阅读说明：本技术 一种选择性高效提取富硒植物中硒代氨基酸组分的方法 (Method for selectively and efficiently extracting seleno-amino acid components from selenium-rich plants ) 是由 丛欣 李洁 刘海远 徐波 祝振洲 程水源 于 2021-09-14 设计创作，主要内容包括：本发明属于有机硒提取技术领域,公开了一种选择性高效提取富硒植物中硒代氨基酸组分的方法,包括：将富硒植物粉与水混合,调节pH至8.5～9.5后超声处理；加入由碱性蛋白酶、胰蛋白酶和蛋白酶E组成的复合酶进行酶解,所述碱性蛋白酶、胰蛋白酶和蛋白酶E的质量比为1～2:1:1。通过特定的复合酶解体系,配合超声,提高了提取物中蛋白质含量和硒含量,并选择性提高了产物中硒代氨基酸的比例,且保证了提取物中硒代氨基酸的稳定性和活性。(The invention belongs to the technical field of organic selenium extraction, and discloses a method for selectively and efficiently extracting seleno-amino acid components from selenium-enriched plants, which comprises the following steps: mixing the selenium-rich plant powder with water, adjusting the pH value to 8.5-9.5, and then carrying out ultrasonic treatment; and adding a complex enzyme consisting of alkaline protease, trypsin and protease E for enzymolysis, wherein the mass ratio of the alkaline protease to the trypsin to the protease E is 1-2: 1: 1. The protein content and the selenium content in the extract are improved by a specific complex enzymatic hydrolysis system and ultrasound, the proportion of seleno-amino acid in the product is selectively improved, and the stability and the activity of the seleno-amino acid in the extract are ensured.)

1. A method for selectively and efficiently extracting seleno-amino acid components from selenium-rich plants is characterized by comprising the following steps:

s1, mixing the selenium-rich plant powder with water, adjusting the pH value to 8.5-9.5, and then carrying out ultrasonic treatment;

s2, adding a complex enzyme composed of alkaline protease, trypsin and protease E for enzymolysis, wherein the using amount of the complex enzyme is 1-10 wt% of the selenium-rich plant powder, and the mass ratio of the alkaline protease, the trypsin and the protease E is 1-2: 1: 1.

2. The method according to claim 1, wherein the mass ratio of the selenium-rich plant powder to water in step S1 is 1: 10-20.

3. The method according to claim 1, wherein in the ultrasonic treatment of step S1, the solution temperature is 50-60 ℃, the ultrasonic power is 150-200W, and the ultrasonic time is 10-30 min.

4. The method of claim 1, wherein the enzymatic hydrolysis process of step S2 is: stirring and reacting for 0.5-2.5 h at 50-60 ℃.

5. The method as claimed in claim 1, wherein in the step S2, after the enzymolysis reaction solution is centrifuged and filtered, HPLC-AFS detection is directly performed to monitor the contents of the components in various forms of selenium in the enzymolysis reaction solution in real time.

6. The method according to claim 1 or 5, characterized in that the method further comprises the steps of:

after the enzymolysis reaction is finished, carrying out enzyme deactivation treatment on the enzymolysis reaction liquid, and centrifuging to obtain supernatant; adding activated carbon into the supernatant for treatment, and removing inorganic salts from the filtered supernatant by using a nanofiltration membrane; the filtrate was concentrated in vacuo and spray dried.

7. The method of claim 6, wherein the enzyme deactivation process is: quickly heating the enzymolysis reaction liquid to 80-90 ℃, keeping the temperature for 10-30 min, then quickly cooling to room temperature, and carrying out high-speed low-temperature centrifugation to obtain supernatant; the centrifugal temperature is 4 ℃, and the centrifugal speed is 4000-5000 r/min.

8. The method according to claim 6, wherein the adding amount of the activated carbon is 1-2.5 wt% of the mass of the supernatant, and the treatment specifically comprises the following steps: stirring and reacting for 10-30 min at 50-60 ℃; the filtration is performed using a filter plate containing diatomaceous earth.

