A method for extracting solanine from potato organs

Abstract

The present invention belongs to the technical field of compound extraction, and specifically relates to a method for extracting solanine from potato organs. The method of the present invention uses biological enzymatic hydrolysis to assist ultrasonic extraction of solanine from potato organs. The biological enzyme degradation technology can decompose the plant cell wall in a very short time, promote the outflow of internal natural active ingredients, and greatly improve the extraction rate of the target component. Ultrasonic technology uses vibration cavitation, mechanical crushing, thermal mechanism, stirring and other effects to cause the collapse of cavitation bubbles instantly generated by plant tissues in solvents, resulting in cell rupture, which is conducive to the penetration of solvents into plant cells, allowing intracellular components to enter the solvent, and accelerating mutual penetration and dissolution. The biological enzyme enzymatic hydrolysis technology is effectively combined with the ultrasonic technology to achieve a synergistic effect. Compared with the solanine extraction method using a single technical means, it has the advantages of high yield, short time consumption, green and safe, and is more suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of compound extraction, and particularly relates to a method for extracting solanine from potato organs. Background Art

[0002] Potatoes are the fourth largest food crop in the world after rice, wheat and corn. However, in most areas, the aboveground stems and leaves of potatoes are usually discarded or burned when they are harvested, resulting in a waste of resources.

[0003] Potatoes contain a class of steroidal glycoside alkaloids, commonly known as solanine. The main components of solanine in potatoes are α-solanine and α-chaconine, accounting for more than 95% of the total solanine. Solanine is distributed in all tissues of potatoes, with 0.25% in green leaves, 0.5% in buds, 0.73% in flowers, 1.0% in fruits, and only 0.004% in mature tubers. Solanine is a natural toxic secondary metabolite produced during plant growth, which has certain pharmacological activities, including anti-tumor, antibacterial, and anti-inflammatory effects. At the same time, it has the defensive characteristics of protecting plants from viruses, bacteria, fungi, insects, etc. For plants, solanine is the best defensive weapon and can be used to resist the invasion of pests and diseases. It is a "green pesticide" to protect plants. If the above-ground stems and leaves of potatoes can be used as raw materials for solanine extraction, the comprehensive utilization efficiency of potatoes can be greatly improved, the economic value can be increased, and resource recycling can be achieved, turning waste into treasure. Therefore, successfully extracting solanine from potato stems and leaves and applying it in the fields of food, pesticides and pharmacy has very important strategic significance for promoting the development of the potato industry.

[0004] There are many methods for extracting solanine, such as ultrasonic extraction, leaching, and multi-solvent extraction. Ultrasonic extraction is the most commonly used method at this stage. Ultrasonic technology facilitates the penetration of solvents into plant cells, allowing the components in the cells to enter the solvent, accelerating mutual penetration and dissolution. This method has the advantages of short extraction time, saving solvents, and reducing the effect of temperature on the extract. However, most extraction methods for solanine, including ultrasonic extraction, have problems with low extraction efficiency and yield, which have not yet been effectively solved. Summary of the invention

[0005] The object of the present invention is to provide a method for extracting solanine from potato organs, which can effectively improve the extraction yield of solanine and has higher extraction efficiency.

[0006] The present invention provides a method for extracting solanine from potatoes, comprising the following steps:

[0007] 1) mixing a potato powder sample with cellulase, and performing ultrasound-assisted enzymolysis in an acidic aqueous solution to obtain an enzymolysis reaction solution;

[0008] 2) performing enzyme inactivation treatment on the enzymolysis reaction liquid to obtain an enzyme inactivated enzymolysis reaction liquid;

[0009] 3) mixing the enzyme-inactivated enzymatic reaction liquid with anhydrous ethanol until the volume fraction of ethanol is 70%, and then standing for extraction to obtain an extract;

[0010] 4) filtering the extract under reduced pressure, and concentrating the obtained filtrate to obtain a concentrate;

[0011] 5) dissolving the concentrate with an acidic aqueous solution, and filtering the obtained solution again under reduced pressure to obtain a reduced pressure filtered solution;

[0012] 6) adjusting the pH value of the vacuum filtered solution to 10.0-10.5, and allowing to stand under refrigeration to obtain a standing mixture;

[0013] 7) subjecting the standing mixture to solid-liquid separation, washing the obtained precipitate with an alkaline aqueous solution until the washing liquid is clear and transparent, and the washed precipitate contains the solanine.

