Matrine-bacterial cellulose gel prepared through in-situ growth, preparation method of matrine-bacterial cellulose gel, matrine-bacterial cellulose sustained-release particles and application of matrine-bacterial cellulose sustained-release particles

Abstract

The invention provides a method for preparing sophocarpidine-bacterial cellulose gel through in-situ growth, which comprises the following steps: crushing fresh yellow wine vinasse by using a stirrer, adding water, boiling, cooling to 60 DEG C, then adding glucoamylase, fully and uniformly stirring, carrying out enzymolysis, and filtering by using gauze after the enzymolysis is finished to obtain vinasse enzymatic hydrolysate; adding cane sugar and black tea into water, heating to boil, cooling to room temperature, filtering, taking filtrate, adding the wall-broken hibiscus manihot flavone extract into the filtrate, and stirring for dissolving to obtain a fermentation aid; taking 200ml of vinasse enzymatic hydrolysate, adjusting the pH value to 4, then adding a fermentation auxiliary agent and sophocarpidine, inoculating acetobacter gluconoxylinum, standing and culturing for 7 days at the constant temperature of 25 DEG C, and then washing and draining to obtain the sophocarpidine-bacterial cellulose gel. Meanwhile, the invention further provides the prepared matrine-bacterial cellulose gel, sustained-release particles and application of the matrine-bacterial cellulose gel and the sustained-release particles. The problems that in the prior art, the workload is large and the cost is high due to the fact that matrine needs to be frequently applied many times are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of matrine embedding, and in particular to a matrine-bacterial cellulose gel prepared by in-situ growth and a preparation method thereof, matrine-bacterial cellulose slow-release particles and applications thereof. Background Art

[0002] Matrine is a water-soluble alkaloid extracted from the leguminous plants Sophora flavescens and Sophora alopecuroides. It paralyzes the insect's nerve center, causing protein coagulation and clogging of its pores, leading to suffocation and death. It is currently the most widely registered and best-selling botanical insecticide. Currently, matrine pesticide formulations, including powders, aqueous solutions, and emulsifiable concentrates, all suffer from poor stability, a short half-life, and a slow onset of action, resulting in poor efficacy (the effect is halved after 5 days, and the pest is dead after 3 days). This results in the need for frequent and repeated applications, resulting in significant pollution, heavy workload, high costs, and poor market competitiveness. Summary of the Invention

[0003] In view of the shortcomings of the existing technology, the present invention provides a method for preparing matrine-bacterial cellulose gel by in situ growth, which solves the problem that matrine needs to be applied multiple times and frequently in the existing technology, resulting in large workload and high cost.

[0004] According to an embodiment of the present invention, a method for preparing matrine-bacterial cellulose gel by in situ growth comprises the following steps:

[0005] Preparation of liquid culture medium: fresh rice wine lees were crushed with a blender, boiled with water, cooled to 60°C, and then glucoamylase was added and stirred thoroughly for enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the enzymatic hydrolyzed lees solution was obtained by filtering with gauze.

[0006] Preparation of fermentation aid: adding sucrose and black tea to water, heating to boil, cooling to room temperature, filtering and collecting the filtrate, adding the wall-broken sunflower flavonoids extract to the filtrate, stirring and dissolving to obtain the fermentation aid;

[0007] Take 200ml of distiller's grains enzymatic hydrolysate and adjust the pH to 4, then add 20ml of fermentation aid and 25g of matrine, inoculate 12ml (bacteria concentration is 10 8 The Gluconobacter xylylene was cultured at a constant temperature of 25°C for 7 days, and then washed with clean water and drained to obtain matrine-bacterial cellulose gel.

[0008] Matrine is added to a liquid culture medium, and matrine is embedded in the three-dimensional spatial structure of bacterial nanocellulose through an in situ growth method. After molding, matrine-bacterial cellulose gel is obtained. The matrine inside the matrine-bacterial cellulose gel is not easily affected by oxygen and moisture in the air and microorganisms in the soil, and can be slowly released through the degradation of bacterial nanocellulose, thereby achieving a long-lasting and lasting insecticidal effect. This solves the problem in the prior art that matrine requires multiple and frequent applications, resulting in a large workload and high cost.

[0009] Furthermore, when preparing the liquid culture medium, 200 g of rice wine lees was used, 600 ml of water was added, and 0.5% of the total mass fraction of glucoamylase (enzyme activity was 260,000 U / ml) was added based on the total mass fraction of rice wine lees and water.

[0010] Furthermore, the enzymatic hydrolysis condition is to keep the mixture at 60°C for 3 hours.

