CN112956498A - Multi-bacterium fermentation liquor with sterilization effect and preparation method and application thereof - Google Patents
Multi-bacterium fermentation liquor with sterilization effect and preparation method and application thereof Download PDFInfo
- Publication number
- CN112956498A CN112956498A CN202110178436.XA CN202110178436A CN112956498A CN 112956498 A CN112956498 A CN 112956498A CN 202110178436 A CN202110178436 A CN 202110178436A CN 112956498 A CN112956498 A CN 112956498A
- Authority
- CN
- China
- Prior art keywords
- parts
- fermentation
- fermentation broth
- concentration
- effect
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/40—Liliopsida [monocotyledons]
- A01N65/48—Zingiberaceae [Ginger family], e.g. ginger or galangal
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Agronomy & Crop Science (AREA)
- Biotechnology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to the technical field of fermentation, in particular to a multi-bacterium fermentation liquid with a sterilization effect, and a preparation method and application thereof. The invention aims to provide a multi-bacterium fermentation broth with a sterilization effect, and aims to provide a preparation method of the multi-bacterium fermentation broth and application of the multi-bacterium fermentation broth in preparation of a bactericide or a pesticide.
Description
Technical Field
The invention relates to the technical field of fermentation, in particular to a multi-bacterium fermentation liquid with a sterilization effect, and a preparation method and application thereof.
Background
The ferment is a beverage rich in secondary metabolites, which is obtained by using fruits and vegetables as raw materials and performing multi-strain composite fermentation. The enzyme is derived from Japan, is originally meant as enzyme in Japanese, and is researched and developed into fermentation liquor fermented by a multi-strain compound microorganism population through the Nippon microorganism expert Ishiki sense family.
At present, the research on the bacteriostatic activity of multi-bacterium fermentation liquor is less, and the bacteriostatic activity on fungi is more rare, but theoretically, the multi-bacterium fermentation liquor belongs to a fermentation type product, has strong acidity and contains bacteriostatic substances such as organic acid, phenols, active enzyme and the like. The strong acid environment can cause the denaturation of pathogenic bacteria protein, the phenolic compound can destroy the cell structure, the content of soluble protein and extracellular nucleic acid of the strain can be increased, and the pathogenic bacteria can die. The zymolyte can generate polypeptide micromolecules with bacteriostatic activity, and the micromolecule substances have natural antibacterial property. The organic acid can permeabilize the outer membrane of bacteria, improve the intracellular osmotic pressure and inhibit the synthesis of biological macromolecules, thereby achieving the bacteriostatic effect.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims at providing a multi-bacterium fermentation broth with a sterilization effect, and aims at providing a preparation method of the multi-bacterium fermentation broth and application of the multi-bacterium fermentation broth in preparation of a bactericide or a pesticide.
In order to achieve the purpose, the technical scheme of the invention is as follows, the multi-bacterium fermentation liquor with the sterilization effect comprises the following raw materials in parts by weight:
3-5 parts of dried chili, 2-4 parts of chinaberry, 4-6 parts of tea seed cake, 3-5 parts of tobacco leaf, 2-4 parts of Chinese wampee bark, 3-5 parts of tripterygium wilfordii, 2-4 parts of Chinese honeylocust fruit, 3-5 parts of radix euphorbiae lantu, 2-4 parts of oleander, 1-3 parts of sargentgloryvine stem, 1-3 parts of macleaya cordata, 1-3 parts of cephalotaxus fortunei, 0.25-0.75 part of orange peel, 0.5-1.5 parts of selenium malt, 0.5-1.5 parts of sweet wormwood herb, 0.5-1.5 parts of cinnamon, 0.5-1.5 parts of vanilla, 0.5-1.5 parts of star anise, 0.5-1.5 parts of pepper, 0.25-0.75 part of fennel, 0.25-0.75 part of rutabaga, 1-3 parts of gelsemium elegans, 1-3 parts of ginger, 1-3 parts of purple onion, 4-6 parts of garlic, 1-3 parts of black bean and.
Further, the feed comprises the following raw materials in parts by mass:
4 parts of dried chili, 3 parts of chinaberry, 5 parts of tea seed cake, 4 parts of tobacco leaf, 3 parts of Chinese wampee leaf, 4 parts of tripterygium wilfordii, 3 parts of saponin, 4 parts of radix euphorbiae lantu, 3 parts of oleander, 2 parts of sargentgloryvine stem, 2 parts of macleaya cordata, 2 parts of cephalotaxus fortunei, 0.5 part of orange peel, 1 part of selenium malt, 1 part of sweet wormwood, 1 part of cinnamon, 1 part of vanilla, 1 part of star anise, 1 part of pepper, 0.5 part of fennel, 0.5 part of rue grass, 2 parts of gelsemium elegans, 2 parts of ginger, 2 parts of purple onion, 5 parts of garlic, 2 parts of black bean and 2 parts.
Further, the multi-bacterium fermentation liquid is obtained by fermenting the raw materials with fermentation strains, and comprises the following steps:
step one, pretreatment of raw materials: drying and grinding the raw materials, and sieving to prepare a mixed material;
step two, adding 20-60g of carbon source and 60-100g of the mixed material obtained in the step one into each liter of water, uniformly mixing, and sterilizing at high temperature and high pressure to prepare a fermentation substrate;
step three, inoculating fermentation strains into the fermentation substrate obtained in the step two, wherein the fermentation conditions are as follows: fermenting at 28-30 deg.C and 200rpm for 20 days at pH 4.5-6.5 under anaerobic condition; the fermentation strain is as follows: mixing saccharomyces cerevisiae, lactobacillus pentosus and lactobacillus japonicas according to the volume ratio of 1:1: 1; the inoculum size was 3%.
Further, in the step one, the raw materials are dried and ground at 55 ℃ and then sieved by a 80-mesh sieve to prepare a mixed material.
Further, in the second step, 40g of carbon source and 80g of the mixed material obtained in the first step are added into each liter of water.
Further, the carbon source is one or more of glucose, fructose, fructo-oligosaccharide, maltose, maltodextrin, mannose, arabinose, pullulan, brown sugar, sucrose and starch.
Further, in the third step, the fermentation temperature is 28 ℃.
The invention also requests to protect the application of the multi-bacterium fermentation liquor in the preparation of bactericides or pesticides.
The invention has the beneficial effects that:
1. the invention obtains the fermentation liquor with the bacteriostatic action by carrying out multi-bacteria fermentation on fruit grains and various bacteriostatic and bactericidal plants, the raw materials are easily available and convenient to obtain, the main components and the relative content of the fermentation liquor are obtained by carrying out qualitative and quantitative analysis on the fermentation liquor through ultra-high performance liquid chromatography and tandem mass spectrometry, the obtained fermentation liquor is rich in nutrient substances and effective active substances, and has two sources, namely all components of the used raw materials and new components generated after microbial fermentation conversion, and the content of a plurality of effective active substances is increased to a certain extent through microbial fermentation. The ingredients of the fermentation liquor are qualitatively and quantitatively analyzed through high performance liquid chromatography and mass spectrometry, and the result shows that 624 substances are totally contained in the fermentation liquor, wherein the types of flavonoid compounds are 146 at most, the relative content of phenolic acid compounds is 29.38% at most, in addition, the organic acid content is 26.79%, the alkaloid content is 7.74%, and the amino acid and derivatives content is 12.07%, the fermentation liquor contains a large amount of phenols, alkaloids and other substances, and the bacteriostatic activity of the fermentation liquor to common plant pathogenic bacteria is particularly excellent.
