A pickled mustard cabbage extract, and a method of extracting and use thereof

Abstract

This invention provides a preserved mustard greens extract, its extraction method, and its application. During extraction, chopped dried preserved mustard greens are first soaked in ascorbic acid solution and then steamed with vinegar. The steamed dried mustard greens are then frozen at low temperature and ground into powder. The powder is then soaked in ascorbic acid-acetic acid solution under microwave stirring. Anhydrous calcium chloride and dichloromethane are added, and extraction is performed with ultrasonic assistance. The powder is filtered through a Buchner funnel. The extract is concentrated to obtain the primary extract, which is then processed using C... 18 The column was eluted, the eluent was collected and concentrated to dryness, then precipitated with alcohol, filtered through a Buchner funnel, and the filtrate was subjected to column chromatography. After concentration, dialysis was performed to remove salts, and the filtrate was freeze-dried to obtain the preserved mustard greens extract. The preserved mustard greens extract obtained in this application, through acidification, grinding, microwave treatment, ultrasonic extraction, two concentrations, purification, filtration, and chromatography, showed a significantly increased content of indole compounds. This preserved mustard greens extract can effectively improve plant growth rate and inhibit plant pathogenic microorganisms, thus benefiting plant growth.

Description

Technical Field

[0001] The present invention relates to the field of extraction and application of preserved mustard greens, and particularly to an extract of preserved mustard greens, its extraction method and application. Background Art

[0002] Preserved mustard greens are one of the popular ingredients in Guangdong region. It needs to go through multiple processes such as scalding, fermentation, sun-drying, and steam cooking. It has a golden color, a unique aroma, and rich dietary fiber. It can be paired with various ingredients to make delicious dishes, and has the effects of enhancing digestion and relieving greasy. Preserved mustard greens have a history of thousands of years in the diet. On this basis, Wang Chao from Xiangtan University studied the extract of preserved mustard greens and found that it has antioxidant and antibacterial properties, has antibacterial effects on four microorganisms, namely Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Aspergillus niger, and has a fresh-keeping effect on pork.

[0003] At present, the research on preserved mustard greens is only limited to the application in food. The usual extraction method is to crush the dried preserved mustard greens into powder and then carry out heating extraction with anhydrous ethanol or use the Soxhlet extraction method to obtain the ethanol extract of dried preserved mustard greens. Then, through suction filtration and reduced-pressure concentration, a brown extract paste is obtained. The extract paste is dissolved with distilled water and ultrasonic-assisted dispersion is used. The obtained suspension is successively subjected to fractional extraction with two organic solvents of different polarities, namely petroleum ether and ethyl acetate. The petroleum ether phase, ethyl acetate phase extraction liquid and raffinate are collected. Each extraction liquid and raffinate are concentrated and dried by rotary evaporation at 40 °C for standby. Due to the roughness of the traditional extraction method, many components in the sample cannot be effectively extracted. Therefore, the main component in the extracted preserved mustard greens extract is plant polyphenols. However, dried preserved mustard greens not only contain plant polyphenols, but also other organic components such as indole glucosinolate degradation products. Therefore, it is necessary to develop a new extraction method for preserved mustard greens extract, which can greatly improve the retention rate of effective components in the preserved mustard greens extract and is conducive to expanding the application field of the preserved mustard greens extract. Summary of the Invention

[0004] The present invention aims at the problems existing in the above-mentioned prior art and provides an extract of preserved mustard greens, its extraction method and application.

[0005] In order to achieve the above purpose, the present invention adopts the following technical measures:

[0006] An extraction method of an extract of preserved mustard greens, comprising the following steps:

[0007] Step 1: Cut the sun-dried dried preserved mustard greens into pieces, rinse them twice with distilled water, soak them in a 3% ascorbic acid solution for 15 - 60 s, and expose the dried preserved mustard greens to acetic acid vapor at 45 °C - 60 °C for 5 - 60 min;

[0008] Step 2: Place the steamed dried mustard greens in a low temperature environment of -120℃~-196℃ for 18~24h, take them out and grind them into mustard green powder at low temperature. Soak the mustard green powder in ascorbic acid-acetic acid solution and stir in a microwave environment of 300MHz~300kMHz for 1~5h.

[0009] Step 3: Add anhydrous calcium chloride and an equal volume of dichloromethane to the ascorbic acid-acetic acid solution. After ultrasonic extraction for 1-3 hours, filter using a Buchner funnel, wash the filter residue with dichloromethane, and obtain the extract.

[0010] Step 4: The extract is first concentrated in a vacuum concentrator to recover dichloromethane. Then, the concentration temperature is increased for a second concentration to obtain an extract paste. The paste is then dried with nitrogen to obtain the initial extract.

[0011] Step 5: Activate C with 15 mL of methanol 18 The column was equilibrated with deionized water. The initial extract was fully dissolved in petroleum ether:ethyl acetate (V:V=9.1:0.9), washed three times, and poured into the sample. The eluent was discarded. The sample was then eluted with petroleum ether:ethyl acetate (V:V=2:8), the eluent was collected and concentrated to dryness, and then precipitated with 95% ethanol for 5-10 hours. After filtration through a Buchner funnel, an ethanol solution of the extract was obtained.

[0012] Step 6: Filter the obtained extract ethanol solution through a microporous membrane with a pore size of 0.45 μm. Perform column chromatography on the filtrate, elute with 0.1 mol / L NaCl, collect the eluent in the elution peak region, concentrate it, dialyze to remove salt and freeze dry to obtain the preserved mustard greens extract.

[0013] In step 1, the preserved mustard greens are slightly acidified by a 3% ascorbic acid solution and acetic acid vapor before extraction to enhance their antioxidant properties and reduce microbial contamination. Furthermore, the indole thioglycoside degradation products in preserved mustard greens contain indole compounds (such as 7-hydroxyindole, 3,3-diindolemethane, and 3-indoleacetonitrile) and intermediates of indole thioglycoside degradation. These intermediates are unstable and difficult to extract. They can generate ascorbic acidogen and indole-3-methanol under the action of water and ascorbic acid. Under heating and weakly acidic conditions, indole-3-methanol can generate stable dimers such as 3,3-diindolemethane and other trimers, thus facilitating extraction and recovery.

[0014] In step 1, the fumigation temperature of the preserved mustard greens is 45℃~60℃. The temperature is not too high, which would destroy the indole compounds. It can also promote the condensation of intermediate products of indole glucosinolate degradation to form dimers and trimers. Using fumigation instead of blanching can reduce the loss of indole glucosinolate degradation products.

[0015] Specifically, the preserved mustard greens can be laid out in a single layer in a steamer. The steamer below the steamer contains a 36-40% acetic acid aqueous solution. By heating at 45℃-60℃, the acetic acid is accelerated to evaporate. The evaporated acetic acid can adhere to the surface of the preserved mustard greens, causing the degradation intermediates of indole glucosinolates in the preserved mustard greens to condense into dimers and trimers.

[0016] Furthermore, the dried plum vegetables mentioned in step 1 are prepared using the following steps:

[0017] Step A: Blanch the cleaned mustard greens in a 3% ascorbic acid solution at 40℃~60℃ for 5~10 seconds, then remove and air dry for 0.5~2 hours. Repeat this step three times.

[0018] Step B: Place the blanched mustard greens in a steamer and steam for 3-15 minutes. Then transfer them to a sealed container to ferment for 4-8 hours. Place the fermented mustard greens in the sun to dry for 1-5 days.

[0019] Step C: Place the pickled mustard greens obtained in step B in a steamer and steam for 50-80 minutes. Then place the pickled mustard greens in the sun to dry for 1-2 days. Repeat this step three times.

[0020] In this application, the pickled mustard greens are blanched for 5-10 seconds each time. This short blanching time, under acidic conditions, and at a temperature of 40℃-60℃, prevents the complete destruction of the fiber tissue and enzyme activity of the pickled mustard greens in a single blanching. The endogenous myrosinase in the pickled mustard greens is activated, which degrades glucosinolates and generates indole glucosinolate degradation products. Through multiple blanching and drying, sufficient reaction conditions and time are provided for the glucosinolate degradation of the pickled mustard greens. The intermediate products of indole glucosinolate degradation react with ascorbic acid to form indole dimers and trimers.

[0021] Furthermore, in step 2, the dried plum vegetables can be placed in a liquid nitrogen environment for low-temperature freezing for 18-24 hours.

[0022] Furthermore, in step 2, the ascorbic acid-acetic acid solution is prepared by dissolving 10g of ascorbic acid in 100mL of deionized water, then adding 100mg of EDTA and 30mL of glacial acetic acid and shaking well.

[0023] Furthermore, in step 2, the mixture of preserved mustard greens powder and ascorbic acid-acetic acid is transferred to an XH-100A+ microwave catalytic synthesis extractor and stirred for 1-5 hours in a microwave environment of 300MHz~300kMHz. Under the influence of the microwave field, the ascorbic acid-acetic acid solution undergoes strong oscillations, leading to the relaxation of hydrogen bonds between preserved mustard greens powder molecules, the breakdown of cell fiber structures, accelerated dissolution of the extractable components, and increased penetration of solution molecules. Simultaneously, it allows the degradation intermediates of indole thioglycosides to undergo a complete condensation reaction.

[0024] Furthermore, in step 2, the volume ratio of preserved mustard green powder to ascorbic acid-acetic acid solution is 1:10~50.

[0025] Furthermore, in step 3, the volume ratio of anhydrous calcium chloride to dichloromethane is 1:2~3.

[0026] Furthermore, in step 4, the temperature for the first concentration is 39°C and the concentration pressure is 45~50 kPa; the temperature for the second concentration is 95~100°C and the concentration pressure is 0.05~0.075 MPa.

[0027] Furthermore, in step 6, the molecular weight cutoff of the dialysis bag for dialysis is 3500 Da.

[0028] Furthermore, in step 6, column chromatography is performed using a DEAE-52 column or a Sephadex G-50 dextran gel column, eluted with 0.1 mol / L NaCl at a flow rate of 2 mL / min.

[0029] Furthermore, a preserved mustard greens extract is obtained using the aforementioned method for extracting preserved mustard greens.

[0030] Furthermore, the preserved mustard greens extract can be used in any of the following applications:

[0031] (1) Regulating plant growth rate;

[0032] (2) Preparation of formulations for regulating plant growth rate;

[0033] (3) Inhibit plant pathogenic microorganisms;

[0034] (4) Prepare agents for inhibiting plant pathogenic microorganisms.

[0035] Furthermore, this invention provides the application of plum vegetable extract in regulating plant growth rate:

[0036] Method 1: Apply by drenching: Dissolve the preserved mustard greens extract in 50-200 times the amount of water and spray it on the soil where the plants are planted. Spray evenly once every 2-3 months. Compared with watering, the plant growth rate is increased by more than 30%.

[0037] Method Two: Hydroponics. Dissolve the preserved mustard greens extract in 50-200 times its volume of water to obtain a preserved mustard greens extract solution. Soak the seeds in water at 20℃ for 10 minutes, then immerse the seeds in the preserved mustard greens extract solution for cultivation. Seeds can be from crops such as wheat, rice, barley, buckwheat, sorghum, and oats; legumes such as soybeans, broad beans, mung beans, red beans, and peanuts; and root vegetables such as sweet potato vines, potato tubers, and onions. Compared to traditional hydroponic media, using preserved mustard greens extract solution as the culture medium increases seed germination rate by more than 20% and promotes more developed root growth.

[0038] Method 3: Foliar spraying: Dissolve the preserved mustard greens extract in 50-200 times the amount of water to obtain a preserved mustard greens extract solution. Spray the preserved mustard greens extract solution on the leaves and branches of the crop for more vigorous crop growth.

[0039] Furthermore, the present invention provides the use of plum vegetable extract in the preparation of formulations for regulating plant growth rate: the formulation also includes excipients, which may be common excipients in fertilizers, or excipients commonly added when preparing plant growth promoters.

[0040] Specific auxiliary materials can be selected from at least one of the following: straw ash, organic fertilizer, humic acid, glucose, potassium fertilizer, nitrogen fertilizer, and phosphate fertilizer.

[0041] Furthermore, this formulation can be prepared in the form of powder, granules, capsules, liquid, tablets, pills, paste, emulsion, film, etc.

[0042] Furthermore, this invention provides the application of plum vegetable extract in inhibiting plant pathogenic microorganisms:

[0043] Method 1: Dissolve the preserved mustard greens extract in 50 to 200 times its volume of water, then irrigate the soil surface with the solution, and cover the soil surface with a transparent film. Using this method, the inhibition rate of pathogenic microorganisms in the soil can reach more than 65%.

[0044] Method 2: After crushing the preserved mustard greens extract into powder, spread it evenly on the soil surface. Apply 30 kg per acre, then turn the soil over and irrigate with water until the soil is infiltrated. Using this method, the inhibition rate of pathogenic microorganisms in the soil can reach more than 55%.

[0045] Method 3: Dissolve the preserved mustard greens extract in 50 to 200 times its volume of water to obtain a preserved mustard greens extract solution, and spray the preserved mustard greens extract solution on the leaves and branches of the crop.

[0046] Furthermore, the present invention provides the use of plum vegetable extract in the preparation of formulations for inhibiting plant pathogenic microorganisms: the formulation also includes excipients, which may be common excipients in sterilizing agents.

[0047] Furthermore, this formulation can be prepared in the form of powder, granules, capsules, liquid, tablets, pills, paste, emulsion, film, etc.

