CN113598198A - Preparation method of disease-resistant protein glycoconjugate for crop bacterial soft rot, product and application thereof - Google Patents

Preparation method of disease-resistant protein glycoconjugate for crop bacterial soft rot, product and application thereof Download PDF

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CN113598198A
CN113598198A CN202110895895.XA CN202110895895A CN113598198A CN 113598198 A CN113598198 A CN 113598198A CN 202110895895 A CN202110895895 A CN 202110895895A CN 113598198 A CN113598198 A CN 113598198A
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disease
solution
soft rot
liquid
preparing
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赵国忠
郭婷
姚云平
周新运
丁凯丽
王新涛
王新宇
呼振豪
王凤军
王昊
李旭
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Tianjin University of Science and Technology
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Abstract

The invention discloses a preparation method of disease-resistant protein glycoconjugate for crop bacterial soft rot, a product and application thereof, comprising the steps of preparing organic copolymer; preparing an activity-induced lysozyme solution; preparing fermentation waste stress lectin liquid; preparing stabilized protamine liquid; sequentially adding an activity-induced lysozyme solution and a fermentation waste stress lectin solution into the organic copolymer at the temperature of 4-6 ℃, homogenizing and copolymerizing, then mixing with a stabilized protamine solution, and stirring and copolymerizing to obtain the disease-resistant glycoprotein glycoconjugate for the bacterial soft rot of the crops. The disease-resistant protein sugar conjunct for the crop bacterial soft rot disease prepared by the invention can obviously reduce the infection probability of the field bacterial soft rot disease, the cure rate of the soft rot disease of an experimental group applying the protein sugar conjunct is increased, a plant is obviously more upright and sturdy than a diseased plant by applying an application plant of the protein sugar conjunct, the bulb is larger than the diseased plant, and the disease-resistant protein sugar conjunct for the crop bacterial soft rot disease can achieve the effect of increasing the yield.

Description

Preparation method of disease-resistant protein glycoconjugate for crop bacterial soft rot, product and application thereof
Technical Field
The invention belongs to the technical field of biological antibiosis, and particularly relates to a preparation method of a disease-resistant protein glycoconjugate for crop bacterial soft rot, a product and application thereof.
Background
Bacterial soft rot is an infectious disease which has serious harm to the growth of crops, and is easy to get ill when wounds appear on the surfaces of the crops. The bacterial soft rot disease is caused by carrot soft rot pathogenic infection, harmful bacteria live through winter in soil, and garlic plants are induced to get ill after spring sowing in the next year, so that crops are soft rotted, and large-area plants die in severe cases. Nowadays, the invasion of bacterial soft rot in the growth process of crops becomes one of the main reasons for influencing the yield and quality of agricultural products.
Therefore, the bacterial soft rot disease is a problem which needs to be solved urgently in the current crop planting, and a preparation method and a product of disease-resistant protein glycoconjugate of the bacterial soft rot disease of the crops are urgently needed in the field.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made keeping in mind the above and/or other problems occurring in the prior art.
Therefore, the invention aims to overcome the defects in the prior art and provide a preparation method of the disease-resistant protein glycoconjugate for the bacterial soft rot of crops.
In order to solve the technical problems, the invention provides the following technical scheme: a method for preparing disease-resistant protein glycoconjugate for bacterial soft rot of crops comprises,
preparing an organic copolymer;
preparing an activity-induced lysozyme solution;
preparing fermentation waste stress lectin liquid;
preparing stabilized protamine liquid;
sequentially adding an activity-induced lysozyme solution and a fermentation waste stress lectin solution into the organic copolymer at the temperature of 4 ℃, homogenizing and copolymerizing, then mixing with a stabilized protamine solution, and stirring and copolymerizing to obtain the disease-resistant glycoprotein glycoconjugate of the crop bacterial soft rot;
wherein the total weight of the raw materials is calculated in percentage by mass, the organic copolymer accounts for 20-30%, the activity-induced lysozyme liquid accounts for 15-25%, the fermentation waste stress lectin liquid accounts for 15-25%, and the stabilized protamine liquid accounts for 10-20%.
As a preferred scheme of the preparation method of the disease-resistant protein glycoconjugate for the bacterial soft rot of the crops, the preparation method comprises the following steps: the preparation of the organic copolymer comprises the following steps,
according to the weight parts, 1-2 parts of polyethylene glycol aqueous solution, 1-2 parts of sodium alginate, 1-2 parts of beta-glucan and 1-2 parts of phosphate buffer are mixed, treated at 130-140 ℃ for 50-60 min, the pH of the system is adjusted to 6.5-7.5, an organic copolymer is formed through electrostatic interaction, and the organic copolymer is obtained after cooling to room temperature.
As a preferred scheme of the preparation method of the disease-resistant protein glycoconjugate for the bacterial soft rot of the crops, the preparation method comprises the following steps: the preparation of the activity-inducing lysozyme solution comprises,
injecting the enriched and activated lactobacillus bacterial liquid as an activity inducer into the egg white part of a fresh egg, mixing and inducing for 5-8 h, and extracting the egg white to obtain the activity-induced lysozyme liquid; wherein the content of the first and second substances,
according to the weight portion, the viscous antibacterial liquid is 6-7 parts, and the active lactobacillus liquid is 3-5 parts.
As a preferred scheme of the preparation method of the disease-resistant protein glycoconjugate for the bacterial soft rot of the crops, the preparation method comprises the following steps: adding waste liquid fermentation yeast as inducer into large yellow croaker culture box (culture density of 25 pieces/m)2Culturing water not over the surface of the fish body), mixing and stressing for 4-6 h, filtering and collecting fish body surface mucus and water mixture as fermentation waste stress lectin liquid; wherein the content of the first and second substances,
the fish stress agent is 4-5 parts by weight, and the fish surface mucus and water mixture is 5-6 parts by weight.
As a preferred scheme of the preparation method of the disease-resistant protein glycoconjugate for the bacterial soft rot of the crops, the preparation method comprises the following steps: the preparation of the stabilized protamine liquid comprises,
adding EDTA into a protamine solution (the protamine powder and water are prepared into a solution according to a ratio of 1:1000, the concentration of the solution is 0.1%), adjusting the pH value of the solution to 7-7.5, and collecting the solution as a stabilizing solution; wherein, the protamine solution accounts for 5-6 parts by weight, and the EDTA accounts for 0.5-1 part by weight.
As a preferred scheme of the preparation method of the disease-resistant protein glycoconjugate for the bacterial soft rot of the crops, the preparation method comprises the following steps: and (3) carrying out homogeneous copolymerization, wherein the homogeneous rotation speed is 1500-2000 rpm, and the homogeneous copolymerization time is 10-15 min.
As a preferred scheme of the preparation method of the disease-resistant protein glycoconjugate for the bacterial soft rot of the crops, the preparation method comprises the following steps: and stirring and copolymerizing, wherein the stirring rotating speed is 500-1000 rpm, and the stirring and copolymerizing time is 5-10 min.
As a preferred scheme of the preparation method of the disease-resistant protein glycoconjugate for the bacterial soft rot of the crops, the preparation method comprises the following steps: the lactic acid bacteria include one or more non-pathogenic lactic acid bacteria selected from Lactobacillus (Lactobacillus), Streptococcus (Streptococcus), Leuconostoc (Leuconostoc), Bifidobacterium (Bifidobacterium) and Pediococcus (Pediococcus).
As a preferred scheme of the preparation method of the disease-resistant protein glycoconjugate for the bacterial soft rot of the crops, the preparation method comprises the following steps: the liquid fermentation waste yeast comprises one or more of beer yeast, wine yeast, pichia pastoris, kluyveromyces lactis and the like.
Therefore, a further object of the present invention is to overcome the defects in the prior art, and to provide an application of a disease-resistant protein glycoconjugate prepared by a preparation method of a disease-resistant protein glycoconjugate for crop bacterial soft rot in resisting plant bacterial soft rot, wherein the application comprises,
adding 8-10 parts by volume of purified water into the disease-resistant protein glycoconjugate for dilution, applying to the root, and applying the density of 140-160 mL/m2。
The invention also aims to overcome the defects in the prior art and provide the application of the disease-resistant protein glycoconjugate for the bacterial soft rot of the crops as a soil remediation agent in the soil,
the invention has the beneficial effects that:
the invention provides a preparation method of a disease-resistant protein sugar conjunct for crop bacterial soft rot, which can obviously reduce the infection probability of the bacterial soft rot in the field. The field test shows that the cure rate of the soft rot disease of the experimental group applied with the protein-sugar union is increased, meanwhile, the plant applied with the protein-sugar union is obviously more upright and stronger than the diseased plant, and the bulb is larger than the diseased plant, which indicates that the disease-resistant protein-sugar union for the bacterial soft rot disease of the crops can achieve the effect of increasing the yield.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is a comparison of the bacteriostatic effect of the anti-disease protein glycoconjugate against Erwinia carotovora of bacterial soft rot of crops in example 2 of the present invention.
FIG. 2 is a diagram illustrating an embodiment of the present invention.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The polyethylene glycol is a polyethylene glycol 3350 aqueous solution, the concentration of the polyethylene glycol aqueous solution is 15-20%, and the polyethylene glycol aqueous solution is purchased from a Haian petrochemical plant in Jiangsu province; beta-glucan, available from Shandong Riche Biotech, Inc.;
the activated lactobacillus bacterial liquid of the invention has the activation method that: using 130rpm of a liquid MRS culture medium shaking table to activate for 8 hours to obtain activated bacterial liquid, wherein the number of strains in the activated bacterial liquid is 109CFU/mL, lactobacillus is Lactobacillus plantarum, purchased from CICC China Industrial microbial cultures Collection management center, with the strain preservation number CICC 21794;
liquid fermentation waste yeast, purchased from beer fermentation waste of Jinwei beer (Tianjin) GmbH;
other raw materials, which are not specifically described, are all commercially available.
Example 1
The embodiment provides a preparation method of disease-resistant protein glycoconjugate for crop bacterial soft rot, which comprises the following steps:
(1) preparation of organic copolymer: processing 2 parts of polyethylene glycol, 2 parts of sodium alginate, 2 parts of beta-glucan and 2 parts of phosphate buffer solution for 60min at 130 ℃, controlling the pH to be 7.0, forming an organic copolymer through electrostatic interaction, and cooling to room temperature for later use;
(2) preparing an activity induction lysozyme solution: injecting the enriched and activated lactobacillus bacterial liquid as an activity inducer into egg white in fresh eggs, mixing and inducing for 6 hours, and extracting the egg white to obtain the activity-induced lysozyme liquid; wherein the content of the first and second substances,
the egg white is 6 parts, and the activated lactobacillus bacterial liquid is 3 parts;
(3) preparing fermentation waste stress lectin liquid: adding liquid fermentation waste yeast as an inducer into a large yellow croaker culture box, mixing for 6h, filtering and collecting fish body surface mucus and water mixture as fermentation waste stress lectin liquid; wherein the content of the first and second substances,
according to the parts by weight, the inducer is 4 parts, and the mixture of the mucus on the surface of the fish body and the water is 5 parts;
the large yellow croaker culture box has the culture density of 25 strips/m2The water consumption for breeding is not over the surface of the fish body;
(4) preparing a stabilized protamine solution: adding EDTA into protamine solution, adjusting pH value to 7, and collecting to obtain stabilizing solution; wherein, the protamine solution accounts for 5 parts by weight, and the EDTA accounts for 1 part by weight;
the protamine solution is prepared by protamine powder and water according to the ratio of 1:1000, and the concentration of the solution is 0.1%;
(5) preparing disease-resistant protein glycoconjugate for bacterial soft rot of crops: sequentially adding an activity-induced lysozyme solution and a fermentation waste stress lectin solution into the organic copolymer at 4 ℃, carrying out homogeneous copolymerization for 10min at 2000rpm, then adding a stabilized protamine solution, and carrying out stirring copolymerization for 5min at 1000rpm to obtain the crop bacterial soft rot disease-resistant glycoprotein glycoconjugate 1;
wherein, the total weight of the raw materials is calculated in percentage by mass, the organic copolymer accounts for 30 percent, the activity-induced lysozyme liquid accounts for 25 percent, the fermentation waste stress lectin liquid accounts for 25 percent, and the stabilized protamine liquid accounts for 20 percent;
(6) adding 1 part of glycoprotein conjunct into 4 parts of purified water 9 parts by weight, and applying the mixture to the root with the density of 150mL/m2The addition is performed periodically according to actual conditions.
Example 2
The embodiment provides a preparation method of disease-resistant protein glycoconjugate for crop bacterial soft rot, which comprises the following steps:
(1) preparing an activity induction lysozyme solution: injecting the enriched and activated lactobacillus bacterial liquid as an activity inducer into egg white in fresh eggs, mixing and inducing for 6 hours, and extracting the egg white to obtain the activity-induced lysozyme liquid; wherein the content of the first and second substances,
the egg white is 6 parts, and the activated lactobacillus bacterial liquid is 3 parts;
(2) preparing fermentation waste stress lectin liquid: adding liquid fermentation waste yeast as an inducer into a large yellow croaker culture box, mixing for 6h, filtering and collecting fish body surface mucus and water mixture as fermentation waste stress lectin liquid; wherein the content of the first and second substances,
according to the parts by weight, the inducer is 4 parts, and the mixture of the mucus on the surface of the fish body and the water is 5 parts;
the large yellow croaker culture box has the culture density of 25 strips/m2The water consumption for breeding is not over the surface of the fish body;
(3) preparing a stabilized protamine solution: adding EDTA into protamine solution, adjusting pH value to 7, and collecting to obtain stabilizing solution; wherein, the protamine solution accounts for 5 parts by weight, and the EDTA accounts for 1 part by weight;
the protamine solution is prepared by protamine powder and water according to the ratio of 1:1000, and the concentration of the solution is 0.1%;
(4) preparing disease-resistant protein glycoconjugate for bacterial soft rot of crops: sequentially adding an activity-induced lysozyme solution and a fermentation waste stress lectin solution at 4 ℃, carrying out homogeneous copolymerization for 10min at 2000rpm, then mixing stabilized protamine, and carrying out stirring copolymerization for 5min at 1000rpm to obtain the disease-resistant protein glycoconjugate 2 for the bacterial soft rot of the crops;
wherein, the total weight of the raw materials is hundred percent, and the mass percent of the raw materials is that the activity inducing lysozyme liquid is 35 percent, the fermentation waste stress lectin liquid is 35 percent, and the stabilized protamine liquid is 30 percent.
(6) Adding 2 parts of glycoprotein conjunct into 4 parts of purified water by weight, adding 9 parts of purified water to compound roots, and applying the mixture with the density of 150mL/m2The addition is performed periodically according to actual conditions.
Experiments according to the methods of example 1 and example 2 show that the organic copolymer can remarkably improve the stability of the protein glycoconjugate (p < 0.05).
The two methods were set up as two experimental groups a and B, 3 in parallel per experimental group.
Group A is added with glycoprotein concatemer 1, and group B is added with glycoprotein concatemer 2. Blank in the measurement of light absorption value is that the protein glycoconjugate 1 and the protein glycoconjugate 2 are added into PDA culture medium. The prepared glycoprotein conjuncts 1 and 2 are equally divided into four parts, and are respectively placed at normal temperature for 7 days, 14 days, 21 days and 28 days for test. 30mL of the diluted glycoprotein glycoside 1 and the diluted glycoprotein glycoside 2 which are placed for different days are added into a PDA liquid culture medium, and the activated Erwinia carotovora is inoculated for 24 hours by a shaking table with 180rpm at the temperature of 28 ℃.
The bacteriostasis condition of the disease-resistant protein sugar conjunct for the bacterial soft rot disease of crops to Erwinia carotovora is judged according to the average value of the absorbance of the bacterial liquid at the end of the culture, and finally the result shows that the protein sugar conjunct 1 carried by the organic copolymer has the activity of 36.84% of the activity at 0 day after 30 days, the absorbance of the group B reaches 0.84 at the end of the experiment, and the higher density of the bacterial liquid shows that the protein sugar conjunct 2 can not reach the bacteriostasis effect at the moment, so the organic copolymer can stably carry the protein sugar conjunct to ensure the activity according to the experiment result, and the result is shown in figure 1.
Example 3
The embodiment provides a preparation method of disease-resistant protein glycoconjugate for crop bacterial soft rot, which comprises the following steps:
mixing egg white, large yellow croaker mucus, water mixture, and protamine liquid to obtain viscous antibacterial liquid.
Wherein the total weight of the raw materials is calculated in percentage by mass, the egg white accounts for 35 percent, the large yellow croaker mucus and water mixture accounts for 35 percent, and the protamine liquid accounts for 30 percent
According to the parts by weight, 4 parts of viscous antibacterial liquid are added into 9 parts of purified water to be applied to the root of the plant, and the application density is 150mL/m2The addition is performed periodically according to actual conditions.
The experimental results of the embodiment 2 and the embodiment 3 show that the viscous antibacterial liquid and the liquid culture medium of the protein glycoconjugate 2 prepared by a series of treatment raw materials have different antibacterial effects, wound surface expansion rates and field yield, and the experimental results of the protein glycoconjugate 2 are higher than those of the viscous antibacterial liquid. Experiments prove that the antibacterial activity can be increased by the pretreatment of the raw materials, but the results are still different from those shown in example 1.
Example 4
The embodiment provides a preparation method of disease-resistant protein glycoconjugate for crop bacterial soft rot, which comprises the following steps:
(1) preparing an activity induction lysozyme solution: injecting the enriched and activated lactobacillus bacterial liquid as an activity inducer into egg white in fresh eggs, mixing and inducing for 6 hours, and extracting the egg white to obtain the activity-induced lysozyme liquid; wherein, the egg white is 6 parts, and the activated lactobacillus bacterial liquid is 3 parts;
(2) the active induced lysozyme solution accounts for 100 percent by weight of the raw materials, and the disease-resistant protein glycoconjugate 3 for the crop bacterial soft rot is prepared by homogenizing the raw materials for 10min at 4 ℃ and 2000rpm of a homogenizer.
(3) Adding 3 parts of glycoprotein conjunct into 4 parts of purified water by weight, adding 9 parts of purified water to compound roots, and applying the mixture with the density of 150mL/m2The addition is performed periodically according to actual conditions.
Example 5
The embodiment provides a preparation method of disease-resistant protein glycoconjugate for crop bacterial soft rot, which comprises the following steps:
(1) preparing fermentation waste stress lectin liquid: adding liquid fermentation waste yeast as an inducer into a large yellow croaker culture box, mixing for 6h, filtering and collecting fish body surface mucus and water mixture as fermentation waste stress lectin liquid; wherein the content of the first and second substances,
according to the parts by weight, the inducer is 4 parts, and the mixture of the mucus on the surface of the fish body and the water is 5 parts;
the large yellow croaker culture box has the culture density of 25 strips/m2The water consumption for breeding is not over the surface of the fish body;
(2) the total weight of the raw materials is calculated in percentage by mass, the activity induction lysozyme solution is 100 percent, and the raw materials are homogenized for 10min at the temperature of 4 ℃ by a homogenizer at 2000rpm to prepare the protein-sugar conjunct body 4.
(3) 4 portions of glycoprotein conjuncted body and 9 portions of purified water are added according to the weight portionApplied with root part at a density of 150mL/m2The addition is performed periodically according to actual conditions.
Example 6
The embodiment provides a preparation method of disease-resistant protein glycoconjugate for crop bacterial soft rot, which comprises the following steps:
(1) preparing a stabilized protamine solution: adding EDTA into protamine solution, adjusting pH value to 7, and collecting to obtain stabilizing solution; wherein, the protamine solution accounts for 5 parts by weight, and the EDTA accounts for 1 part by weight;
the protamine solution is prepared by protamine powder and water according to the ratio of 1:1000, and the concentration of the solution is 0.1%; (ii) a
(2) The total weight of the raw materials is calculated in percentage by mass, the stabilized protamine liquid is 100 percent, and the mixture is homogenized for 10min at the temperature of 4 ℃ by a homogenizer at 2000rpm, so as to prepare the protein-sugar conjunct body 5.
(3) According to the weight portion, 5 portions of glycoprotein conjuncted body and 4 portions of purified water are added into 9 portions of compound root portion, the applied density is 150mL/m2The addition is performed periodically according to actual conditions.
Example 7
In this example, compared with example 1, the total weight of the raw materials is calculated as hundred percent, and the raw materials include, by mass percent, 20% of the organic copolymer, 29% of the activity-inducing lysozyme solution, 29% of the fermentation waste stress lectin solution, 22% of the stabilized protamine solution, and 4 ℃ homogenizing at 2000rpm for 10min by a homogenizer to prepare the glycoprotein union 6, and other preparation processes are the same as those in example 1.
Example 8
In this example, compared with example 1, the total weight of the raw materials is hundred percent, and the raw materials include, by mass percent, 5% of the organic copolymer, 35% of the activity-inducing lysozyme solution, 35% of the fermentation waste stress lectin solution, 25% of the stabilized protamine solution, and 4 ℃ homogenizing at 2000rpm for 10min by a homogenizer to prepare the glycoprotein union 7, and other preparation processes are the same as those in example 1.
Example 9
In this example, compared with example 1, the total weight of the raw materials is hundred percent, and the raw materials include, by mass percent, 1% of the organic copolymer, 35% of the activity-inducing lysozyme solution, 35% of the fermentation waste stress lectin solution, 29% of the stabilized protamine solution, and the mixture is homogenized at 4 ℃ and 2000rpm for 10min by a homogenizer to prepare the glycoprotein union 8, and other preparation processes are the same as those in example 1.
Example 10
In this example, compared with example 1, the total weight of the raw materials is hundred percent, and the raw materials include, by mass percent, 40% of the organic copolymer, 30% of the fermentation waste stress lectin solution, 30% of the stabilized protamine solution, and 10min of homogenization with a homogenizer at 2000rpm at 4 ℃ to prepare the glycoprotein glycoside 9, and other preparation processes are the same as those in example 1.
Example 11
In this example, compared with example 1, the organic copolymer 40%, the activity-inducing lysozyme solution 30%, and the stabilized protamine solution 30% were homogenized at 4 ℃ for 10min at 2000rpm by a homogenizer to obtain the glycoprotein conjugate 10, and the other preparation processes were the same as those of example 1.
Example 12
In this example, compared with example 1, the organic copolymer 37.5 wt%, the activity-inducing lysozyme solution 31.25 wt%, the fermentation waste stress lectin solution 31.25 wt%, and the mixture was homogenized at 4 ℃ and 2000rpm for 10min by a homogenizer to obtain the glycoprotein conjugate 11, and other preparation processes are the same as those of example 1.
The inhibition determination of carrot soft rot Erwinia comprises the following steps: the activated Erwinia carotovora is cultured for 24h by using a PDA liquid culture medium at 28 ℃ and 180rpm in a shaking table, and 3 groups of parallel experiments are set in each experiment group. And judging the bacteriostasis condition of the disease-resistant protein glycoconjugate of the bacterial soft rot disease of the crops to the Erwinia carotovora according to the average value of the absorbance of the bacterial liquid at the end of the culture.
The invention relates to a method for measuring the spread degree of garlic sprout leaf diseases: healthy uninfected garlic shoot leaves with a mass of about 5g were selected and 3 parallel experiments were set up for each experimental group. Preparing wound surface with diameter of 2.5mm at the approximate position of each garlic sprout leaf, and soaking 10 mm-3The culture solution of carrot soft rot Erwinia is coated on the woundAnd (3) spraying different experimental histone glycoconjugates on the wound surface, taking the garlic sprout leaves sprayed with purified water as blank control, measuring the expansion degree of the wound surface at 28 ℃ after 5 days, and calculating the diffusion rate.
The field garlic yield change of the invention is as follows: 120m in 2019 for Jinxiang county, Jining City in Shandong province2The test field is tested, 12 test areas are arranged in total, and each test area is 10m2The medicine concentration is configured according to the size of each square meter, and the change of the garlic yield per mu is observed after the medicine is applied.
The results of the experiments in inventive examples 1 to 12 are shown in table 1.
TABLE 1
Examples Absorbance (OD value) Wound surface expansion Rate (%) Field yield (kg/mu)
Example 1 0.21 29.76 3250
Example 2 0.57 50.02 3012
Example 3 0.89 77.40 2586
Example 4 0.69 62.74 2798
Example 5 0.72 53.28 2890
Example 6 0.69 69.91 2730
Example 7 0.83 53.63 2655
Example 8 0.50 58.15 2846
Example 9 0.64 53.44 2844
Example 10 0.66 50.39 2869
Example 11 0.69 54.56 2775
Example 12 0.66 65.32 2766
As can be seen from Table 1, the OD value of example 1 was the smallest, 0.21; example 3 was maximal, reaching 0.89, with the remainder between the two. The difference of OD values can indicate that the developed protein glycoconjugate can inhibit the growth and the propagation of Erwinia carotovora to different degrees. The expansion rate of the wound surface reflects the recovery condition of the affected part after the medicine is applied, the expansion rate of example 1 is the minimum, and only 29.76% of the expansion rate is obtained, so that the condition of the affected part can be improved and effectively prevented from being diffused by the protein-sugar conjunct body, and other test groups show the prevention and treatment effects of different degrees, but the expansion rates are higher than 50%, and the effects are different from those of example 1. The field test results show that the yield of the example 1 is obviously higher than that of other groups (P <0.05), and the other yields are different from 2000-3000 kg/mu, which can show that the proper proportion of the glycoprotein and the glycoconjugate can achieve a certain yield increase effect. By integrating the results of laboratory tests and field tests, the protein glycoconjugate in the example 1 can effectively inhibit the growth of pathogenic bacteria, prevent the affected wound from expanding and improve the field yield under the condition of controlling variables as much as possible.
Example 13
70m in 2020 to Jinxiang county, Jining City in Shandong province2The test field is tested, 7 test areas are arranged in total, and each test area is 10m2The drug concentration is configured per square meter. The investigation was conducted on days 7 and 14 after the application, 5 plants per spot were collected in the same area using a five-spot sampling method, and the total number of plants, the number of diseased plants, and the number of disease stages were recorded. Grading standard: 0 grade, no disease of the whole plant; grade 1, the top leaf is taken as the 1 st leaf, the 4 th leaf and the following diseases; class 3: the first2 leaf and following onset of disease; grade 5, onset of disease of the whole plant.
Disease index (%) ═ Σ (number of diseased plants at each stage × the disease value)/(total number of investigated plants × 5) × 100; the preventing and treating effect (%) is (disease index of blank control area-disease index of treatment area)/disease index of blank control area x 100.
The glycoprotein, the activity-inducing lysozyme solution, the fermentation waste stress lectin solution, and the stabilized protamine solution were prepared under the conditions described in example 1, and the results of the pharmacological analyses were shown in Table 2.
TABLE 2
Figure BDA0003197875080000111
Note: different letters indicate significant differences (P <0.05)
The experimental result shows that the protein carbohydrate is applied by 150mL/m2The difference is most obvious, the prevention and treatment effect is most outstanding, and the prevention and treatment effect reaches 68.06% after the application for 14 days. In contrast, the glycoprotein concatemers were applied at 50, 100, 200mL/m2150mL/m of activity-inducing lysozyme solution2150mL/m of fermentation waste stress lectin solution2Stabilized protamine solution 150mL/m2The disease prevention effect of each group is relatively common.
The activity-induced lysozyme solution, the fermentation waste stress lectin solution and the stabilized protamine solution all have antibacterial and bactericidal activities, and can play a role when being used alone at proper concentration, but the antibacterial effect is not obvious.
The results of synergistic experiments show that the single use of the raw materials can relieve the morbidity of the soft rot to different degrees, but the effect is poor; by using the developed protein-sugar conjunct with moderate proportional concentration, the antibacterial effect is obviously improved, the bacterial soft rot disease state of plants is improved, and the garlic quality is improved to different degrees.
Example 14
2021 year old, 50m for Jinxiang county, Jining City in Shandong province2The test field is tested, 5 test areas are arranged in total, and each test area is 10m2The drug concentration is given per squareAnd configuring the size of the rice. The investigation was conducted on days 7 and 14 after the application, 5 plants per spot were collected in the same area using a five-spot sampling method, and the total number of plants, the number of diseased plants, and the number of disease stages were recorded. Grading standard: 0 grade, no disease of the whole plant; grade 1, the top leaf is taken as the 1 st leaf, the 4 th leaf and the following diseases; grade 3, incidence of 2 nd leaf and the following; grade 5, onset of disease of the whole plant.
Disease index (%) ═ Σ (number of diseased plants at each stage × the disease value)/(total number of investigated plants × 5) × 100; the preventing and treating effect (%) is (disease index of blank control area-disease index of treatment area)/disease index of blank control area x 100.
Under the conditions of the embodiment 1, the proportions of the organic copolymer, the activity-induced lysozyme solution, the fermentation waste stress lectin solution and the stabilized protamine solution in the disease-resistant protein glycoconjugate for preparing the bacterial soft rot of the crops are different, and specifically: 6:5:5:4, 4:3:3:2, 1:1:1:1, 4:5:5: 4;
sequentially adding 4 parts of glycoprotein and 9 parts of purified water to the mixed root, and applying the mixture with the density of 150mL/m2The different proportions of the protein-sugar conjuncts are analyzed for the drug effect, which is shown in Table 3.
TABLE 3
Figure BDA0003197875080000121
Note: different letters indicate significant differences (P <0.05)
As can be seen from Table 3, the optimal mixture ratio of the raw materials is found by controlling the raw material ratio of the protein-sugar union to achieve the optimal antibacterial effect, the total parts are controlled to be 100 parts, the ratios of the 4 raw materials are controlled to be different, and the antibacterial effect is optimal under the same condition and the best control effect can be achieved by verifying that the ratio is 6:5:5: 4.
FIG. 2 is a comparison graph of an experimental group and diseased plants to which the glycoprotein glycoconjugate 1 is applied, and it can be seen that the plants are obviously more upright and sturdy than the diseased plants, and the bulbs are larger than the diseased plants, indicating that the disease-resistant glycoprotein glycoconjugate can achieve the effect of increasing the yield of crops.
The organic copolymer of the present invention: the organic copolymer raw materials used in the excellent carrier compound have good intermiscibility with most organic matter components, can carry biomacromolecules such as polysaccharide, protein and the like, and can form a viscous outer mold on the surface of the molecules to protect active substances from being isolated from the outside and maintain the stability of the active substances.
Activity-inducing lysozyme solution: lysozyme is a natural antibacterial agent, can be combined with peptidoglycan of microbial cell walls to destroy the stable structure of the cell walls, so that the lysozyme is cracked and flows out of contents to kill microbes. However, the lysozyme activity of the egg white is not high enough, and the lysozyme activity is obviously increased after induction (p is less than 0.05).
Fermenting waste to stress lectin liquid: lectin is glycobindin with remarkable diversity, and large yellow croaker can secrete body surface mucus containing C-type lectin. The C-type lectin used herein is a mannose-binding protein, and can bind to mannose in the surface layer of Erwinia carotovora to cause an antibacterial reaction, and the microorganism with lectin agglutination will not adhere to and invade its host. However, the lectin content of the extract is low and the ability to attach microorganisms is weak. The content of the agglutinin is obviously increased (p is less than 0.05) and the adhesion capability is increased by using the fermentation waste yeast for stress.
Stabilizing the protamine liquid: protamine has bactericidal activity against Erwinia carotovora. The result of single use shows that the bacteriostasis concentration needs to be more than 186 mu g/mL.
Organic copolymer: an excellent carrier complex. The used organic copolymer raw materials have good intermiscibility with most organic matter components, can carry biomacromolecules such as polysaccharide, protein and the like, and can form a viscous outer mold on the surface of the molecule to protect active substances from being isolated from the outside and maintain the stability of the active substances.
Activity-inducing lysozyme solution: lysozyme is a natural antibacterial agent, can be combined with peptidoglycan of microbial cell walls to destroy the stable structure of the cell walls, so that the lysozyme is cracked and flows out of contents to kill microbes. However, the lysozyme activity is not high enough when the lysozyme is used in the early stage, and the lysozyme activity is obviously increased after induction (p < 0.05).
Fermenting waste to stress lectin liquid: lectins are carbohydrate-binding proteins with a significant diversity. The C-type lectin used herein is a mannose-binding protein, and can bind to mannose in the surface layer of Erwinia carotovora to cause an antibacterial reaction, and the microorganism with lectin agglutination will not adhere to and invade its host. However, the lectin content of the extract is low and the ability to attach microorganisms is weak. The content of the agglutinin is obviously increased (p is less than 0.05) and the adhesion capability is increased by using the fermentation waste to stress the extraction.
Stabilizing the protamine liquid: the extracted protein liquid has bactericidal activity on carrot soft rot Erwinia. The result of single use shows that the bacteriostasis concentration needs to be more than 186 mu g/mL.
Compared with the originally used viscous antibacterial liquid, the optimized protein-glycomer antibacterial rate, stability and field yield of the pretreated raw materials show obvious superiority under the condition of carrier compound copolymerization.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims (10)

1. A preparation method of disease-resistant protein glycoconjugate for crop bacterial soft rot is characterized by comprising the following steps: comprises the steps of (a) preparing a mixture of a plurality of raw materials,
preparing an organic copolymer;
preparing an activity-induced lysozyme solution;
preparing fermentation waste stress lectin liquid;
preparing stabilized protamine liquid;
sequentially adding an activity-induced lysozyme solution and a fermentation waste stress lectin solution into the organic copolymer at the temperature of 4-6 ℃, homogenizing and copolymerizing, then mixing with a stabilized protamine solution, and stirring and copolymerizing to obtain the disease-resistant glycoprotein glycoconjugate of the bacterial soft rot of the crops;
wherein the total weight of the raw materials is calculated in percentage by mass, the organic copolymer accounts for 20-30%, the activity-induced lysozyme liquid accounts for 15-25%, the fermentation waste stress lectin liquid accounts for 15-25%, and the stabilized protamine liquid accounts for 10-20%.
2. The method for preparing disease-resistant protein glycoconjugate for crop bacterial soft rot as claimed in claim 1, which is characterized in that: the preparation of the organic copolymer comprises the following steps,
according to the weight parts, 1-2 parts of polyethylene glycol, 1-2 parts of sodium alginate, 1-2 parts of beta-glucan and 1-2 parts of phosphate buffer are mixed, treated at 130-140 ℃ for 50-60 min, the pH value of the system is adjusted to 6.5-7.5, an organic copolymer is formed through electrostatic interaction, and the organic copolymer is obtained after cooling to room temperature.
3. The method for preparing disease-resistant protein glycoconjugate for crop bacterial soft rot as claimed in claim 1, which is characterized in that: the preparation of the activity-inducing lysozyme solution comprises,
injecting enriched and activated lactobacillus bacterial liquid as an activity inducer into egg white in fresh eggs, mixing and inducing for 5-8 h, and extracting the egg white to obtain the activity-induced lysozyme liquid; wherein the content of the first and second substances,
the egg white is 6-7 parts by weight, and the activated lactobacillus liquid is 3-5 parts by weight.
4. The method for preparing disease-resistant protein glycoconjugate for crop bacterial soft rot as claimed in claim 1, which is characterized in that: the preparation of the fermentation waste stress lectin solution comprises the following steps,
adding liquid fermentation waste yeast serving as an inducer into a large yellow croaker culture box, mixing for 4-6 h, filtering and collecting mucus on the surface of a fish body and a water mixture serving as fermentation waste stress lectin liquid; wherein the content of the first and second substances,
4-5 parts of an inducer and 5-6 parts of a mixture of mucus and water on the surface of the fish body by weight;
the large yellow croaker culture box has the culture density of 25 strips/m2And the water consumption for cultivation is not over the surface of the fish body.
5. The method for preparing disease-resistant protein glycoconjugate for crop bacterial soft rot as claimed in claim 1, which is characterized in that: the preparation of the stabilized protamine liquid comprises,
adding EDTA into the protamine solution, adjusting the pH value of the solution to 7-7.5, and collecting the solution as a stabilizing solution; wherein, the protamine solution accounts for 5-6 parts by weight, and the EDTA accounts for 0.5-1 part by weight;
the protamine solution is prepared by protamine powder and water according to the ratio of 1:1000, and the concentration of the solution is 0.1%.
6. The method for preparing the disease-resistant protein glycoconjugate for the bacterial soft rot disease of the crops as claimed in any one of claims 1 to 5, which is characterized in that: the homogeneous copolymerization is carried out, wherein the homogeneous rotation speed is 1500-2000 rpm, and the homogeneous copolymerization time is 10-15 min; and stirring and copolymerizing, wherein the stirring rotating speed is 500-1000 rpm, and the stirring and copolymerizing time is 5-10 min.
7. The method for preparing disease-resistant protein glycoconjugates for bacterial soft rot of crops as claimed in claim 3, wherein the method comprises the following steps: the lactic acid bacteria include one or more non-pathogenic lactic acid bacteria selected from Lactobacillus (Lactobacillus), Streptococcus (Streptococcus), Leuconostoc (Leuconostoc), Bifidobacterium (Bifidobacterium) and Pediococcus (Pediococcus).
8. The method for preparing disease-resistant protein glycoconjugates for bacterial soft rot of crops as claimed in claim 4, wherein the method comprises the following steps: the liquid fermentation waste yeast comprises one or more of beer yeast, wine yeast, pichia pastoris, kluyveromyces lactis and the like.
9. The application of the disease-resistant protein sugar conjunct prepared by the preparation method of any one of the claims 1 to 8 in the resistance of garlic bacterial soft rot is characterized in that: the use of said composition, comprising,
adding 3-5 parts by volume of purified water into 1 part of the disease-resistant protein glycoconjugate, diluting, applying to the root, and applying the density of 140-160 mL/m2。
10. The use of the disease-resistant glycoprotein glycoconjugate prepared by the preparation method of any one of claims 1-8 as a soil remediation agent in soil.
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0374823A2 (en) * 1988-12-21 1990-06-27 HAARMANN &amp; REIMER CORP. Control of microbial growth with nisin/lysozyme formulations
US5458876A (en) * 1988-12-21 1995-10-17 Haarman & Reimer Corp. Control of microbial growth with lantibiotic/lysozyme formulations
WO1996006532A1 (en) * 1994-09-01 1996-03-07 Novo Nordisk A/S A basic protein composition for killing or inhibiting microbial cells
JPH08289770A (en) * 1995-04-20 1996-11-05 Asama Kasei Kk Preservative for food
US5888501A (en) * 1994-07-26 1999-03-30 Auburn University Induced systemic resistance of plants to pathogenic microorganisms
CN101165171A (en) * 2007-09-30 2008-04-23 华中农业大学 Bacillus subtilis BSn5 with bacteriostasis active to carrot soft rot Erwinia and bacteriostasis protein APn5
US20200197451A1 (en) * 2017-08-18 2020-06-25 Baylor College Of Medicine Lectin-bound bacteria and uses thereof

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0374823A2 (en) * 1988-12-21 1990-06-27 HAARMANN &amp; REIMER CORP. Control of microbial growth with nisin/lysozyme formulations
US5458876A (en) * 1988-12-21 1995-10-17 Haarman & Reimer Corp. Control of microbial growth with lantibiotic/lysozyme formulations
US5888501A (en) * 1994-07-26 1999-03-30 Auburn University Induced systemic resistance of plants to pathogenic microorganisms
WO1996006532A1 (en) * 1994-09-01 1996-03-07 Novo Nordisk A/S A basic protein composition for killing or inhibiting microbial cells
JPH08289770A (en) * 1995-04-20 1996-11-05 Asama Kasei Kk Preservative for food
CN101165171A (en) * 2007-09-30 2008-04-23 华中农业大学 Bacillus subtilis BSn5 with bacteriostasis active to carrot soft rot Erwinia and bacteriostasis protein APn5
US20200197451A1 (en) * 2017-08-18 2020-06-25 Baylor College Of Medicine Lectin-bound bacteria and uses thereof

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