9. The method of claim 6, wherein the vacuum concentration is: concentrating under-0.06-0.08 MPa and 60-70 ℃ until the solid content is 25-30%.

10. The method of claim 6, wherein the spray drying conditions are: the air inlet temperature is 140-170 ℃, the air outlet temperature is 85-100 ℃, and the feeding speed is controlled at 5.5-7L/h.

Technical Field

The invention belongs to the technical field of organic selenium extraction, and particularly relates to a method for selectively and efficiently extracting high-activity seleno-amino acid components in selenium-enriched plants.

Background

Selenium is a trace element necessary for human bodies, is not stored in the bodies and cannot be synthesized and produced by self, and must be supplemented by external intake. Research shows that selenium not only participates in the metabolism of various enzymes and proteins of the body, but also has the effects of resisting oxidation, maintaining normal immunity and the like, and can reduce the incidence rate of coronary heart disease, cancer and the like of people; in the field of animal feeding, the feed additive can also improve the farrowing rate and milk yield of sows, enhance the activity of boar sperms, improve the meat quality while enriching selenium, and further directly or indirectly improve the human health. The selenium form in nature mainly comprises inorganic selenium and organic selenium, the poisoning phenomenon can be caused by excessive intake of the inorganic selenium, and the organic selenium has high safety and better absorption rate than the inorganic selenium under the condition of smaller intake. Therefore, organic selenium-rich products of plant origin have become a hot spot for the deep processing of selenium foods.

The steady state obtaining premise of the plant source organic selenium active components such as selenium-rich polypeptide, small peptide and the like is to obtain selenium-rich plant protein efficiently, a common extraction method is a solvent extraction method, and stirring, ultrasound or freeze thawing, pulsed electric field assistance and the like are used as auxiliary materials, but the high-strength long-time pulse or freeze thawing treatment can damage the selenium protein structure, even reduce the content of high-activity seleno-amino acid, or reduce the content of organic selenium, and is not beneficial to the maintenance of the activity of selenium-containing protein. In addition, in the selection of the extraction solvent, alkali, acid, alcohol, salt and the like can also influence the properties of the selenoprotein and the stability of the selenoprotein component to different degrees. When water is used as an extractant, the activity of the protein changes little, but the extraction efficiency is low, and only free water-soluble protein can be extracted.

Compared with a solvent extraction method, the enzyme extraction method has the following advantages: the reaction condition is mild, the specificity is strong, the side reaction is less, and the hydrophilic capability of the enzymolysis product is improved; the peptide chain length is shortened, and the molecular weight of the polypeptide is reduced; the molecular conformation of the protein is changed, and hydrophobic groups in the molecule are exposed, so that the extraction purity of the protein is high.

In addition, selenium is mainly bound to proteins in plants, and usually exists in the form of selenocysteine/selenocysteine, selenomethionine, selenomethylselenocysteine, selenoethethionine and other selenium-amino acid derivatives, and mostly exists in the form of selenocysteine, selenomethionine and the like in animals, wherein physiological activities such as antioxidation and the like are mainly generated in the form of selenoprotein (selenase), an active center is selenocysteine, and a regulatory selenium repository is formed, and selenomethionine exists in a non-regulatory selenium repository. Among the currently discovered physiologically active selenoproteins, those capable of performing physiological functions in the human or animal body are limited to selenocysteine-containing proteins such as glutathione peroxidase. Selenocysteine is called the 21 st amino acid of human body, can be specifically involved in selenoprotein synthesis in human bodies and animal bodies, and becomes an active center of oxidation-reduction reaction of glutathione peroxidase.

However, the prior art is still in the exploration stage of the high-efficiency extraction and separation and detection pretreatment technology for the high-activity seleno-amino acids (including selenocysteine/selenocysteine) organic selenium forms of selenium-rich plants. In the exploration of the method for detecting the enzymolysis pretreatment of the selenium-enriched plant selenium form, the inventor finds that different complex enzyme hydrolysis systems have different stability on selenocysteine/selenocysteine components.