[0014] Preferably, the dosage of the cellulase is 1%-3% of the mass of the potato powder sample, and the enzyme activity of the cellulase is ≥50000 U / g.

[0015] Preferably, the temperature of the ultrasound-assisted enzymatic hydrolysis is 50-55° C., the power is 250-300 W, and the pH value is 5; the total duration of the ultrasound-assisted enzymatic hydrolysis is 1.0-2.0 h, and the ultrasound action time is 30-50 min.

[0016] Preferably, the static extraction time in step 3) is 10-18 hours.

[0017] Preferably, the acidic aqueous solution in step 5) is an aqueous sulfuric acid solution, and the volume fraction of the aqueous sulfuric acid solution is 5%.

[0018] Preferably, in step 6), the standing time is 12-24 hours; and the refrigeration temperature is 4-6°C.

[0019] Preferably, the solid-liquid separation method includes centrifugation, the centrifugal speed is 4000r / min, and the time is 20min; the alkaline aqueous solution includes an ammonia solution, and the volume concentration of the ammonia solution is 1%-1.5%.

[0020] Preferably, the method further comprises purifying the washed precipitate, the purification method comprises solid phase extraction, and the solid phase extraction column comprises a C18 solid phase extraction column.

[0021] Preferably, the activation of the solid phase extraction includes methanol activation and distilled water activation performed in sequence; the elution is distilled water elution; and the elution is methanol elution and isopropanol elution performed in sequence, and the volume ratio of methanol to isopropanol is 4:1.

[0022] Preferably, the potato powder sample is derived from one or more of sprouts, stems and leaves of potatoes.

[0023] Beneficial effects:

[0024] The invention provides a method for extracting solanine from potatoes, comprising the following steps: 1) mixing a potato powder sample with cellulase, performing ultrasonic-assisted enzymolysis in an acidic aqueous solution to obtain an enzymolysis reaction liquid; 2) performing enzyme inactivation treatment on the enzymolysis reaction liquid to obtain an enzyme-inactivated enzymolysis reaction liquid; 3) mixing the enzyme-inactivated enzymolysis reaction liquid with anhydrous ethanol until the volume fraction of ethanol is 70%, and then performing static extraction to obtain an extract; 4) performing vacuum filtration on the extract, and concentrating the obtained filtrate to obtain a concentrate; 5) dissolving the concentrate with an acidic aqueous solution, and performing vacuum filtration on the obtained solution again to obtain a vacuum filtration solution; 6) adjusting the pH value of the vacuum filtration solution to 10.0-10.5, and then performing static extraction under refrigeration conditions to obtain a static mixture; 7) performing solid-liquid separation on the static mixture, and washing the obtained precipitate with an ammonia aqueous solution until the washing liquid is clear and transparent, and the washed precipitate contains the solanine. The method of the present invention uses biological enzymatic hydrolysis to assist ultrasonic extraction of solanine in potato organs. The biological enzyme degradation technology can decompose the plant cell wall in a very short time, promote the outflow of internal natural active ingredients, and greatly improve the extraction rate of the target component. Ultrasonic technology utilizes vibration cavitation, mechanical crushing, thermal mechanism, stirring and other effects to collapse the cavitation bubbles instantly generated by plant tissue in the solvent, causing cell rupture, which is beneficial for the solvent to penetrate into the inside of the plant cell, so that the intracellular components enter the solvent, and accelerate mutual penetration and dissolution. The biological enzyme enzymatic hydrolysis technology is effectively combined with the ultrasonic technology to achieve a synergistic effect. Compared with the single solanine extraction method, it has the advantages of high yield, short time consumption, green and safe, and is more suitable for industrial production. At the same time, the present invention can make full use of the aboveground stems and leaves of potatoes, turn waste into treasure, and rationally utilize resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0026] Figure 1 is the standard curve of α-solanine in Example 1;

[0027] Figure 2 is the α-chaconine standard curve in Example 1;

[0028] Figure 3 The effects of different extraction methods on the solanine content in potato peels in Example 1 and Comparative Examples 1-3 are shown;

[0029] Figure 4 The effect of the amount of cellulase added on the amount of solanine extracted in Example 3;

[0030] Figure 5 The effect of enzymatic hydrolysis time on the solanine extraction amount in Example 3;

