CN109628321B - Trichoderma strain prepared by solid fermentation of stevia rebaudiana residue and preparation method thereof - Google Patents
Trichoderma strain prepared by solid fermentation of stevia rebaudiana residue and preparation method thereof Download PDFInfo
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Abstract
The invention provides a solid fermentation trichoderma strain by utilizing stevia rebaudiana residue and a preparation method thereof, belonging to the field of bioengineering. The formula is 50% of stevia rebaudiana residue and 50% of rice bran, the pH value of the material is adjusted to 2.5-3.0 by acid hydrolysis of amino acid, and the water content is adjusted to 60% by tap water. The sterilization and inoculation technology is that a solid sterilization tank is sterilized for 40 minutes at 115 ℃, after the materials are cooled, a seed transfer pipeline is adopted to inoculate 10% of trichoderma liquid strains into the materials in the sterilization tank from a strain liquid fermentation tank, and a sterilization tank stirring system is utilized to uniformly mix the strains and the materials. The tray fermentation conditions comprise material thickness of 3cm, temperature of 26-28 deg.C, humidity of 70-90%, ten thousand-stage clean room culture for 4 days, and trichoderma spore amount reaching dry weight of 1.75 × 1010cfu/g. The trichoderma strains produced by the method have obvious growth promoting effect on crops by being matched with organic fertilizer for application.
Description
Technical Field
The invention belongs to the field of bioengineering, and relates to a technology for solid fermentation of trichoderma harzianum strain by utilizing stevia rebaudiana residue.
Background
Trichoderma fungi (Trichoderma, hereinafter referred to as Trichoderma) are an important soil inhabitation bacterium, spore germination and hypha growth of the Trichoderma fungi can adapt to a wider soil temperature and humidity and pH range, and good biocontrol effects generated by various mechanisms such as heavy parasitic action, antibiotic action, competitive action, induced resistance and the like become important development objects of biological antifungal preparations and biological fertilizers.
A large number of studies show that solid fermentation can significantly increase the number of spores of Trichoderma species due to its growth habit closer to that of Trichoderma species than liquid fermentation. Solid fermentation of trichoderma harzianum relates to liquid culture of trichoderma harzianum, selection and treatment of solid raw materials and control of culture environment. At present, some researchers have conducted some studies on solid fermentation of trichoderma species, for example, chinese patent CN105237146A discloses solid fermentation of trichoderma using sisal dregs; CN103468629A discloses fermentation of trichoderma using orange peel solids; CN104059860A discloses solid fermentation of Trichoderma using wheat bran and sawdust; CN1055862779A discloses solid fermentation of trichoderma using crop straw. The trichoderma fermentation technology reported above mainly relates to raw materials and raw material treatment, culture temperature, culture time and the like, but does not consider the fermentation environment of trichoderma, and if the fermentation environment is not clean, it is difficult to ensure that each batch of trichoderma can reach qualified quality.
Stevia rebaudiana (Stevia rebaudiana) contains abundant stevioside, a third natural sugar source following sucrose and beet sugar. Stevia rebaudiana residue is a product obtained after the extraction of stevioside, and is rich in crude fiber and crude protein. At present, the method for producing the trichoderma strains by using the stevia rebaudiana residues is not reported, and the trichoderma strains are subjected to solid fermentation by using the stevia rebaudiana residues as main raw materials, so that high-quality trichoderma strains can be obtained, and the problem of resource utilization of the stevia rebaudiana residues can be effectively solved. In addition, the patent gives detailed descriptions on the sterilization mode of the solid raw material, the aseptic inoculation method of trichoderma liquid strains, the environmental cleanliness of the trichoderma strain tray fermentation and the like, and has important guiding significance on the large-scale industrial production of the trichoderma strains.
Disclosure of Invention
The main purpose of the research is to provide a method for solid fermentation of trichoderma strains by utilizing stevia rebaudiana dregs aiming at the defects of the prior art; another purpose of the research is to obtain high-quality trichoderma strains which can be further applied to the preparation of bio-organic fertilizers.
