CN118077719B - Application of a microbial preparation in Platycodon grandiflorum cultivation - Google Patents

Abstract

The present invention provides an application of a microbial preparation in Platycodon grandiflorum planting, particularly for increasing the content of platycodon saponin D, total saponins and platycodon polysaccharides in Platycodon grandiflorum planting. The strain in the microbial preparation is selected from tkm‑1, which is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee with a deposit number of CGMCC No.14950. In the Platycodon grandiflorum planted with the microbial preparation, the content of platycodon saponin D is increased by nearly 20%, the total saponins are increased by more than 25%, and the platycodon polysaccharides are increased by nearly 30%.

Description

Application of a microbial preparation in Platycodon grandiflorum cultivation

Technical Field

The invention relates to the field of microorganisms, and in particular to application of a microbial preparation in platycodon grandiflorum planting.

Background technique

Platycodon grandiflorum (Jacq.) A. DC is a perennial herb with other names such as baggage flower, bell flower, doraki, and monk's mane flower. It is one of the major traditional Chinese medicinal materials in my country and has the effects of clearing the lungs, relieving throat and expectoration, and draining pus. The root of Platycodon grandiflorum is used as medicine. In addition to its medicinal value, Platycodon grandiflorum is also an edible and ornamental plant. The main components of Platycodon grandiflorum are saponins, polysaccharides, amino acids, phenols, etc. It is reported that Platycodon grandiflorum saponins have anti-inflammatory, antioxidant, antibacterial, and anti-tumor effects. It is reported that Platycodon grandiflorum saponin D has the most significant effects in anti-inflammatory and anti-tumor effects. Platycodon grandiflorum polysaccharides have certain antioxidant effects and can remove oxygen free radicals in the body. There are also reports that the hypoglycemic effect of Platycodon grandiflorum may be related to the improvement of insulin levels and its antioxidant capacity. Platycodon grandiflorum polysaccharides were tested on streptozotocin-induced diabetic rats at low, medium and high doses, and their insulin levels, glucose tolerance, and liver tissue superoxide dismutase and malondialdehyde were measured. The results showed that Platycodon grandiflorum polysaccharides have certain antioxidant and hypoglycemic abilities.

Due to the limited arable land area in the authentic production area, people have been planting Platycodon grandiflorum for many years in pursuit of the economic benefits of planting Platycodon grandiflorum, which has led to serious occurrence of diseases and insect pests, and reduced yield and quality. Planting Platycodon grandiflorum also has a serious inhibitory effect on the growth and yield formation of subsequent crops, resulting in the land after planting Platycodon grandiflorum can only be planted with gramineous crops. At present, the common way for farmers to solve the problem of continuous cropping is crop rotation, and Platycodon grandiflorum must be rotated with gramineous crops, and the rotation cycle should be 3 years or more.

In recent years, in the research on overcoming continuous cropping obstacles, soil improvement methods based on microbial agents or organic fertilizers have received much attention. Studies have shown that the repair effect of Bacillus subtilis on soil with continuous cropping obstacles of greenhouse vegetables is achieved by reducing the number of plant pathogens-Fusarium oxysporum in soil with continuous cropping obstacles, increasing the number of bacteria and actinomycetes in the soil, reducing the number of total fungi, and adjusting the soil microbial flora structure and the activity of sucrase, urease, phosphatase and catalase in the soil. Ji Yuliang et al. found that Bacillus subtilis PS-8 can improve the microecological environment of soil with continuous cropping obstacles of Platycodon grandiflorum, promote the growth of Platycodon grandiflorum, and have a repairing effect on soil with continuous cropping obstacles. It can be further used to develop a soil conditioner for continuous cropping obstacles in Platycodon grandiflorum field production (Journal of Shangluo College, Vol. 36, No. 6, 2022, 15-25).