[0031] Figure 6 The effect of enzymatic hydrolysis temperature on the solanine extraction amount in Example 3;

[0032] Figure 7 The figure shows the effect of using sprouts, stems and leaves of different potato varieties as extraction materials on the solanine content in Example 3. DETAILED DESCRIPTION

[0033] The present invention provides a method for extracting solanine from potatoes, comprising the following steps:

[0034] 1) mixing a potato powder sample with cellulase, and performing ultrasound-assisted enzymolysis in an acidic aqueous solution to obtain an enzymolysis reaction solution;

[0035] 2) performing enzyme inactivation treatment on the enzymolysis reaction liquid to obtain an enzyme inactivated enzymolysis reaction liquid;

[0036] 3) mixing the enzyme-inactivated enzymatic reaction liquid with anhydrous ethanol until the volume fraction of ethanol is 70%, and then standing for extraction to obtain an extract;

[0037] 4) filtering the extract under reduced pressure, and concentrating the obtained filtrate to obtain a concentrate;

[0038] 5) dissolving the concentrate with an acidic aqueous solution, and filtering the obtained solution again under reduced pressure to obtain a reduced pressure filtered solution;

[0039] 6) adjusting the pH value of the vacuum filtered solution to 10.0-10.5, and allowing to stand under refrigeration to obtain a standing mixture;

[0040] 7) subjecting the standing mixture to solid-liquid separation, washing the obtained precipitate with an aqueous ammonia solution until the washing liquid is clear and transparent, and the washed precipitate contains the solanine.

[0041] The present invention mixes a potato powder sample with cellulase, performs ultrasound-assisted enzymolysis in an acidic aqueous solution, and obtains an enzymolysis reaction liquid. The potato powder sample of the present invention is preferably derived from one or more of the buds, stems and leaves of potatoes, and more preferably from the stems and leaves of potatoes. The preparation step of the potato powder sample of the present invention preferably includes: drying the fresh potato sample at a constant temperature to a constant weight, crushing and sieving the obtained dry product, taking the sieve under the sieve, and obtaining the potato powder sample. The temperature of the constant temperature drying of the present invention is preferably 70°C; the aperture of the sieve is preferably 60 meshes. The present invention does not specifically limit the variety of the potato, and any potato variety sample can be used to extract solanine by the method of the present invention, but there are differences in the content of solanine between different potato varieties. Further, those skilled in the art can conventionally select potato varieties as extraction raw materials according to needs. The dosage of the cellulase of the present invention is preferably 1.0%-3.0% of the mass of the potato powder sample, further preferably 1.5%-2%, more preferably 1.5% or 2.0%; the enzymatic activity of the cellulase is preferably ≥50000U / g. The temperature of the ultrasonic-assisted enzymatic hydrolysis of the present invention is preferably 50-55°C, more preferably 50°C; the power of the ultrasonic-assisted enzymatic hydrolysis is preferably 250-300W, more preferably 300W; the pH value of the ultrasonic-assisted enzymatic hydrolysis is preferably 5; the total duration of the ultrasonic-assisted enzymatic hydrolysis is preferably 1.0-2.0h, further preferably 1.5-2.0h, more preferably 1.5h, wherein the ultrasonic action time is preferably 30-50min, more preferably 30min. The acidic aqueous solution of the present invention is preferably a hydrochloric acid aqueous solution, that is, the pH of the enzymatic hydrolysis reaction system is adjusted to 5 with a hydrochloric acid aqueous solution.

[0042] After obtaining the enzymatic hydrolysis reaction liquid, the present invention performs enzyme inactivation treatment on the enzymatic hydrolysis reaction liquid to obtain an enzyme inactivated enzymatic hydrolysis reaction liquid. The enzyme inactivation treatment method of the present invention preferably includes heating the enzymatic hydrolysis reaction liquid to 95° C., and the time of maintaining the temperature at 95° C. is preferably 5 minutes.

[0043] After obtaining the enzyme-killed enzymolysis reaction liquid, the present invention mixes the enzyme-killed enzymolysis reaction liquid with anhydrous ethanol until the volume fraction of ethanol is 70%, and then performs static extraction to obtain an extract. The function of maintaining the volume fraction of ethanol at 70% during static extraction is to fully dissolve solanine. The static extraction time of the present invention is preferably 10-18 hours, and more preferably 15-18 hours.

