A method for extracting and determining selenoprotein from selenium-rich lentinula edodes mycelium

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to a method for extracting and determining selenoprotein from selenium-rich lycoperdon pygmaeum mycelium, wherein the lycoperdon pygmaeum mycelium fermented by a 20L biological culture tank is dried in a vacuum drying box, ground into standard powder through a 100-mesh sieve, 0.5g of the standard powder is weighed, a 0.05mol / L NaOH solution is added, ultrasonic-assisted extraction is performed, then centrifugation is performed at a speed of 3000r / min for 15min, the supernatant is adjusted to an isoelectric point, centrifugation is performed again to obtain a protein precipitate, and finally the protein precipitate is dried in a freeze dryer to obtain a protein sample. The present application is dedicated to using the selenium-rich lycoperdon pygmaeum mycelium as raw material, adopting ultrasonic extraction, optimally optimizing the extraction process of selenoprotein, quantitatively analyzing the selenium content in the protein, detecting the change of the type and quantity of amino acids before and after selenium enrichment through a thin layer chromatography test, and understanding the influence of selenium on the amino acids in the lycoperdon pygmaeum mycelium.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a method for extracting and determining selenium protein from selenium-rich Lyophyllum decastes mycelium. BACKGROUND

[0002] Selenium is an essential trace mineral element for living organisms, and plays an extremely important role in human health. China is a large selenium-deficient country, and the main source of selenium for the human body is through food intake. However, it is difficult for people to intake selenium from local natural food to achieve the purpose of selenium supplementation. In recent years, people have improved the selenium content in food through artificial exogenous selenium cultivation technology. Edible fungi have the ability to absorb selenium from the growth substrate, and through the metabolism of mycelium, inorganic selenium is converted into organic selenium, which mainly exists in the form of selenium protein, selenium polysaccharide, selenium nucleic acid, selenium-containing polypeptide, selenium flavone, etc., among which selenium-containing protein is the main one. After selenium-enriched edible fungi, their respective functions are improved, and they can be better absorbed by the human body, and can be used to produce new food supplements with high selenium.

[0003] Lyophyllum decastes, also known as fragrant pine mushroom or delicious jade mushroom, is a kind of rare edible fungus with good market prospect in the market, and is also a kind of medicinal fungus. Studies have shown that the balance of protein in Lyophyllum decastes mycelium is better than that of fruiting body, and the nutritional value is higher than that of fruiting body, and it has high production and edible value. Lyophyllum decastes also has strong selenium-enriching ability, and the selenium-enriched mycelium can greatly improve its nutritional value and application range. In recent years, with the change of selenium research direction from total selenium to organic selenium, selenium protein has become one of the focuses of people's research. Therefore, it is of great significance to study the extraction process of selenium protein for further study of the structure and functional factors of selenium protein. Amino acids are the basic building blocks of proteins, and they are closely related to life and various forms of life activities. Every cell and all important components in the body are involved in protein. In theory, the closer the content and proportion of essential amino acids in food to the human body's essential amino acid pattern, the higher the quality of the protein composed.

[0004] At present, there are few reports on selenium protein of Lyophyllum decastes at home and abroad. SUMMARY

[0005] The purpose of the present application is to provide a method for extracting selenium protein from selenium-rich Lyophyllum decastes mycelium, and also to provide a method for accurately determining the selenium content in selenium protein.

[0006] In order to achieve the above purpose, the technical scheme provided by the present application is as follows:

[0007] A method for extracting and determining selenium protein from selenium-rich Lyophyllum decastes mycelium, characterized in that it comprises the following steps:

[0008] Step 1: Selenium-rich Lyophyllum decastes is obtained by selenium-rich fermentation culture of Lyophyllum decastes in a 20L bioreactor;

[0009] Step 2: The selenium-rich Lyophyllum decastes mycelium obtained in Step 1 is dried in a vacuum drying oven;

[0010] Step 3: The dried selenium-rich Lyophyllum decastes mycelium obtained in Step 2 is ground and sieved through a 100-mesh sieve to obtain standard powder;

[0011] Step 4: 0.5g of the standard powder prepared in Step 3 is weighed and added to a 0.05mol / L NaOH solution, and ultrasonic extraction is performed 1-4 times under the conditions of extraction time 20-60min, solid-liquid ratio 1:100-1:400, and extraction temperature 40-80℃;

[0012] Step 5: After extraction in Step 4, centrifugation is performed at 3000r / min for 15min to obtain supernatant;

[0013] Step 6: The pH value of the supernatant obtained in Step 5 is adjusted to the isoelectric point, and the selenium-containing protein precipitate is obtained by centrifugation again;

[0014] Step 7: The selenium-containing protein precipitate obtained in Step 6 is dried in a freeze dryer to obtain a protein sample;

[0015] Step 8: A standard curve of selenium content versus absorbance is drawn, and the selenium content and the selenium content in the protein per gram of mycelium are calculated according to the selenium standard curve;

[0016] Step 9: 0.1g of the selenium-containing protein extracted in Step 7 is placed in a hydrolysis tube, 10-15mL of 6mol / L hydrochloric acid is added, the hydrolysis tube is tightly sealed and placed in a constant temperature drying oven at (110±1)℃ for hydrolysis for 22h, and then cooled;

[0017] Step 10: The hydrolysis tube is opened after cooling in Step 9, the hydrolysis solution is transferred into a 25mL volumetric flask, and the volume is adjusted to the mark with deionized water. 1mL of the filtrate is placed in a 25mL beaker and dried in a vacuum drying oven at 40-50℃. If there is residue, dissolve it with 1-2mL of deionized water and dry again. Add 5mL of 0.02mol / L hydrochloric acid solution to dissolve as the test solution;

[0018] Step 11: The test solution obtained in Step 10 is determined by thin layer chromatography (TLC) to qualitatively analyze the amino acids in the selenium-rich Lyophyllum decastes mycelium protein;

[0019] Step 12: The amino acids in the test solution obtained in Step 10 are quantitatively analyzed, and the specific steps are as follows:

[0020] The standard samples with large mobility value difference were mixed together, 10 μL of the mixed standard sample was taken by a micro-injection device, 20 μL of the hydrolysate of the protein in the selenium-rich Laetiporus and the hydrolysate of the protein in the Laetiporus without selenium enrichment were taken by a micro-injection device, and the samples were spotted in batches and dried by alternately blowing cold and hot air each time. After the chromatography, the content of the amino acid was calculated according to the area ratio.

[0021] Preferably, in step 4, the extraction time is 60 min, the solid-liquid ratio is 1:200 g / mL, the extraction temperature is 60°C, and the extraction times are 2.

[0022] Preferably, in step 6, the pH value is 3.5.

[0023] Compared with the prior art, the present application has the following advantages: the present application is committed to taking the selenium-rich Laetiporus mycelium as a raw material, adopting ultrasonic extraction, optimally optimizing the extraction process of the selenium protein, quantitatively analyzing the content of selenium in the protein, detecting the changes in the types and quantities of the amino acids before and after the selenium enrichment through a thin layer chromatography test, understanding the influence of selenium on the amino acids in the Laetiporus mycelium, providing certain theoretical basis for the research on the influence of the nutritional value of the selenium-rich edible fungi, providing theoretical basis for the development and utilization of the selenium-rich Laetiporus mycelium, and having practical significance and important prospects. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly understand the present application, the present disclosure is further introduced by combining the drawings in the specification and the illustrative examples, and the drawings and examples are used to explain and illustrate, but do not constitute a limitation on the disclosure.

[0025] Figure 1 It is a graph for the determination of the isoelectric point of the protein in Example 1 of the present application;

[0026] Figure 2 It is a graph for the influence of the extraction time on the extraction amount of the selenium protein in Example 2 of the present application;

[0027] Figure 3 It is a graph for the influence of the solid-liquid ratio on the extraction amount of the selenium protein in Example 2 of the present application;

[0028] Figure 4 It is a graph for the influence of the temperature on the extraction amount of the selenium protein in Example 2 of the present application;

[0029] Figure 5 It is a graph for the influence of the extraction times on the extraction amount of the selenium protein in Example 2 of the present application;

[0030] Figure 6 It is a response surface graph for the interaction of various factors on the extraction rate of the Laetiporus mycelium protein in Example 3 of the present application;

[0031] Figure 7One-dimensional thin layer chromatogram of standard amino acids, selenium-enriched mycelium and non-selenium-enriched mycelium hydrolysate in Example 5 of the present application;

