CN112129864A - Method for determining selenium form in selenium-rich plant dry powder - Google Patents
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a method for determining the form of selenium in selenium-enriched plant dry powder, belonging to the field of analysis and test. Ultrasonic-assisted extraction of free selenium-containing substances in the selenium-rich plant dry powder is adopted, an extraction solution containing Sodium Dodecyl Sulfate (SDS) is added into a precipitate after centrifugation, and a double-enzyme method is adopted for enzymolysis twice to extract combined selenium-containing substances. Collecting the supernatant obtained in each step, passing through a membrane, injecting the concentrated solution into a GPC system for separation, collecting small molecular fractions, concentrating, and determining the form content of each selenium by using a liquid chromatography-atomic fluorescence spectrometer (HPLC-AFS) or a high performance liquid chromatography-inductively coupled plasma mass spectrometer (HPLC-ICP-MS). The method has the advantages of short extraction time, high extraction efficiency, low cost of enzyme, and stable selenium form during treatment.
Description
Technical Field
The invention relates to a method for determining the form of selenium in selenium-enriched plant dry powder, belonging to the field of analysis and test.
Background
Selenium is an essential trace element of the human body and is listed as one of 15 nutrients. Selenium has antioxidant, antitumor, antiinflammatory, and antiaging effects. A plurality of countries and regions are in a state of selenium deficiency, seven regions in China are in a selenium deficiency zone, and the daily intake of selenium for adults is reported to be lower than the recommended dose of the Chinese society of nutrition. When the selenium in a human body is deficient, a plurality of pathological changes can be caused, so that daily selenium supplement is very necessary. The development of safe and effective selenium supplement products is the key point of selenium-rich health care products in China, and the utilization value of a plurality of selenium supplement products is not only related to the content of selenium but also related to the chemical form of the selenium. Selenium in nature exists mainly in three forms of inorganic selenium, organic selenium and simple substance selenium. The different forms of selenium also differ in their functionality, with inorganic selenium being highly toxic and of low biological effectiveness. Organic selenium is considered to be high-quality selenium due to its low toxicity and high bioavailability.
Plants are an indispensable key link of the selenium ecological chain, and can absorb inorganic selenium and convert the inorganic selenium into organic selenium compounds, so that the plants are considered as biochemical factories for synthesizing natural organic selenium. The organic selenium in the plant accounts for more than 80 percent of the total selenium, mainly consists of selenoprotein and small molecular selenide existing in the form of seleno-amino acid and derivatives thereof, and the plant source selenium-containing compound becomes an important way for obtaining the organic selenium. Therefore, the selenium nutrition mode of daily food intake is established by converting selenium-rich crops into organic selenium, and the selenium nutrition mode becomes one of the research hotspots of the current functional food.
Due to the complex matrix of the plant sample, the various forms of the selenium-containing compound, instability and very low content of the selenium-containing compound, the pretreatment method not only considers higher recovery rate but also keeps the original form from being transformed when the selenium sample is subjected to selenium form analysis. The water extraction method and the weak acid extraction method are mainly suitable for extracting small molecular selenium-containing compounds, and selenium form transformation or organic selenium degradation may occur in the extraction process. For the selenium-containing compound bound to the protein, the enzymolysis method can not only effectively release the selenium-containing compound, but also reduce the morphological transformation to a smaller extent by mild extraction conditions. The existing method for analyzing and determining the form of the plant-derived organic selenium is not perfect, wherein the pretreatment of a sample is a main technical difficulty. The existing selenium form separation method has long reaction time and is easy to cause the conversion of the selenium form; and the selected protease has high cost and is limited to be popularized in application. Therefore, it is necessary to develop a selenium-enriched plant dry powder pretreatment and selenium form analysis method with low cost and high efficiency.
Disclosure of Invention
[ problem ] to
The existing method for analyzing and determining the form of the plant-derived organic selenium is not perfect, wherein the pretreatment of a sample is a main technical difficulty. The existing method for separating the selenium form has long reaction time and is easy to cause the conversion of the selenium form; and the selected protease has high cost and is limited in application and popularization.
