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Abstract Ultrasoundassisted extraction method was applied to extract the anthocyanins from seleniumenriched purple potato， and the optimum extraction conditions for anthocyanins in seleniumenriched purple potato were obtained by response surface methodology： the phosphoric acid concentration of 1.0%， the solidliquid ratio at 5， and the extraction time of 19 min. Under these conditions， the yield of anthocyanins in seleniumenriched purple potato reached （0.409±0.010） mg/g （RSD=2.51%， n=3）. The extraction of anthocyanins from seleniumrich fresh purple potato with dilute phosphoric acid has the advantages of less acid consumption and good extraction effect.
Key words Selenium; Purple potato; Anthocyanin; Extraction
Selenium is one of the essential trace elements in human body and animal body. It has the effects of resisting oxidation， enhancing immunity， antagonizing heavy metals， enhancing reproductive function and regulating protein synthesis and absorption of nutrients， and is praised as "the anticancer king" and "the protective agent of life" among the trace elements in human body[1].
Potato is one of the worlds top ten nutritious foods. It has strong growth adaptability and high yield and is the fourth largest staple food in China. The skin or flesh of purple potato is purpleblack， which contain 98% of acylated anthocyanins[2-3]. Anthocyanins are natural antioxidants and are the most potent free radical scavengers ever discovered. They are brightly colored and have no toxic side effects. They are widely used in the food， pharmaceutical and health care industries[4-9]. The nutritional and commercial value of seleniumenriched purple potato is higher than that of ordinary potato because of their selenium and anthocyanins. At present， the research on purple potato anthocyanins remains in the composition analysis[10-11]and the antioxidation and free radicalscavenging activity[12-13]. The extraction methods of anthocyanins differ due to the extraction materials， the purposes of extraction and the types of anthocyanins. At present， the main extraction methods include the solvent extraction method[14]， ultrasonic assisted method[15-16]， microwave assisted method[17]， highvoltage pulsed electric fieldassisted method[18]and so on. In order to obtain anthocyanins closer to the natural state and to avoid acylation of anthocyanins， weak citric acid is often used as an extractant[19-20]. Citric acid is a polybasic medium strong acid， which has a good colorprotecting effect on anthocyanins， but the price is high， resulting in high production cost of anthocyanin pigments. Phosphoric acid is also a polybasic acid and is similar to citric acid， but at a low price. Therefore， in this study， the technical parameters of ultrasonicassisted extraction of anthocyanins from seleniumenriched purple potato were investigated， aiming to provide a basis for further research and development. Materials and Methods
Experimental materials
Seleniumrich purple potato was provided by the Agricultural Resource and Environment Research Institute， Guangxi Academy of Agricultural Sciences/Guangxi Selenium Rich Agricultural Research Center. Phosphoric acid， acetic acid， citric acid， hydrochloric acid， and anhydrous ethanol were all chemical reagents of Sinopharm Group.
SB2512DTN ultrasonic cleaning machine， Ningbo Xinzhi Biotechnology Co.， Ltd.; RE52AA rotary evaporator， Shanghai Yarong Biochemical Instrument Factory; UNICO 2000 visible spectrophotometer， Unico （Shanghai） Co.， Ltd.; AX200 0.000 1 gelectronic balance， Japan Shimadzu Philippines.
Experimental methods
Analysis of anthocyanin content
The anthocyanin content was analyzed by the pH differential method in reference[21]. An appropriate amount of the extract was diluted to a volume of 10 ml with buffer solutions of pH=1.0 and pH=4.5， respectively， and the absorbance was measured at the wavelengths of 520 and 700 nm， respectively. The anthocyanin yield was calculated by the following formula：
Anthocyanin yield （mg/g）=A·MW·DF·V/ε·L·m
Wherein A=（A520-A700）pH=1-（A520-A700）pH=4.5; MW is the molar mass of cyanidine3galacoside （449.2 g/mol）; ε is the molar extinction coefficient of cyanidine3galacoside （26 900L/mol·cm）; V is the solution volume （ml）; L is the cuvette thickness （1 cm）; DF is the dilution factor; and m is the mass of purple potato （g）.
