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Abstract [Objectives] The aims were to optimize the extraction process of selenoproteins from selenium-enriched rice in Guangxi and provide references for the intensive processing and comprehensive utilization of selenium protein resources. [Methods] Selenium-enriched rice was used as materials to extract selenoproteins by phosphate buffer extraction method and to optimize the extraction process of selenoproteins by using the orthogonal experiment. Proteins and selenium content was measured by Coomassie Brilliant Blue G-250 reagent and AFS （atomic fluorescence spectrometry） respectively. [Results] The most significant factor affecting extraction of rice Selenoproteins was extraction NaOH concentration， followed by the ratio of solid-liquid， temperature and then extraction time. The optimum extraction conditions of selenoproteins from rice were extraction temperature of 50 ℃， NaOH concentration of 0.14 mol/L， extraction time of 5 h， and solid-liquid ratio of 1∶30. [Conclusions] The alkali extraction process optimized by orthogonal test could effectively improve the extraction rate of selenoproteins， and the optimized process parameters could be popularized and applied in practical production.
Key words Selenium-enriched rice； Selenoproteins； Orthogonal experiment； Extraction efficiency
Trace elements selenium is an essential nutrient element for human body. According to survey data of WHO， about 1 billion people are currently facing the problem of selenium malnutrition. The “hidden hunger” caused by selenium deficiency is threatening human health[1]. Scientific and rational selenium supplementation has become an important way to enhance physical fitness of human body. Rice is one of the main grains in China. It can be used as an intermediate carrier for selenium enrichment， and the extracted rice proteins can be used as a new selenium-enriched nutrition product with high nutritional value. Research and development of selenoproteins in selenium-enriched rice will be a new idea to solve the problem of lack of domestic protein industry in China， which is of practical significance for the effective use of the added value of selenium-enriched rice. Zhang[2] found that selenium-containing proteins were the main form of selenium conferred in rice， accounting for 73% of total selenium， and the selenium contained in glutelin accounted for 48% of total selenium. Fang et al.[3] found that the total selenium content in selenium-enriched rice was as high as 206.71 μg/kg， and organic selenium was the main form storing in rice， accounting for 81.70% of total selenium. Alkali-soluble selenoproteins were the main form of organic selenium， accounting for 53.40%， polysaccharide combined selenium accounted for 9.28%， and RNA combined selenium accounted for 1.86%. Li[4] made systematic investigation to the selenoproteins in Xinchun No.36 Triticale in Xinjiang using single factor and response surface analysis， and found that the content of selenoproteins was （240.52 ± 0.3） mg/g. The optimum conditions for the extraction of alkali soluble selenium proteins were obtained as follows： 0.17 mol/L NaOH， pH 4.7， ammonium sulfate saturation of 66.0%， solid-liquid ratio of 1∶10， and the extraction rate of the alkali-soluble selenoproteins was 76.7 %. Wang et al.[5] optimized the selenoproteins extraction process assisted by repeated freeze-thaw method， and the results showed that the protein extraction yield increased by 20% in the experimental group with 3 times of freezing-thawing repetition under the same conditions of 0.10 mol/L NaOH， extraction time of 3 h， extraction times of twice， extraction temperature of 70 ℃， solid-liquid ratio of 1∶60. Zou et al.[6] extracted rice barn protein by ultrasonic-assisted method， and the optimum extraction conditions were determined as follows： ultrasonic treatment time of 50 min， ultrasonic power of 380 W， and solid-liquid ratio of 1∶16， when the rice barn protein yield reached up to 51.66%. Gergely[7] extracted selenoproteins from mushrooms with 3 extraction media of 0.1 mol/L NaOH， 30 mM Tris-HCL and enzymatic digestion. At present， most of the methods for the extraction of plant proteins are the alkaline， enzymatic and compound extraction methods. However， due to the different factors such as the raw material processing method and test environmen， the obtained research conclusions are also different. In addition， there are few reports on the optimization and confirmation of the extracting process and extracting conditions of selenoproteins from selenium-enriched rice in China. Therefore， the effect of alkaline extraction of selenoproteins from selenium-enriched rice was explored to obtain the optimum extraction conditions， so as to improve the exploitation and utilization of selenoprotein resources and to provide strong supports for the increase of the added value， diversity and convenience of nutritious foods.