Growth regulators are widely used in agricultural production and can effectively regulate the growth process of crops to achieve the purpose of stabilizing and increasing production. CN106747721A discloses a special composite microbial fertilizer for Platycodon grandiflorum and a preparation method thereof. The special composite microbial fertilizer for Platycodon grandiflorum is prepared from the following raw materials by weight: 52-58 parts of organic fertilizer, 38-46 parts of inorganic fertilizer, 3-5 parts of soil active functional bacteria, 0.1-0.2 parts of growth regulator, the soil active functional bacteria include brown nitrogen-fixing bacteria, Bacillus licheniformis, Bacillus subtilis, Bacillus megaterium, Bacillus mucilaginosus and Streptomyces jingyangensis, the growth regulator is diethylaminoethanol hexanoate, the above-mentioned composite fertilizer can effectively inhibit and resist various diseases and insect pests in the growth process of Platycodon grandiflorum, and can promote the synthesis of total saponins of Platycodon grandiflorum; the special composite microbial fertilizer for Platycodon grandiflorum can significantly improve the growth and quality of Platycodon grandiflorum.

Summary of the invention

The invention provides an application of a microbial preparation in platycodon planting, in particular, the microbial preparation and strigolactone GR24 are used in platycodon planting in combination, so as to increase the content of effective ingredients platycoside D, total saponins and platycodon polysaccharides in platycodon planting.

The strain in the microbial preparation is selected from Bacillus Subtilis tkm-1, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with the deposit number CGMCC No.14950 and has been disclosed in patent CN108865949A.

Inoculate Bacillus subtilis Tkm-1 into LB medium and culture at 30-32°C for 24-48 hours to obtain Bacillus subtilis Tkm-1 culture solution.

The LB medium consists of 5 g/L yeast powder, 10 g/L peptone, 10 g/L sodium chloride, 20 g/L glucose and 20 g/L agar powder.

The microbial preparation also includes shrimp and crab shells, collagen, sodium chloride, compound amino acids, carbohydrates, and compound fertilizers. The weight ratio of the bacterial culture solution to the shrimp and crab shells, collagen, sodium chloride, compound amino acids, carbohydrates, and compound fertilizers is (0.1~1): (0.1~10): (0.01~1): (0.01~9): (1~50): (1~20): (1~10), preferably 1:6:1:0.6:30:20:10.

The shrimp and crab shells are derived from any chitin-rich aquatic animal shells in rivers, lakes and seas. The dried product is crushed and passed through a 20-100 mesh sieve to form a fine granular powder. Preferably, the powder is shrimp shell powder, crab shell powder or shrimp and crab powder.

The collagen may be one or more of pig skin collagen, fish skin collagen or cow skin collagen.

The main components of complex amino acids are glycine, alanine, leucine, isoleucine, valine, cystine, cysteine, methionine, threonine, serine, phenylalanine, tyrosine, tryptophan, proline, hydroxyproline, glutamic acid, and aspartic acid.

The carbohydrate is selected from one or more of glucose, fructose and sucrose.

The weight ratio of nitrogen, phosphorus and potassium in the compound fertilizer is (10~20):(10~20):(10~20), preferably 15:15:15.

The preparation method of the microbial preparation comprises:

(1) adding Bacillus subtilis culture solution, shrimp and crab shells, collagen, and sodium chloride into water, mixing, fermenting at a temperature of 25 to 35° C. for 3 to 5 days, bubbling and ventilating, and obtaining a fermentation solution;

(2) Adding compound amino acids, carbohydrates and compound fertilizers to the fermentation liquid of step (1), fermenting at a temperature of 25-35° C. for 3-5 days, and performing bubbling aeration to obtain a microbial preparation.

The number of viable bacteria in the microbial preparation obtained in step (2) is not less than 1×10 ⁹ to 2×10 ¹⁰ cells/ml, preferably not less than 1×10 ¹⁰ cells/ml.

The method of applying the microbial preparation is to dilute the microbial preparation 1 to 10 times with water to obtain a microbial preparation dilution, and then directly irrigate the roots of Platycodon grandiflorum with the microbial preparation dilution. The amount of microbial preparation dilution used is 100L~1000L/mu.

Compared with the clear water group, the content of platycodon saponin D in platycodon grown with this microbial preparation increased by nearly 20%, the total saponins increased by more than 25%, and the platycodon polysaccharides increased by nearly 30%.

The present invention also uses the above-mentioned microbial preparation in combination with strigolactone GR24, adding strigolactone GR24 to the microbial preparation diluent at a concentration of 1-10 μmol/L, preferably 5 μmol/L.

Compared with the results without adding strigolactone, the addition of strigolactone GR24 reduced the number of Platycodon lateral roots. It was unexpectedly found that the combination of strigolactone GR24 and Bacillus subtilis tkm-1 could significantly increase the content of Platycodon polysaccharides by nearly 50%, while increasing the content of platycodon saponin D by nearly 30% and the total saponins by nearly 40%.

Detailed ways

Example 1

Preparation of dilutions of microbial preparations

0.5kg of Bacillus subtilis tkm-1 culture solution, 3kg of crab powder, 0.5kg of pig skin collagen, 0.3kg of sodium chloride, 15kg of complex amino acids, 10kg of fructose, and 5kg of compound fertilizer (nitrogen, phosphorus and potassium ratio of 15:15:15).

Bacillus subtilis tkm-1 was obtained from Beijing Techmei High-Tech Co., Ltd., and other components were purchased from the market.

Preparation method of microbial preparation:

(1) Add Bacillus subtilis culture medium, crab powder, collagen, and sodium chloride to 1000 L of water, culture with bubbling aeration, control the culture temperature at 28°C, and culture for 4 days to obtain fermentation liquid;

(2) Add compound amino acids, fructose and compound fertilizer to the fermentation liquid in step (1), continue culturing with bubbling aeration at a temperature of 32° C. for 3 days, and obtain the microbial preparation for future use.

The obtained microbial preparation was tested for bacterial density, which was 2.3×10 ¹¹ cfu/ml.

The obtained microbial preparation was diluted 10 times with water to obtain a microbial preparation dilution solution.

Example 2

Investigation of the concentration of strigolactone

Select full, neat and uniform Platycodon grandiflorum seeds, soak them in 29° C. water for 7 hours, sow them in a culture dish covered with two layers of moist filter paper, and place them in a 29° C. constant temperature incubator for culture. After the seed embryo grows to about 1 cm, select seedlings with similar growth potential and move them to test tubes filled with the culture solution of Example 1 for culture. Two seedlings are cultured in each test tube. The temperature is controlled at 25° C. The concentrations of strigolactone GR24 in the culture solution are 1, 5, and 10 μmol/L, respectively. A blank control group (without adding strigolactone GR24) is set up. Each group has 10 test tubes. The growth of the seedlings is observed and recorded. The Platycodon grandiflorum seedlings are collected after 4 weeks.

Ten Platycodon grandiflorum seedlings were selected from each group, the roots were rinsed clean, the length of the main root was measured with a ruler, and the number of lateral roots was observed and counted with the naked eye.

Table 1 Effects of strigolactone concentration on roots of Platycodon grandiflorum seedlings

Streptolactone concentration Mean taproot length (cm) The mean number of lateral roots 0μmol/L 4.18±0.33 7.8±2.15 1μmol/L 5.40±0.41 6.5±2.80 5μmol/L 5.87±0.50 4.9±2.18 10μmol/L 6.01±0.55 5.2±1.93

The results showed that strigolactone could significantly promote the growth of Platycodon grandiflorum's main root and significantly reduce the number of lateral roots.

Example 3

Platycodon grandiflorum planting

Select Platycodon grandiflorum seeds, soak them in 29℃ warm water for about 7 hours, take them out and put them on a wet cloth, then place them in an environment of about 28℃, cover them with moist hemp slices to achieve the purpose of germination, then mix the seeds with sand, and then sow them on the bed surface, use a broom to lightly sweep the seeds to cover them and then slightly press them down. After the seedlings emerge, in addition to conventional fertilization and watering, select 100 seedlings from each group, and implement root irrigation according to Table 2, 3 times a year, and observe for 3 years.

Table 2 Root irrigation solution and volume

Group Root irrigation Root irrigation volume Shimizu Group Shimizu 50ml/plant Group 1 Example 1 diluent 50ml/plant Group 2 Example 1 diluent (diethylaminoethanol hexanoate 0.1% w/w) 50ml/plant Group 3 Example 1 Dilution (Strigolactone concentration 5 μmol/L) 50ml/plant

Ten Platycodon grandiflorum plants were selected from each group, the roots were rinsed clean, the length of the main root was measured with a ruler, the root diameter was measured with a vernier caliper, and the number of lateral roots was visually observed and counted. The results are shown in Table 3.

Table 3 Effects of different root irrigation solutions on Platycodon grandiflorum roots

Group Mean taproot length (cm) Mean root diameter (mm) Number of lateral roots Shimizu Group 24.06±2.45 20.53±2.83 9.30±1.34 Group 1 25.26±3.22 23.59±2.16 9.90±1.79 Group 2 27.35±2.49 25.79±3.63 10.50±2.64 Group 3 29.32±2.50 25.00±3.23 8.60±1.65

The results showed that compared with the water group, Groups 1 to 3 could significantly increase the length and diameter of the main root of Platycodon grandiflorum. Compared with Groups 1 and 2, Group 3 could significantly reduce the number of lateral roots after using strigolactone.

Test Example 1 Determination of the content of active ingredients in Platycodon grandiflorum

The contents of total saponins, platycodon saponin D and platycodon polysaccharide in the platycodon obtained in Example 3 were determined.

(1) Determination of total saponin content

Preparation of test solution: Dry and crush fresh Platycodon grandiflorum, take about 1.0 g of dry powder (sieve No. 2), weigh accurately, put it in a Soxhlet extractor, add an appropriate amount of 70% methanol, soak overnight, heat and reflux for extraction for 6 hours, put the filtrate in an evaporating dish, evaporate to dryness in a water bath, dissolve the residue with 10ml water, slowly pass the obtained solution through the treated AB-8 macroporous adsorption resin column, repeat the sample loading 5 times, elute with 150ml water, discard the water, and then elute with 300ml 60% ethanol, collect the 60% ethanol eluate, and evaporate to dryness. Dissolve the residue with 70% methanol, transfer to a 10ml volumetric flask, make up to volume, shake well, and obtain.

Preparation of Platycodon saponin D reference solution: Accurately weigh an appropriate amount of Platycodon saponin D reference solution and place it in a volumetric flask. Add an appropriate amount of 70% methanol to dissolve it into a solution containing 3.5 mg per 1 ml.

Drawing of standard curve: Accurately pipette 20, 40, 80, 160, 200 μl of Platycodon saponin D reference solution into a 10 ml stoppered ground-mouth test tube, evaporate to dryness in a 70°C water bath, accurately add 0.5 ml of 10% vanillin test solution and 5 ml of 60% sulfuric acid, shake well, heat in a 60°C water bath for 15 min, cool in an ice water bath for 3 min, use the blank solvent treated in parallel with the same method as the blank, and measure the absorbance at 472 nm. Draw a standard curve with the standard concentration C (μl/ml) as the horizontal axis and the absorbance A as the vertical axis to obtain the regression equation.

Accurately measure 50 μl of the test solution and place it in a 10 ml ground-mouth test tube with a stopper. Evaporate to dryness in a 70°C water bath. Accurately add 0.5 ml of 10% vanillin test solution and 5 ml of 60% sulfuric acid. Shake well. Heat in a 60°C water bath for 15 min. Cool in an ice water bath for 3 min. Measure the absorbance at 472 nm.

The content of total saponins in the test sample Platycodon grandiflorum was calculated according to the regression equation.

(2) Determination of the content of platycodon saponin D

Octadecylsilane bonded silica gel was used as filler; acetonitrile-water (25:75) was used as mobile phase; evaporative light scattering detector was used for detection. The theoretical plate number calculated based on the platycodon saponin D peak should not be less than 3000.

ELSD detection conditions: drift tube temperature 90 °C, air flow rate 2.8 L/min.

Preparation of reference solution: Take an appropriate amount of Platycodon saponin D reference substance, weigh it accurately, and add methanol to make a solution containing 0.5 mg per 1 ml.

Preparation of test solution: Dry and crush fresh Platycodon grandiflorum, take about 2g of powder (pass through No. 2 sieve), accurately weigh, accurately add 50ml of 50% methanol, weigh, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with 50% methanol; shake well, filter, accurately measure 25ml of the filtrate, evaporate to dryness on a water bath, add 20ml of water to the residue, heat slightly to dissolve, shake and extract 3 times with n-butanol saturated with water, 20ml each time, combine the n-butanol solutions, wash with 50ml of ammonia test solution, discard the ammonia solution, wash with 50ml of water saturated with n-butanol, discard the water solution, evaporate the n-butanol solution to dryness, add methanol 3 ml to dissolve, add 0.5 g of silica gel and mix well, evaporate to dryness in a water bath, add to a silica gel column [100-120 mesh, 10 g, inner diameter 2 cm, wet-packed with a chloroform-methanol (9:1) mixed solution], elute with 50 ml of a chloroform-methanol (9:1) mixed solution, discard the eluent, then elute with 100 ml of a chloroform-methanol-water (60:20:3) mixed solution, discard the eluent, and then elute with 100 ml of a chloroform-methanol-water (60:29:6) mixed solution, collect the eluent, evaporate to dryness, dissolve the residue in methanol, transfer to a 5 ml volumetric flask, add methanol to the mark, shake well, filter, and obtain.

Determination method: Accurately pipette 5μl and 10μl of reference solution and 10-15μl of test solution respectively, inject into liquid chromatograph, determine, and calculate with external standard two-point method logarithmic equation to obtain the result.

(3) Determination of Platycodon grandiflorum polysaccharide content

Preparation of test solution: Dry and crush fresh Platycodon grandiflorum, pass through a 60-mesh sieve, take 10g, add 250ml of water, extract 3 times at 80℃, 1 hour each time, combine the extracts, filter, concentrate to 1/5 of the original volume, centrifuge at 3000r/min for 20 minutes, and remove the impurities and precipitates that appear during the concentration process. Take the supernatant and add ethanol to 80% alcohol content, let it stand at 4℃ for 24 hours; centrifuge to remove the precipitate, heat it with water until it is completely dissolved, concentrate, add ethanol to 80% alcohol content, let it stand at 4℃ for 24 hours, dissolve the precipitate with distilled water and transfer it to a 500 ml volumetric flask, add distilled water to the scale, shake well, and you are done.

Preparation of glucose reference solution: Accurately weigh 12.5 mg of glucose reference dried to constant weight and place in a 100 ml volumetric flask, add water to dissolve and dilute to the scale.

Preparation of phenol solution: Take 100g of phenol, add 0.1g of aluminum sheet and 0.05g of sodium bicarbonate, collect the fraction at 182℃ by atmospheric distillation, weigh 5g of this fraction, put it in a 100ml brown volumetric flask, add distilled water to the scale, shake well, and you have it.

Preparation of glucose standard solution: Accurately pipette 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 ml of glucose reference solution into 10 ml volumetric flasks respectively, and dilute to the scale with distilled water.

Drawing of standard curve: Accurately pipette 1.0 ml of each glucose standard solution into a stoppered test tube, add 1 ml of 5% phenol respectively, shake well, quickly drop 5.0 ml of concentrated sulfuric acid, shake well and leave for 10 min, heat in a boiling water bath for 15 min, take out and cool to room temperature. Add distilled water to another test tube and perform the same operation as above as a blank control. Measure the absorbance at 490 nm on a spectrophotometer, perform a linear regression of the absorbance against the glucose concentration, draw a standard curve, and find the regression equation.

Take 1.0 ml of the test solution and measure the absorbance in the same way, and substitute it into the regression equation to calculate the content of the test Platycodon grandiflorum polysaccharide.

(4) Results of determination of active ingredient content in samples

The results of the determination of the contents of Platycodon saponin D, total saponins and Platycodon polysaccharides in the samples are shown in Table 4:

Table 4 Determination results of the contents of Platycodon saponin D, total saponins and Platycodon polysaccharides

Group Platycodon saponin D (mg/g) Total saponins (w/w) Platycodon polysaccharide (w/w) Shimizu Group 1.42 6.3% 20.3% Group 1 1.68 7.9% 26.1% Group 2 1.72 8.2% 25.9% Group 3 1.81 8.7% 29.7%

(5) Data analysis

Compared with the water group, the percentage increase of effective ingredients is shown in Table 5:

Table 5 Compared with the water group, the percentage increase of effective ingredients

Group Platycodon saponin D Total saponins Total polysaccharides (Group 1-Clear Water Group)/Clear Water Group 18.3%# 25.4% 28.6% (Group 2-Clear Water Group)/Clear Water Group 21.1% 30.2% 27.6% (Group 3-Clear Water Group)/Clear Water Group 27.5% 38.1% 46.3%

# (1.68-1.42) ÷ 1.42 × 100% = 18.3%

As shown in Table 5, compared with the water group, groups 1-3 can significantly increase the content of effective ingredient platycoside D, total saponins and total polysaccharides in Platycodon grandiflorum; compared with groups 1 and 2, group 3 can significantly increase the content of total polysaccharides in Platycodon grandiflorum.

The above content describes the preferred embodiments of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications. These simple modifications all belong to the protection scope of the present invention.

It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims (4)

1. An application of a microbial preparation in improving the content of effective ingredients in Platycodon grandiflorum in Platycodon grandiflorum cultivation, characterized in that the microbial preparation and strigolactone GR24 are used in Platycodon grandiflorum cultivation in combination, the strain in the microbial preparation is Bacillus subtilis, the Bacillus subtilis is tkm-1, and the deposit number is CGMCC No.14950, the Bacillus subtilis is inoculated into LB culture medium, and cultured at 30-32°C for 24-48h to obtain Bacillus subtilis culture solution, the microbial preparation also includes shrimp and crab shells, collagen, sodium chloride, compound amino acids, carbohydrates, and compound fertilizers, the weight ratio of Bacillus subtilis culture solution to shrimp and crab shells, collagen, sodium chloride, compound amino acids, carbohydrates, and compound fertilizers is 1:6:1:0.6:30:20:10, and the microbial preparation is prepared by the following steps: (1) adding Bacillus subtilis culture solution, shrimp and crab shells, collagen, and sodium chloride into water, mixing, fermenting at a temperature of 25 to 35° C., fermenting for 3 to 5 days, and bubbling to obtain a fermentation solution; (2) adding the compound amino acid, carbohydrate and compound fertilizer to the fermentation liquid of step (1), fermenting at a temperature of 25 to 35° C. for 3 to 5 days, and bubbling and aerating to obtain; The application concentration of the strigolactone GR24 is 1-10 μmol/L, and the effective ingredients are platycodon saponin D, total saponins and total polysaccharides. 2. The use according to claim 1 is characterized in that the microbial preparation is diluted 1 to 10 times with water to obtain a microbial preparation dilution, and the microbial preparation dilution is used to irrigate the roots of Platycodon grandiflorum. 3. The use according to claim 2, characterized in that the strigolactone GR24 is added to the microbial preparation dilution and the platycodon grandiflorum is root-irrigated together. 4. The use according to claim 3, characterized in that the administration concentration of strigolactone GR24 is 5 μmol/L.