Screening of extraction solvent
The fresh potato was washed with water and cut into 2 mm pieces after the surface was naturally airdried. Eight parts （1 g each） of seleniumenriched purple potato pieces were added into conical flasks， respectively， into which 10 ml of 0.25% hydrochloric acid， 0.50% hydrochloric acid， 0.75% phosphoric acid， 1.50% phosphoric acid， 2.0% citric acid， 3.0% citric acid， 3.0% acetic acid and 4.0% acetic acid were added， respectively. Ultrasonic extraction was performed at room temperature for 20 min， followed by filtration and collection of filtrate. The anthocyanin content was determined， and the yield was calculated.
Single factor experiment
The effects of phosphoric acid concentration （0.25， 0.5， 0.75， 1.0， 1.25， 1.5%）， ultrasonic time （5， 7.5， 10， 15， 20， 25， 30 min） and materialtoliquid ratio （1∶2， 1∶3， 1∶4， 1∶5， 1∶6， 1∶7， 1∶8 g/ml） on the extraction of anthocyanins from seleniumenriched purple potato were investigated.
Response surface optimization According to the results of single factor experiment， with phosphoric acid concentration （A）， ultrasonic time （B） and materialtoliquid ratio （C） as three factors and the anthocyanin yield as the response value， the experiment was designed by BoxBehnken method in the DesignExpert 8.0.6 software. The levels of factors and codes of the factors are shown in Table 1.
Verification experiment
Three parallel experiments on the extraction of anthocyanins from seleniumrich purple potato were carried out according to the response surface method.
Results and Analysis
Single factor experiment
Screening of extraction solvent
Based on the extraction technique of anthocyanins from purple sweetpotato in previous studies[21]， the extraction effects of such four kinds of extraction solvents as hydrochloric acid （ 0.25%， 0.50% by weight）， phosphoric acid （0.75%， 1.50%）， citric acid （2.0%， 3.0%） and acetic acid （3.0%， 4.0%） on anthocyanins from seleniumrich purple potato were investigated at first under the materialtoliquid ratio of 1∶10 and ultrasonic time of 20 min. The results are shown in Fig. 1.
It can be seen from Fig. 1 that the extraction rate of anthocyanins from seleniumrich purple potato can reach 15% with 0.25%hydrochloric acid， but considering the volatility of hydrochloric acid， it is not suitable for industrial production. Acetic acid requires a high concentration to achieve a better extraction effect. Industrially， 3.0% citric acid aqueous solution is commonly used to extract anthocyanins. Although the effect is good， the production cost is high. The effects of extracting anthocyanins with 1.50% phosphoric acid and 3.0% citric acid aqueous solutions were better. In addition， the acidity of phosphoric acid （ka1=7.11×10-3， ka2=6.23×10-8， ka3=4.50×10-13） is similar to citric acid （ka1=7.44×10-4， ka2=1.73×10-5， Ka3=4.02×10-7）， and both have good complexing ability. Therefore， phosphoric acid extraction was selected.
Factors affecting the extraction of anthocyanins from phosphoric acid solution
In order to initially determine the ranges of extraction conditions for response surface optimization， a single factor experiment was first performed. The experimental results are shown in Table 2， Table 2 and Table 3.
The results from Table 2， Table 3 and Table 4 showed that with the materialtoliquid ratio at 1∶10 and extraction time of 20 min， the content of anthocyanins in the extract first rose and then decreased with the increase of phosphoric acid concentration. The anthocyanin yield reached a maximum at the phosphoric acid concentration of 1%. Then， at the phosphoric acid concentration of 1% and ultrasonic time of 20 min， the materialtoliquid ratio was set to be 1∶2， 1∶3， 1∶4， 1∶5， 1∶6， 1∶7 and 1∶8 g/ml. The experiment was carried out， and the anthocyanin extraction yield peaked at the ratio of 1∶5. Finally， under 1% phosphoric acid and the materialtoliquid ratio at 1∶5， the effect of ultrasonic time on the extraction of anthocyanins in purple potato was investigated. The results showed that the extraction rate of anthocyanins increased first with the increase of ultrasonic time. The yield of anthocyaninsin the seleniumrich purple potato reached the highest level when the ultrasonic time was 20 min， and then became stable. Optimization of extraction of anthocyanins from purple potato by BoxBehnken design
Response surface methodology to optimize experimental results
Based on the results of the single factor experiment， with the phosphoric acid concentration （A）， ultrasonic time （B） and materialtoliquid ratio （C） as independent variables and anthocyanin yield （Y） as the response value， the experimental deign was optimized by BoxBehnken method. The experimental design and results are shown in Table 5.
Response surface regression model and analysis of variance
The DesignExpert software was used to perform regression fitting on the experimental data of Table 5， and the regression equation with the anthocyanin yield （Y） as the response value was obtained.
Deming LIU et al. Optimization of Extraction Process of Anthocyanins from Seleniumenriched Purple Potato by Response Surface Methodology
The variance analysis of the regression model is shown in Table 6.
As can be seen from Table 6， the regression model P<0.000 1， indicated that the regression model was highly significant; the missing term P=0.255 9>0.05， indicated that the lack of fit of the model was not significant. The model correlation coefficient R2=0.994 8， indicated that the model can explain 99.48% of the change， and the model fitted well with the experiment， so the model can be used to analyze and predict the extraction process of anthocyanins in seleniumenriched purple potato. Combined with the F value of each factor， the effects of the three factors on the extraction of anthocyanins in seleniumenriched purple potato ranked as ultrasonic time>phosphoric acid concentration>materialtoliquid ratio.
Factor interaction analysis
The analysis was carried out on the interaction between the factors in the extraction of anthocyanins from seleniumenriched purple potato by ultrasonic aqueous phosphoric acid solution. The results are shown in Fig. 2.
Fig. 2 showed the interaction of the three factors on the extraction of anthocyanins from seleniumenriched purple potato. It can be seen from Fig. 2（a） that the change of the time surface was steeper， indicating that the effect of ultrasonic time on anthocyanin yield was more significant than the effect of phosphoric acid concentration. When the materialtoliquid ratio was constant， the anthocyanin yield had a maximum value when the ultrasonic time was 15-20 min and the phosphoric acid concentration was 0.85%-1.15%. It can be seen from Fig. 2（b） that the concentration of phosphoric acid had a significant effect on the yield of anthocyanins compared with the materialtoliquid ratio when the ultrasonic time was controlled constant. When the materialtoliquid ratio was 1∶3-1∶7g/ml， the anthocyanin yield had a maximum value when the concentration of phosphoric acid was at 0.85%-1.15%. It can be seen from Fig. 2（c） that when controlling the materialtoliquid ratio constant， the anthocyanin yield had a maximum value when the phosphoric acid concentration was 0.85%-1.15% and the ultrasonic time was 15-25 min. Verification experiment
The optimal extraction process parameters of anthocyanins in seleniumenriched purple potato were obtained by response surface optimization： the concentration of phosphoric acid of 0.98%， the materialtoliquid ratio at 4.96， and the extraction time of 18.92 min. The actual experiment adjusted them as follows： the extraction time of 19 min， the materialtoliquid ratio at 5 and the phosphoric acid concentration of 1.0%. Three parallel experiments were carried out under these conditions， and the average yield of anthocyanins was （0.409±0.010） mg/g （RSD=2.51%， n=3）， which was not much different from the model predicted value of 0.411 mg/g， indicating the validity of the equation.
Conclusions
In this study， the extraction process of anthocyanins in seleniumenriched purple potato was optimized by response surface methodology： the phosphoric acid concentration of 0.98%， the materialtoliquid ratio at 4.96 and the ultrasonic time of 18.92 min. The results of the verification experiment were in good agreement with the model predictions. The replacement of citric acid with phosphoric acid to extract anthocyanins also has the characteristics of low acid concentration， which is beneficial to the reduction of the production cost， which provides a reference for the deep development of anthocyanins from seleniumrich purple potato.