The purpose of the invention can be realized by the following technical scheme:
a method for preparing Trichoderma strain by solid fermentation of stevia rebaudiana residue comprises adding 10-15% Trichoderma liquid strain into sterilized tray fermentation material, mixing, and culturing in ten thousand clean rooms at 26-28 deg.C and 70-90% humidity for 4 days; the tray fermentation material is prepared by using a mixture of stevia rebaudiana residue and rice bran as a raw material, adjusting the pH of the material to 2.5-3.0 by adding acid hydrolysis amino acid, and adjusting the water content of tap water to 60-65%.
The tray fermentation material is preferably prepared by using 50% of stevia rebaudiana residue and 50% of rice bran as raw materials, adjusting the pH of the material to 2.5-3.0 by acid hydrolysis of amino acid, and adjusting the water content of tap water to 60%.
The method preferably comprises the following steps: sterilizing the tray fermentation material for 40 minutes at 115 ℃ by using a solid sterilization tank, cooling, inoculating 10% of trichoderma liquid strain into the sterilization tank material from a strain liquid fermentation tank by using a transfer pipeline, and uniformly mixing the strain and the material by using a sterilization tank stirring system. The solid sterilization tank is provided with stirring equipment, so that on one hand, the materials can be sufficiently sterilized through stirring; on the other hand, after the materials are cooled to 30 ℃, liquid strains are inoculated through a seed transferring pipeline, and the strains and the materials can be fully and uniformly stirred.
The thickness of the tray fermentation material is preferably 2-4cm, more preferably 2-3cm, and still more preferably 3 cm.
The stevia rebaudiana dregs are dried, the water content is less than 10%, and the grain size is less than 5 mm.
The content of free amino acid in the acid hydrolysis amino acid is 15 percent, and the concentration of free hydrogen ions is 3M.
The preparation method of the acid hydrolysis amino acid is shown in patent of the invention which is previously granted by the applicant: a liquid amino acid complex prepared from protein of pig dead of disease and its application (ZL201410042218.3) are provided.
The Trichoderma solid strain prepared by the method of the present invention, wherein,the number of trichoderma spores reaches 1.75 multiplied by 10 in dry weight10cfu/g。
The invention provides a solid fermentation trichoderma strain by utilizing stevia rebaudiana residue and a preparation method thereof. Compared with the reported technology, the method is more comprehensive, and particularly, detailed descriptions are made on methods such as sterilization, inoculation, culture and the like, so that the method is more suitable for large-scale industrial production of trichoderma strains.
Compared with the products on the current market, the product has the following advantages:
1) the solid fermentation of the Trichoderma species by stevia rebaudiana is not only limited to the production of spores on the surface of the material (the raw materials such as straw and the like are usually only used for producing spores on the surface, and the number of the spores is 2 multiplied by 109cfu/g or so), so the density of the trichoderma spore produced by the method is high.
2) The trichoderma strains produced by the method are carried out in a clean room, so that the pollution of mixed bacteria can be effectively avoided, and the quality of the trichoderma spores produced by the method is good.
3) The test result shows that the product can obviously promote the growth of corn plants by being matched with the application of the organic fertilizer in the corn pot experiment.
Drawings
FIG. 1 is a sectional view of a strain of Trichoderma fermented by stevia rebaudiana.
FIG. 2 Effect of different solid feed carriers on solid fermentation of Trichoderma species.
FIG. 3 the effect of stevia on solid fermentation of Trichoderma species with different rice bran addition ratios.
FIG. 4 effect of different tray fermentation thicknesses on solid fermentation of Trichoderma species.
FIG. 5 Effect of different tray fermentation moisture content on solid fermentation of Trichoderma species.
FIG. 6 Effect of different tray fermentation inoculum sizes on solid fermentation of Trichoderma species.