[ solution ]
Aiming at the problems, the invention provides a method for determining the selenium form in selenium-enriched plant dry powder, and in the process of pretreating the selenium-enriched plant dry powder, the invention adopts a method of sequentially carrying out enzymolysis on double enzymes, wherein the double enzymes are flavourzyme and protease E, and the residual selenium-containing substances in the precipitate are further extracted through secondary enzymolysis, so that the extraction effect and the stability of selenium-containing compounds are ensured. The pretreatment method has the advantages of high extraction efficiency, short time, simple operation and the like.
The invention provides a method for determining selenium form in selenium-enriched plant dry powder, which adopts the following technical scheme:
(1) extraction of free selenium species in the sample: taking a selenium-rich plant dry powder sample, adding water for dissolving, extracting, and taking a supernatant to obtain a supernatant 1;
(2) enzymolysis extraction of bound selenium substances in a sample: dissolving the precipitate obtained by centrifugation in the step (1) in an extraction solution containing Sodium Dodecyl Sulfate (SDS), sequentially adding flavourzyme and protease E step by step for enzymolysis, centrifuging after enzymolysis, and taking the supernatant to obtain a supernatant 2;
(3) and (3) carrying out secondary enzymolysis on the sample: and (3) taking the precipitate obtained after centrifugation in the step (2), repeating the operation in the step (2) for enzymolysis, centrifuging after enzymolysis, and taking the supernatant to obtain a supernatant 3.
(4) Sample detection: collecting the supernatant obtained in each step, passing through a membrane, injecting the concentrated solution into a Gel Permeation Chromatography (GPC) system for separation, collecting small molecular fractions, and measuring each selenium form after concentration.
In one embodiment of the invention, the selenium-rich plant dry powder sample is weighed in the step (1), dissolved by adding water, extracted by ultrasound assistance, and centrifuged after the ultrasound is finished to obtain the supernatant 1.
In one embodiment of the invention, the mass-volume ratio of the selenium-rich plant dry powder to water in the step (1) is 1: (50-70), and performing ultrasonic treatment at the temperature of 35-50 ℃ for 15-25min at 30-50 KHz.
In one embodiment of the present invention, the extraction solution in step (2) is deionized water or a 20mM Tris-HCl solution with pH 7.5.
In one embodiment of the present invention, SDS is added in the amount of 0.05-0.15 wt% of the extraction solution in step (2).
In one embodiment of the invention, the mass-to-volume ratio of the selenium-enriched plant dry powder precipitate subjected to ultrasonic treatment in the step (2) to the extraction solution containing SDS is 1: (50-70).
In one embodiment of the invention, the mass ratio of the flavourzyme and protease E in the step (2) to the selenium-enriched plant dry powder precipitate after ultrasonic treatment is 1: (10-40), the enzymolysis temperature is 40-50 ℃, and the enzymolysis time is 4-5 hours.
In one embodiment of the present invention, the order of adding the flavourzyme and the protease E in step (2) is to add the flavourzyme for enzymolysis for 4-5 hours first, and then add the protease E for enzymolysis for 4-5 hours.
In one embodiment of the invention, the supernatant obtained in each step in step (4) is collected and passed through a membrane, the concentrated solution is injected into a Gel Permeation Chromatography (GPC) system for separation, and small molecule fractions are collected and concentrated to determine each selenium form.
In one embodiment of the present invention, the specific parameters of the GPC separation in step (4) are: packing of a chromatographic column: superdex 75 gel filtration packing; eluent: deionized water or 20mM Tris-HCl solution with pH 7.5; flow rate: 1.0 mL/min.
In one embodiment of the present invention, the selenium form is determined by high performance liquid chromatography-hydride generation-atomic fluorescence spectroscopy (HPLC-HG-AFS) or high performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP MS) after concentration in step (4).
In one embodiment of the present invention, the method for determining the content of each form of selenium by HPLC-HG-AFS in step (4) comprises: a chromatographic column: hamilton PRP-X100 anion exchange column (250 mm. times.4 mm, 10 μm); mobile phase: mobile phase A: 40mmol/L (NH)4)2HPO4The pH value is 6.0; mobile phase B: 60mmol/L (NH)4)2HPO4(ii) a Sample introduction volume: 100 mu L of the solution; flow rate: 1.0 mL/min.