[0044] After obtaining the extract, the present invention performs vacuum filtration on the extract, and concentrates the obtained filtrate to obtain a concentrate. The present invention preferably uses a rotary evaporator for the concentration.

[0045] After obtaining the concentrate, the present invention dissolves the concentrate with an acidic aqueous solution, and the obtained solution is filtered under reduced pressure again to obtain a reduced pressure filtered solution. The acidic aqueous solution of the present invention is preferably a sulfuric acid aqueous solution, and the volume fraction of the sulfuric acid aqueous solution is preferably 5%.

[0046] After obtaining the vacuum filtered solution, the present invention adjusts the pH value of the vacuum filtered solution to 10.0-10.5, and then allows it to stand under refrigerated conditions to obtain a standing mixture. The present invention preferably uses concentrated ammonia water to adjust the pH value, and the pH value environment has the effect of precipitating solanine under alkaline conditions. The refrigeration temperature of the present invention is preferably 4-6°C, more preferably 4°C; the standing time is preferably 12-24h, more preferably overnight. The present invention has the effect of precipitating solanine as much as possible by standing under refrigerated conditions.

[0047] After obtaining the standing mixture, the present invention performs solid-liquid separation on the standing mixture, washes the obtained precipitate with an alkaline aqueous solution until the washing liquid is clear and transparent, and obtains the washed precipitate. The solid-liquid separation method of the present invention preferably includes centrifugation, and the rotation speed of the centrifugation is preferably 4000r / min, and the time is preferably 20min. The alkaline aqueous solution of the present invention preferably includes an ammonia solution, and the volume concentration of the ammonia solution is preferably 1%-1.5%, more preferably 1%. The present invention does not specifically limit the washing conditions, and the conventional washing conditions in the art can be used. The washed precipitate of the present invention is a crude solanine extract.

[0048] After obtaining the crude solanine extract, the present invention preferably purifies the crude solanine extract to obtain solanine. The purification method of the present invention preferably includes solid phase extraction, and the extraction column of the solid phase extraction is preferably a C18 solid phase extraction column. The present invention preferably performs solid phase extraction on the crude solanine extract to obtain the eluent, and performs rotary evaporation and drying, and the obtained powder is the solanine. The activation of the solid phase extraction of the present invention preferably includes methanol activation and distilled water activation in sequence; elution is preferably distilled water elution; elution is preferably methanol elution and isopropanol elution in sequence, and the volume ratio of methanol to isopropanol is preferably 4:1. The present invention preferably further includes vacuumizing the extraction column after the activation, elution and elution. The present invention adopts C18 solid phase extraction column purification technology, and does not need to use a large amount of organic reagents for repeated extraction. Therefore, there is no problem that the solvent is difficult to handle, the toxicity is strong and the pollution is large. It has the technical advantages of convenient operation, low energy consumption, simple process, no secondary pollution, and greatly improves the extraction efficiency. The solanine of the present invention preferably includes α-chaconine and α-solanine.

[0049] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0050] Example 1

[0051] A method for extracting solanine from potato organs, comprising the following steps:

[0052] 1. Experimental materials: When potatoes enter the maturity stage, take their above-ground stems and leaves, cut the stems into stem segments of about 2 cm in length, transfer the stem segments and leaves to a drying oven and dry them at a constant temperature of 70°C until the sample maintains a constant weight. After taking them out, grind them in a grinder and pass them through a 60-mesh sieve. Take the sieved material to obtain the stem and leaf experimental sample - potato stem and leaf powder, which is stored in a sealed bag for later use.

[0053] 2. Solanine extraction: Accurately weigh 5 g of pretreated potato stem and leaf powder and 1.5% cellulase (enzyme activity ≥ 50,000 U / g) of the potato stem and leaf powder, add 40 mL of acid water with a pH value of 5 (adjust distilled water to pH 5 with 0.02 mol / L hydrochloric acid to make acid water), vortex mix, extract in a water bath at 50°C for 1.5 h, and perform ultrasonic treatment at the beginning of the extraction. The ultrasonic conditions are: power 300 W, action time 30 min. After the enzymatic hydrolysis is completed, the solution is heated to 95°C to inactivate the enzyme, anhydrous ethanol is added to a volume fraction of 70%, and the extract is allowed to stand for 18 hours. The extract is filtered under reduced pressure, the filtrate is concentrated in a rotary evaporator, and the concentrate is dissolved with 30 mL of 5% H2SO4 by volume. After reduced pressure filtration, the pH value of the filtrate is adjusted to 10.0-10.5 with concentrated ammonia water, and the filtrate is placed in a 4°C refrigerator overnight, and then centrifuged at 4000r / min for 20 minutes. The precipitate is washed several times with 1% ammonia water by volume until the washing solution is clear and transparent, and the precipitate is the crude solanine extract.