[0032] Figure 8 Two-dimensional thin layer chromatogram of standard amino acids, non-selenium-enriched mycelium (BL) and selenium-enriched mycelium (Sa) protein hydrolysate in Example 5 of the present application;

[0033] Figure 9 Quantitative one-dimensional thin layer chromatogram of mixed standard amino acids, selenium-enriched and non-selenium-enriched hydrolysate in Example 6 of the present application. DETAILED DESCRIPTION

[0034] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some but not all of the embodiments of the present application. The components of the embodiments of the present application described and shown in the drawings herein can be arranged and designed in various different configurations.

[0035] Therefore, the following detailed description of the embodiments of the present application provided in the drawings is not intended to limit the scope of the claimed present application, but only represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application.

[0036] Example 1 Determination of isoelectric point of selenium-containing protein

[0037] As shown in Figure 1 The content of protein in the extract solution presents a concave shape with the increase of pH value. The content of protein decreases sharply with the increase of pH value from 2.5 to 3.5, and then starts to increase slowly after 3.5, and rapidly increases after 4.5, and reaches the maximum value of 0.2233 mg / mL at 5.0. It is shown that the solubility of protein in the supernatant is lower at pH value of 3.5-4.5, and the content of protein is higher outside this range. The residual concentration of protein in the supernatant is lower within the range of pH value of 3.5-4.5, and the charge of protein is zero at 3.5, and the solubility is the lowest. Therefore, pH 3.5 is selected as the optimal extraction pH value.

[0038] Example 2 Selection of selenium-containing protein extraction conditions

[0039] Accurately weigh 0.5000g sample, select different extraction time (20, 40, 60 min and 80 min), different liquid ratio (1:100, 1:200, 1:300 and 1:400), different temperature (40, 50, 60, 70 ℃ and 80 ℃) and different extraction times (1, 2, 3 times and 4 times) single factor, study the influence of these single factors on the extraction rate of selenoprotein.

[0040] 1, the influence of different extraction time on the extraction amount of selenoprotein

[0041] As Figure 2 shown, with the increase of extraction time, the content of extracted protein increases, when the extraction time is 60 min, it reaches the maximum, which is 1.08 mg / mL, after the extraction time exceeds 60 min, with the extension of extraction time, the protein content presents a slow downward trend, when the extraction time is 100 min, the protein content increases slowly with the increase of extraction time. It may be that after the increase of extraction time, the easily soluble protein has been dissolved, resulting in the decrease of content, which is similar to the result of extracting protein in the cap of Coprinus comatus by Su Yuchun. Therefore, 60 min is selected as the best extraction time.

[0042] 2, the influence of different liquid ratio on the extraction of selenoprotein

[0043] As Figure 3 shown, with the increase of liquid ratio, the content of extracted protein also increases, when the liquid ratio is 1:200, it reaches 0.625 mg / mL, then with the increase of liquid ratio, the content of protein does not increase obviously. It may be because with the increase of solvent, the concentration difference in solid-liquid phase increases, the dissolution rate of solute increases, so that the protein yield increases. However, if the liquid ratio is too large, it will cause burden to the subsequent concentration step, considering the use amount of solvent in the process and reducing the production cost, the liquid ratio is determined as 1:200 g / mL.

[0044] 3, the influence of different temperature on the extraction of selenoprotein

[0045] As Figure 4 shown, with the increase of extraction temperature, the content of protein also increases, the higher the temperature, the better the alkali extraction effect. But when the temperature exceeds 60 ℃, the trend of protein content change with temperature decreases, and there are documents reported that the denaturation temperature of protein is 70-80 ℃, so the actual extraction temperature is determined as 60 ℃. This is similar to the extraction temperature determined by Tian Minjue and Li Hao in the study of extraction process of selenoprotein in selenium-rich Hericium erinaceus.

[0046] 4, the influence of different extraction times on the extraction amount of protein

[0047] As Figure 5As shown in the extraction of protein, the more the extraction times, the less the protein content remaining in the residue, the residue after 2 times of alkali extraction, the protein content detection value is negative, which shows that 2 times of alkali extraction can effectively extract the protein, and the extraction times is selected as 2 times in the extraction process.

[0048] Example 3 Response surface test of selenoprotein extraction process

[0049] According to the single factor test results, the Design-expert 8.0 software system is used, the Box-Behnken response surface design method is used to select the four factors affecting the protein content, i.e. extraction temperature, extraction time, solid-liquid ratio and extraction times, which are represented by A, B, C and D respectively, and a 4-factor 3-level test is designed, and the factor level table is shown in Table 1.

[0050] Table 1 Factors and levels of response surface analysis

[0051]

[0052] According to the central composite design principle of Box-Behnken, three levels of four factors are selected in the test, and a response surface analysis test of 29 test points of four factors and three levels is designed, and the response surface test results are shown in Table 2. The data in Table 2 is subjected to multiple regression analysis by Design-Expert statistical analysis software, and the main analysis results are shown in Table 3. According to the variance analysis, the effects of the first and second terms of the equation are significant, and the loss of fit term is not significant (P>0.05), which indicates that the equation fitting is good and the model is stable.

[0053] Table 2 Response surface test design and results

[0054]

[0055] Table 3 Variance analysis results of response surface experiment

[0056]

[0057]

[0058] Note: * indicates P<0.05 reaches the significant difference level; ** indicates P<0.01 reaches the extremely significant difference level

[0059] The experimental results are analyzed by using software Design Expert 8.0 to obtain a multiple quadratic regression equation: Y1=-1029.65035+13.35583A+14.36027B+0.99689C+130.50677D-0.013646AB-5.29493×10 - 3AC-0.16322AD-2.23932×10 -3 BC-0.32921BD-0.061462CD-0.086737A 2 -0.10292B 2 -9.59921×10 -4 C 2 -18.98566D 2

[0060] In the formula, Y1 represents the protein extraction rate, and A, B, C, and D are the encoded values ​​of the four variables mentioned above. The coefficient of determination R0 2 =0.9291, indicating a good regression fit. P < 0.0001, indicating the equation is highly significant. The values ​​of A, D, and B in the model are... 2 C 2 D 2 Reaching a highly significant level, C, CD, A 2 The effect of F is significant, while the effects of B, AB, AC, AD, and BD are not significant. Based on the F value, the main factors affecting protein extraction rate are, in descending order: D > A > C > B, i.e., extraction times > extraction temperature > solid-liquid ratio > extraction time.

[0061] Using Design Expert 8.0 software, response surface methodology and corresponding contour plots can be obtained for the four factors. The shape of the contour lines reflects the strength of the interaction effect; ellipses indicate a significant interaction between the two factors, while circles indicate the opposite. The steeper the slope of the response surface, the greater the impact of changes in the factors on the response value. Figure 6 As shown, the effects of various factors on the protein extraction rate of lotus leaf pleated umbrella buds are reflected. Comparing the six sets of graphs, it can be seen that: the number of extractions has a greater impact on the protein extraction rate, with a steeper curve; extraction temperature and material-to-liquid ratio have the next greatest impact; extraction time has the least impact on the protein extraction rate, with a relatively gentle curve change, which is consistent with the results of the analysis of variance.

[0062] Example 4 Verification Test

[0063] 1. Data analysis using Design-Expert 8.0 statistical analysis software revealed the optimal extraction conditions for selenoprotein from *Agaricus peltata* mycelium: extraction temperature 64.05℃, extraction time 59.63 min, solid-liquid ratio 1:198.62 g / mL, and 2.32 extractions. Theoretically, the protein extraction rate was 76.90%. Considering experimental feasibility, the optimal conditions were adjusted to an extraction temperature of 64℃, extraction time of 60 min, solid-liquid ratio of 1:200 g / mL, and 2 extractions. Under these conditions, selenoprotein was extracted from *Agaricus peltata* mycelium. Through three parallel experiments, the protein extraction rate was measured to be 75.13%, with a relative error of 2.36%, further validating the correctness of the regression model.

[0064] A standard curve was plotted on absorbance versus selenium content, yielding Y = 0.0151X - 0.0014, and a correlation coefficient R. 2 =0.9934, the accuracy meets the requirements.

[0065] 2. Selenium content determination method: The selenium content is determined according to existing methods. Selenium standard solution: Accurately weigh 0.10 g of selenium standard and dissolve it in 2 mL of nitric acid. Heat at low temperature until dry, dissolve in 1:1 hydrochloric acid, and make up to 1 L. Dilute to 1 μg / mL before use.