In one embodiment of the present invention, the method for determining the content of each selenium form by HPLC-ICPMS in step (4) comprises: thermo Scientific Hypersil GOLD C8(250 mm. times.4.6 mm, 5 μm); mobile phase: 20mmol/L potassium dihydrogen phosphate (containing 0.05 wt.% heptafluorobutyric acid, 3 wt.% methanol); the sample injection volume is 15 mu L; flow rate: 1.2 mL/min.
[ advantageous effects ]:
(1) when the ultrasonic extraction of the free selenium is carried out, the invention destroys the compact fiber tissue structure of the selenium-rich plant dry powder, and provides better conditions for enzymolysis. The addition of SDS destroys the protein structure in the selenium-enriched plant dry powder, and the protein-combined selenium-containing substance is more beneficial to being extracted.
(2) The selected proteases are flavourzyme and proteinase E, which are cheap with frequently used proteinase K and proteinase XIV reported in the literature, and reduce the pretreatment cost of the sample.
(3) The enzymolysis means in the invention adopts a method of double enzyme enzymolysis in sequence, and secondary enzymolysis further extracts the residual selenium-containing substances in the precipitate, thereby ensuring the extraction effect and stability of the selenium-containing compound. The method has the advantages of high extraction efficiency, short time, simple operation and the like.
Drawings
FIG. 1 is a chromatogram of a standard solution of selenium of different forms in example 1.
FIG. 2 shows the selenium extraction rate for different enzyme types under different enzymatic hydrolysis time conditions in comparative example 1.
FIG. 3 shows the selenium extraction yield for the different enzyme addition modes of comparative example 2.
Fig. 4 shows the selenium extraction rate for different enzymatic hydrolysis times in comparative example 3.
Detailed Description
[ example 1 ]
(1) Dissolving 0.1g cardamine hirsute dry powder in 5mL of deionized water, controlling the temperature at 40 ℃, performing 40KHz ultrasonic treatment for 15min, centrifuging the obtained mixture at 4000rpm for 15min after the ultrasonic treatment is finished, and taking supernatant to obtain supernatant 1;
(2) dissolving 1mg of Sodium Dodecyl Sulfate (SDS) in 7mL of Tris-HCL with pH value of 7.5, dissolving all precipitates obtained after centrifugation in the step (1) in the Tris-HCL, adding 10mg of Flavourzyme (Flavourzyme500MG, Novoxil Biotech Co., Ltd.) into the precipitates, stirring for enzymolysis for 4 hours, adding 10mg of protease E (Pronase E, Type XIV, not less than 3.5units/mg, Sigma-aldrich Co., Ltd.) into the precipitates, stirring for enzymolysis for 4 hours, wherein the enzymolysis temperature is 40 ℃, centrifuging at 4000rpm after enzymolysis for 15min, and taking supernatant 2;
(3) taking the precipitate centrifuged in the step (2), repeating the operation in the step (2) for enzymolysis, centrifuging at 4000rpm for 15min after enzymolysis, and taking the supernatant to obtain a supernatant 3;
(4) collecting the supernatant obtained in each step, filtering by a 0.45 mu m nylon filter head, injecting the solution after rotary evaporation and concentration into a GPC system for separation, and collecting small molecular fraction. The specific parameters of the GPC separation are: packing of a chromatographic column: superdex 75 gel filtration packing; eluent: deionized water or 20mM Tris-HCl solution with pH 7.5; flow rate: 1.0 mL/min. After vacuum concentration, HPLC-HG-AFS is adopted to determine the content of each selenium form. The chromatograms of the standard solutions of different forms of selenium are shown in FIG. 1, and FIG. 1 is the chromatogram of five selenium standards (SeCys2, MeSeCys, Se (IV), SeMet, Se (VI)). The content of five forms of selenium in cardamine hirsute is calculated by an external standard method, and the calculation formula is as follows:
the selenium extraction rate in cardamine hirsute in the embodiment is calculated to be 83.8%.