[0054] 3. Detection of solanine content: Use methanol to fully dissolve the solanine crude extract, then use activated carbon to remove the pigment, and finally concentrate it into 5mL methanol solution, and measure the absorbance at a wavelength of 300nm. Figure 1 ) and α-chaconine standard curve ( Figure 2 ), the absorbance was measured at a wavelength of 300 nm using a spectrophotometer, and the solanine content was calculated. Figure 3 And as shown in Table 3.

[0055] The method for preparing the standard curve is as follows:

[0056] α-Solanine standard solution: Accurately weigh 5.000 mg of α-solanine standard and dissolve it in 5 mL of methanol solution. Prepare different concentrations of solanine standard solutions according to Table 1 and measure the absorbance at a wavelength of 300 nm.

[0057] α-chaconine standard solution: Accurately weigh 5.000 mg of α-chaconine standard and dissolve it in 5 mL of methanol solution. Prepare different concentrations of solanine standard solutions according to Table 2 and measure the absorbance at a wavelength of 300 nm.

[0058] Table 1 Different concentrations of α-solanine standard solutions and absorbance values

[0059] α-Solanine standard solution (mL) 0 0.2 0.4 0.6 0.8 1.0 Methanol 2 1.8 1.6 1.4 1.2 1 α-Solanine 0 0.1 0.2 0.3 0.4 0.5 <![CDATA[OD 300 ]]> 0 0.043 0.057 0.09 0.14 0.147

[0060] Table 2 Different concentrations of α-chaconine standard solutions and absorbance values

[0061] α-Chaconine standard solution (mL) 0 0.2 0.4 0.6 0.8 1.0 Methanol 2 1.8 1.6 1.4 1.2 1 α-Chaconine 0 0.1 0.2 0.3 0.4 0.5 <![CDATA[OD 300 ]]> 0 0.03 0.06 0.08 0.11 0.14

[0062] Comparative Example 1

[0063] The acid water extraction method is used to extract solanine. The steps are as follows:

[0064] The pH value of distilled water was adjusted to 5 with 0.02 mol / L hydrochloric acid to prepare acid water, 40 mL of the acid water solution was measured, 5.0 g of potato stem and leaf powder was added, vortexed and mixed, water bathed at 50°C for 1.5 h, centrifuged at 4000 r / min for 20 min, the supernatant was taken, the residue was added with acid water and extracted once (the extraction time was 2 h), the two extracts were combined, and after cooling, anhydrous ethanol was added to the alcohol volume fraction of 70%, and the extract was allowed to stand for 18 h. The extract was filtered under reduced pressure, the filtrate was concentrated in a rotary evaporator, and the concentrate was dissolved with 30 mL of 5% H2SO4 by volume. After filtering under reduced pressure, the pH value of the filtrate was adjusted to 10.0-10.5 with concentrated ammonia water, and placed in a 4°C refrigerator overnight, and then centrifuged at 4000 r / min for 20 min. The precipitate was washed several times with 1% ammonia water until the washing liquid was clear and transparent, and the precipitate was the crude extract.

[0065] The crude extract was fully dissolved in methanol, and then the pigment was removed with activated carbon. Finally, it was concentrated into 5 mL of methanol solution. The absorbance was measured at a wavelength of 300 nm. The solanine content was calculated according to the method in step 3 of Example 1. The results are as follows: Figure 3 And as shown in Table 3.

[0066] Comparative Example 2

[0067] The acid enzyme solution is used to extract solanine, and the steps are as follows:

[0068] Accurately weigh 5g potato stem and leaf powder and 1.5% cellulase by weight of potato powder, add 40mL of acid water (preparation method is the same as comparative example 1, acid water pH = 5), vortex mix, and extract in a water bath at 50°C for 1.5h. After the enzymolysis is completed, the solution is heated to 95°C to inactivate the enzyme, anhydrous ethanol is added to an alcohol volume fraction of 70%, and it is allowed to stand for 18h. The extract is filtered under reduced pressure, the filtrate is concentrated in a rotary evaporator, the concentrate is dissolved with 30mL of 5% H2SO4 by volume, filtered under reduced pressure, and the pH value of the filtrate is adjusted to 10.0-10.5 with concentrated ammonia water, placed in a 4°C refrigerator overnight, and then centrifuged at 4000r / min for 20min, and the precipitate is washed several times with 1% ammonia water until the washing liquid is clear and transparent, and the precipitate is the crude extract.