[0048] The beneficial effects of this invention are as follows:

[0049] This invention provides a preserved mustard greens extract, its extraction method, and its application. The preserved mustard greens extract obtained through acidification, grinding, microwave treatment, ultrasonic extraction, two concentrations, purification, filtration, and chromatography has a significantly increased content of indole compounds. This preserved mustard greens extract can effectively improve plant growth rate and inhibit plant pathogenic microorganisms, which is beneficial to improving plant growth. Attached Figure Description

[0050] Figure 1 The high-performance liquid chromatogram of 3-indoleacetonitrile is shown.

[0051] Figure 2 The high-performance liquid chromatogram of the plum vegetable extract from Example 1 is shown below.

[0052] Figure 3 The high-performance liquid chromatogram of the preserved mustard greens extract from Example 2 is shown below.

[0053] Figure 4 The high-performance liquid chromatogram of the plum vegetable extract in Example 3 is shown below.

[0054] Figure 5 The high-performance liquid chromatogram of the plum vegetable extract of Comparative Example 1 is shown.

[0055] Figure 6 The high-performance liquid chromatogram of the preserved mustard greens extract of Comparative Example 2 is shown below.

[0056] Figure 7 This is a comparison of bacterial colonies in different groups of culture dishes during the antibacterial test;

[0057] Figure 8 This is a comparison chart of the growth of bean seedlings in each planting pot from the second to the fourth day in the growth rate experiment. Detailed Implementation

[0058] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0059] In the embodiments of this application, the ascorbic acid-acetic acid solution is prepared by dissolving 10g of ascorbic acid in 100mL of deionized water, then adding 100mg of EDTA and 30mL of glacial acetic acid and shaking well.

[0060] Microwave catalytic synthesis extraction instrument: Xianghu XH-100AXH-100A computer-controlled microwave catalytic synthesis extraction instrument provided to Beijing Xianghu Technology Development Co., Ltd.

[0061] Low-temperature grinding: The JXFSTPRP-I low-temperature grinding machine provided by Shanghai Jingxin Industrial Development Co., Ltd. was used.

[0062] Ultra-low temperature industrial freezer: TF-B60-1500L deep freezer, seafood preservation box, and experimental industrial freezer provided to Hangzhou Aipu Instrument Equipment Co., Ltd.

[0063] Example 1:

[0064] A method for extracting a plum vegetable extract, comprising the following steps:

[0065] Step 1: Cut the dried mustard greens into small pieces, rinse twice with distilled water, soak in 3% ascorbic acid solution for 15 seconds, and expose the dried mustard greens to acetic acid vapor at 60℃ for 20 minutes.

[0066] Step 2: Place the steamed dried plum vegetables in a liquid nitrogen environment at -196℃ for 18 hours. After taking them out, grind them into plum vegetable powder at low temperature. Soak the plum vegetable powder in an ascorbic acid-acetic acid solution with a volume ratio of 1:30. Transfer the mixture to a microwave catalytic synthesis extractor and stir it in a microwave environment at 500MHz for 2 hours.

[0067] Step 3: Add anhydrous calcium chloride and an equal volume of dichloromethane to the ascorbic acid-acetic acid solution. The volume ratio of anhydrous calcium chloride to dichloromethane is 1:2. After ultrasonic-assisted extraction for 2 hours, filter using a Buchner funnel and wash the filter residue with dichloromethane to obtain the extract.

[0068] Step 4: The extract is placed in a vacuum concentrator for initial concentration at a temperature of 39°C and a pressure of 45 kPa. After recovering dichloromethane, the concentration temperature is increased for secondary concentration at a temperature of 95°C and a pressure of 0.05 MPa. The resulting extract is dried under nitrogen to obtain the initial extract.

[0069] Step 5: Activate C with 15 mL of methanol 18 The column was then equilibrated with deionized water. The initial extract was fully dissolved in petroleum ether:ethyl acetate (V:V=9.1:0.9), washed three times, and poured into the sample. The eluent was discarded. The sample was then eluted with petroleum ether:ethyl acetate (V:V=2:8), the eluent was collected and concentrated to dryness, and then precipitated with 95% ethanol for 6 hours. After filtration through a Buchner funnel, an ethanol solution of the extract was obtained.

[0070] Step 6: Filter the obtained extract ethanol solution through a microporous membrane with a pore size of 0.45 μm. Perform column chromatography on a Sephadex G-50 dextran gel column, elute with 0.1 mol / L NaCl at a flow rate of 2 mL / min, collect the eluent in the elution peak region, concentrate it, dialyze it with a dialysis bag with a molecular weight cutoff of 3500 Da to remove salt, and freeze-dry it to obtain the preserved mustard greens extract.

[0071] In this embodiment, the preserved mustard greens in step 1 are prepared using the following steps:

[0072] Step A: Blanch the cleaned mustard greens in a 3% ascorbic acid solution at 50℃ for 8 seconds, then remove and air dry for 1 hour. Repeat this step three times.