Materials and Methods
Materials
The selenium-enriched rice was collected from Baima Village， Bama， Guangxi. The average selenium content in the soil in this area was （0.43 ± 1.24） mg/kg. The rice was naturally cultivated in the field and managed routinely. The harvested rice was dried in a blast air oven at 60 ℃， and then crushed using a grinder until all passed through the 100-mesh sieve. And then， the prepared rice was sealed in a dryer for later use.
Instruments and reagents
The main instruments included AFS dual-channel atomic fluorescence photometer； TDZ5-WS high-speed refrigerated centrifuge； TD-5-A centrifuge， ETHOS PLUS microwave digestion system； UV-visible spectrophotometer， SHZ-88 water bath oscillator. The major reagents were NaOH， NaCl， HCl， all of which were guaranteed reagents. The water for the test was ultra-pure water.
Test program
Screening and extraction process of selenoproteins from selenium-enriched rice The solid-liquid volume ratio of the rice powder and the extracting solution was controlled at 1∶20， and the solvents included 0.1 mol/L NaCl， NaOH， HCl and water. After vibrated extraction at normal temperature for 3 h， the extracting solution was centrifuged at 25 ℃ under 4 000 r/min for 20 min. Then， a certain amount of supernatant was taken， which was added with ice acetone of 4 times of the solution volume in ice bath， and then placed in the refrigerator at -24 ℃ for 12 h to precipitate proteins. And then after centrifugation at 4 ℃ under 10 000 r/min for 15 min， the supernatant was discarded and the acetone was volatilized in the fuming cupboard， obtaining the crude proteins. The contents of selenium were measured to choose the best solvent. Single factor test On the basis of obtaining the best solvent， single factor tests were carried out to the extracting agent to analyze the effects of different solid-liquid volume ratio （1∶10， 1∶20， 1∶30， 1∶40， 1∶50， 1∶60）， NaOH concentration （0.02， 0.05， 0.08， 0.10， 0.14， 0.20 mol/L）， extraction temperature （20，30，40，50 and 60 ℃）， extraction time （1，2，3，4，5 and 6 h） on the extraction of selenoproteins.
Orthogonal test Based on the results of single factor tests， L9 （34） orthogonal design was conducted to the 4 factors of solid-liquid ratio， NaOH concentration， extraction temperature and extraction time to determine the optimal extraction conditions. Each factor was conducted with 3 repetitions of horizontal combination tests. The orthogonal factor levels were shown in Table 1.
Results and Analysis
Selection of extracting solution
As shown Fig. 1， with the extraction rate of selenium as the indicator， NaOH had the best effect of extracting selenoproteins， and the extraction rate was 52.21%， which was 7 times of that of water. The selenoprotein extraction rates of the 4 solvents from high to low were in the order of NaOH> NaCl > HCl> H2O， therefore， NaOH was chosen as the extractant.
Single factor tests
Effect of different solid-liquid ratios on the extraction of selenoproteins from rice
Tests were carried out by only changing the solid-liquid ratios with other conditions remaining unchanged （extractant concentration of 0.1 mol/L， normal extraction temperature and extraction time of 3 h）. As shown in Fig. 2， with the increase of solid-liquid ratio， the extraction rates of selenoproteins from rice first increased and then decreased. The extraction rate reached the maximum value when the solid-liquid ratio increased to 1∶40， when the content of selenium was 1.08 μg/g， while when the solid-liquid ratio increased from 1∶50 to 1∶60， the extraction rate dropped rapidly. Therefore， 1∶40 g/mL was the optimal solid-liquid ratio from the single-factor optimization.
Effect of extraction time on the extraction of selenoproteins from rice
Tests were carried out by only changing the extraction time with other conditions remaining unchanged （extractant concentration of 0.1 mol/L， normal extraction temperature and solid-liquid ratio of 1∶20）. As shown in Fig.3， under certain conditions， the extraction time-extraction rate curve first increased and then decreased. The extraction rate reached 15.45% at 4 h， when the content of selenium also reached the maximum of 1.11 μg/g. After 4 h， the extraction rate of proteins showed a downward trend， which may because of the decrease of dissolution rate caused by the swelling of protein molecules. Therefore， 4 h was the optimal extraction time from the single factor optimization. Effect of extraction temperature on the extraction of selenoproteins from rice
Tests were carried out by only changing the extraction temperature with other conditions remaining unchanged （extractant concentration of 0.1 mol/L， extraction time of 3 h and solid-liquid ratio of 1∶20）. As shown in Fig. 4， when the extraction temperature increased from 20 ℃ to 40 ℃ ， The extraction rate of rice protein increased with the increase of temperature and reached the maximum at 40 ℃ with an increase of 10.07% from that at 20 ℃. With the further increase of extraction temperature， the extraction rate decreased. Therefore， 40 ℃ was the optimal temperature for the extraction of selenoproteins from the single factor optimization.
Effect of alkali concentration on the extraction of selenoproteins from rice
Tests were carried out by only changing the concentration of the extractant with other conditions remaining unchanged （extraction time of 3 h， normal extraction temeprature and solid-liquid ratio of 1∶20）. As shown in Fig.5， with the increase of the concentration of extractant， the selenoprotein extraction rate first increased slowly and then dropped rapidly. The extraction rate changed little in the concentration range of 0.02-0.08 mol/L. The extraction rate changed significantly in the range of 0.08-0.14 mol/L. The extraction rate reached the highest value of 28.07% at concentration of 0.14 mol/L， while the extraction rate was the lowest at the alkali concentration of 0.2 mol/L. Therefore， 0.14 mol/L was the optimal concentration of the alkali solution from the single factor optimization.
Orthogonal test results
As shown in Table 2， the range difference R values showed that the concentration of NaOH was the most important factor affecting the extraction rate， followed by the solid-liquid ratio， and the extraction rate was less affected by the extraction temperature and extraction time. The extraction rate of combination A1B3C3D3 was the highest， that is， solid-liquid ratio of 1∶30 g/mL， NaOH concentration of 0.14 mol/L， extraction time of 50 ℃ and extraction time of 5 h， when the extraction rate was 57.11%.
Discussion
In this study， the best extractant was NaOH， which was consistent with previous studies[9]. This may be related to the hydrolysis of hydrogen bonds， amide bonds and disulfide bonds in rice at high concentrations of alkali， thereby releasing more proteins[10-12]. Fang et al.[13] analyzed the content of selenium in proteins by extracting 4 kinds of proteins from rice， finding that selenium mainly existed in the alkali-soluble glutelin. Hu et al.[14] found that albumin and gliadin were not the major proteins for selenium， and Se mainly existed in the alkali-soluble glutelin and globulin which had large molecular weights， in which the alkali-soluble selenoproteins were the main components. Zhang et al.[15] also found that the alkali extraction process had the advantages of low cost， high extraction rate and simple extraction method. On the basis of obtaining the best extractant， single factor tests were carried out to the concentration of extractant， solid-liquid ratio， extraction time and extraction temperature. The results showed that extraction rate of selenoproteins increased significantly with the increase of alkali concentration at the beginning， but the increase was not infinite， and the extraction rate began to decrease after the concentration reached 0.14 mol/L. This was similar to the results of Peng[9]. The reason was that the pH value of the solution could directly change the charges on the surface of the amphiphilic protein， which in turn affected the interactions between protein and protein， protein and solvent， thereby changing the protein solubility. It has found that protein solubility significantly increases at pH> 7， and at pH> 12， more than 90% of the proteins are eluted[16]. Different alkali extraction conditions could have different extraction rates of proteins. At the same time， it has found that the concentration of the extractant should not be too high when extracting proteins， otherwise it would lead to the condensation of lysine with alanine or cystine in the protein[17]. Then， it would be difficult to dissolve out the protein， which would reduce the purity and quality of protein， thus affecting the nutritional value and commercial value of protein， resulting in decreased extraction rate[18].
The test found that the extraction rate first increased with the increase of solid-liquid ratio， and after reaching a certain percentage， it began to decline. This was consistent with the findings of Cao[19]， who extracted Cardamine enshiensis using alkaline extraction method. The reason was that the extraction solvent had a high viscosity at a low solid-liquid ratio， while the concentration of protein was high， which made the repulsive force high between protein molecules and the diffusion effect of protein small， so the extraction rate was low. With the increase of solid-liquid ratio， the viscosity of the extractant decreased， the repulsive force between molecules dropped， and the protein had the diffusion effect become larger and constantly dissolve out， so the extraction rate increased. However， with the further increase of solid-liquid ratio， the extraction rate decreased， probably because the high solid-liquid ratio increased the extraction difficulty， resulting the decrease of the extraction rate.
The single factor tests on extraction temperature showed that the extraction rate decreased after the extraction temperature was above 40 ℃. It has found that with the increase of temperature， the three-dimensional structure of protein stretches， increasing its interaction with water molecules， and the diffusion of molecules increases with increasing temperature， accelerating the rate of protein dissolution. However， when the temperature is increased to a certain temperature， the spatial structure of the protein is destroyed and its hydrophobic group is exposed， leading to the decrease of the protein solubility and the decrease of the extraction rate[20-22]. Lv[23] optimized the extraction process of Chinese chestnut protein， and found that the extraction rate of chestnut protein decreased with the increase of temperature because of the occurrence of chestnut starch gelatinization， which decreased the protein dissolution rate by combining with the protein， thus reducing the protein extraction rate. The extraction rate of selenoprotein increased with the extraction time within 1-4 h of extraction time， which may be related to the continuous dissolution of proteins with high selenium binding capacity[24]. However， when the extraction time exceeded 4 h， the protein extraction rate decreased， which may be due to the decrease of dissolution rate caused by the swelling of protein molecules. With reference to the single factor test results， the L9 （34） orthogonal tests were carried out on the 4 factors of solid-liquid ratio， NaOH concentration， extraction temperature and extraction time. The results showed that the protein extraction rates of 9 combinations were all higher than those of single factor test. Because in multi-factor tests， each factor was not alone， and there were interactions between factors， resulting in strong combination effects. By comparing the value of the range difference R values， the effect of each factor on the extraction rate from high to low was in the order of NaOH concentration> solid-liquid ratio> extraction temperature> extraction time. The final analysis of variance obtained the best extraction conditions of A1B3C3D3.
Although the alkaline extraction method has the advantages of low cost， easy operation and control， but requires longer extraction time， and the extraction effect has not reached the best. Therefore， it is also possible to improve the selenoprotein extraction rate by compound extraction methods such as assisting with ultrasonic-assisted extraction， alkaline-enzymatic method， or micro-jet-assisted method. Guangxi is rich in selenium resources. Research becomes deeper on the selenoproteins in rice， including the process of extracting selenoproteins， the basic theoretical characteristics of selenoproteins and the functional and utilization values. Therefore， rice selenoproteins are expected to be widely used in nutrition and health care， medical care and the food industry， which could not only save resources， but also open up new ideas for the development and utilization of abundant rice resources， so that rice can been developed and applied in depth. Moreover， it has important theoretical and practical significance to transform resource advantages into economic advantages.
Conclusion
The main factors affecting the extraction rate of selenoproteins from selenium-enriched rice were in the order of NaOH concentration> solid-liquid ratio> extraction temperature> extraction time. The optimum extraction conditions of selenoproteins from rice were extraction temperature of 50 ℃， NaOH concentration of 0.14mol/L， extraction time of 5 h， and solid-liquid ratio of 1∶30. The alkali extraction process optimized by orthogonal test could effectively improve the extraction rate of selenoproteins， and the optimized process parameters could be popularized and applied in practical production. References
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Key words Selenium-enriched rice； Selenoproteins； Orthogonal experiment； Extraction efficiency
Trace elements selenium is an essential nutrient element for human body. According to survey data of WHO， about 1 billion people are currently facing the problem of selenium malnutrition. The “hidden hunger” caused by selenium deficiency is threatening human health[1]. Scientific and rational selenium supplementation has become an important way to enhance physical fitness of human body. Rice is one of the main grains in China. It can be used as an intermediate carrier for selenium enrichment， and the extracted rice proteins can be used as a new selenium-enriched nutrition product with high nutritional value. Research and development of selenoproteins in selenium-enriched rice will be a new idea to solve the problem of lack of domestic protein industry in China， which is of practical significance for the effective use of the added value of selenium-enriched rice. Zhang[2] found that selenium-containing proteins were the main form of selenium conferred in rice， accounting for 73% of total selenium， and the selenium contained in glutelin accounted for 48% of total selenium. Fang et al.[3] found that the total selenium content in selenium-enriched rice was as high as 206.71 μg/kg， and organic selenium was the main form storing in rice， accounting for 81.70% of total selenium. Alkali-soluble selenoproteins were the main form of organic selenium， accounting for 53.40%， polysaccharide combined selenium accounted for 9.28%， and RNA combined selenium accounted for 1.86%. Li[4] made systematic investigation to the selenoproteins in Xinchun No.36 Triticale in Xinjiang using single factor and response surface analysis， and found that the content of selenoproteins was （240.52 ± 0.3） mg/g. The optimum conditions for the extraction of alkali soluble selenium proteins were obtained as follows： 0.17 mol/L NaOH， pH 4.7， ammonium sulfate saturation of 66.0%， solid-liquid ratio of 1∶10， and the extraction rate of the alkali-soluble selenoproteins was 76.7 %. Wang et al.[5] optimized the selenoproteins extraction process assisted by repeated freeze-thaw method， and the results showed that the protein extraction yield increased by 20% in the experimental group with 3 times of freezing-thawing repetition under the same conditions of 0.10 mol/L NaOH， extraction time of 3 h， extraction times of twice， extraction temperature of 70 ℃， solid-liquid ratio of 1∶60. Zou et al.[6] extracted rice barn protein by ultrasonic-assisted method， and the optimum extraction conditions were determined as follows： ultrasonic treatment time of 50 min， ultrasonic power of 380 W， and solid-liquid ratio of 1∶16， when the rice barn protein yield reached up to 51.66%. Gergely[7] extracted selenoproteins from mushrooms with 3 extraction media of 0.1 mol/L NaOH， 30 mM Tris-HCL and enzymatic digestion. At present， most of the methods for the extraction of plant proteins are the alkaline， enzymatic and compound extraction methods. However， due to the different factors such as the raw material processing method and test environmen， the obtained research conclusions are also different. In addition， there are few reports on the optimization and confirmation of the extracting process and extracting conditions of selenoproteins from selenium-enriched rice in China. Therefore， the effect of alkaline extraction of selenoproteins from selenium-enriched rice was explored to obtain the optimum extraction conditions， so as to improve the exploitation and utilization of selenoprotein resources and to provide strong supports for the increase of the added value， diversity and convenience of nutritious foods.
Materials and Methods
Materials
The selenium-enriched rice was collected from Baima Village， Bama， Guangxi. The average selenium content in the soil in this area was （0.43 ± 1.24） mg/kg. The rice was naturally cultivated in the field and managed routinely. The harvested rice was dried in a blast air oven at 60 ℃， and then crushed using a grinder until all passed through the 100-mesh sieve. And then， the prepared rice was sealed in a dryer for later use.
Instruments and reagents
The main instruments included AFS dual-channel atomic fluorescence photometer； TDZ5-WS high-speed refrigerated centrifuge； TD-5-A centrifuge， ETHOS PLUS microwave digestion system； UV-visible spectrophotometer， SHZ-88 water bath oscillator. The major reagents were NaOH， NaCl， HCl， all of which were guaranteed reagents. The water for the test was ultra-pure water.
Test program
Screening and extraction process of selenoproteins from selenium-enriched rice The solid-liquid volume ratio of the rice powder and the extracting solution was controlled at 1∶20， and the solvents included 0.1 mol/L NaCl， NaOH， HCl and water. After vibrated extraction at normal temperature for 3 h， the extracting solution was centrifuged at 25 ℃ under 4 000 r/min for 20 min. Then， a certain amount of supernatant was taken， which was added with ice acetone of 4 times of the solution volume in ice bath， and then placed in the refrigerator at -24 ℃ for 12 h to precipitate proteins. And then after centrifugation at 4 ℃ under 10 000 r/min for 15 min， the supernatant was discarded and the acetone was volatilized in the fuming cupboard， obtaining the crude proteins. The contents of selenium were measured to choose the best solvent. Single factor test On the basis of obtaining the best solvent， single factor tests were carried out to the extracting agent to analyze the effects of different solid-liquid volume ratio （1∶10， 1∶20， 1∶30， 1∶40， 1∶50， 1∶60）， NaOH concentration （0.02， 0.05， 0.08， 0.10， 0.14， 0.20 mol/L）， extraction temperature （20，30，40，50 and 60 ℃）， extraction time （1，2，3，4，5 and 6 h） on the extraction of selenoproteins.
Orthogonal test Based on the results of single factor tests， L9 （34） orthogonal design was conducted to the 4 factors of solid-liquid ratio， NaOH concentration， extraction temperature and extraction time to determine the optimal extraction conditions. Each factor was conducted with 3 repetitions of horizontal combination tests. The orthogonal factor levels were shown in Table 1.
Results and Analysis
Selection of extracting solution
As shown Fig. 1， with the extraction rate of selenium as the indicator， NaOH had the best effect of extracting selenoproteins， and the extraction rate was 52.21%， which was 7 times of that of water. The selenoprotein extraction rates of the 4 solvents from high to low were in the order of NaOH> NaCl > HCl> H2O， therefore， NaOH was chosen as the extractant.
Single factor tests
Effect of different solid-liquid ratios on the extraction of selenoproteins from rice
Tests were carried out by only changing the solid-liquid ratios with other conditions remaining unchanged （extractant concentration of 0.1 mol/L， normal extraction temperature and extraction time of 3 h）. As shown in Fig. 2， with the increase of solid-liquid ratio， the extraction rates of selenoproteins from rice first increased and then decreased. The extraction rate reached the maximum value when the solid-liquid ratio increased to 1∶40， when the content of selenium was 1.08 μg/g， while when the solid-liquid ratio increased from 1∶50 to 1∶60， the extraction rate dropped rapidly. Therefore， 1∶40 g/mL was the optimal solid-liquid ratio from the single-factor optimization.