References
[1] RAYMAN MP. Selenium and human health[J]. Lancet.2012，379（9822）：1256-1268.
[2] WANG L， DENG FM， ZHAO LY， et al. Research progress in health function and utilization of purple potato[J]. China Brewing， 2015（7）： 117-120. （in Chinese）
[3] FOSSEN T， ANDERSEN OM. Anthocyanins from tubers and shoots of the purple potato， Solanum tuberosum[J]. Journal of Horticultural Science and Biotechnology， 2000， 75（3）： 360-363.
[4] ANNANARYJU D. Sarma， antioxidant ability of anthocyanins against ascorbic acid oxidation[J]. phytochemistry， 1997， 45（4）： 671-674.
[5] WU XZ， PIAO JY， ZHANG XY， et al. Isolation and identification of anthocyanidin from the berries of Lonicera caerulea[J] . Journal of Yanbian University （Natural Science Edition）， 2001， 27（3）： 191-194. （in Chinese）
[6] FANG ZX， NI YY， LI HM. Stability in different conditions of anthocyanins from purple sweet potato[J] . Food and Fermentation Industries， 2002， 28（10）： 31-34. （in Chinese）
[7] ZHAO XL. Research progress of ingredient and healthy function of blueberry[J]. Chinese Wild Plant Resources， 2011， 30（6）： 19-23. （in Chinese） [8] GU RX， HU HL， LIU YC， et al. Extraction technology and antibacterial activity of anthocyanin in purple sweet potato[J]. Shandong Agricultural Sciences， 2012， 44（4）： 107-113. （in Chinese）
[9] HAO WB， JIANG GM， CHE WS. The study on antioxidant and antibacterial activity of anthocyanins from blueberry[J]. Journal of Heihe University， 2014， 5（3）： 123-125. （in Chinese）
[10] FRANCESCA IERI， MARZIA INNOCENTI， LUISA ANDRENELLI， et al. Rapid HPLC/DAD/MS method to determine phenolic acids， glycoalkaloids and anthocyanins in pigmented potatoes （Solanum tuberosum L.） and correlations with variety and geographical origin[J]. Food Chemistry， 2011 125：750-759.
[11] LIZ GUTIRREZQUEQUEZANA， ANSSI L. VUORINEN， HEIKKI KALLIO， et al. Improved analysis of anthocyanins and vitamin C in bluepurple potato cultivars[J]. Food Chemistry， 242（2018） 217-224.
[12] GABRIELA BURGOS， WALTER AMOROS， LUPITA MUNOA， et al. Total phenolic， total anthocyanin and phenolic acid concentrations and antioxidant activity of purplefleshed potatoes as affected by boiling[J]. Journal of Food Composition and Analysis， 2013（30）： 6-12.
[13] AGNIESZKA KITA， ANNA BA KOWSKABARCZAK， GRAZYNA LISI NSKA， et al. Antioxidant activity and quality of red and purple flesh potato chips[J]. LWTFood Science and Technology， 2015（62）： 525-531.
[14] SUN X， ZHAO L， LI YT， et al. Research on the extraction methods of pigment from purple potato[J]. China Food Additives， 2013， 116（1）： 81-87. （in Chinese）
[15] XIAO HJ， MENG LQ， SHI PF. Optimization of ultrasonicassisted extraction for anthocyanins from purple sweet potato by response surface method[J]. Food Research and Development， 2015， 36（7）： 33-38. （in Chinese）
[16] FLIX ADJ， YVES F. LOZANO， PAUL LOZANO， et al. Optimization of anthocyanin， flavonol and phenolic acid extractions from Delonix regia tree flowers using ultrasoundassisted water extraction[J]. Industrial Crops and Products， 2010， 3（32）： 439-444.