Disclosure of Invention

Aiming at the problems in the prior art, the invention realizes the selective and efficient extraction of the seleno-amino acid, especially selenocysteine/selenocysteine, by a specific complex enzymatic hydrolysis system, and ensures the stability and activity of the seleno-amino acid in the extract.

The technical scheme of the invention is as follows:

a method for selectively extracting seleno-amino acid components from selenium-rich plants specifically comprises the following steps:

s1, mixing the selenium-rich plant powder with water, adjusting the pH value to 8.5-9.5, and then carrying out ultrasonic treatment;

s2, adding a complex enzyme composed of alkaline protease, trypsin and protease E for enzymolysis, wherein the amount of the complex enzyme is 1-10 wt% of the selenium-rich plant powder, and the mass ratio of the alkaline protease, the trypsin and the protease E is 1-2: 1: 1.

Further, in the above technical scheme, the mass ratio of the selenium-rich plant powder and water in step S1 is 1: 10-20, the pH regulator is sodium hydroxide or potassium hydroxide, or a Tris-HCl buffer solution with a pH meeting the above conditions may be directly used.

Further, in the above technical scheme, during the ultrasonic treatment in the step S1, the temperature of the solution is 50-60 ℃, the ultrasonic power is 150-200W, and the ultrasonic time is 10-30 min.

Further, in the above technical solution, the enzymolysis process in step S2 is: stirring and reacting for 0.5-2.5 h at 50-60 ℃.

Further, in the above technical solution, in the enzymolysis process in step S2, after taking the enzymolysis reaction solution and performing centrifugal filtration, HPLC-AFS detection is directly performed to monitor the content of the seleno-amino acid component in real time. When HPLC-AFS is adopted for detection, the detection conditions are the same as T/CHC 1001-2019 appendix A of the plant-derived high organic selenium food raw material. It can be understood that the extraction method provided by the invention can be directly used for detecting the selenium form in a sample, and thus, the real-time detection of the content of the seleno-amino acid component in the enzymolysis process is realized.

Further, in the above technical solution, the enzymolysis reaction solution obtained in step S2 is further processed to obtain a product rich in seleno-amino acid components, and the processing process is as follows:

after the enzymolysis reaction is finished, carrying out enzyme deactivation treatment on the enzymolysis reaction liquid, and centrifuging to obtain supernatant;

adding activated carbon into the supernatant for treatment, and removing inorganic salts from the filtered supernatant by using a nanofiltration membrane;

the filtrate was concentrated in vacuo and spray dried.

Further, the enzyme deactivation treatment process specifically comprises the following steps: quickly heating the enzymolysis reaction liquid to 80-90 ℃, keeping the temperature for 10-30 min, then quickly cooling to room temperature, and carrying out high-speed low-temperature centrifugation to obtain supernatant; furthermore, the centrifugal temperature is 4 ℃, and the centrifugal speed is 4000-5000 r/min.

Furthermore, the adding amount of the activated carbon is 1-2.5 wt% of the mass of the supernatant, the treatment temperature is 50-60 ℃, and the reaction is carried out for 10-30 min under the stirring condition; the filtration employs a filter plate coated with diatomaceous earth.

Further, the parameters of the vacuum concentration are: vacuum degree of-0.06 to-0.08 MPa, 60 to 70 ℃, and concentration to solid content of 25 to 30 percent.

Furthermore, the spray drying adopts a spray dryer, and the working parameters of the spray dryer are as follows: the air inlet temperature is 140-170 ℃, the air outlet temperature is 85-100 ℃, and the feeding speed is controlled at 5.5-7L/h.

The invention has the beneficial effects that:

1) through the combination of the specific complex enzyme, the selenium-rich plant protein components in the plant cells can be fully dissolved out by enzymolysis under the mild condition of a water phase system, and compared with solvent extraction, the content of the selenium-containing protein can be increased to more than 40 percent; meanwhile, the selenium-containing protein of the plant is cut into selenocysteine/selenocysteine (calculated by selenocysteine), so that the content of the selenium-containing protein is increased from about 10-20 percent to about 40 percent, and the industrial application value is greatly improved.