2. The inhibition effect experiment of the multi-fungus fermentation liquor on the hypha growth of 13 fungi shows that the fermentation liquor has a remarkable inhibition effect on the hypha growth of the 13 fungi, the inhibition effect is also remarkably enhanced along with the increase of the concentration of the fermentation liquor, and the inhibition effect on rhizopus stolonifer is particularly ideal in the inhibition experiment on putrefying fungi, wherein the half-effect concentration EC50 is 8.60%, and the minimum inhibitory concentration MIC is 14%; in addition, the multi-bacterium fermentation liquor has excellent bacteriostatic activity on plant pathogenic fungi, is most ideal for corynespora casseliflava, alternaria mali, colletotrichum schinense, setaria apiacea and fusarium graminearum, and has the EC50 for inhibiting the growth of mycelium of 3.45%, 4.04%, 3.78%, 2.74% and 3.67% respectively, and the MIC for inhibiting spore germination of 8%, 7%, 5%, 4% and 8% respectively. The results show that the multi-bacterium fermentation liquor has the potential of being used as an antifungal pesticide.
3. Through the experiment of the inhibitory effect of multi-bacterium fermentation liquor on bacteria, the total inhibitory effect of the concentration of the fermentation liquor on the bacteria shows the tendency of promoting and inhibiting after the bacteria, after the fermentation liquor reaches a certain concentration, the inhibitory effect on other strains except vibrio parahaemolyticus is obvious along with the rise of the concentration of the fermentation liquor, the minimum inhibitory concentration MIC of the fermentation liquor on tested strains is lower, and respectively comprises 10% of Escherichia coli MIC, 18% of vibrio parahaemolyticus MIC, 14% of Listeria MIC, 10% of Salmonella MIC, 12% of aeromonas hydrophila MIC and 10% of Shigella MIC. Therefore, the fermentation liquor has stronger inhibition effect on bacteria.
4. Therefore, the multi-bacterium fermentation liquor has potential application value in agricultural industry and can be used as a substitute of some pesticides harmful to the environment. In addition, the multi-bacterium fermentation liquor is rich in nutrition and contains a plant growth regulator, and the efficacy of a foliar fertilizer can be considered.
Drawings
FIG. 1 is a graph of the effect of different addition levels of fermentation broth on the bacteriostatic activity of Aspergillus niger;
FIG. 2 is a graph of the log C-P (log fermentation broth concentration-several-value) standard curve of the inhibitory activity of fermentation broth against Aspergillus niger;
FIG. 3 is a graph showing the effect of different addition amounts of fermentation broth on the bacteriostatic activity of Penicillium citrinum;
FIG. 4 is a graph showing the log C-P (log-several values of fermentation broth concentration) standard curve of the inhibitory activity of fermentation broth on Penicillium citrinum;
FIG. 5 is a graph of the effect of different addition levels of fermentation broth on the bacteriostatic activity of Aspergillus flavus;
FIG. 6 is a log C-P (log-several values of fermentation broth concentration) standard curve of the inhibition of Aspergillus flavus by fermentation broth;
FIG. 7 is a graph of the effect of different addition levels of fermentation broth on the bacteriostatic activity of Aspergillus versicolor;
FIG. 8 is a graph of the log C-P (log of fermentation broth concentration-several values) standard curve of the inhibitory activity of fermentation broth against Aspergillus versicolor;
FIG. 9 is the effect of different addition amounts of fermentation broth on the bacteriostatic activity of M.oxysporum;
FIG. 10 is a graph showing the log C-P (log of fermentation broth concentration-several values) standard curve of the inhibitory activity of a fermentation broth against Cladosporium oxysporum;
FIG. 11 is a graph of the effect of different addition levels of fermentation broth on the bacteriostatic activity of Rhizopus stolonifer;
FIG. 12 is a log C-P (log fermentation broth concentration-several-degree value) standard curve of the inhibitory activity of fermentation broth against Rhizopus stolonifer;
FIG. 13 is a graph of the effect of different addition amounts of fermentation broth on the bacteriostatic activity of E.sanguiensis;
FIG. 14 is a graph showing the log C-P (log of fermentation broth-several values) standard curve of inhibitory activity of fermentation broth on E.sanguiensis;
FIG. 15 is a graph of the effect of different addition levels of fermentation broth on the bacteriostatic activity of Higgins colletotrichum;
FIG. 16 is a graph of log C-P (log fermentation broth concentration-log value) calibration of inhibitory activity of fermentation broth against Colletotrichum schinseng;
FIG. 17 is a graph of the effect of different addition levels of fermentation broth on the bacteriostatic activity of Alternaria mali;
FIG. 18 is a graph showing the log C-P (log of fermentation broth-several values) standard curve of the inhibitory activity of fermentation broth on Alternaria mali;
FIG. 19 is a graph of the effect of different addition levels of fermentation broth on the bacteriostatic activity of Alternaria graminearum;
FIG. 20 is a graph of the log C-P (log fermentation broth concentration-value) standard curve of the inhibitory activity of the fermentation broth on Alternaria graminearum;
FIG. 21 is a graph of the effect of different addition levels of fermentation broth on the bacteriostatic activity of potato anthracnose pathogen;
FIG. 22 is a graph of the log C-P (log fermentation broth concentration-several-degree value) standard curve of the inhibitory activity of fermentation broth against potato anthracnose pathogen;
FIG. 23 is a graph of the effect of different addition levels of fermentation broth on the bacteriostatic activity of Neurospora apiacea;
FIG. 24 is a graph showing the log C-P (log of fermentation broth concentration-several values) standard curve of the inhibitory activity of fermentation broth on Larix apiiformis;
FIG. 25 is a graph of the effect of different addition levels of fermentation broth on the bacteriostatic activity of Fusarium oxysporum;
FIG. 26 is a graph showing the log C-P (log fermentation broth concentration-several value) standard curve of the inhibitory activity of fermentation broth against Fusarium oxysporum;
FIG. 27 is a graph showing the effect of different addition levels of fermentation broth on the bacteriostatic activity of A-Aspergillus niger, B-Fusarium graminearum, C-Colletotrichum solani, and D-Pseudobulbus lablab;
FIG. 28 is a hairC-OD of influence of concentration of yeast liquid on growth amount of escherichia coli620(fermentation broth concentration-biomass) graph;
FIG. 29 is a graph of the log C-P (log fermentation broth concentration-several value) standard curve of the inhibitory activity of fermentation broth on E.coli;
FIG. 30 is a C-OD of the effect of fermentation broth concentration on Vibrio parahaemolyticus growth620(enzyme concentration-biomass) plot;
FIG. 31 is C-OD of the effect of fermentation broth concentration on Listeria growth620(enzyme concentration-biomass) plot;
FIG. 32 is a C-OD of the effect of fermentation broth concentration on Salmonella growth620(enzyme concentration-biomass) plot;
FIG. 33 is C-OD of the effect of fermentation broth concentration on growth of Aeromonas hydrophila620(enzyme concentration-biomass) plot;
FIG. 34 is a log C-P (log fermentation broth concentration-several value) standard curve of inhibitory activity of fermentation broth against Aeromonas hydrophila;
FIG. 35 is C-OD of effect of fermentation broth concentration on shigella growth620(enzyme concentration-biomass) plot;
FIG. 36 is a log C-P (log fermentation broth-several value) standard curve of shigella inhibitory activity of fermentation broth.
Detailed Description
The technical solution of the present invention will be described clearly and completely in the following with reference to the embodiments of the present invention, which are only a part of the embodiments of the present invention, but not all of the embodiments.
First, fermentation process
The raw materials in parts by mass are as follows:
3-5 parts of dried chili, 2-4 parts of chinaberry, 4-6 parts of tea seed cake, 3-5 parts of tobacco leaf, 2-4 parts of Chinese wampee bark, 3-5 parts of tripterygium wilfordii, 2-4 parts of Chinese honeylocust fruit, 3-5 parts of radix euphorbiae lantu, 2-4 parts of oleander, 1-3 parts of sargentgloryvine stem, 1-3 parts of macleaya cordata, 1-3 parts of cephalotaxus fortunei, 0.25-0.75 part of orange peel, 0.5-1.5 parts of selenium malt, 0.5-1.5 parts of sweet wormwood herb, 0.5-1.5 parts of cinnamon, 0.5-1.5 parts of vanilla, 0.5-1.5 parts of star anise, 0.5-1.5 parts of pepper, 0.25-0.75 part of fennel, 0.25-0.75 part of rutabaga, 1-3 parts of gelsemium elegans, 1-3 parts of ginger, 1-3 parts of purple onion, 4-6 parts of garlic, 1-3 parts of black bean and.
As further preferred, the raw material formula is:
4 parts of dried chili, 3 parts of chinaberry, 5 parts of tea seed cake, 4 parts of tobacco leaf, 3 parts of Chinese wampee leaf, 4 parts of tripterygium wilfordii, 3 parts of saponin, 4 parts of radix euphorbiae lantu, 3 parts of oleander, 2 parts of sargentgloryvine stem, 2 parts of macleaya cordata, 2 parts of cephalotaxus fortunei, 0.5 part of orange peel, 1 part of selenium malt, 1 part of sweet wormwood, 1 part of cinnamon, 1 part of vanilla, 1 part of star anise, 1 part of pepper, 0.5 part of fennel, 0.5 part of rue grass, 2 parts of gelsemium elegans, 2 parts of ginger, 2 parts of purple onion, 5 parts of garlic, 2 parts of black bean and 2 parts.
Pretreatment: drying and grinding the raw materials at 55 ℃, and then sieving the raw materials with a 80-mesh sieve to prepare a mixed material.
Fermentation substrate: 40g glucose +80g mixed material +1000mL purified water.
And (3) sterilization conditions: sterilizing at high temperature and high pressure, and keeping the temperature at 121 ℃ for 10 min.
Fermentation strain: saccharomyces cerevisiae + Lactobacillus pentosus Lactplantibibacillus pentosus + Lactobacillus sakazakii.
Inoculation amount: respectively culturing the three strains until the logarithmic phase is reached, mixing the three strains according to the volume ratio of 1:1:1 to obtain mixed strains, and inoculating the mixed strains for 3%.
Fermentation conditions are as follows: 30 ℃, 200rpm, 20 days, natural pH (5.3 +/-0.3), anaerobic fermentation.
Second, component detection and analysis
1. The method comprises the steps of performing multi-bacterium fermentation on plants containing effective bacteriostatic components to obtain fermentation liquor, qualitatively and quantitatively analyzing the components of the fermentation liquor through high performance liquid chromatography and mass spectrometry to find that 624 substances are totally contained in the fermentation liquor, wherein the types of flavonoid compounds are the most (146), the relative content of phenolic acid compounds is the highest (29.38%), and in addition, organic acid (26.79%), alkaloid (7.74%) and amino acid and derivatives (12.07%) are also main components, and the substance components contained in the fermentation liquor are summarized, and the results are shown in the following table 1.
TABLE 1 fermentation liquor of the types and ratios of the components
2. The bacteriostatic activity of the multi-bacterium fermentation liquor on fungi.
2.1 principle of the experiment (colony growth Rate method)
The colony growth rate method is slightly modified to determine the antifungal activity of the multi-bacterium fermentation liquid. Adding fermentation broth into sterilized PDA culture medium, cooling to 45 deg.C, making into antibacterial PDA plate with concentration (V)Fermentation liquor/VGeneral assembly) 0% to 30%, 0% as blank control, three replicates per concentration. Placing a circular filter paper (diameter of 5mm) in the center of the bacteriostatic PDA plate, and sucking 5 μ l spore suspension (10 μ l)8spores/mL) was dropped onto a filter paper sheet and cultured in the dark at the optimum temperature in an incubator. When the diameter of the fungal colony in the blank control is greater than 50mm, the colony diameter is measured by vernier caliper criss-cross. The concentration at which the fungi did not grow on the bacteriostatic PDA plates after 3 weeks of culture was MIC. The bacteriostatic rate was calculated as follows:
in the above formula, CK represents the average colony diameter of the control group fungus, and EG represents the average colony diameter of the treatment group fungus. Drawing (logarithm of concentration-several-degree value) logC-P standard curve and obtaining virulence regression equation, calculating half-maximal Effect Concentration (EC) for inhibiting mycelium growth50) And its corresponding 95% confidence limit.
2.2 Experimental methods
Configuring an antibacterial PDA culture dish: the addition amounts of the multi-strain fermentation liquid are respectively 0% (CK) and n%.
② placing the filter paper sheet in the middle of the culture dish, and dripping 5ul of mycelium spore suspension on the filter paper sheet by a liquid transfer device.
And culturing in an inverted mode for a certain time at a certain temperature and in a weak light.
Fourthly, measuring the diameter of the bacterial colony by adopting a vernier caliper cross method.
Calculating the bacteriostatic rate as follows:
sixthly, the concentration is converted into logarithm, the inhibition percentage is converted into a value of several degrees, a logC-P graph is drawn, a toxicity regression equation is calculated, and EC is obtained50。
2.3 results of the experiment
2.3.1 bacteriostatic Activity of Multi-Strain fermentation broths on spoilage fungi
2.3.1.1 bacteriostatic activity of the multi-strain fermentation liquid on Aspergillus niger.
Culturing at constant temperature of 35 deg.C for 3d, and obtaining MIC (minimum inhibitory concentration) according to the experimental result shown in figure 1; measuring the diameter by vernier caliper cross method, processing data to obtain logC-P (fermentation broth concentration logarithm-several value) as shown in FIG. 2, and obtaining virulence regression equation and half-effect concentration EC50。
2.3.1.2 antibacterial activity of multiple-strain fermentation liquid on Penicillium citrinum.
Culturing at constant temperature of 26.5 deg.C for 6d, and obtaining MIC according to the experimental result shown in FIG. 3; measuring the diameter by vernier caliper cross method, processing data to obtain logC-P as shown in FIG. 4, and obtaining virulence regression equation and EC50。
2.3.1.3 antibacterial activity of multiple-strain fermentation liquid on Aspergillus flavus.
Culturing at constant temperature of 26.5 deg.C for 6d, and obtaining MIC according to the experimental result shown in FIG. 5; measuring the diameter by vernier caliper cross method, processing data to obtain logC-P as shown in FIG. 6, and obtaining virulence regression equation and EC50。
2.3.1.4 antibacterial effect of multiple-strain fermentation liquid on Aspergillus versicolor.
Culturing at constant temperature of 26.5 deg.C for 7d, and obtaining MIC according to the experimental result shown in FIG. 7; measuring the diameter by vernier caliper cross method, processing data to obtain logC-P as shown in FIG. 8, and obtaining virulence regression equation and EC50。
2.3.1.5 antibacterial activity of the fermentation liquid of multiple strains on Acremonium acutum.
Culturing at constant temperature of 26.5 deg.C for 7d, and obtaining MIC according to the experimental result shown in FIG. 9; measuring the diameter by vernier caliper cross method, processing data to obtain logC-P as shown in FIG. 10, and obtaining virulence regression equation and EC50。
2.3.1.6 summary of the bacteriostatic activity of the fermentation broth against spoilage fungi, the results are shown in Table 2.
TABLE 2 summary of the bacteriostatic activity of the multi-strain fermentation broth on putrefying fungi
| Spoilage bacteria | Regression equation of virulence | R2 | EC50(％) | MIC(％) |
| Aspergillus niger | Y＝2.477x+1.992 | 0.9993 | 16.37 | 26 |
| Penicillium citrinum | Y＝2.124x+2.343 | 0.9994 | 17.82 | 30 |
| Aspergillus flavus | Y＝2.112x+2.260 | 0.9993 | 19.82 | 26 |
| Aspergillus versicolor | Y＝3.879x+0.1740 | 0.9936 | 17.54 | 22 |
| Cladosporium oxysporum (Fr.) Kuntze | Y＝3.310x+0.9941 | 0.9941 | 8.86 | 12 |
2.3.2 bacteriostatic activity of Multi-bacterium fermentation broth on plant-derived pathogenic fungi
2.3.2.1 antibacterial effect of multi-strain fermentation liquid on Rhizopus stolonifer.
Culturing at constant temperature of 26.5 deg.C for 2d, and obtaining MIC according to the experimental result shown in FIG. 11; measuring the diameter by vernier caliper cross method, processing data to obtain logC-P as shown in FIG. 12, and obtaining virulence regression equation and EC50。
2.3.2.2 antibacterial activity of multiple-strain fermentation liquid on Pseudobulbus Cremastrae seu pleiones.
Culturing at constant temperature of 26.5 deg.C for 7d, and obtaining MIC according to the experimental result shown in FIG. 13; measuring the diameter by vernier caliper cross method, processing data to obtain logC-P as shown in FIG. 14, and obtaining virulence regression equation and EC50。
2.3.2.3 bacteriostatic activity of Bacillus thuringiensis C.kikuchi in multi-strain fermentation broth.
Culturing at constant temperature of 26.5 deg.C for 6d, and obtaining MIC according to the experimental result shown in FIG. 15; measuring the diameter by vernier caliper cross method, processing data to obtain logC-P as shown in FIG. 16, and obtaining virulence regression equation and EC50。
2.3.2.4 antibacterial activity of multiple-strain fermentation liquid on Alternaria mali.
Culturing at constant temperature of 26.5 deg.C for 6d, and obtaining MIC as shown in FIG. 17; the diameter is measured by a vernier caliper cross method, logC-P is obtained after data processing as shown in figure 18, and a virulence regression equation and EC50 are obtained.
2.3.2.5 the bacteriostatic activity of the multi-bacterium fermentation liquor on the alternaria graminearum.
Culturing at constant temperature of 26.5 deg.C for 4d, and obtaining MIC according to the experimental result shown in FIG. 19; the diameter is measured by a vernier caliper cross method, logC-P is obtained after data processing as shown in figure 20, and a virulence regression equation and EC50 are obtained.
2.3.2.6 antibacterial activity of multiple-strain fermentation liquid on potato anthracnose pathogen.
Culturing at constant temperature of 26.5 deg.C for 4d, and obtaining MIC according to the experimental result shown in FIG. 21; the diameter is measured by a vernier caliper cross method, logC-P is obtained after data processing as shown in figure 22, and a virulence regression equation and EC50 are obtained.
2.3.2.7 antibacterial activity of multiple bacteria fermentation liquid on Neisseria apiacea.
Culturing at constant temperature of 26.5 deg.C for 7d, and obtaining MIC according to the experimental result shown in FIG. 23; the diameter is measured by a vernier caliper cross method, logC-P is obtained after data processing as shown in figure 24, and a virulence regression equation and EC50 are obtained.
2.3.2.8 antibacterial activity of multiple-strain fermentation liquid on Fusarium oxysporum.
Culturing at constant temperature of 26.5 deg.C for 7d, and obtaining MIC according to the experimental result shown in FIG. 25; measuring the diameter by vernier caliper cross method, processing data to obtain logC-P as shown in FIG. 26, and obtaining virulence regression equation and EC50。
2.3.2.9 summary of the bacteriostatic activity of the multi-strain fermentation broth against pathogenic fungi of plant origin, the results are shown in Table 3 below.
TABLE 3 summary of the bacteriostatic activity of the multi-strain fermentation broth against plant-derived pathogenic fungi
| Pathogenic bacteria | Regression equation of virulence | R2 | EC50(％) | MIC(％) |
| Rhizopus stolonifer (pers.: Fr.) Kuntze | Y＝4.127X+1.143 | 0.9811 | 8.60 | 14 |
| Pseudobulbus sennae | Y＝3.904X+2.899 | 0.9818 | 3.45 | 8 |
| Colletotrichum schikinsonii | Y＝6.635X+1.169 | 0.9892 | 3.78 | 5 |
| Alternaria mali | Y＝2.544X+3.458 | 0.9964 | 4.04 | 7 |
| Alternaria gramineara | Y＝3.519X+3.011 | 0.9987 | 3.67 | 8 |
| Colletotrichum solani | Y＝5.397X+0.9903 | 0.9921 | 5.53 | 10 |
| Lasiosphaera Seu Calvatia | Y＝3.986X+3.253 | 0.9890 | 2.74 | 4 |
| Fusarium oxysporum | Y＝3.448X+1.771 | 0.9955 | 8.63 | 13 |
2.4 Total analysis of the bacteriostatic activity of the fungi by the multi-strain fermentation broth.
The results of the bacteriostatic activity of the fermentation broth on A-Aspergillus niger, B-Fusarium graminearum, C-Colletotrichum solani, and D-Pseudobulbus sennae are shown in FIG. 27.
As can be seen from tables 2 and 3, the multi-fungus fermentation broth has a significant inhibitory effect on the hyphal growth of 13 fungi. FIG. 27 shows the inhibitory effect of the fermentation broth on the hyphal growth of several fungi tested, and it can be seen that the inhibitory effect is also significantly enhanced with the increase in the concentration of the fermentation broth.
As can be seen from the results, the fermentation broth had an excellent inhibitory activity against Rhizopus stolonifer (EC)508.60%, MIC 14%). As shown in Table 3, the fermentation broth was used for the hypha of 8 common plant pathogenic fungiHas excellent antibacterial activity for growth, and has strong inhibition (EC) on 6 plant pathogenic bacteria of Pseudobulbus sennae, Alternaria Malabarici, Colletotrichum Hikinsonii, Cereus, Anemonium celeriaceum, Colletotrichum graminearum and potato colletotrichum50Less than or equal to 5 percent), wherein the inhibition on the cercospora apiacea is strongest (EC)50＝2.74％、MIC＝4％)。
The growth speed of the hypha of the tested fungus is slowed down along with the increase of the concentration of the fermentation liquor, and the size of the colony is obviously inhibited by different degrees. In fact, the inhibition of fungi by the fermentation broth based on the method of the present experiment is divided into two parts, inhibition of spore germination and inhibition of mycelium growth, and therefore the EC here50In fact, it refers to the half-maximal effect concentration of the broth on the inhibition of mycelium growth, while the MIC actually refers to the lowest concentration of the broth on the inhibition of spore germination. From experimental results, the inhibitory properties of the fermentation liquor on the growth of mycelia and the germination of spores are consistent, the inhibitory effect on the mycelia is strong, and the inhibitory effect on the germination of the spores is ideal, so that the mycelia and the spores of the test bacteria have similar tolerance to the fermentation liquor, the fermentation liquor contains a large amount of phenols, alkaloids and other substances, and the bacteriostatic activity of the fermentation liquor on common plant pathogenic bacteria is particularly excellent.
3. The bacteriostatic activity of the multi-bacterium fermentation liquor on bacteria.
3.1 principle of the experiment (constant broth method)
Minimum Inhibitory Concentration (MIC) was determined using 96-well plate microtissues. Adding 190 μ l MHB culture medium (corresponding to the optimum pH of the test bacteria) and 10 μ l test bacteria liquid cultured to logarithmic phase in NB culture medium into the 1 st to 11 th wells, wherein the concentration of fermentation liquid in the culture medium (V)Fermentation liquor/VGeneral assembly) 0%, 1% -20%, the 1 st well is a blank control without fermentation broth, the 12 th well is a reference zeroing well containing only 200 μ l MHB medium, and each concentration is measured in triplicate. In addition, a negative control well (200. mu.l of MHB medium containing fermentation broth at the corresponding concentration) was placed in each well containing fermentation broth. Culturing at optimum growth temperature for test bacteria for 12h, and measuring OD with enzyme-labeling instrument620. Taking the concentration of the fermentation liquid as the abscissa and OD620The ordinate represents the inflection point of the curve, and the minimum inhibitory concentration is the inflection point of the curve.
3.2 Experimental methods
The test strain is cultured in MHB culture medium for 12 hr.
② 35mL test tubes are used as culture containers, the liquid loading capacity is 5mL, and MHB culture medium is selected as the culture medium.
Thirdly, adding multi-bacterium fermentation liquor into the culture medium, then adding the test bacterium suspension, culturing for 12h at constant temperature, and taking MHB culture medium as reference, and using the ultraviolet visible light spectrophotometer OD620Measurement of the absorbance, A1。
③ 0 percent of addition amount of fermentation liquor and an ultraviolet-visible spectrophotometer OD620Measurement of the absorbance, A2。
③ no test bacteria solution is added, and the ultraviolet-visible spectrophotometer OD620Measurement of the absorbance, A3。
Sixthly, adding amount of fermentation liquor: 1-10%, and n is 1%; 10-20%, and n is 2%.
Adding amount of the bacterial suspension: until the concentration of each tube of bacterial suspension is 0.5, the concentration of each McLeod tube;
the bacteriostatic rate was calculated as follows:
converting the concentration into logarithm, converting the inhibition percentage into several values, drawing logC-P diagram, calculating toxicity regression equation to obtain EC50。
3.3 results of the experiment
3.3.1 bacteriostatic activity of the multi-bacterium fermentation liquor on pathogenic bacteria of animal origin.
3.3.1.1 antibacterial activity of the multi-bacterium fermentation liquid on Escherichia coli.
Culturing at 35 deg.C and 180r/min for 12h at constant temperature, and processing the obtained absorbance data to obtain C-OD620(fermentation broth concentration-biomass) is shown in FIG. 28 and log C-P is shown in FIG. 29.
3.3.1.2 bacteriostatic activity of the multi-bacterium fermentation broth on vibrio parahaemolyticus.
Culturing at 35 deg.C and 180r/min for 12h at constant temperature, processing the obtained absorbance data,obtaining C-OD620(fermentation broth concentration-biomass) is shown in FIG. 30.
3.3.1.3 antibacterial activity of Listeria monocytogenes fermented liquid.
Culturing at 35 deg.C and 180r/min for 12h at constant temperature, and processing the obtained absorbance data to obtain C-OD620(fermentation broth concentration-biomass) is shown in FIG. 31.
3.3.1.4 bacteriostatic activity of the multi-strain fermentation liquid on salmonella.
Culturing at 35 deg.C and 180r/min for 12h at constant temperature, and processing the obtained absorbance data to obtain C-OD620(fermentation broth concentration-biomass) is shown in FIG. 32.
3.3.1.5 antibacterial activity of multiple-strain fermentation liquid against Aeromonas hydrophila.
Culturing at 28 deg.C and 180r/min for 12h at constant temperature, and processing the obtained absorbance data to obtain C-OD620(fermentation broth concentration-biomass) is shown in FIG. 33 and log C-P is shown in FIG. 34.
3.3.1.6 antibacterial activity of multiple-strain fermentation liquid on Shigella.
Culturing at 35 deg.C and 180r/min for 12h at constant temperature, and processing the obtained absorbance data to obtain C-OD620(fermentation broth concentration-biomass) is shown in FIG. 35 and log C-P is shown in FIG. 36.
3.3.1.7 summary of the bacteriostatic activity of the multi-bacterial ferments on pathogenic bacteria of animal origin, the results are shown in Table 4 below.
TABLE 4 summary of the bacteriostatic activity of the multi-bacterial fermented enzymes on pathogenic bacteria of animal origin
3.4 Total analysis of bacterial inhibition by the multi-strain fermentation broth.
The experimental result shows that the bacteriostatic activity of the multi-bacterium fermentation liquor on bacteria generally shows the tendency of promoting before inhibiting. When the fermentation liquor is in low concentration, the fermentation liquor has no obvious promotion or inhibition effect on escherichia coli, has a slight inhibition effect on aeromonas hydrophila, and has an obvious growth promotion effect on vibrio parahaemolyticus, listeria, salmonella and shigella; when the fermentation liquor reaches a certain concentration, the inhibition effect on other strains except the vibrio parahaemolyticus is obvious along with the increase of the concentration of the fermentation liquor. The minimum inhibitory concentration of the fermentation liquor to the bacteria is more than or equal to 10 percent, and the minimum inhibitory concentrations are respectively 10 percent of escherichia coli MIC, 18 percent of vibrio parahaemolyticus MIC, 14 percent of Listeria MIC, 10 percent of salmonella MIC, 12 percent of aeromonas hydrophila MIC and 10 percent of shigella MIC.
The MHB culture medium used in the bacteria bacteriostasis experiment mainly comprises acid hydrolyzed casein, beef powder and soluble starch, and both carbon source and nitrogen source are macromolecules which are difficult to directly utilize by bacteria in the first time. Numerous studies have shown that the strategy for mixed substrate utilization by microorganisms is to preferentially utilize readily absorbed and transformed small molecular nutrients, such as large amounts of D-glucose, D-fructose and various amino acids and derivatives in the fermentation broth. When the fermentation liquor is added into the culture medium, nutrient substances which can be directly utilized by microorganisms such as monosaccharide, amino acid and the like in the culture medium are increased, the growth and reproduction speed of bacteria is relatively accelerated, but meanwhile, antibacterial substances in the culture medium are also increased, so that the fermentation liquor shows an obvious inhibition effect on the growth of the bacteria after reaching a certain concentration.
As mentioned above, the fermentation liquor contains a large amount of phenols (flavonoids, phenolic acids and tannins), alkaloids, terpenoids and other substances, wherein galangin has an obvious bacteriostatic action on bacillus cereus, bacillus pumilus, bacillus subtilis, staphylococcus aureus and enterobacter cloacae, and the MIC of the galangin is 0.1-0.5 mg/ml; gallic acid has obvious bacteriostatic effect on pseudomonas putida (MIC 2.5mg/mL, MBC 10mg/mL) and the killing time of the gallic acid under 2.5, 5 and 10mg/mL for pseudomonas putida is 12, 6 and 1h respectively; the capsaicin shows obvious bacteriostatic activity on colibacillus, pseudomonas aeruginosa, klebsiella pneumoniae, staphylococcus aureus, enterococcus faecalis bacillus subtilis and candida albicans, and the MIC of the capsaicin is 2-8 mu g/ml. In addition, the pH of the fermentation liquor is reduced to about 3.25 by a large amount of organic acid contained in the fermentation liquor, the pH of the culture medium is also reduced along with the increase of the concentration of the fermentation liquor, and the growth of the test bacteria can be inhibited when the pH is not in the optimal range of the test bacteria. Therefore, the promotion effect of the nutrient substances in the fermentation liquid on the growth of the test bacteria is larger than the inhibition effect of the bacteriostatic substances and the pH on the growth of the test bacteria at low concentration, and the inhibition effect is larger than the promotion effect when the fermentation liquid in the culture medium reaches the critical concentration between promotion and inhibition. The difference of each test bacterium in tolerance to the bacteriostatic substance and pH results in the difference of critical concentration and MIC, while Escherichia coli and Aeromonas hydrophila have lower tolerance to the bacteriostatic substance or pH, so that the phenomenon that fermentation liquor promotes growth does not occur from the beginning.
And thirdly, bacteriostasis effect under different conditions.
The indicator bacteria used were: collybia graminearum (ACCC 37687).
1. Influence of different sterilization temperatures and fermentation modes on antibacterial effect
A fermentation mode: composite fermentation and natural fermentation
Carbon fermentation substrate: 20g of brown sugar, 20g of mixed materials and 1000mL of purified water
Fermentation days: 20 days
Fermentation conditions are as follows: 30 ℃, 200rpm, 20 days, natural pH (5.3 +/-0.3), anaerobic fermentation.
And (3) sterilization temperature: normal temperature (no sterilization), 65 deg.C (30min), 80 deg.C (10min), 100 deg.C (10min), 115 deg.C (10min), 121 deg.C (10min)
The results are shown in Table 5:
| sterilization temperature (. degree.C.) | At normal temperature | At normal temperature | 65 | 65* | 80 | 80* | 100 | 100* | 115 | 115* | 121 | 121* |
| MIC(％) | 23 | 26 | 18 | 20 | 14 | 18 | 17 | \ | 18 | \ | 14 | \ |
Note: natural fermentation (no added bacterial species); \\ no bacteriostatic effect; MIC-minimum inhibitory concentration
From the experimental results, it can be known that under the condition of no external strain, the heat preservation sterilization at 100 ℃, 115 ℃ and 121 ℃ for 10min has no bacteriostatic effect, and the control group with the external strain has the bacteriostatic effect. This indicates that all the primary microorganisms in the fermentation substrate can be killed by 10min of heat-insulation sterilization at 100 ℃. In addition, the contrast with the added strain has a bacteriostatic effect, which shows that the substances in the solution obtained by leaching the water solvent have no bacteriostatic ability, and the substances with the bacteriostatic effect can be generated only by further metabolizing the mixed material substrate and the extract in the solution through microbial fermentation.
In the experimental groups of normal temperature, 65 ℃ and 80 ℃, the fermentation substrate carries native microorganisms, and sterilization can not be completely carried out at 65 ℃ and 80 ℃, so that the antibacterial effect is achieved. From the results, the bacteriostatic effect of the added strain is superior to that of natural fermentation. In addition, the bacteriostatic effect is better along with the rise of the sterilization temperature, because more substances in the substrate are leached out and enter the metabolism process of the fermentation strain.
From the results, the bacteriostasis effect of the heat preservation and sterilization at 80 ℃ for 10min is consistent with the bacteriostasis effect of the heat preservation and sterilization at 115 ℃ for 10min, but is lower than the bacteriostasis effect of the heat preservation and sterilization at 121 ℃ for 10 min. Although the energy consumption of 121 ℃ is more than 80 ℃, the whole process is not influenced by the difference of the primary microorganisms contained in different batches of bottom species in consideration of adding a specific strain for fermentation after complete sterilization, so the sterilization condition of heat preservation and sterilization at 121 ℃ for 10min is preferred.
2. Influence of different carbon sources on the bacteriostatic effect
Fermentation strain: saccharomyces cerevisiae + Lactobacillus pentosus + Lactobacillus japonicas
Inoculation amount: mixing the bacteria in a volume ratio of 1:1:1, and inoculating the mixed bacteria for 3%.
Carbon fermentation substrate: 20g of carbon source, 20g of mixed material and 1000mL of purified water
And (3) sterilization temperature: sterilizing at 121 deg.C for 10 min.
Fermentation conditions are as follows: 30 ℃, 200rpm, 20 days, natural pH (5.3 +/-0.3), anaerobic fermentation.
Carbon source: glucose, fructose, fructo-oligosaccharide, maltose, maltodextrin, mannose, arabinose, pullulan, brown sugar, sucrose, starch
The results are shown in Table 6:
note: MIC-minimum inhibitory concentration
As a result, arabinose showed the best effect, and glucose was preferred in view of cost.
3. Influence of glucose addition on antibacterial effect
Fermentation strain: saccharomyces cerevisiae + Lactobacillus pentosus + Lactobacillus japonicas
Inoculation amount: mixing the bacteria in a volume ratio of 1:1:1, and inoculating the mixed bacteria for 3%.
Carbon fermentation substrate: glucose, 20g of mixed material and 1000mL of purified water
And (3) sterilization temperature: sterilizing at 121 deg.C for 10 min.
Fermentation conditions are as follows: 30 ℃, 200rpm, 20 days, natural pH (5.3 +/-0.3), anaerobic fermentation.
Glucose addition amount (g/L): 0. 10, 20, 30, 40, 50, 60, 70, 80, 90, 100
The results are shown in Table 7:
note: MIC-minimum inhibitory concentration
As a result, it was found that the bacteriostatic effects were the same for 20g/L, 30g/L and 40g/L, and that the fermentation time was optimized in consideration of the later stage, preferably 40 g/L.
4. Influence of mixed material addition on antibacterial effect
Fermentation strain: saccharomyces cerevisiae + Lactobacillus pentosus + Lactobacillus japonicas
Inoculation amount: mixing the bacteria in a volume ratio of 1:1:1, and inoculating the mixed bacteria for 3%.
Carbon fermentation substrate: 40g of glucose, 80 mixed materials and 1000mL of purified water
And (3) sterilization temperature: sterilizing at 121 deg.C for 10 min.
Fermentation conditions are as follows: 30 ℃, 200rpm, 20 days, natural pH (5.3 +/-0.3), anaerobic fermentation.
The addition amount (g/L) of the mixed material is as follows: 0. 10, 20, 30, 40, 50, 60, 70, 80, 90, 100
The results are shown in Table 8:
| addition amount (g/L) | 0 | 10 | 20 | 30 | 40 | 50 | 60 | 70 | 80 | 90 | 100 |
| MIC(％) | \ | 8.5 | 7.5 | 7.0 | 6.5 | 6.5 | 6.5 | 6.0 | 5.5 | 5.5 | 5.5 |
Note: \\ no bacteriostatic effect; MIC-minimum inhibitory concentration
As a result, it was found that the antibacterial effect was not obtained by adding the kneaded material, but 80g/L, 90g/L and 100g/L were the most effective, and 80g/L was preferable in view of cost.
5. Influence of different fermentation initiation pH values on bacteriostatic effect
Fermentation strain: saccharomyces cerevisiae + Lactobacillus pentosus + Lactobacillus japonicas
Inoculation amount: mixing the bacteria in a volume ratio of 1:1:1, and inoculating the mixed bacteria for 3%.
Carbon fermentation substrate: 40g of glucose, 80g of mixed material and 1000mL of purified water
And (3) sterilization temperature: sterilizing at 121 deg.C for 10 min.
Fermentation conditions are as follows: anaerobic fermentation at 30 deg.C and 200rpm for 20 days.
Initial pH: 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, natural pH (5.3)
The results are shown in Table 9:
note: \\ no bacteriostatic effect; MIC-minimum inhibitory concentration
As is clear from the results of the experiment, the pH at the end of the fermentation was about 3.5 regardless of the initial pH. Since the fermentation strain contains lactic acid bacteria, lactic acid is produced, and when the pH is lowered to about 3.5, the growth and acid production of microorganisms are inhibited, which is also confirmed by the fact that the initial pH is 3.5 and no bacteriostatic effect is obtained. The bacteriostatic effect is the same when the pH is between 4.5 and 6.5, and the natural pH is also in the interval, so the natural pH is preferably the fermentation initial pH.
6. Influence of fermentation temperature on bacteriostatic effect
Fermentation strain: saccharomyces cerevisiae + Lactobacillus pentosus + Lactobacillus japonicas
Inoculation amount: mixing the bacteria at a ratio of 1:1:1, and inoculating the mixed bacteria at a ratio of 3%.
Carbon fermentation substrate: 40g of glucose, 80g of mixed material and 1000mL of purified water
And (3) sterilization temperature: sterilizing at 121 deg.C for 10 min.
Fermentation conditions are as follows: 200rpm, 20 days, anaerobic fermentation, natural pH.
Fermentation temperature (. degree. C.): 14. 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40
The results are shown in Table 10:
| temperature (. degree.C.) | 14 | 16 | 18 | 20 | 22 | 24 | 26 | 28 | 30 | 32 | 34 | 36 | 38 | 40 |
| MIC(％) | \ | \ | \ | 10.0 | 8.5 | 6.0 | 6.0 | 5.0 | 5.5 | 6.0 | 7.0 | \ | \ | \ |
Note: \\ no bacteriostatic effect; MIC-minimum inhibitory concentration
As a result, it was found that the microorganisms did not grow at 18 ℃ or lower and at 36 ℃ or higher, and the effect was the best at 28 ℃.
It should be noted that the above-mentioned embodiments are only for illustrating the principle and the implementation manner of the present invention, but the present invention is not limited to the above-mentioned embodiments, and any simple modification, equivalent change and modification made to the above-mentioned embodiments according to the technical essence of the present invention fall within the protection scope of the present invention.
Claims (8)
1. A multi-bacterium fermentation broth with a sterilization effect is characterized by comprising the following raw materials in parts by weight:
3-5 parts of dried chili, 2-4 parts of chinaberry, 4-6 parts of tea seed cake, 3-5 parts of tobacco leaf, 2-4 parts of Chinese wampee bark, 3-5 parts of tripterygium wilfordii, 2-4 parts of Chinese honeylocust fruit, 3-5 parts of radix euphorbiae lantu, 2-4 parts of oleander, 1-3 parts of sargentgloryvine stem, 1-3 parts of macleaya cordata, 1-3 parts of cephalotaxus fortunei, 0.25-0.75 part of orange peel, 0.5-1.5 parts of selenium malt, 0.5-1.5 parts of sweet wormwood herb, 0.5-1.5 parts of cinnamon, 0.5-1.5 parts of vanilla, 0.5-1.5 parts of star anise, 0.5-1.5 parts of pepper, 0.25-0.75 part of fennel, 0.25-0.75 part of rutabaga, 1-3 parts of gelsemium elegans, 1-3 parts of ginger, 1-3 parts of purple onion, 4-6 parts of garlic, 1-3 parts of black bean and.
2. The multi-bacterium fermentation broth with bactericidal effect according to claim 1, which is characterized by comprising the following raw materials in parts by weight:
4 parts of dried chili, 3 parts of chinaberry, 5 parts of tea seed cake, 4 parts of tobacco leaf, 3 parts of Chinese wampee leaf, 4 parts of tripterygium wilfordii, 3 parts of saponin, 4 parts of radix euphorbiae lantu, 3 parts of oleander, 2 parts of sargentgloryvine stem, 2 parts of macleaya cordata, 2 parts of cephalotaxus fortunei, 0.5 part of orange peel, 1 part of selenium malt, 1 part of sweet wormwood, 1 part of cinnamon, 1 part of vanilla, 1 part of star anise, 1 part of pepper, 0.5 part of fennel, 0.5 part of rue grass, 2 parts of gelsemium elegans, 2 parts of ginger, 2 parts of purple onion, 5 parts of garlic, 2 parts of black bean and 2 parts.
3. The method for preparing multi-bacterium fermentation liquid with bactericidal effect according to claim 1, wherein the multi-bacterium fermentation liquid is obtained by fermenting the raw material with fermentation strains, and comprises the following steps:
step one, pretreatment of raw materials: drying and grinding the raw materials, and sieving to prepare a mixed material;
step two, adding 20-60g of carbon source and 60-100g of the mixed material obtained in the step one into each liter of water, uniformly mixing, and sterilizing at high temperature and high pressure to prepare a fermentation substrate;
step three, inoculating fermentation strains into the fermentation substrate obtained in the step two, wherein the fermentation conditions are as follows: fermenting at 28-30 deg.C and 200rpm for 20 days at pH 4.5-6.5 under anaerobic condition; the fermentation strain is as follows: mixing saccharomyces cerevisiae, lactobacillus pentosus and lactobacillus japonicas according to the volume ratio of 1:1: 1; the inoculum size was 3%.
4. The method for preparing multi-bacterium fermentation liquor with bactericidal effect according to claim 3, characterized in that in step one, the raw materials are dried and ground at 55 ℃ and then sieved by a 80-mesh sieve to prepare a mixed material.
5. The method for preparing a multi-bacteria fermentation broth with bactericidal effect as claimed in claim 3, wherein in step two, 40g carbon source and 80g mixed material obtained in step one are added into each liter of water.
6. The method according to claim 5, wherein the carbon source is one or more of glucose, fructose, fructo-oligosaccharide, maltose, maltodextrin, mannose, arabinose, pullulan, brown sugar, sucrose and starch.
7. The method for preparing a multi-bacteria fermentation broth with bactericidal effect according to claim 3, wherein the fermentation temperature is 28 ℃ in step three.
8. Use of a multi-bacterial fermentation broth according to any one of claims 1-7 for the preparation of a fungicide or pesticide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110178436.XA CN112956498B (en) | 2021-02-09 | 2021-02-09 | Multi-bacterium fermentation liquor with sterilization effect and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110178436.XA CN112956498B (en) | 2021-02-09 | 2021-02-09 | Multi-bacterium fermentation liquor with sterilization effect and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112956498A true CN112956498A (en) | 2021-06-15 |
| CN112956498B CN112956498B (en) | 2022-01-04 |
Family
ID=76284536
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110178436.XA Active CN112956498B (en) | 2021-02-09 | 2021-02-09 | Multi-bacterium fermentation liquor with sterilization effect and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112956498B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113767933A (en) * | 2021-08-25 | 2021-12-10 | 华南农业大学 | Chinese honeylocust fruit fermentation product and application thereof in preventing and treating plant diseases and insect pests and promoting growth |
| CN117343883A (en) * | 2023-12-06 | 2024-01-05 | 山东健源生物科技有限公司 | Lactobacillus plantarum and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120263699A1 (en) * | 2011-04-13 | 2012-10-18 | Nzymsys, Inc. | Compositions and Methods for Preservation of Fresh Produce |
| CN109221296A (en) * | 2018-10-25 | 2019-01-18 | 黑龙江惊哲森林农业科技开发有限责任公司 | A kind of plant degerming bio-fermented liquid and preparation method thereof |
| CN110269069A (en) * | 2019-08-05 | 2019-09-24 | 深圳大学 | The new application of Fructus Foeniculi volatile oil |
| CN111328838A (en) * | 2020-03-18 | 2020-06-26 | 珠海市自然之旅生物技术有限公司 | Composite plant extract with antiseptic and bacteriostatic functions and preparation method and application thereof |
| CN111990415A (en) * | 2020-08-08 | 2020-11-27 | 山西农业大学 | Botanical fungicide and preparation method and application thereof |
| CN112293436A (en) * | 2020-11-06 | 2021-02-02 | 苏州天纯农业科技有限公司 | Special enzyme for reducing soil diseases and insect pests |
-
2021
- 2021-02-09 CN CN202110178436.XA patent/CN112956498B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120263699A1 (en) * | 2011-04-13 | 2012-10-18 | Nzymsys, Inc. | Compositions and Methods for Preservation of Fresh Produce |
| CN109221296A (en) * | 2018-10-25 | 2019-01-18 | 黑龙江惊哲森林农业科技开发有限责任公司 | A kind of plant degerming bio-fermented liquid and preparation method thereof |
| CN110269069A (en) * | 2019-08-05 | 2019-09-24 | 深圳大学 | The new application of Fructus Foeniculi volatile oil |
| CN111328838A (en) * | 2020-03-18 | 2020-06-26 | 珠海市自然之旅生物技术有限公司 | Composite plant extract with antiseptic and bacteriostatic functions and preparation method and application thereof |
| CN111990415A (en) * | 2020-08-08 | 2020-11-27 | 山西农业大学 | Botanical fungicide and preparation method and application thereof |
| CN112293436A (en) * | 2020-11-06 | 2021-02-02 | 苏州天纯农业科技有限公司 | Special enzyme for reducing soil diseases and insect pests |
Non-Patent Citations (1)
| Title |
|---|
| 中国土农药志编辑委员会编著: "《中国土农药志》", 31 May 1959, 科学出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113767933A (en) * | 2021-08-25 | 2021-12-10 | 华南农业大学 | Chinese honeylocust fruit fermentation product and application thereof in preventing and treating plant diseases and insect pests and promoting growth |
| CN117343883A (en) * | 2023-12-06 | 2024-01-05 | 山东健源生物科技有限公司 | Lactobacillus plantarum and application thereof |
| CN117343883B (en) * | 2023-12-06 | 2024-02-20 | 山东健源生物科技有限公司 | Lactobacillus plantarum and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112956498B (en) | 2022-01-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2479253B1 (en) | Antagonistic bacteria for preventing and treating panama wilt disease of continuously planted banana and microorganism organic fertilizer thereof | |
| CN104673682B (en) | Aspergillus niger and its application in aflatoxin biology prevention and control | |
| Abdel-Azeem et al. | Aspergillus: Biodiversity, ecological significances, and industrial applications | |
| CN112956498B (en) | Multi-bacterium fermentation liquor with sterilization effect and preparation method and application thereof | |
| Hiroko Hassegawa et al. | Growth and antibacterial activity of Lentinula edodes in liquid media supplemented with agricultural wastes | |
| US20070231311A1 (en) | Method for preserving grain by-products | |
| CN102690755A (en) | Compound microbial bacterial preparation for degrading crop straw and preparation method and application of compound microbial bacterial preparation | |
| CN110396028B (en) | Biological organic fertilizer suitable for rape and application thereof | |
| Wu et al. | In vitro responses of Fusarium oxysporum f. sp. niveum to phenolic acids in decaying watermelon tissues | |
| Abdel-Kareem et al. | Novel marine yeast strains as plant growth-promoting agents improve defense in wheat (Triticum aestivum) against Fusarium oxysporum | |
| Chebotar’ et al. | Antifungal and phytostimulating characteristics of Bacillus subtilis Ch-13 rhizospheric strain, producer of bioprepations | |
| Gnonlonfoun et al. | Inhibition of the Growth of Fusarium tricinctum and Reduction of Its Enniatin Production by Erwinia gerunden sis Isolated from Barley Kernels | |
| CN107988271A (en) | A kind of processing method of wheat containing vomitoxin and application | |
| CN111777472A (en) | Gamma-PGA-rich organic fertilizer produced by semi-solid fermentation of waste lees of white spirit and production method thereof | |
| Rodrigues et al. | Biosurfactants as biocontrol agents against mycotoxigenic fungi | |
| KR101348435B1 (en) | Lactobacillus plantarum YML007 having anti-fungal activitiy and its use | |
| CN102757907B (en) | Endosulfan degradation stain and application thereof in soil remediation | |
| El-Taher et al. | Biosecurity for reducing ochratoxin A productivity and their impact on germination and ultrastructures of germinated wheat grains | |
| KR101873978B1 (en) | COMPOSITION FOR PREVENTING PLANT PATHOGENS COMPRISING EXTRACT FROM SPENT SUBSTRATE OF Lentinula edodes AND METHOD FOR PREPARING THE SAME | |
| CN109456900B (en) | Composite biological preparation and application thereof | |
| Ogidi et al. | Fruit preservation with bioethanol obtained from the fermentation of brewer's spent grain with Saccharomyces carlsbergensis | |
| Mitrović et al. | Effect of agitation rate on the production of antifungal metabolites by Streptomyces hygroscopicus in a lab-scale bioreactor | |
| Auerbach | Mould growth and mycotoxin contamination of silages: sources, types and solutions. | |
| El Sayed et al. | Abd EAAM. Investigation of fungus associated within co-occurrence of aflatoxins and ochratoxin a in cereals from Egypt | |
| CN114574384A (en) | Biopesticide extracted from microbial plants and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |