A method for extracting selenoprotein from Cucurbita sphaerocarpa

Abstract

The present invention provides a method for extracting selenoprotein from ground melon, and belongs to the technical field of plant selenoprotein extraction. The present invention mixes ground melon powder with alkali solution, extracts by shaking, and obtains ground melon selenoprotein solution by centrifugation. The present invention uses ground melon fruit as raw material, destroys the secondary structure and hydrogen bonds in the protein molecules by alkali extraction, causes the charge type on its surface to be consistent, accelerates the dissociation between the two, improves the solubility of the protein molecules, and significantly improves the extraction efficiency of ground melon selenoprotein. The extracted selenoprotein content is 5.925 mg / g. The extraction method of the present invention is simple, has low extraction cost, and has broad market promotion value.

Description

Technical Field

[0001] The invention belongs to the technical field of plant selenoprotein extraction, and in particular relates to a method for extracting selenoprotein from Cucurbita sphaerocarpa. Background Art

[0002] Selenium (Selenium, Se) is a necessary nutrient element in animals and a beneficial nutrient element in plants. It is also an essential trace element for the human body. Relevant studies have shown that selenium has important physiological effects on animals and humans. It has a positive effect in many aspects such as anti-cancer and cancer prevention, human immunity regulation, prevention of cardiovascular and cerebrovascular diseases, and anti-aging. At the same time, selenium is also widely used in industrial production. Selenium has a detoxification effect on heavy metals, radiation, and microwaves. Selenium, like other elements, cannot be synthesized by the body, so to ensure the human body's demand for selenium, it is necessary to take in a certain amount of selenium every day. Insufficient selenium intake can cause diseases such as Keshan disease, Kashin-Beck disease, cancer, diabetes, cardiovascular and cerebrovascular diseases, and hypertension syndrome. Although selenium plays an irreplaceable role in the human body, we should also pay attention to selenium poisoning caused by excessive selenium. At present, the common ways to supplement selenium are mainly through inorganic sodium selenite and food intake. Inorganic sodium selenite is a highly toxic substance with high toxicity, and animals have low absorption and utilization rate of inorganic selenium. The safe dietary selenium intake of humans is 50-500μg / d, which is close to the toxic dose (750μg / d). Therefore, using sodium selenite as a direct source of selenium for animals and humans is risky; organic selenium bound to biological macromolecules is safe, has good biological activity, and has a high absorption rate, so the development and research of organic selenium supplement products have received widespread attention. Studies have shown that taking selenium from natural foods through food chain transformation is a safe way to supplement selenium. The main form of selenium in plants is organic selenium, accounting for more than 80% of the total, of which protein selenium is the main one. Selenoproteins refer to a class of proteins that are formed by selenium in the form of selenocysteine ​​(Sec) bound to a peptide chain through amino acid dehydration condensation and exhibit selenium physiological activity.

[0003] Cynanchum thesioides (Freyn) K. Schum and Cynanchum thesioides (Freyn) K. Schum. var. australe (Maxim.) Tsiang et PTLi. are perennial xerophytic upright semishrubs of the genus Cynanchum in the Apocynaceae family. They are a plant that integrates feeding, medicinal, edible and industrial raw materials. Cynanchum thesioides is a variant of Cynanchum thesioides. In recent years, Cynanchum thesioides has attracted more and more attention as a wild vegetable. Cynanchum thesioides has strong drought resistance, disease resistance and environmental adaptability. It can grow in dry valleys, roadsides and desertified land, and can also grow on hillsides at an altitude of 200-2000m. It is a pioneer plant for windbreak, sand fixation and soil and water conservation. The whole plant and fruit of the ground melon can be used as medicine, with the effects of anti-cancer and anti-tumor, improving immunity, invigorating qi, and promoting lactation. It can be used externally to treat warts and genital warts. It has important value in the research and development and utilization of Chinese medicine resources and is a common characteristic plant resource for both food and medicine. The ground melon has a high nutritional value. Its fruit is rich in milk, contains more nutrients and a variety of mineral elements that are beneficial to the human body. It tastes crisp, tender, sweet, and exudes a light milky aroma. And the way to eat it is simple. It can be eaten fresh, cold, or stir-fried. It is mostly eaten as a green wild vegetable by the people and is deeply favored by residents in agricultural and pastoral areas. The ground melon is a natural selenium-rich fruit and vegetable. The selenium content in the dried whole herb sample of the ground melon is much higher than the average selenium content of 104 vegetables in the food composition table.

[0004] At present, the main extraction methods of selenoproteins include column separation technology, biological methods, auxiliary extraction technology, etc. The existing extraction methods are costly, the processing technology is complicated, and there is also the problem of serious loss of selenoproteins. Therefore, the study of a method for efficient extraction of selenoproteins has broad market promotion value. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a method for extracting selenoprotein from Cucurbita oleracea to overcome the problems of high cost, complex treatment process and serious loss of selenoprotein in the prior art.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a method for extracting selenoprotein from Cucurbita chinensis, comprising the following steps: mixing selenoprotein powder with alkali solution, extracting by shaking, and centrifuging to obtain a selenoprotein solution from Cucurbita chinensis.

[0008] Preferably, the preparation method of the ground melon powder comprises the following steps: drying and crushing the ground melon to obtain the ground melon powder.

[0009] Preferably, the drying temperature is 50-70° C., the drying time is 12-18 h, and the mesh size of the ground melon powder after crushing is 60-100 mesh.

[0010] Preferably, the alkali solution is NaOH solution.

[0011] Preferably, the concentration of the alkali solution is 0.05-0.25 mol / L.

[0012] Preferably, the mixing ratio of the ground melon powder and the alkali solution is 1g:20-40mL.

[0013] Preferably, the extraction temperature is 30-50°C.

[0014] Preferably, the extraction time is 1-3 hours.

[0015] Preferably, the oscillation speed is 80-120 r / min.

[0016] Preferably, the centrifugal temperature is 2-6°C, the centrifugal speed is 4000-6000r / min, and the centrifugal time is 10-20min.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The invention uses the fruit of the ground melon as the experimental material, destroys the secondary structure and hydrogen bond in the protein molecule by alkali extraction, causes the charge type on its surface to be consistent, accelerates the dissociation between the two, improves the solubility of the protein molecule, and significantly improves the extraction efficiency of the ground melon selenoprotein, and the extracted selenoprotein content is 5.925 mg / g. The extraction method of the invention is simple, the extraction cost is low, and it has broad market promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Effects of different extraction times by water extraction on the content of soluble selenoprotein in the fruit of Glechoma longituba;

[0020] Figure 2 Effects of different extraction temperatures on the content of soluble selenoproteins in the fruit of Glechoma longituba;

[0021] Figure 3 Effects of different material-liquid ratios in water extraction on the soluble selenoprotein content in the fruit of Cucurbita sphaerocarpa;

[0022] Figure 4 The three-dimensional response curve and contour map of the interaction between the two factors of water extraction on the soluble selenoprotein content of the fruit of the ground melon;

[0023] Figure 5 Effects of different acid extraction times on the content of soluble selenoprotein in the fruit of Cucurbita sphaerocarpa;

[0024] Figure 6 Effects of different extraction temperatures by acid extraction on the content of soluble selenoprotein in the fruit of Cucurbita sphaerocarpa;

[0025] Figure 7 Effects of different solid-liquid ratios by acid extraction on the soluble selenoprotein content in the fruit of Cucurbita sphaerocarpa;

[0026] Figure 8 Effects of different hydrochloric acid concentrations in acid extraction on the content of soluble selenoproteins in the fruit of Cucurbita sphaerocarpa;

[0027] Fig. 9 Three-dimensional response curve and contour map of the interaction between two factors of acid extraction on the content of soluble selenoprotein in the fruit of Glechoma longituba;

[0028] Fig.10 Effects of different extraction times by alcohol extraction on the content of soluble selenoprotein in the fruit of Glechoma longituba;

[0029] Fig.11 Effects of different extraction temperatures of alcohol extraction on the content of soluble selenoprotein in the fruit of Cucurbita sphaerocarpa;

[0030] Fig.12 Effects of different material-liquid ratios in alcohol extraction on the soluble selenoprotein content in the fruit of Cucurbita sphaerocarpa;

[0031] Fig.13 Effects of different ethanol concentrations in alcohol extraction on the content of soluble selenoproteins in the fruit of Cucurbita sphaerocarpa;

[0032] Fig.14 Three-dimensional response curve and contour map of the effect of the interaction of two factors in alcohol extraction on the soluble selenoprotein content in the fruit of Glechoma longituba;

[0033] Fig.15 Effects of different extraction times by alkaline extraction on the content of soluble selenoprotein in the fruit of Glechoma longituba;

[0034] Fig.16 Effects of different extraction temperatures of alkaline extraction on the content of soluble selenoprotein in the fruit of Cucurbita sphaerocarpa;

[0035] Fig.17 Effects of different material-liquid ratios in alkaline extraction on the soluble selenoprotein content in the fruit of Gastrodia elata.

[0036] Fig.18 Effects of different sodium hydroxide concentrations in alkaline extraction on the content of soluble selenoprotein in the fruit of Cucurbita sphaerocarpa;

[0037] Fig.19 Three-dimensional response curve and contour map of the effect of the interaction between two factors of alkaline extraction method on the soluble selenoprotein content in the fruit of Cucurbita oleifera. DETAILED DESCRIPTION

[0038] The invention provides a method for extracting selenoprotein from Cucurbita chinensis, which comprises the following steps: mixing Cucurbita chinensis powder with alkali solution, extracting by shaking, and obtaining a selenoprotein solution by centrifugation.

[0039] In the present invention, the preparation method of the ground melon powder comprises the following steps: drying and crushing the ground melon to obtain the ground melon powder. The ground melon is a two-year-old ground melon in the maturity period. The ground melon is washed, dried and then dried. The drying temperature is preferably 50-70°C, more preferably 55-65°C; the drying time is preferably 12-18h, more preferably 14-16h; the crushing method is to use a micro-plant crusher for crushing, and the mesh size of the ground melon powder after crushing is 60-100 meshes.

[0040] In the present invention, the alkali solution is NaOH solution; the concentration of the alkali solution is preferably 0.05-0.25 mol / L, and more preferably 0.1-0.2 mol / L; the ratio of the ground melon powder to the alkali solution is preferably 1g:20-40mL, and more preferably 1g:25-35mL; the extraction temperature is preferably 30-50°C, and more preferably 35-45°C; the extraction time is preferably 1-3h, and more preferably 1.5-2.5h; the oscillation method is to oscillate in a water constant temperature oscillator, and the oscillation speed is preferably 80-120r / min, and more preferably 90-110r / min; the centrifugal temperature is preferably 2-6°C, and more preferably 3-5°C, the centrifugal speed is preferably 4000-6000r / min, and more preferably 4500-5500r / min, and the centrifugal time is preferably 10-20min, and more preferably 12-18min.

[0041] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0042] Example 1

[0043] The two-year-old mature ground melon collected from the teaching farm of the College of Horticulture and Plant Protection of Inner Mongolia Agricultural University was washed, dried, and dried at 60°C to constant mass. The ground melon powder was obtained by a micro-plant grinder and passed through an 80-mesh sieve. 1 g of the ground melon fruit powder and 23 mL of a 0.25 mol / L NaOH solution were accurately weighed in a centrifuge tube. The centrifuge tube was placed in a water constant temperature oscillator (speed 100 r / min) at a temperature of 44.6°C for sufficient extraction, and then taken out and refrigerated centrifuged (4°C centrifugation for 15 min, speed 5000 r / min) to obtain the ground melon selenoprotein solution.

[0044] Example 2

[0045] The two-year-old mature ground melon collected from the teaching farm of the College of Horticulture and Plant Protection of Inner Mongolia Agricultural University was washed, dried and dried at 50°C to constant mass, crushed with a micro-plant grinder and passed through a 70-mesh sieve to obtain the ground melon powder, accurately weighed 1g of the ground melon fruit powder and 20mL of a 0.20mol / L NaOH solution in a centrifuge tube, placed the centrifuge tube in a water constant temperature oscillator (speed 80r / min) at a temperature of 50°C for sufficient extraction, and then taken out and refrigerated centrifuged (5°C centrifugation for 20min, speed 4500r / min) to obtain the ground melon selenoprotein solution.

[0046] Example 3

[0047] The two-year-old mature ground melon collected from the teaching farm of the College of Horticulture and Plant Protection of Inner Mongolia Agricultural University was washed, dried, and dried at 70°C to constant mass. The ground melon powder was obtained by a micro-plant grinder and passed through a 90-mesh sieve. 1 g of the ground melon fruit powder and 30 mL of a 0.15 mol / L NaOH solution were accurately weighed in a centrifuge tube. The centrifuge tube was placed in a water constant temperature oscillator (speed 120 r / min) at a temperature of 40°C for sufficient extraction, and then taken out and refrigerated centrifuged (3°C for 10 min, speed 5500 r / min) to obtain the ground melon selenoprotein solution.

[0048] Experimental Example 1

[0049] Extraction of selenoprotein from the fruit of melon by water extraction:

[0050] Ultrapure water was used as the extraction solvent, and the extraction time, extraction temperature and solid-liquid ratio were selected as the investigation objects. A single factor experiment was used to explore the influence of each factor on the selenoprotein content in the selenoprotein extract of the ground melon. The investigation level of each factor is shown in Table 1.

[0051] Table 1 Factors for extracting soluble selenoprotein from radix scoparia fruit by water extraction

[0052]

[0053]

[0054] Effects of different extraction times on the extraction of selenoprotein from Glehnia littoralis:

[0055] Accurately weigh 0.1 g of the fruit powder of the radix ophiopogonis fruit into a centrifuge tube, and set the extraction conditions as follows: temperature 40°C, solid-liquid ratio 1:30, extraction times of 1.0 h, 1.5 h, 2.0 h, 2.5 h, and 3.0 h, respectively, and repeat 3 times.

[0056] Experimental results: Figure 1 As shown. Figure 1It can be seen that when other extraction conditions remain unchanged, the content of soluble selenoprotein in the extract of the fruit of the ground melon first increases and then decreases with time. When the extraction time reaches 2h, the soluble selenoprotein content reaches a maximum of 1.27mg / g. Therefore, 1.5h, 2h, and 2.5h are selected as the response center values.

[0057] Effects of different extraction temperatures on the extraction of selenoprotein from Cucurbita sphaerocarpa:

[0058] Accurately weigh 0.1 g of the fruit powder of the radix ophiopogonis fruit into a centrifuge tube, and set the extraction conditions as follows: time 2.0 h, solid-liquid ratio 1:30, extraction temperatures of 30°C, 35°C, 40°C, 45°C, and 50°C, respectively, and repeat 3 times.

[0059] Experimental results: Figure 2 As shown. Figure 2 It can be seen that when other extraction conditions are fixed, the content of soluble selenoprotein in the fruit extract of the ground melon gradually decreases with the increase of temperature. When the extraction temperature is 30℃, the maximum content of soluble selenoprotein is 0.81 mg / g. Therefore, the temperatures of 25℃, 30℃ and 35℃ are selected as the response center values.

[0060] Effects of different material-liquid ratios on the extraction of selenoprotein from Gastrodia elata:

[0061] Accurately weigh 0.1 g of the fruit powder of the radix ophiopogonis fruit into a centrifuge tube, and set the extraction conditions as follows: time 2.0 h, temperature 40°C, solid-liquid ratios of 1:20, 1:25, 1:30, 1:35, and 1:40, respectively, and repeat 3 times.

[0062] Experimental results: Figure 3 As shown. Figure 3 It can be seen that when other conditions are fixed, the content of soluble selenoprotein in the extract of the fruit of the ground melon gradually decreases with the decrease of the solid-liquid ratio. When the solid-liquid ratio is 1:20, the soluble selenoprotein content is the highest at 2.42 mg / g. Therefore, the solid-liquid ratios of 1:15, 1:20, and 1:25 are selected as the response center values.

[0063] Optimization of extraction technology of selenoprotein from Cucurbita sphaerocarpa by response surface methodology:

[0064] Taking the selenoprotein content in the extract of Cucurbita oleracea fruit as the response value, the optimal level range was determined through experiments and a significance analysis was performed. Taking extraction time, solid-liquid ratio and extraction temperature as factors, the Design-Expert 13 software was used to design a three-factor three-level BBD response surface method to select the optimal extraction process of selenoprotein in Cucurbita oleracea fruit. The setting levels of each factor are shown in Table 2.

[0065] Table 2 Factors and levels of response surface design

[0066]

[0067] Experimental results: As shown in Table 3 and Figure 4 shown.

[0068] Table 3 Response surface analysis scheme and results of soluble selenoproteins in radix scoparia fruit

[0069]

[0070]

[0071] Verification of the optimal process for extracting selenoprotein from the fruit of Cucurbita oleifera by water extraction:

[0072] The optimal extraction process of selenoprotein from ground melon by water extraction is: extraction time 2.29h, solid-liquid ratio 1:15.87, extraction temperature 28.64℃, and theoretical selenoprotein content is 2.741mg / g. In order to facilitate the operability of the experiment, the process parameters were set as: extraction time 2.3h, solid-liquid ratio 1:15.9, temperature 28.6℃, and the experimental selenoprotein content was 2.738mg / g, which is close to the theoretical predicted value, indicating that this model and extraction process are stable and highly operable, and can be used for the extraction of selenoprotein from ground melon fruit.

[0073] Table 4 Verification experimental results

[0074]

[0075] Experimental Example 2

[0076] Effect of acid extraction on selenoprotein content in Gastrodia elata fruit:

[0077] Extraction time, extraction temperature, solid-liquid ratio and hydrochloric acid concentration were selected as the investigation objects, and single factor experiments were used to explore the influence of each factor on the selenoprotein content in the selenoprotein extract of Cucurbita oleracea. The investigation level of each factor is shown in Table 5.

[0078] Table 5 Factors investigated in the extraction of soluble selenoprotein from radix scoparia fruit by acid extraction

[0079]

[0080]

[0081] Effects of different extraction times on the extraction of selenoprotein from Glehnia littoralis:

[0082] Accurately weigh 0.1 g of the fruit powder of the radix ophiopogonis into a centrifuge tube, and set the extraction conditions as follows: temperature 40°C, solid-liquid ratio 1:30, solvent concentration 0.15 mol / L, extraction times of 1.0 h, 1.5 h, 2.0 h, 2.5 h, and 3.0 h, respectively. The experiment was repeated three times.

[0083] Experimental results: Figure 5 As shown. Figure 5 It can be seen that when other extraction conditions are fixed, the content of soluble selenoprotein in the extract of the fruit of the ground melon first gradually increases with time, reaching a maximum value of 1.63 mg / g at 2.5 h, and then its content decreases. Therefore, the times of 2h, 2.5h, and 3h are selected as the response center values.

[0084] Effects of different extraction temperatures on the extraction of selenoprotein from Cucurbita sphaerocarpa:

[0085] Accurately weigh 0.1 g of the fruit powder of the radix ophiopogonis into a centrifuge tube, and set the extraction conditions as follows: time 2.0 h, solid-liquid ratio 1:30, solvent concentration 0.15 mol / L, extraction temperatures at 30°C, 35°C, 40°C, 45°C, and 50°C, respectively. The experiment was repeated three times.

[0086] Experimental results: Figure 6 As shown. Figure 6 It can be seen that when other extraction conditions are fixed, the content of soluble selenoprotein in the fruit extract of the ground melon gradually increases with the increase of temperature, reaching a maximum value of 1.79 mg / g at 50°C. Therefore, extraction temperatures of 45°C, 50°C, and 55°C were selected as the response center values.

[0087] Effects of different material-liquid ratios on the extraction of selenoprotein from Gastrodia elata:

[0088] Accurately weigh 0.1 g of the fruit powder of the radix ophiopogonis fruit into a centrifuge tube, and set the extraction conditions as follows: time 2.0 h, temperature 40°C, solvent concentration 0.15 mol / L, and the solid-liquid ratios of 1:20, 1:25, 1:30, 1:35, and 1:40 for extraction. The experiment was repeated three times.

[0089] Experimental results: Figure 7 As shown. Figure 7 It can be seen that when other extraction conditions are fixed, the content of soluble selenoprotein in the extract of the fruit of the ground melon first gradually increases with the decrease of the solid-liquid ratio, reaching a maximum value of 2.02 mg / g at 1:30, and then its content decreases with the decrease of the solid-liquid ratio. Therefore, the solid-liquid ratios of 1:25, 1:30, and 1:35 were selected as the response center values.

[0090] Effects of different hydrochloric acid concentrations on the extraction of selenoprotein from Cucurbita sphaerocarpa:

[0091] Accurately weigh 0.1 g of the fruit powder of the radix ophiopogonis into a centrifuge tube, and set the extraction conditions as follows: time 2.0 h, temperature 40°C, solid-liquid ratio 1:30, and solvent concentrations of 0.05 mol / L, 0.10 mol / L, 0.15 mol / L, 0.20 mol / L, and 0.25 mol / L, respectively, for extraction. The experiment was repeated three times.

[0092] Experimental results: Figure 8 As shown. Figure 8 It can be seen that when other extraction conditions are fixed, the content of soluble selenoprotein in the extract of the fruit of the ground melon first gradually increases with the increase of solvent concentration, reaching a maximum value of 3.56 mg / g at 0.2 mol / L, and then its content decreases. Therefore, solvent concentrations of 0.15 mol / L, 0.2 mol / L, and 0.25 mol / L were selected as the response center values.

[0093] Response surface methodology to optimize the extraction process:

[0094] Taking the selenoprotein content in the extract of Cucurbita oleracea fruit as the response value, the optimal level range was determined through experiments and a significance analysis was performed. Taking the solid-liquid ratio, extraction temperature and solvent concentration as factors, the Design-Expert 13 software was used to design a three-factor three-level BBD response surface method to select the optimal extraction process of selenoprotein in Cucurbita oleracea fruit. The setting levels of each factor are shown in Table 6.

[0095] Table 6 Factors and levels of response surface design

[0096]

[0097] Experimental results: As shown in Table 7 and Fig. 9 shown.

[0098] Table 7 Response surface analysis scheme and results of soluble selenoproteins in radix cucumis fruit

[0099]

[0100]

[0101] Verification of the best process for extracting selenoprotein from the fruit of melon by acid extraction:

[0102] The best extraction process of selenoprotein in the fruit of ground melon by acid extraction is: solid-liquid ratio 1:28.6, extraction temperature 51.603℃, solvent concentration 0.192mol / L, and the theoretical selenoprotein content is 2.762mg / g. In order to facilitate the operability of the experiment, the process parameters were set as follows: solid-liquid ratio 1:29, extraction temperature 51.7℃, solvent concentration 0.19mol / L in the verification experiment. The experimental selenoprotein content was 2.761mg / g, which was close to the theoretical predicted value, indicating that this model and extraction process are stable and highly operable, and can be used for the extraction of selenoprotein in ground melon fruit.

[0103] Table 8 Verification experimental results

[0104]

[0105] Experimental Example 3

[0106] Extraction of selenoprotein from the fruit of melon by ethanol extraction:

[0107] Extraction time, extraction temperature, solid-liquid ratio and ethanol concentration were selected as the investigation objects, and single factor experiments were used to explore the influence of each factor on the selenoprotein content in the selenoprotein extract of Cucurbita oleracea. The investigation level of each factor is shown in Table 9.

[0108] Table 9 Factors investigated in the extraction of soluble selenoprotein from scoparia fruit by ethanol extraction

[0109] Extraction time (h) Extraction temperature (℃) Material-liquid ratio (g / ml) Solvent concentration (%) 1.0 30 1:20 60 1.5 35 1:25 65 2.0 40 1:30 70 2.5 45 1:35 75 3.0 50 1:40 80

[0110] Effects of different extraction times on the extraction of selenoprotein from Glehnia littoralis:

[0111] Accurately weigh 0.1 g of the fruit powder of the radix ophiopogonis was placed in a centrifuge tube, and the extraction conditions were set as follows: temperature 40°C, solid-liquid ratio 1:30, solvent concentration 70%, and extraction times of 1.0 h, 1.5 h, 2.0 h, 2.5 h, and 3.0 h, respectively. The experiment was repeated three times.

[0112] Experimental results: Fig.10 As shown. Fig.10 It can be seen that when other extraction conditions are fixed, the content of soluble selenoprotein in the extract of the fruit of the ground melon first increases gradually with the increase of time, reaching a maximum value of 1.15 mg / g at 2.5 h, and then its content gradually decreases with the increase of time. Therefore, the times of 2h, 2.5h, and 3h are selected as the response center values.