[17] HUANG Q， CHEN C， PENG H， et al. Study on the microwave extraction technology of proanthocyanidins from sweetpotato skins[J]. Food Science and Technology， 2010， 3（35）： 199-203.（in Chinese）
[18] SILVA S， COSTA E， CALHAU C， et al. Anthocyanin extraction from plant tissues： A review[J]. Critical Reviews in Food Science and Nutrition， 2015： 262-275.
[19] WANG RL， ZHOU F， ZHOU QC， et al. Extraction， purification and identification of anthocyanins from purple potato[J]. Food Science， 2011， 32（22）：55-59. （in Chinese）
[20] ZHANG JX， FENG YM， CHEN Q， et al. The extraction process optimization of anthocyanins in purple potatoes from Wuchuan County， Inner Mongolia[J]. Journal of Inner Mongolia Agricultural University， 2013， 34（6）：115-119. （in Chinese）
[21] LIU W， WANG HP， YANG CN， et al. Study on extraction of pigment from purple sweet potato by phosphoric acid aqueous solution[J]. Guangzhou Chemical Industry， 2017， 45（18）：71-74. （in Chinese）
Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU
Key words Selenium; Purple potato; Anthocyanin; Extraction
Selenium is one of the essential trace elements in human body and animal body. It has the effects of resisting oxidation， enhancing immunity， antagonizing heavy metals， enhancing reproductive function and regulating protein synthesis and absorption of nutrients， and is praised as "the anticancer king" and "the protective agent of life" among the trace elements in human body[1].
Potato is one of the worlds top ten nutritious foods. It has strong growth adaptability and high yield and is the fourth largest staple food in China. The skin or flesh of purple potato is purpleblack， which contain 98% of acylated anthocyanins[2-3]. Anthocyanins are natural antioxidants and are the most potent free radical scavengers ever discovered. They are brightly colored and have no toxic side effects. They are widely used in the food， pharmaceutical and health care industries[4-9]. The nutritional and commercial value of seleniumenriched purple potato is higher than that of ordinary potato because of their selenium and anthocyanins. At present， the research on purple potato anthocyanins remains in the composition analysis[10-11]and the antioxidation and free radicalscavenging activity[12-13]. The extraction methods of anthocyanins differ due to the extraction materials， the purposes of extraction and the types of anthocyanins. At present， the main extraction methods include the solvent extraction method[14]， ultrasonic assisted method[15-16]， microwave assisted method[17]， highvoltage pulsed electric fieldassisted method[18]and so on. In order to obtain anthocyanins closer to the natural state and to avoid acylation of anthocyanins， weak citric acid is often used as an extractant[19-20]. Citric acid is a polybasic medium strong acid， which has a good colorprotecting effect on anthocyanins， but the price is high， resulting in high production cost of anthocyanin pigments. Phosphoric acid is also a polybasic acid and is similar to citric acid， but at a low price. Therefore， in this study， the technical parameters of ultrasonicassisted extraction of anthocyanins from seleniumenriched purple potato were investigated， aiming to provide a basis for further research and development. Materials and Methods
Experimental materials
Seleniumrich purple potato was provided by the Agricultural Resource and Environment Research Institute， Guangxi Academy of Agricultural Sciences/Guangxi Selenium Rich Agricultural Research Center. Phosphoric acid， acetic acid， citric acid， hydrochloric acid， and anhydrous ethanol were all chemical reagents of Sinopharm Group.
SB2512DTN ultrasonic cleaning machine， Ningbo Xinzhi Biotechnology Co.， Ltd.; RE52AA rotary evaporator， Shanghai Yarong Biochemical Instrument Factory; UNICO 2000 visible spectrophotometer， Unico （Shanghai） Co.， Ltd.; AX200 0.000 1 gelectronic balance， Japan Shimadzu Philippines.