[0066] 3. Protein content was determined according to the biuret method.

[0067] Biuret reagent: Weigh 1.5g CuSO4 and 6.0g potassium sodium tartrate, dissolve them in 500mL of water, slowly add 300mL of 10% NaOH solution while stirring, and dilute with water to 1000mL.

[0068] Calculations based on the selenium standard curve showed that the selenium content per gram of mycelium was 0.062 mg. Under the same conditions, the protein content in non-selenium-enriched mycelium was 52.11%, and the selenium content was not detectable.

[0069] Example 5: Qualitative analysis of amino acids in selenium-enriched lotus leaf agaric mycelium proteins

[0070] 1. Single-phase thin-layer chromatography analysis of amino acids in proteins before and after selenium enrichment

[0071] like Figure 7 As shown in the figure: Y is tyrosine, D is aspartic acid, L is leucine, Sa is a selenium-enriched sample, BL is a non-selenium-enriched sample, C is cysteine, A is alanine, and F is phenylalanine.

[0072] Five colored spots were observed in the hydrolysates of *Agaricus peltata* mycelium samples before and after selenium enrichment, but their sizes differed, indicating different amino acid contents. Color spots with Rf values ​​close to those of the standard amino acids included aspartic acid, tyrosine, alanine, phenylalanine, and leucine. Both selenium-enriched and non-selenium-enriched *Agaricus peltata* mycelium hydrolysates showed adjacent colored spots after chromatography, indicating the presence of other amino acids not indicated in the standard, in addition to the aforementioned amino acids. To further understand the amino acid composition of the protein hydrolysates from selenium-enriched and non-selenium-enriched *Agaricus peltata* mycelium, two-dimensional thin-layer chromatography was performed.

[0073] 2. Two-dimensional chromatography analysis of amino acid types in selenium-enriched and non-selenium-enriched lotus leaf pleated capillaries

[0074] like Figure 8As shown, six amino acid standards were mixed and subjected to two-dimensional chromatography, while hydrolysates of selenium-enriched and non-selenium-enriched lotus leaf agaric mycelium were also subjected to two-dimensional chromatography.

[0075] Thirteen colored spots were observed in the hydrolysis products of selenium-enriched *Amanita muscaria* mycelium and the blank hydrolysis products of non-selenium-enriched *Amanita muscaria* mycelium. However, the size and appearance of some colored spots were different. Figure 8 The difference in specific color development location is determined by measuring the Rf value at each point.

[0076] Table 4. Mobility of standard amino acids, unenriched (BL), and selenium-enriched mycelial protein hydrolysate (Sa) by two-dimensional chromatography.

[0077]

[0078]

[0079] Six colorimetric spots in the standard amino acid sample corresponded to positions in the protein hydrolysate of both selenium-enriched and non-selenium-enriched *Agaricus nitidum* mycelium. However, there were additional colorimetric spots for seven amino acids (a1, a2, a3, a4, a5, a6, and a7) that did not correspond to the positions in the standard sample. Among these, amino acids a3 (0.31, 0.21), a4 (0.37, 0.31), and a6 (0.42, 0.21) were present in the selenium-enriched *Agaricus nitidum* mycelium sample. The migration rates of amino acids a3 (0.39, 0.14), a4 (0.44, 0.24), and a6 (0.45, 0.34) in the selenium-enriched and non-selenium-enriched *Agaricus nitidissima* mycelium (55) differed significantly. a3, a4, and a6 should be different amino acids in the selenium-enriched and non-selenium-enriched *Agaricus nitidissima* mycelium. Therefore, the types of amino acids constituting proteins in *Agaricus nitidissima* mycelium changed after selenium enrichment, as shown in Table 4.

[0080] Example 6 Quantitative Analysis

[0081] like Figure 9 As shown in the figure: Sa is a selenium-enriched mycelium sample; BL is a non-selenium-enriched mycelium sample.

[0082] Standard amino acids with significantly different migration rates (leucine, alanine, and aspartic acid) were mixed to form Standard 1, and (phenylalanine and tyrosine) were mixed to form Standard 2. These Standard 1 and Standard 2 were then subjected to one-dimensional chromatography with hydrolysates of selenium-enriched and non-selenium-enriched *Amanita muscaria* mycelium to roughly determine the differences in the content of various amino acids.

[0083] Table 5. Amino acid content in selenium-enriched and non-selenium-enriched *Agaricus bisporus* mycelium.

[0084]

[0085] The color developing points of mixed standard 1 from top to bottom are leucine, alanine, aspartic acid, and the color developing points of mixed standard 2 from top to bottom are phenylalanine, tyrosine. The points corresponding to the amino acids in the standard sample appear in the mycelium of L. deliciosus enriched with selenium and the mycelium of L. deliciosus not enriched with selenium, and the content of the amino acids is determined according to the ratio of the area size of the two, wherein the content of the color developing point close to aspartic acid Rf value is 2.4 μg / μL and 2.0 μg / μL respectively, the content of the color developing point close to tyrosine Rf value is 3.8 μg / μL and 3.3 μg / μL respectively, the content of the color developing point close to alanine Rf value is 2.5 μg / μL and 2.2 μg / μL respectively, the content of the color developing point close to phenylalanine Rf value is 1.0 μg / μL and 1.0 μg / μL respectively, and the content of the color developing point close to leucine Rf value is 1.2 μg / μL and 1.1 μg / μL respectively (see Table 5). It can be known from the above two-way thin layer chromatography that the amino acids at the positions of the color developing points are not single amino acids but mixtures of several amino acids. In the experiment, the content of single amino acid is used to replace the content of mixed amino acid due to the limitation of experimental conditions, and the change in the amount of amino acids in the protein in the mycelium enriched with selenium and the mycelium not enriched with selenium is approximately determined. According to the above experimental results, compared with the mycelium of L. deliciosus not enriched with selenium, the content of the mixed amino acids at the color developing position of phenylalanine does not change, the content of the mixed amino acids at the color developing position of aspartic acid increases by 0.4 μg / μL, the content of the mixed amino acids at the color developing position of tyrosine increases by 0.5 μg / μL, the content of the mixed amino acids at the color developing position of alanine increases by 0.3 μg / μL, and the content of the mixed amino acids at the color developing position of leucine increases by 0.1 μg / μL. In summary, the total content of amino acids in the mycelium of L. deliciosus enriched with selenium increases. To accurately measure the change in the amount of various amino acids before and after the enrichment of selenium, the determination of the content of amino acids is required.

[0086] Example 7 Conclusion

[0087] Through the optimization experiment, the process conditions for extracting selenium protein from the mycelium of L. deliciosus are determined as follows: time 60 min, temperature 60℃, solid-liquid ratio 1:200 g / mL, and extraction times 2, and the theoretical protein extraction rate is 76.90%. This is different from the research on selenium-enriched edible fungi by Wang Lianfang, Dou Chunxia, etc. in that the protein yield is 30.17%, which indicates that the protein extraction rate in different fungi will be different. The content of selenium in the mycelium protein is determined by 3,3'-diaminobenzidine spectrophotometry to be 0.062 mg / g, which indicates that selenium is distributed in the protein in the mycelium of L. deliciosus.

[0088] The results of one-way chromatography and two-way chromatography experiments show that there are 13 kinds of amino acids in the hydrolysate of the selenium-rich L. deliciosum mycelium and the L. deliciosum mycelium without selenium enrichment, 6 of which have similar mobility to the standard amino acids aspartic acid, tyrosine, alanine, phenylalanine, leucine and cysteine, and the other 7 are unknown amino acids, wherein the mobility values of amino acids a3 (0.31, 0.21), a4 (0.37, 0.31), a6 (0.42, 0.55) in the selenium-rich L. deliciosum mycelium and amino acids a3 (0.39, 0.14), a4 (0.44, 0.24), a6 (0.45, 0.34) in the L. deliciosum mycelium without selenium enrichment are different, so the types of amino acids in the selenium-rich L. deliciosum mycelium are changed compared with those in the L. deliciosum mycelium without selenium enrichment. Through preliminary quantitative analysis, the total content of amino acids in the selenium-rich L. deliciosum mycelium is increased.

[0089] The mathematical model between the factors and the response values is established by the response surface analysis method, the interaction between different factors can be directly observed, the test times can be reduced and the efficiency can be improved by adjusting the factors, the method has wide application value in actual production and can improve the protein extraction rate. The selenium-rich culture is carried out by adding Na2SeO3 into the L. deliciosum mycelium fermentation liquor, the content of the protein in the mycelium is promoted, the selenium element necessary for human life activities is increased, the types of amino acids are changed, and the content of the amino acids is also increased, which provides a reliable theoretical basis for the further development and utilization of the selenium-rich L. deliciosum mycelium, the selenium-rich fermentation improves the edible value of the L. deliciosum mycelium, meanwhile, the additional value of the L. deliciosum can be improved, and the method has wide application prospect.

[0090] It should be noted that similar reference numerals and letters refer to like items in the drawings and thus, once an item is defined in one drawing, it is not necessary to further define and explain it in a following drawing.

[0091] It should be understood by those skilled in the art that the present application is not limited by the above examples, the above examples and the description in the specification are only to illustrate the principles of the present application, and various changes and improvements can be made without departing from the spirit and scope of the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for extracting and determining selenoprotein from the mycelium of the selenium-rich leaf of Lycoperdon sp. characterized by, It comprises the following steps: Step 1: selenium-rich fermentation culture of Lyophyllum decastes is carried out by using a 20L bioreactor to obtain selenium-rich Lyophyllum decastes mycelium; Step 2: the selenium-rich Lyophyllum decastes mycelium obtained in step 1 is dried in a vacuum drying oven; Step 3: the dried selenium-rich Lyophyllum decastes mycelium obtained in step 2 is ground and sieved to obtain 100-mesh standard powder; Step 4: 0.5 g of the standard powder prepared in step 3 is weighed and added into a 0.05 mol / L NaOH solution, and ultrasonic extraction is carried out 1-4 times under the conditions of extraction time of 20-60 min, solid-liquid ratio of 1:100-1:400 and extraction temperature of 40-80℃; Step 5: after extraction in step 4, centrifugation is carried out at a speed of 3000 r / min for 15 min to obtain supernatant; Step 6: the pH value of the supernatant obtained in step 5 is adjusted to the isoelectric point, and the pH value is 3.5, and the selenium-containing protein precipitate is obtained by centrifugation again; Step 7: the selenium-containing protein precipitate obtained in step 6 is dried in a freeze dryer to obtain a selenium-containing protein sample; Step 8: a standard curve of selenium content is drawn according to the absorbance, and the selenium content and protein content per gram of selenium-rich mycelium are calculated according to the selenium standard curve; Step 9: 0.1 g of the protein extracted in step 7 is weighed and placed in a hydrolysis tube, 10-15 mL of 6 mol / L hydrochloric acid is added, the hydrolysis tube is tightly sealed and placed in a constant temperature drying oven at 110±1℃ for hydrolysis for 22 h, and then taken out and cooled; Step 10: the hydrolysis tube is opened after cooling in step 9, the hydrolysis solution is transferred into a 25 mL volumetric flask, and the volume is adjusted to the mark with deionized water, 1 mL of the filtrate is taken into a 25 mL beaker, and the sample is dried in a vacuum drying oven at 40-50℃, if there is residue, it is dissolved with 1-2 mL of deionized water, and then dried, 5 mL of 0.02 mol / L hydrochloric acid solution is added for dissolution as the test solution; Step 11: the test solution obtained in step 10 is determined by thin layer chromatography method to qualitatively analyze the amino acids in the selenium-rich Lyophyllum decastes mycelium protein; Step 12: the amino acids in the test solution obtained in step 10 are quantitatively analyzed, and the specific steps are as follows: The samples with large migration value difference are mixed together, 10µL of the mixed sample is taken by a micro-sampler, 20µL of the protein hydrolysate extracted from the selenium-rich Lyophyllum decastes mycelium and the protein hydrolysate extracted from the Lyophyllum decastes mycelium without selenium enrichment are spotted in batches, and each time the sample is dried by alternately blowing cold and hot air, and the content of amino acids is calculated according to the area ratio after chromatography.

2. The method according to claim 1, wherein the method is characterized in that, In step 4, the extraction time is 60 min, the solid-liquid ratio is 1:200 g / mL, the extraction temperature is 60℃, and the extraction times is 2.

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