[0113] Effects of different extraction temperatures on the extraction of selenoprotein from Cucurbita sphaerocarpa:

[0114] Accurately weigh 0.1 g of the fruit powder of the radix ophiopogonis into a centrifuge tube, and set the extraction conditions as follows: time 2.0 h, solid-liquid ratio 1:30, solvent concentration 70%, extraction temperatures at 30°C, 35°C, 40°C, 45°C, and 50°C, respectively. The experiment was repeated three times.

[0115] Experimental results: Fig.11 As shown. Fig.11 It can be seen that when other extraction conditions are fixed, the content of soluble selenoprotein in the extract of the fruit of the ground melon gradually increases with the increase of the extraction temperature, reaching a maximum value of 1.47 mg / g at 50°C. Therefore, extraction temperatures of 45°C, 50°C, and 55°C were selected as the response center values.

[0116] Effects of different material-liquid ratios on the extraction of selenoprotein from Gastrodia elata:

[0117] Accurately weigh 0.1 g of the fruit powder of the radix ophiopogonis into a centrifuge tube, and set the extraction conditions as follows: time 2.0 h, temperature 40°C, solvent concentration 70%, and solid-liquid ratios of 1:20, 1:25, 1:30, 1:35, and 1:40, respectively. The experiment was repeated three times.

[0118] Experimental results: Fig.12 As shown. Fig.12 It can be seen that when other extraction conditions are fixed, the content of soluble selenoprotein in the extract of the fruit of the ground melon first increases gradually with the decrease of the solid-liquid ratio, reaching a maximum value of 1.45 mg / g at 1:25, and then its content gradually decreases with the decrease of the solid-liquid ratio. Therefore, the solid-liquid ratios of 1:20, 1:25, and 1:30 were selected as the response center values.

[0119] Effects of different ethanol concentrations on the extraction of selenoprotein from Cucurbita sphaerocarpa:

[0120] Accurately weigh 0.1 g of the fruit powder of the radix ophiopogonis fruit into a centrifuge tube, and set the extraction conditions as follows: time 2.0 h, temperature 40°C, solid-liquid ratio 1:30, and solvent concentrations of 60%, 65%, 70%, 75%, and 80% for extraction. The experiment was repeated three times.

[0121] Experimental results: Fig.13 As shown. Fig.13 It can be seen that when other extraction conditions are fixed, the soluble selenoprotein content in the extract of the fruit of the ground melon first increases with the increase of solvent concentration and then gradually increases, reaching a maximum value of 2.06 mg / g at 80 h. Therefore, solvent concentrations of 75%, 80%, and 85% were selected as the response center values.

[0122] Optimization of extraction process by response surface methodology

[0123] Taking the selenoprotein content in the extract of Cucurbita oleracea fruit as the response value, the optimal level range was determined through experiments and a significance analysis was performed. Taking the solid-liquid ratio, extraction temperature and solvent concentration as factors, the Design-Expert 13 software was used to design a three-factor and three-level BBD response surface method to select the optimal extraction process of selenoprotein in Cucurbita oleracea fruit. The setting levels of each factor are shown in Table 10.

[0124] Table 10 Factors and levels of response surface design

[0125]

[0126] Experimental results: As shown in Tables 11, 12 and Fig.14 shown.

[0127] Table 11 Response surface analysis scheme and results of soluble selenoproteins in radix cucumis fruit

[0128]

[0129]

[0130] Table 12 Analysis of variance of regression model equation

[0131]

[0132]

[0133] Taking the selenoprotein content of the fruit extract of the melon as the response value, after regression fitting by the software, the regression equation of the influence of each factor on the response value was obtained as follows:

[0134] Y=-87.944+2728.72×A+1.380×B+140.347×C-15.665×AB-8.235×AC+3.95400×BC-26572.664×A2-0.018×B2-887.850×C2

[0135] The results of variance analysis showed that the P value of the regression model was 0.0009, which reached an extremely significant level (P < 0.01), and the P value of the lack of fit term was 0.1101 (P > 0.05), which showed no significant difference. The coefficient of determination of the model, R2 = 0.9504, indicated that 95.04% of the response value changes could be explained, and the model fitted the actual situation well, which could be used to analyze and predict the results of the soluble selenoprotein content of the fruit of the ground melon extracted by the salt extraction method. The first-order term C, and the second-order terms A2 and C2 of the model all reached a significant level (P < 0.05). From the F value, it can be seen that the main influencing factors on the soluble selenoprotein content in the fruit extract of the ground melon are C > B > A, that is, solvent concentration > extraction temperature > material-liquid ratio.

[0136] Depend on Fig.14 It can be seen that the slopes of AC and BC are relatively steep, and there is a certain slope between AB; the contour maps of AB and BC are closer to ellipses, indicating that there is a good interaction between A and B, and between B and C. Among them, the contour map of the investigated factor AB has a higher density, indicating that factors A and B have a significant effect on the content of selenoprotein in the extract of the fruit of the ground melon.

[0137] Verification of the optimal process for extracting selenoprotein from the fruit of Cucurbita oleifera by alcohol extraction:

[0138] The optimal process for extracting selenoprotein from the fruit of the ground melon by alcohol extraction is: solid-liquid ratio 1:23.3, extraction temperature 52.485℃, solvent concentration 81.37%, and the theoretical selenoprotein content is 3.271mg / g. In order to facilitate the operability of the experiment, the process parameters were set as follows during the verification experiment: solid-liquid ratio 1:23, extraction temperature 52.5℃, solvent concentration 81.4%, and the experimental selenoprotein content was 3.268mg / g, which was close to the theoretical predicted value, indicating that this model and extraction process are stable and highly operable, and can be used for the extraction of selenoprotein from the fruit of the ground melon.

[0139] Experimental Example 4

[0140] Extraction of selenoprotein from the fruit of ground melon by alkali extraction:

[0141] Extraction time, extraction temperature, solid-liquid ratio and NaOH concentration were selected as the investigation objects, and single factor experiments were used to explore the influence of each factor on the selenoprotein content in the selenoprotein extract of Cucurbita oleracea. The investigation level of each factor is shown in Table 13.

[0142] Table 13 Factors investigated in extracting soluble selenoprotein from radix scoparia fruit by alkaline extraction

[0143] Extraction time (h) Extraction temperature (℃) Material-liquid ratio (g / ml) Solvent concentration (mol / L) 1.0 30 1:20 0.05 1.5 35 1:25 0.10 2.0 40 1:30 0.15 2.5 45 1:35 0.20 3.0 50 1:40 0.25

[0144] Effects of different extraction times on the extraction of selenoprotein from Glehnia littoralis:

[0145] Accurately weigh 0.1 g of the fruit powder of the radix ophiopogonis into a centrifuge tube, and set the extraction conditions as follows: temperature 40°C, solid-liquid ratio 1:30, solvent concentration 0.15 mol / L, extraction times of 1.0 h, 1.5 h, 2.0 h, 2.5 h, and 3.0 h, respectively. The experiment was repeated three times.

[0146] Experimental results: Fig.15 As shown. Fig.15 It can be seen that when other extraction conditions are fixed, the content of soluble selenoprotein in the extract of the fruit of the ground melon first increases gradually with time, reaching a maximum value of 4.97 mg / g at 2 h, and then its content gradually decreases. Therefore, the times of 1.5 h, 2 h, and 2.5 h are selected as the response center values.

[0147] Effects of different extraction temperatures on the extraction of selenoprotein from Cucurbita sphaerocarpa:

[0148] Accurately weigh 0.1 g of the fruit powder of the radix ophiopogonis into a centrifuge tube, and set the extraction conditions as follows: time 2.0 h, solid-liquid ratio 1:30, solvent concentration 0.15 mol / L, extraction temperatures at 30°C, 35°C, 40°C, 45°C, and 50°C, respectively. The experiment was repeated three times.

[0149] Experimental results: Fig.16 As shown. Fig.16 It can be seen that when other extraction conditions are fixed, the content of soluble selenoprotein in the extract of the fruit of the ground melon first gradually increases with the increase of temperature, reaching a maximum value of 5.30 mg / g at 40°C, and then its content begins to decrease with the increase of temperature. Therefore, extraction temperatures of 35°C, 40°C, and 45°C were selected as the response center values.

[0150] Effects of different material-liquid ratios on the extraction of selenoprotein from Gastrodia elata:

[0151] Accurately weigh 0.1 g of the fruit powder of the radix ophiopogonis fruit into a centrifuge tube, and set the extraction conditions as follows: time 2.0 h, temperature 40°C, solvent concentration 0.15 mol / L, and the solid-liquid ratios of 1:20, 1:25, 1:30, 1:35, and 1:40 for extraction. The experiment was repeated three times.

[0152] Experimental results: Fig.17 As shown. Fig.17 It can be seen that when other extraction conditions are fixed, the content of soluble selenoprotein in the extract of the fruit of the ground melon first increases and then gradually decreases with the decrease of the solid-liquid ratio, reaching a maximum value of 5.05 mg / g when the solid-liquid ratio is 1:25. Therefore, the solid-liquid ratios of 1:20, 1:25, and 1:30 are selected as the response center values.

[0153] Effects of different sodium hydroxide concentrations on the extraction of selenoprotein from Glehnia littoralis:

[0154] Accurately weigh 0.1 g of the fruit powder of the radix ophiopogonis into a centrifuge tube, and set the extraction conditions as follows: time 2.0 h, temperature 40°C, solid-liquid ratio 1:30, and solvent concentrations of 0.05 mol / L, 0.10 mol / L, 0.15 mol / L, 0.20 mol / L, and 0.25 mol / L, respectively, for extraction. The experiment was repeated three times.

[0155] Experimental results: Fig.18 As shown. Fig.18It can be seen that when other extraction conditions are fixed, the content of soluble selenoprotein in the fruit extract of the ground melon gradually increases with the increase of solvent concentration, reaching a maximum value of 5.40 mg / g when the solvent concentration is 0.2 mol / L. Therefore, solvent concentrations of 0.15 mol / L, 0.2 mol / L, and 0.25 mol / L were selected as the response center values.

[0156] Optimization of extraction process by response surface methodology

[0157] Taking the selenoprotein content in the extract of Cucurbita oleracea fruit as the response value, the optimal level range was determined through experiments and a significance analysis was performed. Taking the solid-liquid ratio, extraction temperature and solvent concentration as factors, the Design-Expert 13 software was used to design a three-factor and three-level BBD response surface method to select the optimal extraction process of selenoprotein in Cucurbita oleracea fruit. The setting levels of each factor are shown in Table 14.

[0158] Table 14 Factors and levels of response surface design

[0159]

[0160]

[0161] Experimental results: As shown in Tables 15, 16 and Fig.19 shown.

[0162] Table 15 Response surface analysis scheme and results of soluble selenoproteins in radix cucumis fruit

[0163]

[0164] Table 16 Analysis of variance of regression model equation

[0165]

[0166]

[0167] Taking the selenoprotein content of the fruit extract of the melon as the response value, after regression fitting by the software, the regression equation of the influence of each factor on the response value was obtained as follows:

[0168] Y=5.47+0.8114×A+0.7951×B-0.6765×C-0.2298×AB+0.8045×AC+0.594×BC-0.8590×A2-0.3325×B2-0.2452×C2

[0169] The results of variance analysis showed that the P value of the regression model was 0.0074, which reached an extremely significant level (P < 0.01), and the P value of the lack of fit term was 0.0967 (P > 0.05), which showed no significant difference. The coefficient of determination of the model, R2 = 0.9056, indicated that 90.56% of the response value changes could be explained, and the model fitted the actual situation well, which could be used to analyze and predict the results of soluble selenoprotein content in the fruit of the ground melon by alkali extraction. The linear terms A and B in the model; the interaction terms AC; and the quadratic term A2 reached a significant level (P < 0.05). From the F value, it can be seen that the main influencing factors on the content of soluble selenoprotein in the fruit extract of the ground melon are A > B > C, that is, the ratio of solid to liquid > extraction temperature > solvent concentration.

[0170] Depend on Fig.19 As shown in the figure, the slopes of AB and AC are relatively steep, and there is a certain slope between BC; the AB contour map is closer to an ellipse, indicating that there is a good interaction between A and B. Among them, the contour map of the investigated factor AB has the highest density, indicating that factors A and B have a significant effect on the content of selenoprotein in the extract of the fruit of the ground melon.

[0171] Verification of the optimal process for extracting selenoprotein from the fruit of melon by alkaline extraction:

[0172] The optimal extraction process of selenoprotein from the fruit of the ground melon by alkali extraction is: solid-liquid ratio 1:22.7, extraction temperature 44.62℃, solvent concentration 0.243mol / L, and the theoretical selenoprotein content is 5.925mg / g. In order to facilitate the operability of the experiment, the process parameters were set as follows: solid-liquid ratio 1:23, extraction temperature 44.6℃, solvent concentration 0.25mol / L in the verification experiment. The experimental selenoprotein content was 5.924mg / g, which was close to the theoretical predicted value, indicating that this model and extraction process are stable and highly operable, and can be used for the extraction of selenoprotein from the fruit of the ground melon.

[0173] Table 17 Verification experimental results

[0174]

[0175] It can be seen from the above embodiments and experimental examples that the content of selenoprotein obtained by alkali extraction is 5.924 mg / g, which is much higher than the content of selenoprotein obtained by water extraction, acid extraction and alcohol extraction.

[0176] The extraction method of the selenoprotein of the ground melon of the present invention significantly improves the extraction efficiency of the selenoprotein of the ground melon. The extraction method of the present invention is simple, has low extraction cost and has broad market promotion value.

[0177] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for extracting selenoprotein from Cucurbita sphaerocarpa, characterized in that: The following steps are involved: The powder of the radix ophiopogonis is mixed with the alkali solution, and then shaken for extraction, and centrifuged to obtain the radix ophiopogonis selenoprotein solution; The concentration of the alkali solution is 0.25 mol / L; The ratio of the ground melon powder to the alkali solution is 1g:23mL; The extraction temperature is 44.6°C; the extraction time is 1-3h.

2. The method for extracting selenoprotein from Cucurbita sphaerocarpa according to claim 1, wherein The preparation method of the ground melon powder comprises the following steps: drying and crushing the ground melon to obtain the ground melon powder.

3. The method for extracting selenoprotein from Cucurbita sphaerocarpa according to claim 2, wherein The drying temperature is 50-70° C., the drying time is 12-18 hours, and the mesh size of the ground melon powder obtained after the crushing is 60-100 meshes.

4. The method for extracting selenoprotein from Cucurbita sphaerocarpa according to claim 1, wherein The alkali solution is NaOH solution.

5. The method for extracting selenoprotein from Cucurbita sphaerocarpa according to claim 1, wherein The rotation speed of the oscillation is 80-120 r / min.

6. The method for extracting selenoprotein from Cucurbita sphaerocarpa according to claim 1, wherein The centrifugal temperature is 2-6°C, the centrifugal speed is 4000-6000r / min, and the centrifugal time is 10-20min.

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