FIG. 7 the effect of different tray fermentation times on solid fermentation of Trichoderma species.
FIG. 8 is a graph of the growth promoting effect of Trichoderma species on potted maize plants.
Detailed Description
1. Obtaining strains and raw materials
Strain: trichoderma guizhou NJAU4742 (Trichoderma guizhou NJAU4742, accession number CGMCC NO.12166) was stored in the laboratory of the plant nutrition issue group of the university resource and environmental sciences college of Nanjing agriculture.
Fermentation raw materials: acidolysis amino acid, stevia rebaudiana residue, rice bran, rice chaff and rice straw powder. The acid hydrolysis amino acid is provided by river-yin combined industry biotechnology limited company, the content of free amino acid is 15 percent, the concentration of free hydrogen ions is 3M, and the acid hydrolysis amino acid is prepared according to the method recorded in ZL 201410042218.3; the stevia rebaudiana dregs are purchased from Totai na Shengte Biotech Co., Ltd, dried, crushed and processed by a 10-mesh screen; the rice bran, the rice chaff and the rice straw powder are obtained by market purchase. The basic physicochemical properties of stevia rebaudiana residue, rice bran, rice hull and rice straw powder are shown in table 1:
TABLE 1 determination of the physico-chemical Properties of the different materials
2. Solid fermentation of trichoderma spp
2.1 Trichoderma liquid culture
Activating trichoderma guizhouense NJAU4742 by a PDA solid culture medium, inoculating the activated trichoderma guizhouense NJAU4742 to a PDA liquid culture medium, performing liquid fermentation culture for 4 days at the temperature of 28 ℃ at 170r/min, transferring the activated trichoderma guichenense NJAU4742 to a liquid fermentation tank, and fermenting for 4 days to obtain trichoderma guizhouense NJAU4742 liquid bacterial liquid.
2.2 solid Medium screening
The solid culture medium is set to 5 treatments, which are respectively 100% of stevia rebaudiana dregs, 70% of stevia rebaudiana dregs and 30% of rice bran, 70% of stevia rebaudiana dregs and 30% of wheat bran, 70% of stevia rebaudiana dregs and 30% of rice chaff and 70% of stevia rebaudiana dregs and 30% of rice straw powder. Initial pH of the optimum solid fermentation of trichoderma guizhouense NJAU4742 is 2.5-3.0, pH of the 5 treatments is adjusted to be 2.5-3.0 by using acid hydrolysis amino acid liquor (the proportion of the added acid hydrolysis amino acid is 6.6%), and water content is adjusted to be 60%. Sterilizing at 115 deg.C for 40 min, cooling, inoculating 10% of the obtained liquid bacterial solution of Trichoderma guizhou NJAU4742, and making tray (material thickness 3cm) at 28 deg.C and 90% humiditySolid fermentation for 4 days. And (3) counting the number of trichoderma spores: 10g of trichoderma solid culture strain is taken, diluted in 90ml of sterile water, fully and uniformly mixed by using a vortex mixer, and the spore concentration is measured by adopting a blood counting plate method. As shown in fig. 2, different material combinations have a significant effect on the spore count of trichoderma fermentations. The fermentation effect of 30% rice bran added to stevia rebaudiana residue is obviously better than that of other treatments, and the number of spores reaches 3.5 multiplied by 109cfu/g FW. The spore amount of the pure stevia rebaudiana residue reaches 1.5 multiplied by 109cfu/g FW, indicating that pure stevia rebaudiana residue is also suitable for solid fermentation of Trichoderma species. Compared with pure stevia rebaudiana residue treatment, 30% of rice chaff and 30% of rice straw powder added to the stevia rebaudiana residue have no obvious influence on the number of trichoderma species spores, and 30% of bran added to the stevia rebaudiana residue obviously reduces the number of trichoderma species spores. The results show that the rice bran, the rice chaff and the rice straw powder can be used as carriers of solid fermentation trichoderma reesei strains of stevia rebaudiana residue, and the effect of adding the rice bran is optimal.
2.3 solid Medium addition ratio
The solid culture medium is added with 5 treatments according to the proportion of 90% stevia rebaudiana dregs + 10% rice bran, 70% stevia rebaudiana dregs + 30% rice bran, 50% stevia rebaudiana dregs +50% rice bran, 30% stevia rebaudiana dregs + 70% rice bran and 10% stevia rebaudiana dregs + 90% rice bran. The initial pH, water content, sterilization mode, inoculum size and culture conditions were the same as 2.2. As shown in fig. 3, the addition of stevia rebaudiana residue with different proportions of rice bran has a significant effect on the number of spores fermented by trichoderma. The fermentation effect of the stevia rebaudiana dregs added with 50 percent of rice bran is obviously better than that of other treatments, and the number of spores reaches 7 multiplied by 10 of the fresh weight9cfu/g. The results show that the stevia rebaudiana residue is added with 50% of rice bran to be the optimal proportion.
2.4 tray fermentation thickness
The thickness of the shallow tray fermentation material is set to 5 treatments, which are 1, 2, 3, 4 and 5cm respectively. The materials are 50% of stevia rebaudiana residue and 50% of rice bran. The initial pH, water content, sterilization mode, inoculum size, temperature, humidity and incubation time were the same as 2.2. As shown in fig. 4, the tray fermentation material thickness has a significant effect on trichoderma fermentation spore count. Within 1-3cm, the number of trichoderma fermentation spores also tends to rise along with the increase of the thickness of the material. However, when the thickness of the material is more than 3cm, the number of trichoderma fermentation spores is reduced along with the increase of the thickness of the material, and the results show that the thickness of the material of 3cm is optimal.
2.5 tray fermentation moisture content
The water content of the tray fermentation is set to 5 treatments, which are respectively 50%, 55%, 60%, 65% and 70%. The materials are 50% of stevia rebaudiana residue and 50% of rice bran. The initial pH, sterilization mode, inoculum size, temperature, humidity, material thickness and incubation time were 2.2. As shown in FIG. 5, the water content of the tray fermentation material has a significant effect on the number of spores of Trichoderma fermentation. When the water content is lower than 60%, the number of trichoderma fermentation spores is in an ascending trend along with the increase of the water content; when the water content of the material is higher than 60%, the number of trichoderma fermentation spores is in a descending trend along with the increase of the water content, and the result shows that the water content of 60% is optimal.
2.6 shallow plate fermentation inoculum size
The tray fermentation inoculum size was set to 5 treatments, 5%, 7.5%, 10%, 12.5% and 15%, respectively. The materials are 50% of stevia rebaudiana residue and 50% of rice bran. The initial pH, water content, sterilization mode, temperature, humidity, material thickness and incubation time were the same as 2.2. As shown in FIG. 6, the proportion of inoculated tray fermentation material had a significant effect on the number of spores fermented by Trichoderma. When the inoculation ratio is lower than 10%, the number of trichoderma fermentation spores is in an ascending trend along with the increase of the inoculation ratio; when the inoculation ratio is higher than 10%, the number of trichoderma fermentative spores has no significant difference with the increase of the inoculation ratio. In order to save the cost of industrial production, 10% is preferably used as the inoculation ratio.
2.7 tray fermentation time
The tray fermentation inoculum size was set for 5 treatments, 2, 3, 4, 5 and 6 days respectively. The materials are 50% of stevia rebaudiana residue and 50% of rice bran. The initial pH, water content, sterilization mode, inoculum size, temperature, humidity and material thickness were 2.2. As shown in fig. 7, tray fermentation time has a significant effect on trichoderma fermentation spore numbers. Within 4 days, the number of trichoderma fermentation spores is in an ascending trend along with the increase of the inoculation time; after 4 days, there was no significant difference in the number of trichoderma fermentative spores as time increased. In order to save time and production costs, we prefer 4 days as fermentation time.
2.8 growth promoting Effect of Trichoderma Carnivorum potted plants
In the pot experiment, 1 control and 5 treatments are set, wherein the treatment is a non-fertilization control treatment (CK); trichoderma species treatment (T1); fertilizer treatment (T2); fertilizer + trichoderma treatment (T3); organic fertilizer treatment (T4); organic fertilizer + trichoderma treatment (T5), wherein the number of trichoderma added to T1, T3 and T5 is consistent, and the nitrogen, phosphorus and potassium nutrients applied to T2, T3, T4 and T5 are consistent. As can be seen from FIG. 8 and Table 2, there was no significant difference between the biomass index bacteria of corn and the only Trichoderma species applied compared to the control treatment; compared with chemical fertilizers, the chemical fertilizer and the trichoderma have no obvious difference on various biomass index bacteria of the corn; compared with organic fertilizers, the organic fertilizer and the trichoderma strains have remarkable promoting effects on the stem thickness, the overground fresh weight and the overground dry weight of the corn. The results show that the trichoderma strains are matched with the organic fertilizer to have good growth promoting effect on crops, and the results have important guiding significance on the use of the trichoderma strain products.
TABLE 2 Effect of different treatments on corn crop Biomass
Note: no fertilization control treatment (CK); trichoderma species treatment (T1); fertilizer treatment (T2); fertilizer + trichoderma treatment (T3); organic fertilizer treatment (T4); organic fertilizer + Trichoderma seed treatment (T5)
Claims (8)
1. A method for preparing Trichoderma strain by solid fermentation of stevia rebaudiana residue is characterized in that 10-15% Trichoderma liquid strain is added into sterilized tray fermentation material, and the mixture is uniformly mixed, wherein the tray fermentation temperature is 26-28 ℃, the humidity is 70-90%, and the cultivation is carried out in a ten thousand-level clean room for 4 days; the tray fermentation material is prepared by using a mixture of stevia rebaudiana residue and rice bran as a raw material, adjusting the pH of the material to 2.5-3.0 by adding acid hydrolysis amino acid, and adjusting the water content of tap water to 60-65%.
2. The method of claim 1, wherein the stevia rebaudiana residue is dried to a moisture content of less than 10% and a particle size of less than 5 mm.
3. The method as claimed in claim 1, wherein the tray fermentation material is prepared by adjusting pH of the material to 2.5-3.0 by acid hydrolysis of amino acid and adjusting water content to 60% with tap water, using 50% stevia rebaudiana residue and 50% rice bran as raw materials.
4. The method of claim 3, wherein the tray fermentation material is sterilized in a solid sterilization tank at 115 ℃ for 40 minutes, after cooling, 10% of Trichoderma liquid strain is inoculated from the strain liquid fermentation tank to the sterilization tank material by using a transfer pipeline, and the strain and the material are mixed by using a sterilization tank stirring system.
5. The method of claim 1, wherein the tray of fermentation material has a thickness of 2-4 cm.
6. The method of claim 5, wherein the tray of fermented material has a thickness of 2-3 cm.
7. The method of claim 6, wherein the tray of fermented material has a thickness of 3 cm.
8. The method as claimed in claim 1, wherein the acid-hydrolyzed amino acid has a free amino acid content of 15% and a free hydrogen ion concentration of 3M.
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| CN110982711B (en) * | 2019-12-04 | 2020-08-07 | 南京农业大学 | Method for open production of trichoderma solid strain |
| CN112745151A (en) * | 2021-01-11 | 2021-05-04 | 南京农业大学 | Composite biological organic fertilizer containing bacillus and trichoderma and preparation method thereof |
| CN113528356B (en) * | 2021-08-17 | 2023-02-17 | 南京农业大学 | Solid trichoderma preparation fermented by tea leaves and application of solid trichoderma preparation in tea seedling raising |
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