Experimental methods
Analysis of anthocyanin content
The anthocyanin content was analyzed by the pH differential method in reference[21]. An appropriate amount of the extract was diluted to a volume of 10 ml with buffer solutions of pH=1.0 and pH=4.5， respectively， and the absorbance was measured at the wavelengths of 520 and 700 nm， respectively. The anthocyanin yield was calculated by the following formula：
Anthocyanin yield （mg/g）=A·MW·DF·V/ε·L·m
Wherein A=（A520-A700）pH=1-（A520-A700）pH=4.5; MW is the molar mass of cyanidine3galacoside （449.2 g/mol）; ε is the molar extinction coefficient of cyanidine3galacoside （26 900L/mol·cm）; V is the solution volume （ml）; L is the cuvette thickness （1 cm）; DF is the dilution factor; and m is the mass of purple potato （g）.
Screening of extraction solvent
The fresh potato was washed with water and cut into 2 mm pieces after the surface was naturally airdried. Eight parts （1 g each） of seleniumenriched purple potato pieces were added into conical flasks， respectively， into which 10 ml of 0.25% hydrochloric acid， 0.50% hydrochloric acid， 0.75% phosphoric acid， 1.50% phosphoric acid， 2.0% citric acid， 3.0% citric acid， 3.0% acetic acid and 4.0% acetic acid were added， respectively. Ultrasonic extraction was performed at room temperature for 20 min， followed by filtration and collection of filtrate. The anthocyanin content was determined， and the yield was calculated.
Single factor experiment
The effects of phosphoric acid concentration （0.25， 0.5， 0.75， 1.0， 1.25， 1.5%）， ultrasonic time （5， 7.5， 10， 15， 20， 25， 30 min） and materialtoliquid ratio （1∶2， 1∶3， 1∶4， 1∶5， 1∶6， 1∶7， 1∶8 g/ml） on the extraction of anthocyanins from seleniumenriched purple potato were investigated.
Response surface optimization According to the results of single factor experiment， with phosphoric acid concentration （A）， ultrasonic time （B） and materialtoliquid ratio （C） as three factors and the anthocyanin yield as the response value， the experiment was designed by BoxBehnken method in the DesignExpert 8.0.6 software. The levels of factors and codes of the factors are shown in Table 1.
Verification experiment
Three parallel experiments on the extraction of anthocyanins from seleniumrich purple potato were carried out according to the response surface method.
Results and Analysis
Single factor experiment
Screening of extraction solvent
Based on the extraction technique of anthocyanins from purple sweetpotato in previous studies[21]， the extraction effects of such four kinds of extraction solvents as hydrochloric acid （ 0.25%， 0.50% by weight）， phosphoric acid （0.75%， 1.50%）， citric acid （2.0%， 3.0%） and acetic acid （3.0%， 4.0%） on anthocyanins from seleniumrich purple potato were investigated at first under the materialtoliquid ratio of 1∶10 and ultrasonic time of 20 min. The results are shown in Fig. 1.
It can be seen from Fig. 1 that the extraction rate of anthocyanins from seleniumrich purple potato can reach 15% with 0.25%hydrochloric acid， but considering the volatility of hydrochloric acid， it is not suitable for industrial production. Acetic acid requires a high concentration to achieve a better extraction effect. Industrially， 3.0% citric acid aqueous solution is commonly used to extract anthocyanins. Although the effect is good， the production cost is high. The effects of extracting anthocyanins with 1.50% phosphoric acid and 3.0% citric acid aqueous solutions were better. In addition， the acidity of phosphoric acid （ka1=7.11×10-3， ka2=6.23×10-8， ka3=4.50×10-13） is similar to citric acid （ka1=7.44×10-4， ka2=1.73×10-5， Ka3=4.02×10-7）， and both have good complexing ability. Therefore， phosphoric acid extraction was selected.
Factors affecting the extraction of anthocyanins from phosphoric acid solution
In order to initially determine the ranges of extraction conditions for response surface optimization， a single factor experiment was first performed. The experimental results are shown in Table 2， Table 2 and Table 3.
