 5 min. Enzymolysis was performed with the addition of α-amylase.
Single factor tests of enzymolysis conditions
（1） Enzymolysis time： A certain amount of Chinese chestnut was cut and cooked for 30 min， and peels were removed quickly， followed by homogenization. During enzymolysis， the material-to-liquid ratio was 1∶4， the addition amount of enzyme was 7 U/g， the temperature of water bath was set at 65 ℃， and enzymolysis was performed at 30， 50， 70， 90 and 110 min， respectively. The values of browning degree were determined and averaged.
（2） Enzymolysis temperature： A certain amount of Chinese chestnut was cut and cooked for 30 min， and peels were removed quickly， followed by homogenization. During enzymolysis， the material-to-liquid ratio was 1∶4， the addition amount of enzyme was 7 U/g， the enzymolysis time was 90 min， and the temperatures of water baths were set at 50， 55， 60， 65 and 70 ℃， respectively. The values of browning degree were determined and averaged. （3） Amount of enzyme： A certain amount of Chinese chestnut was cut and cooked for 30 min， and peels were removed quickly， followed by homogenization. During enzymolysis， the material-to-liquid ratio was 1∶4， the temperature of water bath was set at 65 ℃， the enzymolysis time was 90 min， and the enzyme was added according to 5， 6， 7， 8 and 9 U/g， respectively. The values of browning degree were determined and averaged.
Optimization of enzymatic process
According to the results of single factor tests， enzymolysis time （min）， enzymolysis temperature （℃） and amount of enzyme （U/g） were selected as evaluation factors， and with the browning degree of Chinese chestnut as response value， three-factor three-level response surface method analysis were designed with Design-Expert. The test results were analyzed， and the factors and levels are shown in Table 1.
Determination of browning degree
Chinese chestnut sample was added with 20 ml of 10% trichloroacetic acid solution and subjected to 10 min of vortex oscillation. The sample was stood at 4 ℃ in a refrigerator for 24 h， and centrifuged at 5 000 r/min for 20 min， obtaining supernatant， which was determined with an infrared spectrophotometer for absorbance at 420 nm， and distilled water was also determined as control.
Results and Analysis
Effects of enzymolysis conditions on browning degree of Chinese chestnut serosity
Effect of enzymolysis time on browning degree of Chinese chestnut serosity
It could be seen from Fig. 1 that over time， the browning degree of the enzymolysis liquid became higher and higher， after exceeding 70 min， the increasing trend of the liquid was no longer remarkable， and when the enzymolysis time reached 90 min， the browning degree of the enzymolysis liquid became lower. Therefore， the enzymolysis time was selected as 90 min.
As shown in Fig. 2， the browning degree of Chinese chestnut serosity was lowered with the increase of temperature， and over 55 ℃， the decreasing trend was not remarkable. Due to the effect of high temperature on enzyme activity， taking economic benefit into consideration， the enzymolysis temperature was selected at 55 ℃.
It could be seen from Fig. 3 that the browning degree of chestnut liquid increased with the amount of enzyme increasing， and when the amount of enzyme increased from 7 to 8 U/g， the browning degree of Chinese chestnut serosity decreased remarkably. At the enzyme amount of 8 U/g， the browning degree of Chinese chestnut serosity reached its maximum value， and thus， the amount of enzyme was selected at 8 U/g. Optimization of enzymolysis conditions
According to the results of single factor tests， three factors （enzymolysis time， enzymolysis temperature and amount of enzyme） were selected as evaluation factors， and with the browning degree of chestnut as response value， three-factor three-level response surface method （RSM） analysis were designed with Design-Expert. The test results were analyzed， and the results are shown in Table 2.
Significance testing was performed with modes and regression equations in Design Expert. The effects of the three factors on the browning degree of Chinese chestnut serosity ranked as enzymolysis time>enzyme amount>enzymolysis temperature. The regression equation between the browning degree of Chinese chestnut serosity and enzymolysis was obtained：
R1=1.25+8.500E-003A+0.039B+0.022C+5.000E-004AB+0.022AC-7.250E-003BC+0.049A2+0.018B2-0.013C2
It could be known from the equation， the enzymolysis conditions giving the enzymolysis liquid with the best stability were as follows： enzymolysis time of 75.27 min， enzymolysis temperature at 57.34 ℃， and enzyme amount of 7.02 U/g， under which the browning degree of Chinese chestnut serosity was 1.195.