2) The ultrasonic treatment can rapidly break plant cell walls and destroy plant fiber structures, can effectively remove impurities combined on the selenoprotein, can promote the opening of a protein spiral structure, is beneficial to the rapid enzymolysis of the complex enzyme, greatly reduces the using amount and the enzymolysis time of the complex enzyme, and effectively improves the quality yield of the selenoprotein extract from about 10 percent to over 25 percent; experiments prove that on the basis of ensuring the dissolution efficiency of the selenocysteine-containing component, the normal reaction time of single enzymolysis is shortened from 8-12 h to about 0.5-2.5 h, and the ultrasonic frequency and the ultrasonic time are effectively reduced by the composite enzymolysis reaction, so that the stability of the selenocysteine/selenocysteine component obtained by extraction and conversion is effectively ensured, and the energy consumption of the production process and the industrial application cost are greatly saved.

3) In the enzymolysis process, the enzymolysis reaction liquid can be directly taken, and the characterization identification of the selenocysteine/selenocysteine is carried out after the quick and simple treatment (centrifugation to take supernatant and filtration by using a filter membrane), so that the product control efficiency is effectively improved.

4) Firstly, quickly pretreating by adopting activated carbon and diatomite, and then finely filtering by using a nanofiltration membrane; the filter plate coated with diatomite can efficiently remove various particle impurities in the decolored liquid, and the nanofiltration can remove inorganic salt components in the filtered liquid, thereby effectively improving the proportion of organic selenium and simultaneously ensuring the stability of the plant source selenocysteine/selenocysteine components.

Drawings

FIG. 1 is a chromatogram of a selenium form of a mixed standard solution;

FIG. 2 is a graph showing the effect of complex enzyme enzymolysis time on the stability of a selenium form standard.

Detailed Description

The present invention will be further described with reference to the following examples.

Example 1

The preparation of the selenium-rich product with high content of seleno-amino acid components comprises the following steps:

weighing 10kg of 60-mesh cardamine violifolia dry powder (the total selenium content is 1758mg/kg), and mixing the powder according to a material-liquid ratio of 1:10, uniformly mixing with water, setting the water temperature to be 55 ℃, adjusting the pH to 8.5 by using 1mol/L sodium hydroxide solution, stirring at the speed of 150-200 r/min, setting the ultrasonic power to be 200W, carrying out ultrasonic treatment for 10min, adding 400g of alkaline protease, 200g of protease E and 200g of trypsin, and carrying out enzymolysis reaction for 1 h.

And after the enzymolysis reaction is finished, quickly heating to 85 ℃ for 10min to inactivate enzyme, then quickly cooling to 20-30 ℃, then centrifuging for 3min by adopting a high-speed low-temperature (4 ℃) centrifuge at 5000r/min, and taking supernatant.

Adding 1.5 wt% of 200 mesh food grade active carbon into the supernatant for decolorization, stirring at 150r/min at 55 ℃ for 30min, passing the decolorized solution through a 300 mesh filter paper board uniformly coated with food grade diatomite, and taking the filtered clarified solution.

And (3) circularly desalting and concentrating the filtered and clarified liquid through a nanofiltration membrane of 250Da, and then concentrating and concentrating in vacuum of-0.06 to-0.08 MPa until the solid content is 25 to 30 percent. And finally, drying by using a spray dryer, wherein the working parameters of the spray dryer are as follows: the air inlet temperature is 140-170 ℃, the air outlet temperature is 100 ℃, and the feeding speed is controlled at 5.5-7L/h. The dried product is the plant source organic selenium-rich component extract.

2.82 kg of extract is obtained in total, the mass yield is 28.2%, and the characteristic component detection data are shown in the following table:

composition (I)	Content (wt.)
		Extract component proteins	45％
The total selenium content of the extract	2040mg/kg
		The extract component selenocysteine accounts for	40.5％
The extract component is inorganic selenium	3.9％

Example 2

The preparation of the selenium-rich product with high content of seleno-amino acid components comprises the following steps:

the difference from example 1 is that the enzymatic reaction time is 4 hours, and the rest is the same as example 1.

2.7 kg of extract is obtained in total, the mass yield is 27%, and the detection data of characteristic components are as follows:

composition (I)	Content (wt.)
		Extract component proteins	40.5％
The extract component contains total selenium	1990mg/kg
		The extract component selenocysteine accounts for	28.8％
The extract component is inorganic selenium	4.2％

Example 3

The preparation of the selenium-rich product with high content of seleno-amino acid components comprises the following steps:

the difference from example 1 is that 300g of alkaline protease, 200g of trypsin and 200g of protease E were added, and the rest was the same as example 1.

2.55 kg of extract is obtained altogether, the mass yield is 25.5%, and the characteristic component detection data are as follows:

composition (I)	Content (wt.)
		Extract component proteins	39.8％
The extract component contains total selenium	1966mg/kg
		The extract component selenocysteine accounts for	37.6％
The extract component is inorganic selenium	3.5％

Comparative example 1

The difference from the example 1 is that protease K is used for replacing protease E, the composite enzymolysis reaction time is 4 hours, and the rest is the same as the example 1;

2.45 kg of extract is obtained altogether, the mass yield is 24.5%, and the characteristic component detection data are as follows:

composition (I)	Content (wt.)
		Extract component proteins	38.7％
The extract component contains total selenium	1956mg/kg
		The extract component selenocysteine accounts for	9.6％
The extract component is inorganic selenium	11.5％

Comparative example 2

The method is different from the embodiment 1 in that only ultrasonic-assisted extraction is used, the ultrasonic time is increased to 1h, complex enzyme enzymolysis is not used, and other steps are the same as the embodiment 1.

1.12 kg of extract is obtained altogether, the mass yield is 11.2%, and the characteristic component detection data are as follows:

composition (I)	Content (wt.)
		Extract component proteins	15％
The total selenium content of the extract	1740mg/kg
		The extract component selenocysteine accounts for	10.5％
The extract component is inorganic selenium	4.9％

Comparative example 3

The difference from example 1 is that only a single 4 wt% alkaline protease was used and the other steps were the same as in example 1.

2.41 kg of plant organic selenium extract is obtained, the mass yield is 24.1%, and the characteristic component detection data are as follows:

composition (I)	Content (wt.)
		Extract component proteins	38％
The total selenium content of the extract	1789mg/kg
		The extract component selenocysteine accounts for	20.5％
The extract component is inorganic selenium	9.7％

Comparative example 4

The difference from example 1 is that ultrasound-assisted extraction was not used, and the other steps were the same as in example 1.

1.15 kg of plant organic selenium extract is obtained, the mass yield is 11.5%, and the characteristic component detection data are as follows:

comparative example 5

The difference from example 1 is that proteinase K is used instead of alkaline protease, and the other steps are the same as example 1.

2.35 kg of plant organic selenium extract is obtained, the mass yield is 23.5%, and the characteristic component detection data are as follows:

composition (I)	Content (wt.)
		Extract component proteins	35％
The total selenium content of the extract	1653mg/kg
		The extract component selenocysteine accounts for	18.5％
The extract component is inorganic selenium	9.8％

In the above examples and comparative examples, the total selenium content of cardamine violifolia powder and the total selenium content of the extract components were determined according to the determination standard of selenium in food GB 5009.93-2017, and the content of protein in the extract components was determined by the method of protein determination in food GB 5009.5-2016; the proportion of inorganic selenium and selenocysteine in the extract is carried out by T/CHC 1001-2019 appendix A of plant-derived high organic selenium food raw materials, and specifically comprises the following steps: dissolving a small amount of extract powder in water, detecting by HPLC-AFS to obtain chromatogram of various forms of selenium, and directly calculating the proportion according to the chromatogram.

It can be known from the examples and comparative examples that the content of protein and the ratio of selenocysteine in the extract can be obviously improved through the synergy of ultrasonic extraction and complex enzyme enzymolysis. Compared with single enzyme, the compound protease can improve the content of protein in the extracted components, and can also improve the content of selenocysteine/selenocysteine by enzyme digestion (the selenocysteine is proved by documents to be unstable and is easy to be converted into the selenocysteine, so the compound protease is calculated by the selenocysteine in the invention); and the selenocysteine/selenocysteine has higher stability in the compound enzyme of the invention, and can obtain the selenocysteine/selenocysteine with higher content.

Because the enzyme cutting sites of different enzymes are different, the products obtained by enzymolysis are not used, as shown in a comparative example 1 and a comparative example 5, after the types of the enzymes in the compound enzyme are changed, the extraction rate of the protein is changed, and more importantly, the proportion of the selenocysteine in the products is obviously changed.

In addition, experiments find that the compound enzyme has too long action time, which can cause the reduction of the content of the selenocysteine/selenocysteine, and the concrete steps are as follows:

selenium standard solutions containing various forms of selenium and different concentrations are prepared and detected by HPLC-AFS under the detection conditions (in the standard solution chromatogram shown in FIG. 1, the concentrations of SeCys2, MeSeCys, Se (IV) and Se (VI) are 100ng/ml, and the concentration of SeMet is 200 ng/ml). Adding complex enzyme (the mass ratio of the alkaline protease to the trypsin to the protease E is 2:1:1) into the standard solution, wherein FIG. 2 is a trend graph of the content change of five standard substances along with the increase of enzymolysis time in a complex enzyme system, and the trend graph shows that: the decrease in the amount of selenocysteine with increasing time of the combined enzymatic hydrolysis, i.e. the decrease in stability with increasing time of the enzymatic hydrolysis, is consistent with the results of example 2. The reason is presumed to be: the long-time enzymatic reaction of the compound enzyme causes the form of the selenocysteine to be transformed.

It should be noted that, for comparison, the samples used in the examples and comparative examples are all cardamine violifolia powder, but the method of the present invention is also applicable to other cruciferous plants, such as broccoli, cabbage, etc.

In the extraction method, the selenium-containing protein in the plant can be separated out more efficiently after the sample is subjected to enzymolysis, and meanwhile, the selenium-containing protein is subjected to enzymolysis to form free selenium amino acid which is very stable under the enzymolysis condition, so that the enzymolysis liquid can be directly taken, centrifuged to take the supernatant, diluted, filtered by a filter membrane and the like, and then the supernatant is directly detected, and the real-time monitoring of the enzymolysis process is realized.

It can be understood that the method of ultrasonic extraction and complex enzyme enzymolysis provided by the invention can also be directly used for detecting the content of various selenium forms in a sample, and the following steps can be specifically referred to:

1) mixing the selenium-rich plant powder with water (adjusting the pH to 8.5-9.5), or directly mixing with a Tris-HCl buffer solution (the pH is also 8.5-9.5), and then carrying out ultrasonic treatment for 10-30 min;

2) adding a complex enzyme consisting of alkaline protease, trypsin and protease E for constant-temperature shaking enzymolysis, wherein the mass ratio of the alkaline protease to the trypsin to the protease E is 1-2: 1: 1;

3) transferring the enzymolysis solution to a centrifuge tube, centrifuging at 4000rpm for 30min, sucking supernatant, diluting to a proper time, and filtering through a 0.22 mu m filter membrane.

4) The filtrate was checked by HPLC-AFS.

The technical solution of the present invention is illustrated by the above embodiments, but is not limited to the above embodiments; it will be understood by those skilled in the art that any modification of the present invention, equivalent substitutions for the raw materials of the products of the present invention and the addition of auxiliary components, selection of specific modes, etc., are within the scope of the protection and disclosure of the present invention.