[0011] Furthermore, when preparing the fermentation aid, the amount of water used is 100 ml, sucrose 10 g, black tea 2 g, and 0.2 g of the cracked sunflower flavonoids extract are added.

[0012] Furthermore, the pH of the vinasse enzymatic hydrolyzate was adjusted with 10% by mass dilute hydrochloric acid.

[0013] Furthermore, after inoculating Gluconacetobacter xylylene, 5 ml was poured into each well of the round hole mold, and then the mixture was incubated at a constant temperature.

[0014] According to the embodiment, a matrine-bacterial cellulose gel is also provided, which is prepared by the above method.

[0015] According to the embodiment, a matrine-bacterial cellulose sustained-release granule is provided, which is obtained by drying the matrine-bacterial cellulose gel prepared by the above method, or by drying the above matrine-bacterial cellulose gel, and the drying conditions are: vacuum drying at 45°C for 24 hours.

[0016] According to the embodiment, there is also provided an application of matrine-bacterial cellulose slow-release particles for inhibiting the growth and reproduction of root-knot nematodes.

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

[0018] Matrine is embedded in the three-dimensional spatial structure of bacterial nanocellulose by an in-situ growth method (in the process of glucose molecules polymerizing, secreting, assembling and crystallizing to generate bacterial nanocellulose, matrine is embedded in the three-dimensional spatial structure of bacterial nanocellulose by an in-situ growth method), and a matrine-bacterial cellulose gel is obtained after molding. The matrine inside the matrine-bacterial cellulose gel is not easily affected by oxygen and moisture in the air and microorganisms in the soil, and can be slowly released through the degradation of bacterial nanocellulose, thereby achieving a long-lasting and lasting insecticidal effect. This solves the problem in the prior art that matrine requires multiple and frequent applications, resulting in a large workload and high cost; matrine has biological toxicity and has an inhibitory effect on the growth of Gluconobacter xylonyli. Direct addition of matrine will result in low bacterial nanocellulose film-forming efficiency and a small amount of matrine embedded. The present invention antagonizes the biological toxicity of matrine by adding a flavonoid extract of golden sunflower to the culture medium, promotes the growth of Gluconobacter xylonyli and the formation of nanocellulose, thereby achieving in-situ embedding molding of matrine. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a chromatogram of matrine in an embodiment of the present invention;

[0020] Figure 2 This is the standard curve of matrine in the embodiment of the present invention;

[0021] Figure 3 This is a photo of the matrine-bacterial cellulose gel before drying in an embodiment of the present invention;

[0022] Figure 4 This is an electron micrograph of a bacterial cellulose gel without matrine embedded in an embodiment of the present invention;

[0023] Figure 5 This is an electron micrograph of the matrine-bacterial cellulose gel encapsulating matrine in an embodiment of the present invention. DETAILED DESCRIPTION

[0024] The technical solutions of the present invention are further described below with reference to the accompanying drawings and embodiments.

[0025] The HPLC detection method used in the following examples is:

[0026] (1) Chromatographic conditions: Column: Welch Xtimate™ C18 (4.6 mm × 250 mm, 5 μm); Mobile phase: 0.02 mol / L triethylamine aqueous solution (adjusted to pH = 9.5 with glacial acetic acid): acetonitrile = 60:40; Flow rate: 1.0 ml / min; Detection wavelength: 220 nm; Injection volume: 20 μL; Column temperature: room temperature; Samples were dissolved in acetonitrile: water = 1:1.

[0027] (2) Drawing a standard curve for matrine: 2.000 mg of matrine standard was accurately weighed, and a 2.000 mg / ml matrine solution was prepared with acetonitrile-water (V:V=1:1) as solvent. The solutions were diluted in sequence to obtain 1.000 mg / ml, 0.500 mg / ml, 0.250 mg / ml, and 0.125 mg / ml matrine standard solutions. HPLC detection was performed in sequence, and a standard curve was drawn with the peak area as the ordinate and the concentration as the abscissa. The equation for the standard curve for matrine was calculated as follows: Y=251.178X+0.375(R 2 =0.9995).

[0028] Some of the materials used in the examples were obtained as follows: fresh rice wine lees were obtained from Zhejiang Jiashan Yellow Wine Co., Ltd., matrine was purchased from Maclean Company (content 98%), Gluconobacter xylinum was purchased from Taobao (purchase link: https: / / e.tb.cn / h.6WqCqswp7NiwotC?tk=ab8HevV08ok), and the wall-broken sunflower flavonoids extract was prepared using the method provided in the invention of a Rhizopus arrhizus JHK31 and its application in the assisted extraction of sunflower flavonoids with publication number CN116769612A.

[0029] Example 1

[0030] In an exemplary embodiment, this example provides an in situ growth preparation of matrine-bacterial cellulose gel, which is prepared by the following method:

[0031] 200g of fresh yellow rice wine lees were crushed with a blender, 600ml of water was added and boiled, cooled to 60°C, and then 0.5% of the total mass fraction of yellow rice wine lees and water was added with glucoamylase (enzyme activity of 260000U / ml) and stirred thoroughly, and the mixture was kept at 60°C for 3h for enzymolysis. After the enzymolysis was completed, lees hydrolyzate was obtained by filtering with gauze; 10g of sucrose and 2g of black tea (Jin Junmei black tea) were added to 100ml of water, heated and boiled, cooled to room temperature, filtered, and the filtrate was taken, and 0.2g of broken-wall sunflower flavonoids extract was added to the filtrate, and the fermentation aid was obtained by stirring and dissolving; 200ml of lees hydrolyzate was taken and the pH of the lees hydrolyzate was adjusted to 4 with a mass fraction of 10% dilute hydrochloric acid, and then 20ml of fermentation aid and 25g of matrine were added, and 12ml (bacterial concentration was 10 8 / ml), Gluconobacter xylonyli, 5ml per well was poured into a round hole mold, and then cultured at a constant temperature of 25℃ for 7 days. Finally, it was washed with clean water and placed on gauze to drain for 5 minutes to obtain matrine-bacterial cellulose gel with a wet weight of 172.9g. After drying, matrine-bacterial cellulose sustained-release granules were obtained. The photo of matrine-bacterial cellulose gel before drying is shown in the figure. Figure 3A total of 61.2 ml of the remaining fermentation broth was collected and the matrine content in the remaining fermentation broth was determined to be 7.012 g using the HPLC external standard method. The calculated amount of matrine encapsulated in the bacterial cellulose gel was 17.988 g, and the encapsulation efficiency was 10.4% (encapsulation amount = 25 - 7.012 = 17.988 g, encapsulation efficiency = (17.988 ÷ 172.9) × 100% = 10.4%).

[0032] Example 2

[0033] Compared with Example 1, the following contents are different:

[0034] No 0.2 g of broken-wall sunflower flavonoids extract was added to the filtrate. After the culture was completed, the wet weight of the gel was weighed to be 9.71 g, and the remaining fermentation liquid was 221.8 ml. The remaining amount of matrine in the fermentation liquid was 24.506 g. The encapsulation amount of matrine in the bacterial cellulose gel was calculated to be 0.494 g, and the encapsulation rate was 5.21% (encapsulation amount = 25-24.506 = 0.494 g, encapsulation rate = (0.494÷9.71)×100% = 5.09%).

[0035] Compared with Example 1, the embedding rate of matrine in Example 2 is greatly reduced. This is because matrine has biological toxicity and inhibits the growth of Gluconobacter xylinum. Direct addition of matrine will lead to low bacterial nanocellulose film-forming efficiency and small matrine embedding amount.

[0036] The drying condition in the above Example 1 is vacuum drying at 45° C. for 24 h.

[0037] The matrine-bacterial cellulose gel obtained in Example 1 was dried in the laboratory and compared with the bacterial cellulose gel without matrine (dried in the laboratory under the same conditions), and their microscopic morphologies were observed by scanning electron microscopy. Figure 4 The unencapsulated bacterial cellulose gel has a clear three-dimensional network structure and pores. Figure 5 The matrine-bacterial cellulose gel is embedded with matrine. Due to the addition of matrine, the bacterial celluloses adhere to each other, and the pores in the matrine-bacterial cellulose gel become smaller and fewer. It is speculated that this may be because the oxygen atoms or nitrogen atoms in matrine form hydrogen bonds with the alcohol hydroxyl groups on the bacterial cellulose, making the structure of cellulose tighter.

[0038] 1 g of dried matrine-bacterial cellulose sustained-release granules was placed in 1 L of distilled water. The matrine content released into the water was determined by high performance liquid chromatography. The release rate of matrine in the matrine-bacterial cellulose sustained-release preparation was calculated. The results are shown in Table 1:

[0039] Table 1 Matrine release rate from matrine-bacterial cellulose gel

[0040]

[0041] Potted plant experiment:

[0042] Three-week-old tomato seedlings cultured in sterile soil were placed in 30 320 mm × 200 mm flower pots (divided into 3 groups of 10 pots each) for a blank group and a sample group, and the tomato seedlings were transplanted. The blank group received a hole application of 1 g of oven-dried bacterial cellulose gel (without matrine), while the sample group received a hole application of 1 g of oven-dried matrine-bacterial cellulose sustained-release granules prepared in Example 1. Both the blank and sample groups were also irrigated with 500 ml of water at the root level. The control group received a root application of 500 ml of a diluted matrine solution (5 ml of a 0.3% matrine solution diluted to 1000 ml). Thereafter, every 10 days, the control group received a root application of 500 ml of the diluted matrine solution.

