CN112457997A

CN112457997A - Trichoderma solid fermentation method

Info

Publication number: CN112457997A
Application number: CN202011510687.5A
Authority: CN
Inventors: 粱颁捷; 林智慧; 张珊珊; 顾钢; 肖顺; 林海; 张瀛; 吴荣生; 陈承亮; 周挺
Original assignee: Sanming Tobacco Co Of Fujian Province Tobacco Co
Current assignee: Sanming Tobacco Co Of Fujian Province Tobacco Co
Priority date: 2020-12-18
Filing date: 2020-12-18
Publication date: 2021-03-09
Anticipated expiration: 2040-12-18
Also published as: CN112457997B

Abstract

The invention provides a method for solid fermentation of trichoderma, which comprises the following steps: the method comprises the steps of trichoderma strain activation, preparation of seed fermentation liquor and solid fermentation culture of a porous model. The invention produces trichoderma spores by solid fermentation of a porous model, and the solid substrate is made into a porous shape by a common honeycomb briquette mould, so that the contact area of the solid substrate in unit volume and air is increased, the fermentation condition is improved, and the spore yield of the trichoderma solid fermentation is increased.

Description

Trichoderma solid fermentation method

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to a solid fermentation method of trichoderma.

Background

Trichoderma (Trichodermaspp.) belongs to Deuteromycotina, Hyphomycetes, from the order of Corymonales, from the family of Corymonaceae, Trichoderma, is widely present in nature, is the most potential plant disease biocontrol bacterium in fungi, can control a variety of plant diseases, and has emerged as a commercial Trichoderma preparation abroad. The trichoderma preparation is a live bacterial preparation, and live bacterial spore powder is generally obtained by a solid fermentation mode. In the study of solid fermentation, trichoderma can easily grow through a solid substrate by culturing in a triangular flask or other small container to obtain high spore yield, but when large-scale fermentation is carried out by using a tray, trichoderma usually grows well only on the surface layer of the solid substrate, and the fermentation of trichoderma is insufficient due to insufficient ventilation of the internal culture substrate.

In the prior art, in the large-scale growth of trichoderma, trichoderma usually only grows on the surface layer of a solid substrate, and the internal culture substrate often causes poor growth of the trichoderma due to insufficient ventilation, so that the spore yield of the trichoderma in unit volume is low, namely the conventional large-scale solid fermentation of the trichoderma cannot realize deep full fermentation.

In order to solve the technical problems, the method produces the trichoderma spores through solid fermentation of a porous model, a common honeycomb briquette mold is used for making a solid substrate into the porous solid fermentation model, the contact area of the solid substrate in unit volume and air is increased, the fermentation condition is improved, and the spore yield of the trichoderma solid fermentation is increased. The solid fermentation culture material is made into a homogeneous porous structural form through a honeycomb briquette die, so that sufficient surface area of the solid fermentation culture material can be ensured to be fully contacted with air, the surface area of the solid substrate contacted with the air can be enlarged after the trichoderma is inoculated, and the aerobic condition of trichoderma fermentation is improved, thereby solving the bottleneck problem that the conventional solid fermentation cannot realize deep full fermentation.

Disclosure of Invention

The invention aims to provide a method for solid fermentation of trichoderma.

In order to achieve the purpose, the invention adopts the following technical scheme:

the method comprises the following steps: trichoderma strain activation, preparation of seed fermentation liquid and solid fermentation based on a porous solid model.

Step one, strain activation: the preserved trichoderma strains are inoculated into PDA culture medium (potato dextrose agar culture medium) plates under the aseptic condition and are placed in a constant temperature incubator at 28 ℃ for inverted culture for 4 days.

Step two, preparing seed fermentation liquor: and (3) punching a bacterium dish (phi =10 mm) from the edge of a colony on the plate by using a puncher, taking the bacterium dish as an inoculum, inoculating the inoculum into a PD liquid culture medium (potato glucose), loading 200mL of the PD liquid culture medium in a 500mL triangular flask, inoculating 5 bacterium dishes into each flask, and performing shaking culture on a shaking table at the temperature of 28 ℃ and at the speed of 150 r/min for 96 h to obtain the seed fermentation liquid.

The PD liquid culture medium comprises the following components: 200 g of potato and 20 g of glucose. Cleaning potato, peeling, cutting into pieces, adding 1000 mL of water, boiling for 30 min, filtering with gauze, adding glucose, diluting to 1000 mL, and sterilizing at 121 deg.C for 20 min.

Step three, solid fermentation culture of the honeycomb briquette model: adding a proper amount of water into the solid fermentation substrate, uniformly stirring until the water content is 50-60 wt%, bagging with a polyethylene bag, performing intermittent moist heat sterilization at 121 ℃ for 40min, cooling, taking the seed fermentation liquid as an inoculum of the solid fermentation, wherein the inoculum size is 1.0-2.0 mL/g, punching and molding the solid fermentation substrate inoculated with the fermentation liquid by using a sterilized stainless steel honeycomb briquette mold, wherein the thickness of the mold is about 1-5 cm, and then culturing for 5-7 days under the conditions of 25-28 ℃ and the humidity of more than 80%.

The solid fermentation medium comprises the following components in percentage by mass: 65% of bran, 28% of rice husk, 3% of glucose, 2% of urea and 2% of monopotassium phosphate.

The invention provides a solid fermentation method of trichoderma, which comprises the steps of strain activation, preparation of seed fermentation liquor, solid fermentation culture and the like. The culture medium used for preparing the seed fermentation liquid is PD culture liquid. The culture medium used for solid fermentation consists of bran, rice husk, glucose, urea and potassium dihydrogen phosphate. The solid fermentation substrate is made into a porous shape by a stainless steel honeycomb briquette model, is set with a certain fermentation thickness, and is fermented under the condition of natural ventilation. The fermentation method provided by the invention is adopted to ferment the trichoderma, which is beneficial to improving the spore yield of the trichoderma, the raw materials are cheap and easy to obtain, the cost is low, and the spore-matrix mixture obtained by fermentation can be directly used for preventing and treating crop diseases or processed into a preparation for use after being dried.

The invention has the advantages that:

firstly, the spore yield of trichoderma is improved. The solid matrix is made into a honeycomb briquette ball shape, so that the contact area of the solid matrix in unit volume and air is increased, the fermentation condition is improved, and the spore yield of trichoderma solid fermentation is increased; secondly, the cost is low. The culture medium used for solid fermentation consists of bran, rice husk, glucose, urea and potassium dihydrogen phosphate, the raw materials are cheap and easy to obtain, and the spore-matrix mixture obtained by fermentation can be directly used for preventing and treating crop diseases or processed into a preparation for use after being dried.

Drawings

FIG. 1 shows solid fermentation of Trichoderma strain THGY-01 in porous aeration model.

Detailed Description

In order to make the aforementioned and other features and advantages of the invention more comprehensible, embodiments accompanied with figures are described in detail below. The method of the present invention is a method which is conventional in the art unless otherwise specified.

Example 1 Trichoderma asperellum (T. asperellum) Solid fermentation of THGY-01

1. Activating strains: inoculating the Trichoderma strain THGY-01 into a PDA culture medium plate under aseptic conditions, and placing the PDA culture medium plate in a constant temperature incubator at 28 ℃ for inverted culture for 4 days.

2. Preparing seed fermentation liquor: and (3) punching a bacterium dish (phi =10 mm) from the edge of a bacterial colony on the flat plate by using a puncher, taking the bacterium dish as an inoculum, inoculating the inoculum into a PD liquid culture medium in a 500mL triangular flask, inoculating 5 bacterium dishes into each flask, and performing shaking culture on a shaking table at 28 ℃ and 150 r/min for 96 h to obtain the seed fermentation liquid.

3. Solid fermentation culture: adding appropriate amount of water into the solid fermentation substrate, uniformly stirring until the water content is 50-60 wt%, bagging with polyethylene bags, performing intermittent moist heat sterilization at 121 ℃ for 40min, cooling, taking the seed fermentation broth as an inoculum of the solid fermentation, wherein the inoculum size is 1.0 mL/g, punching and molding the solid fermentation medium inoculated with the fermentation broth by using a sterilized stainless steel honeycomb briquette mold, wherein the mold thickness is 1cm, 3cm and 5cm respectively, and then culturing for 5 days under the conditions of 25 ℃ and 85% humidity. Observing the growth condition of trichoderma hyphae on the porous model substrate every 24 h, after fermenting for 120 h, observing the solid material of the porous model by transversely and longitudinally cutting when the surface layer of the solid substrate of the porous model is full of trichoderma, mashing and uniformly mixing the whole porous model substrate, and measuring the spore quantity by using a blood counting chamber.

4. Test results

Test results show that a large amount of white hyphae can be seen on the solid substrate after 48 hours of inoculation, the color begins to change into light green after 72 hours, and the culture is dark green after 96 hours. The surface layer and the punched part of the solid matrix of the honeycomb briquette model are well fermented, and hypha growth can be seen in the matrix (see figure 1).

When the fermentation is carried out for 120 hours, the spore yield in the substrate with the fermentation thickness of 1cm reaches 3.23 multiplied by 10⁹Per gram; while the spore yield at 1cm position on the substrate surface layer with the fermentation thickness of 3cm is 2.45 multiplied by 10⁹The spore yield at 3cm per gram is 1.95 multiplied by 10⁹The number of spores is 1.71 multiplied by 109 per g; the spore yield at 1cm position on the substrate surface layer with fermentation thickness of 5cm is 2.31 × 10⁹The spore yield at 5cm per gram is 9.1 × 10⁸The total spore yield is 1.43 multiplied by 10⁹Per gram (see Table 1).

TABLE 1 influence of different fermentation thicknesses on solid fermentation spore yield of Trichoderma strain THGY-01 porous model

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims

1. A method for solid fermentation of trichoderma, which is characterized by comprising the following steps:

step one, strain activation: inoculating the trichoderma strains in a PDA culture medium plate under the aseptic condition, and placing the plate in a constant temperature incubator at 28 ℃ for inverted culture for 4 days;

step two, preparing seed fermentation liquor: punching a bacterium dish phi =10mm from the edge of a bacterial colony on a flat plate by using a puncher, taking the bacterium dish phi as an inoculum, inoculating the inoculum into a PD liquid culture medium, filling 200mL of PD liquid culture medium in 500mL of triangular bottles, inoculating 5 bacterium dishes into each bottle, and performing shaking culture on a shaker at 28 ℃ and 150 r/min for 96 hours to obtain seed fermentation liquid;

step three, solid fermentation culture of a porous model: adding a proper amount of water into the solid fermentation substrate, uniformly stirring until the water content is 50-60 wt%, bagging with a polyethylene bag, performing intermittent moist heat sterilization at 121 ℃ for 40min, cooling, taking the seed fermentation liquid as an inoculum of the solid fermentation, wherein the inoculum size is 1.0-2.0 mL/g, punching and molding the solid fermentation substrate inoculated with the fermentation liquid by using a sterilized stainless steel honeycomb briquette mold to prepare a porous solid model, wherein the thickness of the model is 1-5 cm, and then culturing for 5-7 days under the conditions of 25-28 ℃ and the humidity of more than 80%.

2. The method of claim 1, wherein the PD broth is prepared as: cleaning potato 200 g, peeling, cutting into pieces, adding 1000 mL of water, boiling for 30 min, filtering with gauze, adding glucose 20 g, diluting to 1000 mL, and sterilizing at 121 deg.C for 20 min.

3. The method according to claim 1, wherein the solid fermentation medium formulation comprises, in mass percent: 65% of bran, 28% of rice husk, 3% of glucose, 2% of urea and 2% of monopotassium phosphate.