[0069] The crude extract was fully dissolved in methanol solution, and then the pigment was removed by activated carbon. Finally, it was concentrated into 5 mL of methanol solution. The absorbance was measured at a wavelength of 300 nm. The solanine content was calculated according to the method in step 3 of Example 1. The results are as follows: Figure 3 And as shown in Table 3.

[0070] Comparative Example 3

[0071] Ultrasonic assisted extraction of solanine, the steps are as follows:

[0072] Accurately weigh 5g of potato stem and leaf powder, add 40mL of acid water (preparation method is the same as comparative example 1, acid water pH = 5), vortex mix, extract in a water bath at 50°C for 1.5h, and perform ultrasonic treatment at the beginning of extraction, the ultrasonic conditions are: power is 300w, action time is 30min. Add anhydrous ethanol to 70% alcohol volume fraction, and let stand for 18h. Perform vacuum filtration on the extract, concentrate the filtrate in a rotary evaporator, dissolve the concentrate with 30mL of 5% H2SO4 by volume, adjust the pH value of the filtrate to 10.0-10.5 with concentrated ammonia water after vacuum filtration, place in a 4°C refrigerator overnight, then centrifuge at 4000r / min for 20min, wash the precipitate with 1% ammonia water several times until the washing liquid is clear and transparent, and the precipitate is the crude extract.

[0073] The crude extract was fully dissolved in methanol solution, and then the pigment was removed by activated carbon. Finally, it was concentrated into 5 mL of methanol solution. The absorbance was measured at a wavelength of 300 nm. The solanine content was calculated according to the method in step 3 of Example 1. The results are as follows: Figure 3 And as shown in Table 3.

[0074] Table 3 Extraction yield of solanine by different extraction methods in Example 1 and Comparative Examples 1-3

[0075]

[0076] From Table 3 and Figure 3It can be concluded that the extraction efficiency of the four different extraction methods is from high to low: ultrasound combined with enzyme extraction > acid enzyme solution extraction > ultrasonic extraction > acid water extraction. The potato solanine content is the highest when the ultrasound combined with enzyme extraction is used, the content of α-chaconine is 8.949 mg / g dry weight, the content of α-solanine is 8.058 mg / g dry weight, and the extraction rate is 0.850%; while the solanine content extracted by ultrasound alone is significantly lower than that of the present invention, the content of α-chaconine is only 7.133 mg / g dry weight, the content of α-solanine is 6.412 mg / g dry weight, and the extraction rate is 0.677%. Obviously, the effect of biological enzyme combined with ultrasonic treatment is better than that of single ultrasonic treatment. The extraction effect after adding biological enzyme is significantly better than that of non-enzyme extraction method.

[0077] Example 2

[0078] The steps for purification of crude solanine extract are as follows:

[0079] The crude solanine extract prepared in Example 1 was purified using a C18 solid phase extraction column. Before use, 10 mL of methanol was first added to activate the extraction column, followed by vacuuming and discarding the eluent. 10 mL of distilled water was used to activate the extraction column again, and the liquid was discarded. 20 g of solanine crude extract powder was dissolved in 20 mL of methanol, and then 5 mL of the dissolved solanine crude extract solution was added to the filter column, vacuumed, and the waste liquid was discarded. Then 10 mL of distilled water was added for leaching, vacuumed, and the eluent was discarded. A collection tube was placed under the extraction column, 10 mL of methanol was added to the column, and the eluent was collected by vacuuming. 2.5 mL of isopropanol was added again, vacuumed, and the eluent was collected in the same tube. After rotary evaporation, it was dried into powder to obtain 1.4 g of purified solanine extract. The yield of solanine in the separation and purification process was 83% and the purity was 31.18% measured by ultraviolet-visible spectrophotometry based on α-chaconine standard.

[0080] Example 3

[0081] 1 Single factor test

[0082] 1.1 Effect of enzyme addition on solanine extraction

[0083] Five portions of potato stem and leaf powder samples with a mass of 5.000 g were accurately weighed, and an extractant was added. Solanine was extracted from the samples according to the preset method in Example 1. The levels of other factors were kept unchanged, and the effects of different amounts of enzyme added on the amount of solanine extracted from potatoes were investigated. The results are as follows: Figure 4 shown.

[0084] The bio-enzyme degradation method can decompose the plant cell wall in a very short time, promote the outflow of the active ingredients of the natural products inside, and greatly improve the extraction rate of the target ingredients. Therefore, the selection of appropriate cellulase concentration can not only improve the extraction efficiency of solanine, but also reduce the waste of enzymes.

[0085] from Figure 4 It can be seen that with the increase of enzyme concentration, the content of solanine shows a trend of first increasing and then decreasing. When the enzyme addition amount increases from 1% to 2%, the extraction amount of potato solanine increases, and when the enzyme addition amount is 2%, the extraction amount of potato solanine reaches the highest point. When the enzyme addition amount is lower than 1.5%, the enzyme concentration is too low, resulting in limited enzymatic hydrolysis rate. However, there is no significant difference in the extraction amount when the enzyme addition amount is 2.5% and 3%. When the enzyme concentration is too high, due to the competitive inhibition of the enzyme, the enzymatic hydrolysis degree of the cell wall is reduced, resulting in a decrease in intracellular dissolution. Considering the cost issue, it is more reasonable to add 1% to 3% of cellulase. Therefore, the enzyme addition amount of 2% was selected for subsequent experiments.

[0086] 1.2 Effect of enzymatic hydrolysis time on solanine extraction

[0087] Five potato samples with a mass of 5.000 g were accurately weighed, and an extractant was added. The solanine therein was extracted according to the preset method in Example 1. The levels of other factors were kept unchanged, and the effects of different enzymolysis times on the solanine content in potatoes were investigated. The test results are as follows: Figure 5 shown.

[0088] Appropriate enzymatic hydrolysis time can not only improve the extraction efficiency of solanine, but also reduce the waste of experimental time. Figure 5 It can be seen that with the extension of enzymatic hydrolysis time, the content of solanine showed a trend of first increasing and then decreasing. When the enzymatic hydrolysis time increased from 0.5h to 1.5h, the amount of solanine extracted from potatoes gradually increased, and reached the maximum value at the enzymatic hydrolysis time of 1.5h; however, when the enzymatic hydrolysis time was less than 1.5h, the enzymatic hydrolysis rate was limited; when the enzymatic hydrolysis time was 2h and 2.5h, there was no significant difference in the extracted solanine content, which indicated that when the enzymatic hydrolysis time exceeded 2h, the cellulase activity decreased, resulting in a decrease in the amount of extracted solanine in potatoes. Therefore, the enzymatic hydrolysis time of 1.5h was selected for subsequent experiments.

[0089] 1.3 Effect of enzymatic hydrolysis temperature on solanine extraction

[0090] Five potato experimental samples with a mass of 5.0000 g were accurately weighed, and an extractant was added. The solanine therein was extracted according to the preset method in Example 1. The levels of other factors were kept unchanged, and the effects of different enzymatic hydrolysis temperatures on the solanine extraction amount in potatoes were investigated. The experimental results are as follows: Figure 6shown.

[0091] During the enzymatic hydrolysis process, the enzymatic hydrolysis temperature affects the solanine extraction rate by affecting the activity of the enzyme. On the other hand, the increase in temperature accelerates the transfer of substances within the cell and enhances the molecular movement rate of cellulase. Higher temperatures can also moderately soften the cell wall on the surface of the potato peel, thereby affecting the solanine extraction rate. Figure 6 It can be seen that during the extraction process, with the increase of enzymatic hydrolysis temperature, the solanine content showed a trend of first increasing and then decreasing. When the enzymatic hydrolysis temperature was 50°C, the solanine extraction amount reached the highest. When the temperature rose to 65°C, the solanine extraction amount was the lowest, which may be due to the partial inactivation of cellulase due to the high temperature. Therefore, the enzymatic hydrolysis temperature of 50°C was determined to be the optimal treatment time.

[0092] 2Solanine content in aboveground stems and leaves of different potato varieties

[0093] 5.00 g of dry powder of aboveground stems, leaves and buds of "Xisen No. 6" and "Ji Zhang Shu No. 12" were accurately weighed, and solanine was extracted using the optimized ultrasonic combined with enzyme method, i.e., the steps in Example 1 were used, except that the amount of cellulose added was 2% of the mass of the raw material. The solanine content in the stems, leaves and buds of different potato varieties was compared. The results are as follows: Figure 7 As shown in Table 4, the solanine content in the sprouts of the two varieties was significantly higher than that in the aboveground stems and leaves, and the solanine content in the aboveground stems and leaves of "Ji Zhang Shu No. 12" was also significantly higher than that of "Xi Sen No. 6", indicating that there are differences in solanine content among potato varieties.

[0094] Table 4 Solanine content extracted from different organs of different varieties

[0095] variety α-Solanine (mg / gDW) α-Chaconine (mg / gDW) Xisen No.6 Bud 7.784 8.646 Xisen No. 6 stem 1.818 2.064 Xisen No. 6 Leaf 2.435 2.745 Ji Zhang Shu No. 12 Bud 8.813 9.781 Ji Zhang Shu No. 12 Stem 2.847 3.199 Ji Zhang Shu No. 12 Leaf 3.189 3.577

[0096] It can be concluded from the above examples that the solanine extraction method of the present invention has a higher solanine extraction rate and is simpler.

[0097] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for extracting solanine from potatoes, characterized in that: The steps include: 1) mixing a potato powder sample with cellulase, and performing ultrasound-assisted enzymolysis in an acidic aqueous solution to obtain an enzymolysis reaction solution; 2) performing enzyme inactivation treatment on the enzymolysis reaction liquid to obtain an enzyme inactivated enzymolysis reaction liquid; 3) mixing the enzyme-inactivated enzymatic reaction liquid with anhydrous ethanol until the volume fraction of ethanol is 70%, and then standing for extraction to obtain an extract; 4) filtering the extract under reduced pressure, and concentrating the obtained filtrate to obtain a concentrate; 5) dissolving the concentrate with an acidic aqueous solution, and filtering the obtained solution again under reduced pressure to obtain a reduced pressure filtered solution; 6) adjusting the pH value of the vacuum filtered solution to 10.0-10.5, and allowing to stand under refrigeration to obtain a standing mixture; 7) subjecting the standing mixture to solid-liquid separation, washing the obtained precipitate with an alkaline aqueous solution until the washing liquid is clear and transparent, and the washed precipitate contains the solanine.

2. The method according to claim 1, characterized in that The dosage of the cellulase is 1%-3% of the mass of the potato powder sample, and the enzyme activity of the cellulase is ≥50000 U / g.

3. The method according to claim 1 or 2, characterized in that: The temperature of the ultrasound-assisted enzymatic hydrolysis is 50-55° C., the power is 250-300 W, and the pH value is 5; the total duration of the ultrasound-assisted enzymatic hydrolysis is 1.0-2.0 h, and the ultrasound action time is 30-50 min.

4. The method according to claim 1, characterized in that: Step 3) The extraction time is 10-18 hours.

5. The method according to claim 1, characterized in that: Step 5) The acidic aqueous solution is a sulfuric acid aqueous solution, and the volume fraction of the sulfuric acid aqueous solution is 5%.

6. The method according to claim 1, characterized in that Step 6) The standing time is 12-24 hours; the refrigeration temperature is 4-6°C.

7. The method according to claim 1, characterized in that The solid-liquid separation method is centrifugation, the centrifugal speed is 4000r / min, and the time is 20min; the alkaline aqueous solution is an ammonia solution, and the volume concentration of the ammonia solution is 1%-1.5%.

8. The method according to claim 1, characterized in that The method further comprises: purifying the washed precipitate by solid phase extraction, using an extraction column to perform the solid phase extraction, and the extraction column is a C18 solid phase extraction column.

9. The method according to claim 8, characterized in that The activation of the solid phase extraction column includes methanol activation and distilled water activation performed in sequence; the solid phase extraction also includes the steps of rinsing and eluting, the rinsing is distilled water rinsing; the elution is methanol elution and isopropanol elution performed in sequence, and the volume ratio of methanol to isopropanol is 4:

1.

10. The method according to claim 1, characterized in that The potato powder sample is derived from one or more of sprouts, stems and leaves of potatoes.

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