The results from Table 2， Table 3 and Table 4 showed that with the materialtoliquid ratio at 1∶10 and extraction time of 20 min， the content of anthocyanins in the extract first rose and then decreased with the increase of phosphoric acid concentration. The anthocyanin yield reached a maximum at the phosphoric acid concentration of 1%. Then， at the phosphoric acid concentration of 1% and ultrasonic time of 20 min， the materialtoliquid ratio was set to be 1∶2， 1∶3， 1∶4， 1∶5， 1∶6， 1∶7 and 1∶8 g/ml. The experiment was carried out， and the anthocyanin extraction yield peaked at the ratio of 1∶5. Finally， under 1% phosphoric acid and the materialtoliquid ratio at 1∶5， the effect of ultrasonic time on the extraction of anthocyanins in purple potato was investigated. The results showed that the extraction rate of anthocyanins increased first with the increase of ultrasonic time. The yield of anthocyaninsin the seleniumrich purple potato reached the highest level when the ultrasonic time was 20 min， and then became stable. Optimization of extraction of anthocyanins from purple potato by BoxBehnken design
Response surface methodology to optimize experimental results
Based on the results of the single factor experiment， with the phosphoric acid concentration （A）， ultrasonic time （B） and materialtoliquid ratio （C） as independent variables and anthocyanin yield （Y） as the response value， the experimental deign was optimized by BoxBehnken method. The experimental design and results are shown in Table 5.
Response surface regression model and analysis of variance
The DesignExpert software was used to perform regression fitting on the experimental data of Table 5， and the regression equation with the anthocyanin yield （Y） as the response value was obtained.
Deming LIU et al. Optimization of Extraction Process of Anthocyanins from Seleniumenriched Purple Potato by Response Surface Methodology
The variance analysis of the regression model is shown in Table 6.
As can be seen from Table 6， the regression model P<0.000 1， indicated that the regression model was highly significant; the missing term P=0.255 9>0.05， indicated that the lack of fit of the model was not significant. The model correlation coefficient R2=0.994 8， indicated that the model can explain 99.48% of the change， and the model fitted well with the experiment， so the model can be used to analyze and predict the extraction process of anthocyanins in seleniumenriched purple potato. Combined with the F value of each factor， the effects of the three factors on the extraction of anthocyanins in seleniumenriched purple potato ranked as ultrasonic time>phosphoric acid concentration>materialtoliquid ratio.
Factor interaction analysis
The analysis was carried out on the interaction between the factors in the extraction of anthocyanins from seleniumenriched purple potato by ultrasonic aqueous phosphoric acid solution. The results are shown in Fig. 2.
Fig. 2 showed the interaction of the three factors on the extraction of anthocyanins from seleniumenriched purple potato. It can be seen from Fig. 2（a） that the change of the time surface was steeper， indicating that the effect of ultrasonic time on anthocyanin yield was more significant than the effect of phosphoric acid concentration. When the materialtoliquid ratio was constant， the anthocyanin yield had a maximum value when the ultrasonic time was 15-20 min and the phosphoric acid concentration was 0.85%-1.15%. It can be seen from Fig. 2（b） that the concentration of phosphoric acid had a significant effect on the yield of anthocyanins compared with the materialtoliquid ratio when the ultrasonic time was controlled constant. When the materialtoliquid ratio was 1∶3-1∶7g/ml， the anthocyanin yield had a maximum value when the concentration of phosphoric acid was at 0.85%-1.15%. It can be seen from Fig. 2（c） that when controlling the materialtoliquid ratio constant， the anthocyanin yield had a maximum value when the phosphoric acid concentration was 0.85%-1.15% and the ultrasonic time was 15-25 min. Verification experiment
The optimal extraction process parameters of anthocyanins in seleniumenriched purple potato were obtained by response surface optimization： the concentration of phosphoric acid of 0.98%， the materialtoliquid ratio at 4.96， and the extraction time of 18.92 min. The actual experiment adjusted them as follows： the extraction time of 19 min， the materialtoliquid ratio at 5 and the phosphoric acid concentration of 1.0%. Three parallel experiments were carried out under these conditions， and the average yield of anthocyanins was （0.409±0.010） mg/g （RSD=2.51%， n=3）， which was not much different from the model predicted value of 0.411 mg/g， indicating the validity of the equation.