[0043] The nematode inoculation method is to evenly make 8 holes about 2 cm deep around the tomato plants, and use a pipette to add an aqueous solution containing root-knot nematodes (second-instar larvae of southern root-knot nematodes). The root-knot nematode inoculation amount per pot of tomatoes is about 4,000.

[0044] 60 days after transplanting, dig out the tomato roots, gently shake off the rhizosphere soil, and determine the root-knot nematode incidence according to the disease level standards.

[0045] Table 2 Results of potted plant experiments on inhibiting the growth and reproduction of southern root-knot nematodes

[0046]

[0047] As can be seen from Table 2, the root-to-shoot ratio of the tomato plant that received a one-time hole application of matrine-bacterial cellulose gel was as low as 0.02, which was significantly lower than the root-to-shoot ratio of the tomato plant that received multiple root irrigations with 0.3% matrine solution. The root-to-shoot ratios of both groups were lower than those of the blank control group, indicating that the matrine-bacterial cellulose slow-release granules had the strongest inhibitory effect on the growth and reproduction of root-knot nematodes. Moreover, the matrine-bacterial cellulose slow-release granules can achieve a continuous insecticide effect by slowly releasing matrine. During the tomato planting process, only one hole application is required to inhibit the growth of root-knot nematodes throughout its growth cycle, making them more convenient to use than the matrine solution.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for preparing matrine-bacterial cellulose gel by in situ growth, characterized in that: The steps include: Preparation of liquid culture medium: fresh rice wine lees were crushed with a blender, boiled with water, cooled to 60°C, and then glucoamylase was added and stirred thoroughly for enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the enzymatic hydrolyzed lees solution was obtained by filtering with gauze. Preparation of fermentation aid: adding sucrose and black tea to water, heating to boil, cooling to room temperature, filtering and collecting the filtrate, adding the wall-broken sunflower flavonoids extract to the filtrate, stirring and dissolving to obtain the fermentation aid; Take 200ml of distiller's grains enzymatic hydrolysate and adjust the pH to 4, then add 20ml of fermentation aid and 25g of matrine, inoculate 12ml of bacteria at a concentration of 10 8 The Gluconobacter xylonylii at a concentration of 100 μg / ml was cultured at a constant temperature of 25°C for 7 days, and then washed with clean water and drained to obtain matrine-bacterial cellulose gel.

2. The method for preparing matrine-bacterial cellulose gel by in situ growth according to claim 1, characterized in that: When preparing the liquid culture medium, 200 g of yellow rice wine lees is used, 600 ml of water is added, and 0.5% of the mass fraction of glucoamylase accounting for the total mass fraction of yellow rice wine lees and water is added, and the enzyme activity of glucoamylase is 260,000 U / ml.

3. The method for preparing matrine-bacterial cellulose gel by in situ growth according to claim 2, characterized in that: The enzymatic hydrolysis conditions were 60°C for 3 h.

4. The method for preparing matrine-bacterial cellulose gel by in situ growth according to claim 1, wherein: When preparing the fermentation aid, the amount of water used is 100 ml, sucrose 10 g, black tea 2 g, and 0.2 g of the cracked sunflower flavonoids extract are added.

5. The method for preparing matrine-bacterial cellulose gel by in situ growth according to claim 1, characterized in that: The pH of the vinasse enzymatic hydrolyzate was adjusted with 10% by mass dilute hydrochloric acid.

6. The method for preparing matrine-bacterial cellulose gel by in situ growth according to claim 1, characterized in that: After inoculating Gluconobacter xylylene, 5 ml was poured into each well of the round hole mold, and then the mixture was incubated at a constant temperature.

7. A matrine-bacterial cellulose gel, characterized in that: The method according to any one of claims 1 to 6 is adopted to prepare the present invention.

8. A matrine-bacterial cellulose sustained-release granule, characterized in that: The matrine-bacterial cellulose gel prepared by the method of claim 1 is obtained by drying, or the matrine-bacterial cellulose gel as claimed in claim 7 is obtained by drying.

9. The matrine-bacterial cellulose sustained-release granules according to claim 8, characterized in that The drying conditions were: vacuum drying at 45°C for 24 h.

10. The use of the matrine-bacterial cellulose sustained-release granules according to claim 8 or 9, characterized in that: Used to inhibit the growth and reproduction of root-knot nematodes.

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