[0073] Step B: Place the blanched mustard greens in a steamer and steam for 8 minutes, then transfer them to a sealed container to ferment for 8 hours. Place the fermented mustard greens in the sun to dry for 3 days.

[0074] Step C: Place the pickled mustard greens obtained in step B in a steamer and steam for 60 minutes. Then place the pickled mustard greens in the sun to dry for 2 days. Repeat this step three times.

[0075] Example 2

[0076] A method for extracting a plum vegetable extract, comprising the following steps:

[0077] Step 1: Cut the dried mustard greens into small pieces, rinse twice with distilled water, soak in 3% ascorbic acid solution for 30 seconds, and expose the dried mustard greens to acetic acid vapor at 45℃ for 60 minutes.

[0078] Step 2: Place the steamed dried plum vegetables in an ultra-low temperature industrial freezer at -120℃ for 24 hours. After taking them out, grind them into plum vegetable powder at low temperature. Soak the plum vegetable powder in ascorbic acid-acetic acid solution with a volume ratio of 1:20. Transfer the mixture to a microwave catalytic synthesis extractor and stir it in a 300MHz microwave environment for 3 hours.

[0079] Step 3: Add anhydrous calcium chloride and an equal volume of dichloromethane to the ascorbic acid-acetic acid solution. The volume ratio of anhydrous calcium chloride to dichloromethane is 1:3. After ultrasonic extraction for 1.5 hours, filter using a Buchner funnel and wash the filter residue with dichloromethane to obtain the extract.

[0080] Step 4: The extract is first concentrated in a vacuum concentrator at a temperature of 39°C and a pressure of 48 kPa. After recovering dichloromethane, the concentration temperature is increased for a second concentration at 95°C and a pressure of 0.06 MPa. The resulting extract is then dried under nitrogen to obtain the initial extract.

[0081] Step 5: Activate C with 15 mL of methanol 18 The column was then equilibrated with deionized water. The initial extract was fully dissolved in petroleum ether:ethyl acetate (V:V=9.1:0.9), washed three times, and poured into the sample. The eluent was discarded. The sample was then eluted with petroleum ether:ethyl acetate (V:V=2:8), the eluent was collected and concentrated to dryness, and then precipitated with 95% ethanol for 5 hours. After filtration through a Buchner funnel, an ethanol solution of the extract was obtained.

[0082] Step 6: Filter the obtained extract ethanol solution through a microporous membrane with a pore size of 0.45 μm. Perform column chromatography on a Sephadex G-50 dextran gel column, elute with 0.1 mol / L NaCl at a flow rate of 2 mL / min, collect the eluent in the elution peak region, concentrate it, dialyze it with a dialysis bag with a molecular weight cutoff of 3500 Da to remove salt, and freeze-dry it to obtain the preserved mustard greens extract.

[0083] In this embodiment, the preserved mustard greens in step 1 are prepared using the following steps:

[0084] Step A: Blanch the cleaned mustard greens in a 3% ascorbic acid solution at 60℃ for 5 seconds, then remove and air dry for 1.5 hours. Repeat this step three times.

[0085] Step B: Place the blanched mustard greens in a steamer and steam for 15 minutes. Then transfer them to a sealed container to ferment for 6 hours. Place the fermented mustard greens in the sun to dry for 2 days.

[0086] Step C: Place the pickled mustard greens obtained in step B in a steamer and steam for 50 minutes. Then place the pickled mustard greens in the sun to dry for 1 day. Repeat this step three times.

[0087] Example 3

[0088] A method for extracting a plum vegetable extract, comprising the following steps:

[0089] Step 1: Cut the dried mustard greens into small pieces, rinse twice with distilled water, soak in 3% ascorbic acid solution for 60 seconds, and expose the dried mustard greens to acetic acid vapor at 50℃ for 30 minutes.

[0090] Step 2: Place the steamed dried plum vegetables in a liquid nitrogen environment at -196℃ for 20 hours. After taking them out, grind them into plum vegetable powder at low temperature. Soak the plum vegetable powder in an ascorbic acid-acetic acid solution with a volume ratio of 1:10. Transfer the mixture to a microwave catalytic synthesis extractor and stir it in an 800MHz microwave environment for 1 hour.

[0091] Step 3: Add anhydrous calcium chloride and an equal volume of dichloromethane to the ascorbic acid-acetic acid solution. The volume ratio of anhydrous calcium chloride to dichloromethane is 1:2. After ultrasonic-assisted extraction for 3 hours, filter using a Buchner funnel and wash the filter residue with dichloromethane to obtain the extract.

[0092] Step 4: The extract is placed in a vacuum concentrator for initial concentration at a temperature of 39°C and a pressure of 50 kPa. After recovering dichloromethane, the concentration temperature is increased for secondary concentration at a temperature of 100°C and a pressure of 0.075 MPa. The resulting extract is dried under nitrogen to obtain the initial extract.

[0093] Step 5: Activate C with 15 mL of methanol 18 The column was then equilibrated with deionized water. The initial extract was fully dissolved in petroleum ether:ethyl acetate (V:V=9.1:0.9), washed three times, and poured into the sample. The eluent was discarded. The sample was then eluted with petroleum ether:ethyl acetate (V:V=2:8), the eluent was collected and concentrated to dryness, and then precipitated with 95% ethanol for 10 hours. After filtration through a Buchner funnel, an ethanol solution of the extract was obtained.

[0094] Step 6: Filter the obtained extract ethanol solution through a microporous membrane with a pore size of 0.45 μm. Perform column chromatography on a DEAE-52 column, elute with 0.1 mol / L NaCl at a flow rate of 2 mL / min, collect the eluent in the elution peak region, concentrate it, dialyze it with a dialysis bag with a molecular weight cutoff of 3500 Da to remove salt, and freeze-dry it to obtain the preserved mustard greens extract.

[0095] In this embodiment, the preserved mustard greens in step 1 are prepared using the following steps:

[0096] Step A: Blanch the cleaned mustard greens in a 3% ascorbic acid solution at 40℃ for 10 seconds, then remove and air dry for 2 hours. Repeat this step three times.

[0097] Step B: Place the blanched mustard greens in a steamer and steam for 15 minutes. Then transfer them to a sealed container to ferment for 4 hours. Place the fermented mustard greens in the sun to dry for 1 day.

[0098] Step C: Place the pickled mustard greens obtained in step B in a steamer and steam for 70 minutes. Then place the pickled mustard greens in the sun to dry for 2 days. Repeat this step three times.

[0099] Comparative Example 1

[0100] A method for extracting a plum vegetable extract, comprising the following steps:

[0101] (1) The dried preserved mustard greens are pulverized into preserved mustard green powder by a high-speed universal pulverizer;

[0102] (2) Add anhydrous ethanol at a material-to-liquid ratio of 1:5 (w / v), and extract by shaking in a water bath at 60°C for 4 h. Filter the mixture, and repeat the extraction once with the residue under the above conditions. After filtration, combine the two filtrates and evaporate them under reduced pressure at 40°C to obtain a brown plum vegetable ethanol extract.

[0103] (3) Add a certain amount of distilled water and disperse with ultrasonic assistance. The resulting suspension is extracted with dichloromethane. Collect the organic phase extract and concentrate and dry the organic phase extract at 40°C to obtain the plum vegetable extract.

[0104] Specifically, the preserved mustard greens in step 1 are prepared using the following steps:

[0105] Step A: Blanch the cleaned mustard greens in water at 95~100℃ for 10 minutes, then transfer them to a sealed container to ferment for 12 hours. After fermentation, place the mustard greens in the sun to dry for 5 days.

[0106] Step B: Place the pickled mustard greens obtained in Step A in a steamer and steam for 70 minutes, then place the pickled mustard greens in the sun to dry for 5 days.

[0107] Comparative Example 2

[0108] The difference between this comparative example and Comparative Example 1 above is that the preparation method of the preserved mustard greens is different. In step 1, the preserved mustard greens used are those obtained from the preparation steps of Example 1.

[0109] Performance testing

[0110] Determination of active ingredients in extracts

[0111] Experimental Methods: The types and contents of compounds in *Prunus mume* extract were determined using an Agilent 1200 high-performance liquid chromatograph. Each sample was prepared into a 5 mg / ml solution using chromatographic-grade anhydrous methanol, and standards were prepared into a 0.2 mg / ml solution. All samples were filtered through a 0.45 μm membrane filter before injection. The selected chromatographic column was a C24-C24 column. 18Chromatographic column. Mobile phase: methanol-4% acetic acid (35:65), flow rate: 1 ml / min, column temperature: 40℃, injection volume: 10 μL, detection wavelength: 261 nm. The number of peaks in each sample was compared to determine the presence of a standard peak.

[0112] Standard solution: Take 0.02 g of 3-indoleacetonitrile and place it in a 20 ml volumetric flask. Add the mobile phase to dissolve and dilute to the mark. Shake well to prepare a 1.0 mg / ml 3-indoleacetonitrile solution. Take 1 ml of the 3-indoleacetonitrile solution and place it in a 100 ml volumetric flask. Add the mobile phase to dissolve and dilute to the mark to prepare a 20 μg / ml standard solution.

[0113] Sample solution: Take 0.02 g of the preserved mustard greens extract prepared in Examples 1 to 3 and Comparative Examples 1 to 2, place it in a 20 ml volumetric flask, add the mobile phase to dissolve and dilute to the mark, shake well to prepare an extract solution of 1.0 mg / ml, take 1 ml of the extract solution and place it in a 100 ml volumetric flask, add the mobile phase to dissolve and dilute to the mark to prepare a standard solution of 20 μg / ml;

[0114] Test results: Reference Figures 2-6 .

[0115] As shown in the figure, the number of HPLC peaks, retention times, and response values ​​of the samples from Examples 1 to 3 differed from those of Comparative Examples 1 to 2. The number of effective peaks and peak response values ​​of Examples 1 to 3 were greater than those of Comparative Examples 1 and 2, proving that the effective components in the preserved mustard greens extracts obtained from Examples 1 to 3 of this application were greater than those in Comparative Examples 1 and 2, while Comparative Example 1 had the fewest peaks and the lowest peak response values. 3-Indoleacetonitrile was used as the standard (chromatogram reference). Figure 1 Based on the retention time of the peak, the presence of 3-indoleacetonitrile in each sample was identified. It was found that 3-indoleacetonitrile was present in Examples 1 to 3 and Comparative Example 2, but the peak response value of Comparative Example 2 was lower than that of Examples 1 to 3.

[0116] Antibacterial test

[0117] Test method:

[0118] (1) Select the brownish gelatinous substance from the rotten part of the cabbage and inoculate it into 50 ml of LB liquid medium. Incubate at 25℃ for 24 h to obtain a bacterial suspension.

[0119] (2) Take 1 ml of bacterial suspension and add it to 19 ml of sterile physiological saline to dilute. Place it on a shaker and shake at 200 r / min for 10 min. Dispense it into 6 test tubes, each containing 1.5 ml of bacterial suspension. Label the walls of the 6 test tubes with numbers 1 to 6 respectively.

[0120] (3) Mark the lids of the six petri dishes with symbols 7 to 12 respectively, add 20 ml of melted agar medium to each petri dish, sterilize at 121°C for 20 min, cool and solidify, and place on a clean bench.

[0121] (4) Take 1g of each of the preserved mustard green extracts of Examples 1 to 3 and Comparative Examples 1 to 2, dissolve them in 100ml of deionized water to obtain Sample 1 to Sample 5. Add 0.5ml of Sample 1 to Sample 5 to the test tubes labeled 1 to 5 respectively, and add 0.5ml of deionized water to the test tube labeled 6. Shake well.

[0122] (5) Take 1 ml of sample solution from test tubes labeled 1 to 6 and pour it into culture dishes labeled 7 to 12 respectively. Shake the culture dishes to make the sample solution evenly cover the surface of the agar medium.

[0123] (6) Place the petri dishes in an incubator and incubate at 25°C for 48 hours. Calculate the number of colonies in each petri dish and the inhibition rate.

[0124] Test results: Reference Figure 7 .

[0125] As shown in the figure, the culture dish labeled 12 did not contain the preserved mustard greens extract, and its culture medium had a dense distribution of colonies. Although the number of colonies in culture dishes labeled 10 and 11 was less than that in culture dish labeled 12, it was much greater than that in culture dishes labeled 7-9. The number of colonies in each culture dish was calculated, and the results are shown in Table 1 below. It can be seen that the preserved mustard greens extracts of Examples 1-3 have antibacterial effects.

[0126] Table 1 shows the colony counts on each culture dish in the antibacterial test.

[0127]

[0128] Growth rate test

[0129] Test method:

[0130] (1) The pure plant-type cultivation substrate provided by Nanjing Perennial Flower Botanical Garden Co., Ltd. was divided into 4 planting pots, each containing 200g of cultivation substrate, and 20 peas were planted in each of the 4 planting pots.

[0131] (2) Take 100 g of the preserved vegetable extracts of Examples 1 to 2 and Comparative Examples 1 to 2, and dissolve them in 1000 ml of water respectively to obtain hydroponic agents 1 to 4. Arrange 4 planting pots in a "field" shape in two layers. Irrigate 50 ml of hydroponic agent 4 into the right planting pot in the upper layer, 50 ml of hydroponic agent 3 into the left planting pot in the upper layer, 50 ml of hydroponic agent 1 into the right planting pot in the lower layer, and 50 ml of hydroponic agent 2 into the left planting pot in the lower layer. Irrigate once a day, and compare the growth of the mung bean sprouts in the 4 planting pots on the second, third, and fourth days.

[0132] Test results: Refer to Figure 8 .

[0133] As can be seen from the figure, within the same growth time and growth environment, in the planting pots irrigated with hydroponic agents 1 and 2, the germination rate and growth of the peas are significantly better than those of the peas irrigated with hydroponic agents 3 and 4. Thus, it can be seen that the preserved vegetable extract of the present application has an obvious effect on regulating the plant growth rate.

[0134] The above content is a further detailed description of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, and all should be regarded as belonging to the protection scope of the present invention.

Claims

1. A method for extracting a plum vegetable extract, characterized in that, Includes the following steps: Step 1: Cut the dried mustard greens into small pieces, rinse twice with distilled water, soak in 3% ascorbic acid solution for 15-60 seconds, and expose the dried mustard greens to acetic acid vapor at 45℃-60℃ for 5-60 minutes. Step 2: Place the steamed dried mustard greens in a low temperature environment of -120℃~-196℃ for 18~24h, take them out and grind them into mustard green powder at low temperature. Soak the mustard green powder in ascorbic acid-acetic acid solution and stir in a microwave environment of 300MHz~300kMHz for 1~5h. Step 3: Add anhydrous calcium chloride and an equal volume of dichloromethane to the ascorbic acid-acetic acid solution. After ultrasonic extraction for 1-3 hours, filter using a Buchner funnel and wash the filter residue with dichloromethane to obtain the extract. Step 4: The extract is first concentrated in a vacuum concentrator to recover dichloromethane. Then, the concentration temperature is increased for a second concentration to obtain an extract paste. The paste is then dried with nitrogen to obtain the initial extract. Step 5: Activate C with 15 mL of methanol 18 The column was equilibrated with deionized water. The initial extract was fully dissolved in petroleum ether:ethyl acetate (V:V = 9.1:0.9), washed three times, and poured into the sample. The eluent was discarded. The sample was then eluted with petroleum ether:ethyl acetate (V:V = 2:8). The eluent was collected and concentrated to dryness. The sample was then precipitated with 95% ethanol for 5-10 hours. After filtration through a Buchner funnel, an ethanol solution of the extract was obtained. Step 6: Filter the obtained extract ethanol solution through a microporous membrane with a pore size of 0.45 μm, perform column chromatography on the filtrate, elute with 0.1 mol / L NaCl, collect the eluent in the elution peak region, concentrate it, dialyze to remove salt and freeze dry to obtain plum vegetable extract. The dried plum vegetables are prepared using the following steps: Step A: Blanch the cleaned mustard greens in a 3% ascorbic acid solution at 40℃~60℃ for 5~10 seconds, then remove and air dry for 0.5~2 hours. Repeat this step three times. Step B: Place the blanched mustard greens in a steamer and steam for 3-15 minutes. Then transfer them to a sealed container to ferment for 4-8 hours. Place the fermented mustard greens in the sun to dry for 1-5 days. Step C: Place the pickled mustard greens obtained in step B in a steamer and steam for 50-80 minutes. Then place the pickled mustard greens in the sun to dry for 1-2 days. Repeat this step three times.

2. The method for extracting plum vegetable extract according to claim 1, characterized in that, In step 2, the ascorbic acid-acetic acid solution is prepared by dissolving 10g of ascorbic acid in 100mL of deionized water, then adding 100mg of EDTA and 30mL of glacial acetic acid and shaking well.

3. The method for extracting plum vegetable extract according to claim 1, characterized in that, In step 2, the volume ratio of preserved mustard green powder to ascorbic acid-acetic acid solution is 1:10 to 50.

4. The method for extracting plum vegetable extract according to claim 1, characterized in that, In step 3, the volume ratio of anhydrous calcium chloride to dichloromethane is 1:2 to 3.

5. The method for extracting plum vegetable extract according to claim 1, characterized in that, In step 4, the temperature for the first concentration is 39°C and the concentration pressure is 45–50 kPa; the temperature for the second concentration is 95–100°C and the concentration pressure is 0.05–0.075 MPa.

6. The method for extracting plum vegetable extract according to claim 1, characterized in that, In step 5, the alcohol precipitation concentration is 60%–80%, and the alcohol precipitation temperature is 2–6°C.

7. The method for extracting plum vegetable extract according to claim 1, characterized in that, In step 6, the molecular weight cutoff of the dialysis bag is 3500 Da.

8. A plum vegetable extract, characterized in that, The extract was obtained using the extraction method of plum vegetable extract according to any one of claims 1-7.

9. The application of the preserved mustard greens extract according to claim 8 in any of the following: (1) Regulating plant growth rate; (2) Preparation of formulations for regulating plant growth rate; (3) Inhibit plant pathogenic microorganisms; (4) Prepare agents for inhibiting plant pathogenic microorganisms.

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