Effect of extraction time on the extraction of selenoproteins from rice
Tests were carried out by only changing the extraction time with other conditions remaining unchanged （extractant concentration of 0.1 mol/L， normal extraction temperature and solid-liquid ratio of 1∶20）. As shown in Fig.3， under certain conditions， the extraction time-extraction rate curve first increased and then decreased. The extraction rate reached 15.45% at 4 h， when the content of selenium also reached the maximum of 1.11 μg/g. After 4 h， the extraction rate of proteins showed a downward trend， which may because of the decrease of dissolution rate caused by the swelling of protein molecules. Therefore， 4 h was the optimal extraction time from the single factor optimization. Effect of extraction temperature on the extraction of selenoproteins from rice
Tests were carried out by only changing the extraction temperature with other conditions remaining unchanged （extractant concentration of 0.1 mol/L， extraction time of 3 h and solid-liquid ratio of 1∶20）. As shown in Fig. 4， when the extraction temperature increased from 20 ℃ to 40 ℃ ， The extraction rate of rice protein increased with the increase of temperature and reached the maximum at 40 ℃ with an increase of 10.07% from that at 20 ℃. With the further increase of extraction temperature， the extraction rate decreased. Therefore， 40 ℃ was the optimal temperature for the extraction of selenoproteins from the single factor optimization.
Effect of alkali concentration on the extraction of selenoproteins from rice
Tests were carried out by only changing the concentration of the extractant with other conditions remaining unchanged （extraction time of 3 h， normal extraction temeprature and solid-liquid ratio of 1∶20）. As shown in Fig.5， with the increase of the concentration of extractant， the selenoprotein extraction rate first increased slowly and then dropped rapidly. The extraction rate changed little in the concentration range of 0.02-0.08 mol/L. The extraction rate changed significantly in the range of 0.08-0.14 mol/L. The extraction rate reached the highest value of 28.07% at concentration of 0.14 mol/L， while the extraction rate was the lowest at the alkali concentration of 0.2 mol/L. Therefore， 0.14 mol/L was the optimal concentration of the alkali solution from the single factor optimization.
Orthogonal test results
As shown in Table 2， the range difference R values showed that the concentration of NaOH was the most important factor affecting the extraction rate， followed by the solid-liquid ratio， and the extraction rate was less affected by the extraction temperature and extraction time. The extraction rate of combination A1B3C3D3 was the highest， that is， solid-liquid ratio of 1∶30 g/mL， NaOH concentration of 0.14 mol/L， extraction time of 50 ℃ and extraction time of 5 h， when the extraction rate was 57.11%.
Discussion
In this study， the best extractant was NaOH， which was consistent with previous studies[9]. This may be related to the hydrolysis of hydrogen bonds， amide bonds and disulfide bonds in rice at high concentrations of alkali， thereby releasing more proteins[10-12]. Fang et al.[13] analyzed the content of selenium in proteins by extracting 4 kinds of proteins from rice， finding that selenium mainly existed in the alkali-soluble glutelin. Hu et al.[14] found that albumin and gliadin were not the major proteins for selenium， and Se mainly existed in the alkali-soluble glutelin and globulin which had large molecular weights， in which the alkali-soluble selenoproteins were the main components. Zhang et al.[15] also found that the alkali extraction process had the advantages of low cost， high extraction rate and simple extraction method. On the basis of obtaining the best extractant， single factor tests were carried out to the concentration of extractant， solid-liquid ratio， extraction time and extraction temperature. The results showed that extraction rate of selenoproteins increased significantly with the increase of alkali concentration at the beginning， but the increase was not infinite， and the extraction rate began to decrease after the concentration reached 0.14 mol/L. This was similar to the results of Peng[9]. The reason was that the pH value of the solution could directly change the charges on the surface of the amphiphilic protein， which in turn affected the interactions between protein and protein， protein and solvent， thereby changing the protein solubility. It has found that protein solubility significantly increases at pH> 7， and at pH> 12， more than 90% of the proteins are eluted[16]. Different alkali extraction conditions could have different extraction rates of proteins. At the same time， it has found that the concentration of the extractant should not be too high when extracting proteins， otherwise it would lead to the condensation of lysine with alanine or cystine in the protein[17]. Then， it would be difficult to dissolve out the protein， which would reduce the purity and quality of protein， thus affecting the nutritional value and commercial value of protein， resulting in decreased extraction rate[18].
The test found that the extraction rate first increased with the increase of solid-liquid ratio， and after reaching a certain percentage， it began to decline. This was consistent with the findings of Cao[19]， who extracted Cardamine enshiensis using alkaline extraction method. The reason was that the extraction solvent had a high viscosity at a low solid-liquid ratio， while the concentration of protein was high， which made the repulsive force high between protein molecules and the diffusion effect of protein small， so the extraction rate was low. With the increase of solid-liquid ratio， the viscosity of the extractant decreased， the repulsive force between molecules dropped， and the protein had the diffusion effect become larger and constantly dissolve out， so the extraction rate increased. However， with the further increase of solid-liquid ratio， the extraction rate decreased， probably because the high solid-liquid ratio increased the extraction difficulty， resulting the decrease of the extraction rate.
The single factor tests on extraction temperature showed that the extraction rate decreased after the extraction temperature was above 40 ℃. It has found that with the increase of temperature， the three-dimensional structure of protein stretches， increasing its interaction with water molecules， and the diffusion of molecules increases with increasing temperature， accelerating the rate of protein dissolution. However， when the temperature is increased to a certain temperature， the spatial structure of the protein is destroyed and its hydrophobic group is exposed， leading to the decrease of the protein solubility and the decrease of the extraction rate[20-22]. Lv[23] optimized the extraction process of Chinese chestnut protein， and found that the extraction rate of chestnut protein decreased with the increase of temperature because of the occurrence of chestnut starch gelatinization， which decreased the protein dissolution rate by combining with the protein， thus reducing the protein extraction rate. The extraction rate of selenoprotein increased with the extraction time within 1-4 h of extraction time， which may be related to the continuous dissolution of proteins with high selenium binding capacity[24]. However， when the extraction time exceeded 4 h， the protein extraction rate decreased， which may be due to the decrease of dissolution rate caused by the swelling of protein molecules. With reference to the single factor test results， the L9 （34） orthogonal tests were carried out on the 4 factors of solid-liquid ratio， NaOH concentration， extraction temperature and extraction time. The results showed that the protein extraction rates of 9 combinations were all higher than those of single factor test. Because in multi-factor tests， each factor was not alone， and there were interactions between factors， resulting in strong combination effects. By comparing the value of the range difference R values， the effect of each factor on the extraction rate from high to low was in the order of NaOH concentration> solid-liquid ratio> extraction temperature> extraction time. The final analysis of variance obtained the best extraction conditions of A1B3C3D3.
Although the alkaline extraction method has the advantages of low cost， easy operation and control， but requires longer extraction time， and the extraction effect has not reached the best. Therefore， it is also possible to improve the selenoprotein extraction rate by compound extraction methods such as assisting with ultrasonic-assisted extraction， alkaline-enzymatic method， or micro-jet-assisted method. Guangxi is rich in selenium resources. Research becomes deeper on the selenoproteins in rice， including the process of extracting selenoproteins， the basic theoretical characteristics of selenoproteins and the functional and utilization values. Therefore， rice selenoproteins are expected to be widely used in nutrition and health care， medical care and the food industry， which could not only save resources， but also open up new ideas for the development and utilization of abundant rice resources， so that rice can been developed and applied in depth. Moreover， it has important theoretical and practical significance to transform resource advantages into economic advantages.
Conclusion
The main factors affecting the extraction rate of selenoproteins from selenium-enriched rice were in the order of NaOH concentration> solid-liquid ratio> extraction temperature> extraction time. The optimum extraction conditions of selenoproteins from rice were extraction temperature of 50 ℃， NaOH concentration of 0.14mol/L， extraction time of 5 h， and solid-liquid ratio of 1∶30. The alkali extraction process optimized by orthogonal test could effectively improve the extraction rate of selenoproteins， and the optimized process parameters could be popularized and applied in practical production. References
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