It could be seen from Table 3 that the F value of regression model was 33.21， P<0.0001， indicating a very significant difference； the F value of the lack of fit was 0.73. P=0.585 1， indicating a non-significant difference； and the interaction term BC and quadratic terms A2， B2 and C2 exhibited significant effects. Therefore， the fitting effect of the model was good.
By fixing B and C in the model at 0 level， respectively， the response curve of the effect of the interaction between other two factors on the browning degree of the chestnut beverage was obtained， as shown in Fig. 4. According to the analysis of response surface analysis figure， the best parameters could be found out， and interaction between various parameters could be analyzed.
In order to check the reliability of the results obtained by the response surface method， an enzymolysis test should be carried out according to above optimized conditions. According to practical operating conditions， the enzymolysis conditions were corrected to be enzymolysis time of 75 min， enzymolysis temperature at 58 ℃， and enzyme amount of 7 U/g. Under these conditions， three parallel tests were carried out， obtaining the browning degree of 1.228. The relative error between the actual value and the theoretical value was 2.76%， so the model could well reflect the effects of various factors on the stability of Chinese chestnut serosity， demonstrating that the method is feasible for the study on enzymolysis conditions on the stability of Chinese chestnut serosity. Discussion
In this study， single factor tests of enzymolysis conditions were carried out， and combined with response surface method， the optimized conditions were obtained as follows： enzymolysis time of 75 min， enzymolysis temperature at 58 ℃， and enzyme amount of 7 U/g. Under these conditions， the browning degree of Chinese chestnut serosity was 1.228.
References
[1] HAN JC， ZHANG XZ， LIU QX， et al. The progress of the molecular biological studies on Castanea （Tourn.） L.[J]. Molecular Plant Breeding， 2006（1）： 117-124.
[2] WANG XH， SANG JX， ZHANG ZD， et al. Quality analysis of different chestnut[J]. Food Science and Technology， 2004（3）： 95-97.
[3] PENG HZ， CHEN SW， JIANG MD， et al. Difference of amino acid in Castanea mollissima between varieties and yield areas and analysis on its nutrient value[J]. Journal of Zhejiang Forestry Science and Technology， 2000（5）： 30-35.
[4] QI M， YUE CF， LI YM. Medicinal value and development and application of chestnut[J]. Forest By-product and Speciality in China， 1997（03）： 51-52.
[5] MARLENA DUDEK-MAKUCH， EL BIETA STUDZI SKA-SROKA. Horse chestnut-efficacy and safety in chronic venous insufficiency： an overview[J]. Revista Brasileira de Farmacognosia， 2015， 25（5）.
[6] LIANG JL， LIU H， LIU XF， et al. Effect of different processing methods on chestnut fragrance[J]. Food Science and Technology， 2013， 38（07）： 84-88.
[7] SU X， LIU PF， SUN Y， et al. Research of processing characteristics of different species of sugar frying chestnut[J]. Science and Technology of Food Industry， 2012， 33（12）： 128-131.
[8] ZHOU JH， CHANG H， XIE M. Research of processing characteristics of different species of sugar frying chestnut[J]. Food Industry， 2012， 33（10）： 60-62
[9] SU X， GUO LJ， SUN Y， et al. Processing characteristics of different species of chestnut cans[J]. Food and Fermentation Industries， 2012， 38（05）： 129-133.
[10] WU CP. Research of chestnut beverage processing technology[D]. Hefei： Hefei University of Technology， 2015.
[11] PRC Hygiene Ministry. GB/T 24401-2009 α-amylase preparation standards.[S]. Beijing： Standards Press of China， 2004.
[12] WU YH， QI JH， HUANG MQ， et al. Analysis of Substrates and Products of Non-enzymatic Browning in Chinese Chestnut[J]. Chinese Agricultural Science Bulletin， 2012， 28 （30）： 267-271.
[13] ZHOU D， LI YJ， WANG JZ， et al. Changes in activities of polyphenol oxidase and peroxidase activity during enzymatic browning of chestnut[J]. Food Science and Technology， 2014， 39（6）： 47-50.
[14] ZHOU JW， SHAO LY， LIU DH， et al. The kinetic analysis of non-enzymatic browning in blume can during the storage[J]. J Chin Inst Food Sci Tech ， 2015， 15（7）： 18-25.