Wherein, the testing conditions of the HPLC-HG-AFS are as follows:
high performance liquid chromatography conditions
[ example 2 ]
(1) Dissolving 0.1g of cabbage dry powder in 5mL of deionized water, controlling the temperature at 40 ℃, performing 40KHz ultrasonic treatment for 25min, centrifuging the obtained mixture at 4000rpm for 15min after the ultrasonic treatment is finished, and taking supernatant to obtain supernatant 1;
(2) dissolving 1mg of SDS into 7mL of Tris-HCL with pH value of 7.5, dissolving the precipitate obtained after centrifugation in the step (1) into the Tris-HCL, adding 10mg of flavourzyme into the solution, stirring and performing enzymolysis for 4 hours, then adding 10mg of proteinase E, stirring and performing enzymolysis for 4 hours at the enzymolysis temperature of 40 ℃, and centrifuging the mixture for 15min at 4000rpm after enzymolysis to obtain supernatant 2;
(3) taking the precipitate centrifuged in the step (2), repeating the operation in the step (2) for enzymolysis, centrifuging at 4000rpm for 15min after enzymolysis, and taking the supernatant to obtain a supernatant 3;
(4) collecting the supernatant obtained in each step, filtering the supernatant through a 0.45-micron nylon filter head, carrying out rotary evaporation on the concentrated solution, injecting the solution into a GPC system for separation, collecting small molecular fractions, measuring the selenium form content by adopting HPLC-AFS after concentration, calculating the selenium form content by an external standard method, wherein the measuring and calculating methods are the same as those in example 1, and the extraction rate of the selenium in the cabbage dry powder is 85.1%.
[ example 3 ]
(1) Dissolving 0.1g of broccoli dry powder in 7mL of deionized water, controlling the temperature at 40 ℃, performing 40KHz ultrasonic treatment for 15min, centrifuging the obtained mixture at 4000rpm for 15min after the ultrasonic treatment is finished, and taking supernatant to obtain supernatant 1;
(2) dissolving 1mg of SDS into 7mL of Tris-HCL with pH value of 7.5, dissolving the precipitate obtained after centrifugation in the step (1) into the Tris-HCL, adding 10mg of flavourzyme into the solution, stirring and performing enzymolysis for 5 hours, then adding 10mg of proteinase E, stirring and performing enzymolysis for 5 hours at the enzymolysis temperature of 40 ℃, and centrifuging the mixture for 15min at 4000rpm after enzymolysis to obtain supernatant 2;
(3) taking the precipitate centrifuged in the step (2), repeating the operation in the step (2) for enzymolysis, centrifuging at 4000rpm for 15min after enzymolysis, and taking the supernatant to obtain a supernatant 3;
(4) collecting the supernatant obtained in each step, filtering the supernatant through a 0.45-micron nylon filter head, injecting the solution subjected to rotary evaporation and concentration into a GPC system for separation, collecting small molecular fractions, measuring the selenium form content by adopting HPLC-AFS after concentration, calculating the selenium form content by an external standard method, and obtaining the selenium extraction rate of 84.9 percent in the broccoli dry powder by the measuring and calculating methods which are the same as those in the example 1.
Comparative example 1
In order to separate out all selenium in cardamine hirsute, the precipitate of free selenium after ultrasonic extraction is further subjected to enzymolysis to obtain bound selenium in the precipitate. The organic selenium in plants generally exists in a combined state, and the common extraction methods include an acid extraction method, a water extraction method, an enzyme extraction method and the like. The literature proves that the enzyme extraction method has better effect, so that the enzyme extraction method is adopted in the comparative example to separate the combined selenium in cardamine hirsute, and five enzymes are selected to explore the separation efficiency of the enzymes in plants at different times.
(1) Dissolving 0.1g cardamine hirsute dry powder in 5mL of deionized water, controlling the temperature at 40 ℃, performing 40KHz ultrasonic treatment for 25min, centrifuging the obtained mixture at 4000rpm for 15min after the ultrasonic treatment is finished, and taking supernatant to obtain supernatant 1;
(2) and (2) adding 5mL of deionized water into all the precipitates obtained after centrifugation in the step (1), stirring, and respectively adding 10mg of protease E, protease K, neutral protease, flavourzyme and compound protease at the temperature of 40 ℃ for stirring for 2h,4h,8h,10h,12h,16h and 24 h. And centrifuging the obtained mixture at 4000rpm for 15min, taking a supernatant, collecting the supernatant obtained in each step, filtering the supernatant through a 0.45-micrometer nylon filter head, and determining by using HPLC-HG-AFS (high performance liquid chromatography-high-mobility electrophoresis), thereby exploring the influence of different enzymes on the extraction efficiency at different time.
FIG. 2 shows the selenium extraction rate for different enzyme types under different enzymatic hydrolysis time conditions in comparative example 1. As can be seen from FIG. 2, the extraction efficiency of proteinase E is the highest, the extraction efficiency of proteinase K is the second highest, and the extraction efficiencies of the remaining three enzymes are general. The extraction efficiency of several enzymes is obviously improved at 8h, and the change is relatively smooth in the subsequent time period. In addition, as the time exceeds 12 hours, the peak of tetravalent selenium disappears, and as the time increases, the tetravalent selenium is converted into hexavalent selenium, so that the types of selenium cannot be effectively separated. The protease E and the protease K have high selenium extraction efficiency but have poor separation effect on selenomethionine, and the price of the two enzymes is higher. The flavourzyme has obvious extraction effect on the selenomethionine, and the flavourzyme has lower price and can make up the deficiency of the protease E. In view of the above, the selenium form separation efficiency was investigated by stirring for 8 hours using a combined action of protease E and flavourzyme.
Comparative example 2
Effect of enzyme addition on selenium extraction efficiency:
(1) dissolving 0.1g cardamine hirsute dry powder in 5mL of deionized water, controlling the temperature at 40 ℃, performing 40KHz ultrasonic treatment for 25min, centrifuging the obtained mixture at 4000rpm for 15min after the ultrasonic treatment is finished, and taking supernatant to obtain supernatant 1;
(2) and (2) adding 5mL of deionized water into all the centrifuged precipitates obtained in the step (1), and extracting by adopting the following three enzyme adding modes at the temperature of 40 ℃:
1) adding 5mg of protease E, stirring for 4h, adding 5mg of flavourzyme, and stirring for 4 h;
2) adding 5mg of protease E and 5mg of flavourzyme together, and stirring for 8 hours;
3) 5mg of flavourzyme is added and stirred for 4 hours, and then 5mg of protease E is added and stirred for 4 hours.
After the three ways of extraction, the obtained mixture is centrifuged at 4000rpm for 15min, the supernatant is taken, the supernatant obtained in each step is collected, filtered by a 0.45 mu m disposable nylon filter head and then determined by HPLC-HG-AFS.
FIG. 3 shows the selenium extraction yield for the different enzyme addition modes of comparative example 2. As can be seen from FIG. 3, the double-enzyme method has better extraction efficiency than the single-enzyme method, wherein the method of adding the flavourzyme first and then adding the protease E has the highest extraction efficiency, and the extraction efficiency is improved probably because the activity of the protease is reduced with the increase of time, and the extraction efficiency is also influenced by the addition sequence of the two enzymes, which is probably because the composite action of the two protease enzyme cutting sites is different and is in turn better.
Comparative example 3
The influence of the enzymolysis times on the selenium extraction efficiency is as follows:
(1) dissolving 0.1g cardamine hirsute dry powder in 5mL of deionized water, controlling the temperature at 40 ℃, performing 40KHz ultrasonic treatment for 15min, centrifuging the obtained mixture at 4000rpm for 15min after the ultrasonic treatment is finished, and taking supernatant to obtain supernatant 1;
(2) dissolving 1mg of SDS into 7mL of Tris-HCL with pH value of 7.5, dissolving all precipitates obtained after centrifugation in the step (1) into the solution, firstly adding 10mg of flavourzyme into the solution, stirring and performing enzymolysis for 4h, then adding 10mg of proteinase E, stirring and performing enzymolysis for 4h, wherein the enzymolysis temperature is 40 ℃, and centrifuging the solution at 4000rpm for 15min after enzymolysis to obtain supernatant 2;
(3) taking the precipitate centrifuged in the step (2), repeating the operation in the step (2) for enzymolysis, centrifuging at 4000rpm for 15min after enzymolysis, and taking the supernatant to obtain a supernatant 3;
(4) and repeating the enzymolysis process on the precipitate, centrifuging to obtain a supernatant, and combining the supernatants to determine the extraction efficiency of the selenium by enzymolysis for one time, two times and three times.
(5) Collecting the supernatant obtained in each step, filtering with 0.45 μm disposable nylon filter head, injecting the concentrated solution into GPC system for separation, collecting small molecule fraction, concentrating, and determining selenium form content by HPLC-HG-AFS.
The extraction efficiency of selenium is still not very high after one enzymolysis, but the extension time can lead to the conversion of tetravalent selenium, so a multi-enzymolysis method is adopted, namely, the precipitate obtained by the first enzymolysis is subjected to the previous enzymolysis again, and fig. 4 shows the extraction efficiency of selenium under different enzymolysis times in the comparative example 3. It can be seen from fig. 4 that the extraction rate is increased after the second enzymolysis, and the residual selenium in the precipitate can be extracted by carrying out the second enzymolysis. But no obvious change is generated in the third time, and in order to save the experimental time, two enzymolysis methods are finally selected to extract the morphology in the cardamine hirsute.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A method for determining the selenium form in selenium-enriched plant dry powder is characterized by comprising the following steps:
(1) extraction of free selenium species in the sample: weighing selenium-enriched plant dry powder samples, adding water for dissolving, extracting, and taking supernate to obtain supernate 1;
(2) enzymolysis extraction of bound selenium substances in a sample: dissolving the precipitate obtained in the step (1) in an extraction solution containing sodium dodecyl sulfate, sequentially adding flavourzyme and protease E step by step for enzymolysis, centrifuging after enzymolysis, and taking supernatant to obtain supernatant 2;
(3) and (3) carrying out secondary enzymolysis on the sample: taking the precipitate centrifuged in the step (2), repeating the operation in the step (2) for enzymolysis, centrifuging after enzymolysis, and taking the supernatant to obtain a supernatant 3;
(4) sample detection: mixing the supernatants 1, 2, and 3, injecting the concentrated solution into gel permeation chromatographic column system for separation, collecting small molecule fraction, concentrating, and determining selenium forms.
2. The method as claimed in claim 1, wherein the mass volume ratio of the selenium-enriched plant dry powder to the water in the step (1) is 1: (50-70), adding water to dissolve, and then performing ultrasonic-assisted extraction, wherein the ultrasonic temperature is controlled to be 35-50 ℃ and ultrasonic treatment is performed at 30-50 KHz for 15-25 min.
3. The method of claim 1, wherein the extraction solution in step (2) is deionized water or a 20mM Tris-HCl solution with pH 7.5.
4. The method according to claim 1, wherein the sodium lauryl sulfate is added in the amount of 0.05 to 0.15 wt% to the extraction solution in the step (2).
5. The method according to claim 1, wherein the mass to volume ratio of the precipitate to the SDS-containing extraction solution in step (2) is 1: (50-70).
6. The method according to claim 1, wherein the mass ratio of the flavourzyme and protease E to the precipitate in step (2) is 1: (10-40).
7. The method according to claim 1, wherein the enzymolysis temperature in the step (2) is 40-50 ℃ and the enzymolysis time is 4-5 hours.
8. The method as claimed in claim 1, wherein the order of adding the flavourzyme and the protease E in the step (2) is to add the flavourzyme for enzymolysis for 4-5 hours first, and then add the protease E for further enzymolysis for 4-5 hours.
9. The method according to claim 1, wherein the specific parameters of the gel permeation chromatography column for separation in step (4) are as follows: packing of a chromatographic column: superdex 75 gel filtration packing; eluent: deionized water or 20mM Tris-HCl solution with pH 7.5; flow rate: 1.0 mL/min.
10. The method of claim 1, wherein each selenium form is determined in step (4) after concentration using high performance liquid chromatography-hydride generation-atomic fluorescence spectroscopy or high performance liquid chromatography-inductively coupled plasma mass spectrometry.
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| CN113755541B (en) * | 2021-09-14 | 2024-05-07 | 武汉轻工大学 | Method for selectively and efficiently extracting seleno-amino acid component in selenium-enriched plant |
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Address after: 214000 1800 Lihu Avenue, Binhu District, Wuxi, Jiangsu Patentee after: Jiangnan University Patentee after: Enshi Deyuan Selenium Material Engineering Technology Co.,Ltd. Address before: 214000 1800 Lihu Avenue, Binhu District, Wuxi, Jiangsu Patentee before: Jiangnan University Patentee before: ENSHI SERUN HEALTH TECHNOLOGY DEVELOPMENT CO.,LTD. |