Conclusions
In this study， the extraction process of anthocyanins in seleniumenriched purple potato was optimized by response surface methodology： the phosphoric acid concentration of 0.98%， the materialtoliquid ratio at 4.96 and the ultrasonic time of 18.92 min. The results of the verification experiment were in good agreement with the model predictions. The replacement of citric acid with phosphoric acid to extract anthocyanins also has the characteristics of low acid concentration， which is beneficial to the reduction of the production cost， which provides a reference for the deep development of anthocyanins from seleniumrich purple potato.
References
[1] RAYMAN MP. Selenium and human health[J]. Lancet.2012，379（9822）：1256-1268.
[2] WANG L， DENG FM， ZHAO LY， et al. Research progress in health function and utilization of purple potato[J]. China Brewing， 2015（7）： 117-120. （in Chinese）
[3] FOSSEN T， ANDERSEN OM. Anthocyanins from tubers and shoots of the purple potato， Solanum tuberosum[J]. Journal of Horticultural Science and Biotechnology， 2000， 75（3）： 360-363.
[4] ANNANARYJU D. Sarma， antioxidant ability of anthocyanins against ascorbic acid oxidation[J]. phytochemistry， 1997， 45（4）： 671-674.
[5] WU XZ， PIAO JY， ZHANG XY， et al. Isolation and identification of anthocyanidin from the berries of Lonicera caerulea[J] . Journal of Yanbian University （Natural Science Edition）， 2001， 27（3）： 191-194. （in Chinese）
[6] FANG ZX， NI YY， LI HM. Stability in different conditions of anthocyanins from purple sweet potato[J] . Food and Fermentation Industries， 2002， 28（10）： 31-34. （in Chinese）
[7] ZHAO XL. Research progress of ingredient and healthy function of blueberry[J]. Chinese Wild Plant Resources， 2011， 30（6）： 19-23. （in Chinese） [8] GU RX， HU HL， LIU YC， et al. Extraction technology and antibacterial activity of anthocyanin in purple sweet potato[J]. Shandong Agricultural Sciences， 2012， 44（4）： 107-113. （in Chinese）
[9] HAO WB， JIANG GM， CHE WS. The study on antioxidant and antibacterial activity of anthocyanins from blueberry[J]. Journal of Heihe University， 2014， 5（3）： 123-125. （in Chinese）
[10] FRANCESCA IERI， MARZIA INNOCENTI， LUISA ANDRENELLI， et al. Rapid HPLC/DAD/MS method to determine phenolic acids， glycoalkaloids and anthocyanins in pigmented potatoes （Solanum tuberosum L.） and correlations with variety and geographical origin[J]. Food Chemistry， 2011 125：750-759.
[11] LIZ GUTIRREZQUEQUEZANA， ANSSI L. VUORINEN， HEIKKI KALLIO， et al. Improved analysis of anthocyanins and vitamin C in bluepurple potato cultivars[J]. Food Chemistry， 242（2018） 217-224.
[12] GABRIELA BURGOS， WALTER AMOROS， LUPITA MUNOA， et al. Total phenolic， total anthocyanin and phenolic acid concentrations and antioxidant activity of purplefleshed potatoes as affected by boiling[J]. Journal of Food Composition and Analysis， 2013（30）： 6-12.
[13] AGNIESZKA KITA， ANNA BA KOWSKABARCZAK， GRAZYNA LISI NSKA， et al. Antioxidant activity and quality of red and purple flesh potato chips[J]. LWTFood Science and Technology， 2015（62）： 525-531.
[14] SUN X， ZHAO L， LI YT， et al. Research on the extraction methods of pigment from purple potato[J]. China Food Additives， 2013， 116（1）： 81-87. （in Chinese）
[15] XIAO HJ， MENG LQ， SHI PF. Optimization of ultrasonicassisted extraction for anthocyanins from purple sweet potato by response surface method[J]. Food Research and Development， 2015， 36（7）： 33-38. （in Chinese）
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Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU