CN116179371A - Application of apple pomace as resource - Google Patents
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- CN116179371A CN116179371A CN202310486784.2A CN202310486784A CN116179371A CN 116179371 A CN116179371 A CN 116179371A CN 202310486784 A CN202310486784 A CN 202310486784A CN 116179371 A CN116179371 A CN 116179371A
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- trichoderma
- apple pomace
- raw material
- fermentation
- apple
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/50—Treatments combining two or more different biological or biochemical treatments, e.g. anaerobic and aerobic treatment or vermicomposting and aerobic treatment
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- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/80—Separation, elimination or disposal of harmful substances during the treatment
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- C05F5/00—Fertilisers from distillery wastes, molasses, vinasses, sugar plant or similar wastes or residues, e.g. from waste originating from industrial processing of raw material of agricultural origin or derived products thereof
- C05F5/002—Solid waste from mechanical processing of material, e.g. seed coats, olive pits, almond shells, fruit residue, rice hulls
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- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F5/00—Fertilisers from distillery wastes, molasses, vinasses, sugar plant or similar wastes or residues, e.g. from waste originating from industrial processing of raw material of agricultural origin or derived products thereof
- C05F5/006—Waste from chemical processing of material, e.g. diestillation, roasting, cooking
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- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
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- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
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- C12N1/22—Processes using, or culture media containing, cellulose or hydrolysates thereof
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/885—Trichoderma
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
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Abstract
The invention belongs to the field of microorganisms, and particularly relates to a recycling application of apple pomace. The application of apple pomace as fermentation material. According to the invention, firstly, apple pomace is used as a fermentation material, then the apple pomace is naturally piled up and then trichoderma liquid is added for secondary fermentation, so that the trichoderma bio-organic fertilizer is prepared, and the recycling utilization efficiency of the apple pomace is improved. The preparation method has the advantages that the preparation process flow and the raw material formula of the preparation method are relatively simple, the cost is low, the effective viable count of the trichoderma in the obtained organic fertilizer reaches 2 hundred million/g, the organic matter content reaches 60 percent and exceeds the industry standard, the resource utilization of wastes is realized, the economic benefit of the apple processing industry is improved, and the problem of environmental pollution caused by apple residues can be prevented.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a recycling application of apple pomace, which utilizes the apple pomace to prepare trichoderma preparation and trichoderma bio-organic fertilizer.
Background
Crushing and squeezing fresh apples to produce fruit juice, and further extracting pectin from residues to produce pomace. China is a large country for apple planting, and is also a large country for producing apple juice and pectin, and a large amount of apple pomace is produced each year. Apple pomace is not effectively treated and can be rotten and deteriorated in a short time, so that environmental pollution is caused. The smoke table is a main apple producing area in China, is also an important processing and producing area of apple juice and pectin, realizes the recycling utilization of apple pomace, has important significance on the local ecological environment protection, is beneficial to reducing the resource waste, and further improves the economic benefit. At present, apple pomace obtained by juicing and pectin extraction can be used for preparing biomass fuel, and although the problem of environmental pollution of the apple pomace can be relieved to a certain extent, the utilization mode is rough and the benefit is limited. The apple pomace mainly comprises cellulose, hemicellulose and lignin, also contains a certain amount of nutrient components such as mineral substances, soluble sugar, amino acid, vitamins and the like, is suitable for microbial growth, and can be used for producing microbial agents and bio-organic fertilizers with higher values.
Trichoderma is a filamentous fungus belonging to the genus Trichoderma of the family Desmodium of the subdivision Desmodii. Trichoderma is used as a recognized biocontrol bacterium, has antagonism on various plant pathogenic bacteria, especially soil-borne pathogenic fungi such as fusarium and pythium, and is commonly used for biological control of plant diseases. Trichoderma is also a rhizosphere growth promoting bacterium, can enhance nutrient absorption of plants, induce resistance of the plants to stress, and promote plant growth. Therefore, the trichoderma agent and the biological organic fertilizer are effective modes for recycling the apple pomace, and have wide market prospect in the field of agricultural planting.
The trichoderma spores have higher environmental adaptability than mycelia, are more easy to colonize and germinate in soil, and further exert the disease prevention and growth promotion effects, and the key point of preparing the trichoderma preparation is to obtain high spore yield. The solid state fermentation is an important technical joint in the production of trichoderma agents, and the selection of a proper solid state fermentation substrate is beneficial to improving the spore yield, simplifying the operation and reducing the cost. The patent report of successfully preparing trichoderma spore powder by directly using grains such as corn flour, barley and the like as fermentation matrixes is reported. But the organic waste is used as a solid fermentation substrate, so that the production cost of the microbial inoculum can be obviously reduced, the recycling of the waste is realized, the hidden danger of environmental pollution is eliminated, and the microbial inoculum can be more effectively popularized and applied. The Chinese patent No. 201010139041.0 provides a method for preparing trichoderma spore microbial inoculum by using wheat bran, wood dust, vitamins, sucrose and the like as fermentation matrixes and spray drying. Chinese patent No. 201210151677.6 provides a method for producing trichoderma preparation by potato residue. The Chinese patent CN200810011186.5 is prepared by mainly utilizing fungus chaff, adding corn flour, bran, compound micro-vitamins and other auxiliary materials. Chinese patent No. 201610382675.6 provides a method for producing trichoderma preparation by using waste such as mountain Ding Zishe, grape leaf, corn cob, pineapple peel and wheat bran. Chinese patent No. 201310411124.4 reports a method for preparing trichoderma preparation by fermenting orange peel and wheat bran. The method for preparing trichoderma preparation by utilizing waste mostly needs to additionally add more auxiliary materials to provide nitrogen sources or carbon sources so as to ensure the spore yield, and the application link is mainly aimed at crop diseases, and does not pay attention to the application of promoting the growth of saline-alkali soil crops.
Meanwhile, the production of trichoderma bio-organic fertilizer by using organic waste as a matrix through fermentation is also a research hot spot in recent years. Chinese patent No. 201410100137.4 provides a method for producing Trichoderma fertilizer by using pig manure. Chinese patent No. CN201510554088.6 provides a method for producing trichoderma fertilizer by fermentation of waste after sisal fiber extraction. Chinese patent No. 202010670605.7 discloses a method for preparing trichoderma preparation from concentrated molasses alcohol solution and bean paste. Chinese patent No. 201610787853.3 uses green onion leaves as raw materials to prepare trichoderma conidium, and then the trichoderma conidium is mixed with decomposed organic fertilizer to obtain the trichoderma biofertilizer. The Chinese patent No. CN201610003589.X prepares the trichoderma solid ferment by using the straws of corn, wheat and the like and the filter mud of sugar cane sugar making, and mixes the trichoderma solid ferment with the organic fertilizer to obtain the trichoderma bio-organic fertilizer. The Chinese patent CN201610151640.1 utilizes amino acid liquid and straw to prepare a trichoderma solid fermentation product, and then the trichoderma solid fermentation product is mixed with compost to obtain the trichoderma bio-organic fertilizer. The existing preparation method of the trichoderma bio-organic fertilizer is mainly characterized in that organic waste is used for preparing a trichoderma solid fermentation product, and then the trichoderma solid fermentation product is simply mixed with the organic fertilizer to produce the trichoderma bio-organic fertilizer, and the trichoderma bio-organic fertilizer is not produced by directly taking the organic waste as a main raw material and a fermentation carrier. In addition, no report exists on preparing the trichoderma bio-organic fertilizer by utilizing apple pomace at present.
Disclosure of Invention
The invention aims to provide a resource application of apple pomace, which utilizes the apple pomace to prepare trichoderma preparation and trichoderma bio-organic fertilizer.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the application of apple pomace as resource is the application of apple pomace as fermentation material.
The application of the apple pomace in preparing trichoderma agents or trichoderma bio-organic fertilizers as fermentation materials.
The fermentation material is obtained by dehydrating apple pomace after pectin extraction to water content of about 70%, and drying.
Further, the fermentation material is obtained by adding lime with the mass fraction of about 0.15% into apple pomace after pectin extraction, then dehydrating until the water content is about 70%, and drying.
The trichoderma preparation is obtained by inoculating trichoderma liquid into fermentation materials and fermenting;
the trichoderma bio-organic fertilizer is obtained by spraying trichoderma liquid into a fermented material after composting and fermenting.
A trichoderma preparation is prepared by inoculating trichoderma solution to the material of fermented product of apple pomace, and fermenting.
Specifically: the trichoderma spinosum is addedTrichoderma asperellum) Inoculating the bacterial liquid of (a) into a raw material taking apple pomace as a ferment for fermentation treatment at a constant temperature; wherein, potassium dihydrogen phosphate is added into the raw materials to adjust the pH value to be neutral.
A preparation method of trichoderma preparation using apple pomace as raw material comprises mixing trichoderma asperellum with the mixtureTrichoderma asperellum) Inoculating the bacterial liquid of (a) into a raw material taking apple pomace as a ferment for fermentation treatment at a constant temperature; wherein, potassium dihydrogen phosphate is added into the raw materials to adjust the pH value to be neutral.
The trichoderma asperellum is preparedTrichoderma asperellum) The trichoderma asperellum is trichoderma asperellum recorded in Chinese patent No. CN201710035544.5, which is an trichoderma asperellum and application thereof.
Further, in the present description,
lime with the mass percentage of about 0.15% is added into the apple pomace after pectin extraction, and then dehydration is carried out, the dehydration is carried out until the water content is about 70%, and the apple pomace is dried and crushed for later use; then 10% -50% (mass volume fraction) of trichoderma asperellum is added into the dehydrated apple pomaceTrichoderma asperellum) Uniformly stirring the bacterial liquid and 0.5-1.5% (mass fraction) of monopotassium phosphate aqueous solution, spreading a thin layer (within 1 cm) in a sterile culture dish or other sterile vessels, and fermenting at 25-31 ℃ for 2-4 days. Scraping upper spore, oven drying at 36 deg.C, pulverizing, and making into Trichoderma preparation.
Wherein, the adding amount of the monopotassium phosphate is 0.5-1.5g of the monopotassium phosphate per 100g of the pomace.
Trichoderma asperellumTrichoderma asperellum) The bacterial liquid of (2) is obtained by picking Trichoderma asperellum mycelium preserved on agar inclined plane at 4deg.C, inoculating to Potato Dextrose Agar (PDA) plate center, culturing in darkness at 28deg.C for 3-4 days, picking edge mycelium or scraping green spore, transferring mycelium or spore into potato dextrose liquid culture medium (PD), culturing at 28deg.C under shaking (rotation speed 150 rpm) for 4-5 days to form a large amount of white flocculent mycelium, and obtaining Trichoderma liquid；
In order to obtain a large amount of trichoderma liquid, the obtained trichoderma liquid is used as seed liquid, the potato paste powder and the culture medium with glucose as raw materials are used for fermentation in a liquid fermentation tank according to the fermentation conditions of the obtained trichoderma liquid, so that a large amount of trichoderma liquid is prepared.
Preparation of PDA culture medium: 15g of potato soaked powder is weighed, 1000ml of water is added, 20g of glucose and 18g of agar are added, sterilization is carried out for 20 minutes at 121 ℃, and the potato soaked powder is packaged in a culture dish for cooling and then is stored for standby. Preparation of PD culture solution: 15g of potato soaked powder is weighed, 1000ml of water is added, 20g of glucose is added, sterilization is carried out for 20 minutes at 121 ℃, and the potato soaked powder is cooled and stored for standby. Preparing a liquid culture medium by using potato paste powder and glucose as raw materials: 20g of potato paste powder is weighed, 1000ml of water is added, 20g of glucose is added, and sterilization is carried out for 20 minutes at 121 ℃.
A trichoderma bio-organic fertilizer using apple pomace as a raw material: and (3) composting apple pomace serving as a raw material of a fermented product, spraying trichoderma liquid on the apple pomace after the composting treatment, and then fermenting to obtain the finished product.
The method comprises the following steps: the trichoderma spinosum is addedTrichoderma asperellum) Spraying the bacterial liquid of (2) into the raw material of the fermented product of apple pomace after composting treatment, and then fermenting at room temperature;
or, the trichoderma asperellum containingTrichoderma asperellum) Spraying the bacterial liquid of (2) into the raw material of the fermented product of apple pomace after composting treatment, and then fermenting at room temperature; wherein, the raw material of the fermented product is the apple pomace after composting treatment, and wheat bran is added.
The preparation method of the trichoderma bio-organic fertilizer using apple pomace as raw material comprises the steps ofTrichoderma asperellum) Spraying the bacterial liquid of the formula (I) into the raw material of the composted apple pomace serving as a ferment, and then fermenting at room temperature;
or, the trichoderma asperellum containingTrichoderma asperellum) Spraying the bacterial liquid of the formula (I) into the raw material of the composted apple pomace serving as a ferment, and then fermenting at room temperature; wherein the apple pomace after compostingWheat bran is added as a raw material of the fermented product.
Further, in the present description,
the trichoderma spinosum is addedTrichoderma asperellum) The bacterial liquid of the (2) is evenly sprayed into the spread raw material which takes apple pomace as a ferment after composting, and the volume-mass ratio of the inoculation amount of the trichoderma liquid is controlled to be 5-20%. And stacking the materials into a trapezoid stack, turning the stack once every 3 days at room temperature, completing the fermentation of the trichoderma in 7 to 14 days, spreading and airing until the water content is 30%, and crushing to obtain the trichoderma bio-organic fertilizer.
Or, wheat bran is added to the raw material of the composted apple pomace serving as a fermentation product, and the mixture is treated according to the conditions to obtain the trichoderma bio-organic fertilizer.
The trichoderma asperellum is preparedTrichoderma asperellum) The bacterial liquid of (1) is prepared by picking Trichoderma asperellum hyphae preserved on agar inclined planes at 4 ℃, inoculating to the center of a Potato Dextrose Agar (PDA) flat plate, culturing for 3-4 days in darkness at 28 ℃, picking edge hyphae or scraping green spores, transferring the hyphae or spores into a potato dextrose liquid culture medium (PD), and culturing for 4-5 days by shaking (rotating speed 150 rpm) at 28 ℃ to form a large amount of white flocculent hyphae to obtain Trichoderma asperellum liquid for use;
in order to obtain a large amount of trichoderma liquid, the obtained trichoderma liquid is used as seed liquid, the potato paste powder and the culture medium with glucose as raw materials are used for fermentation in a liquid fermentation tank according to the fermentation conditions of the obtained trichoderma liquid, so that a large amount of trichoderma liquid is prepared. Preparation of PDA culture medium: 15g of potato soaked powder is weighed, 1000ml of water is added, 20g of glucose and 18g of agar are added, sterilization is carried out for 20 minutes at 121 ℃, and the potato soaked powder is packaged in a culture dish for cooling and then is stored for standby. Preparation of PD culture solution: 15g of potato soaked powder is weighed, 1000ml of water is added, 20g of glucose is added, sterilization is carried out for 20 minutes at 121 ℃, and the potato soaked powder is cooled and stored for standby. Preparing a liquid culture medium by using potato paste powder and glucose as raw materials: 20g of potato paste powder is weighed, 1000ml of water is added, 20g of glucose is added, and sterilization is carried out for 20 minutes at 121 ℃.
The apple pomace treated by composting is used as a raw material of a ferment, and the mass fraction of the apple pomace after pectin extraction is added into the apple pomaceLime accounting for about 0.15 percent, then dehydrating until the water content is about 70 percent, then naturally composting, namely stacking dehydrated slag into a trapezoid stack shape on an empty site, wherein the height is 1-1.2m, the width is 1.5-2m, the length is set according to the size of the site, and the total volume is ensured to be 1m 3 After natural fermentation for about 3 days, the temperature of the interior of the material is raised to above 50 ℃, composting enters a high-temperature stage and lasts for 10-14 days at 50-60 ℃, then the material is gradually cooled to below 50 ℃, enters a decomposition stage and lasts for about 7 days at 40-50 ℃, and the composting process is completed after the material is cooled to below 40 ℃; in the apple slag composting process, the composting is carried out for 1 time every 3 days, ventilation of materials is promoted, anaerobic fermentation is prevented, and a composting machine or manual composting is adopted according to the volume of the apple slag materials. The temperature in winter is lower, in order to ensure the temperature rise of the composting, the volume of the material can be increased (more than 3m 3 ) And covered with a plastic film or the like. The apple pomace composting process can kill harmful bacteria and weed seeds, and the decomposing stage can further promote nutrients.
The invention has the beneficial effects that:
(1) The apple pomace is used as a fermentation material, the apple pomace is rich in nutrition, auxiliary materials are not needed to be added in the fermentation process to provide a carbon source and a nitrogen source, the operation is relatively simple and feasible, the production cost is reduced, and the recycling utilization of the apple pomace is realized.
(2) The invention prepares the trichoderma spore microbial inoculum by using the apple pomace as a fermentation material, which is beneficial to promoting the recycling of pomace waste, improving economic benefit and eliminating hidden danger of environmental pollution.
(3) According to the invention, firstly, apple pomace is used as a fermentation material, then the apple pomace is naturally piled up and then trichoderma liquid is added for secondary fermentation, so that the trichoderma bio-organic fertilizer is prepared, and the recycling utilization efficiency of the apple pomace is improved. The preparation method has the advantages that the preparation process flow and the raw material formula of the preparation method are relatively simple, the cost is low, the effective viable count of the trichoderma in the obtained organic fertilizer reaches 2 hundred million/g, the organic matter content reaches 60 percent and exceeds the industry standard, the resource utilization of wastes is realized, the economic benefit of the apple processing industry is improved, and the problem of environmental pollution caused by apple residues can be prevented.
(4) The trichoderma adopted by the invention is trichoderma spinosum separated from a tobacco bench orchard, the obtained microbial inoculum and the biological organic fertilizer can effectively prevent and treat continuous cropping diseases of apple seedlings caused by fusarium, the microbial inoculum is convenient to transport, and the microbial inoculum or the organic fertilizer is applied to coastal saline-alkali soil, so that the growth of economic crops in the saline-alkali soil can be promoted, the agricultural development and utilization in coastal saline-alkali areas are facilitated, and the application prospect is wide.
Drawings
FIG. 1 shows the effect of applying trichoderma preparation on the photosynthetic rate of matrimony vine and honeysuckle in saline-alkali soil and dry weight of plants according to the embodiment of the present invention.
Fig. 2 shows the effect of applying trichoderma fertilizer and sterilizing matters thereof on the yield and biomass of medlar dry fruits in saline-alkali soil according to the embodiment of the invention.
FIG. 3 shows the effect of applying Trichoderma fertilizer and partially replacing nitrogen fertilizer on the yield and biomass of dried fruits of Lycium barbarum in saline-alkali soil according to the embodiment of the invention.
Detailed Description
The following description of the embodiments of the present invention is further provided in connection with the accompanying examples, and it should be noted that the embodiments described herein are for the purpose of illustration and explanation only, and are not limiting of the invention.
The strain adopted in the following example is Trichoderma asperellumTrichoderma asperellum) The trichoderma asperellum is%Trichoderma asperellum) The trichoderma asperellum is trichoderma asperellum recorded in Chinese patent No. CN201710035544.5, which is an trichoderma asperellum and application thereof.
The apple pomace is the leftovers of apple pectin manufacturers; namely, the residue obtained after the apple juice was extracted and pectin was extracted.
Example 1
Strain activation: picking up trichoderma asperellum preserved in agar inclined plane at 4 DEG CTrichoderma asperellum) Inoculating to potato glucose agar (PDA) plate center, culturing at 28deg.C in dark for 3-4 days, picking edge mycelium or scraping green spore, transferring mycelium or spore into potato glucose liquid culture medium (PD), culturing at 28deg.C under shaking (rotation speed 150 rpm) for 4-5 days to form a large amount of white flocculent mycelium,obtaining trichoderma fermentation liquor (seed liquor).
Preparation of PDA culture medium: 7.5g potato soaked powder is weighed, 1000ml of water is added, 20g glucose and 18g agar are added, sterilization is carried out for 20 minutes at 121 ℃, and the potato soaked powder is packaged in a culture dish for cooling and then stored for standby. Preparation of PD culture solution: 7.5g of potato soaked powder is weighed, 1000ml of water is added, 20g of glucose is added, sterilization is carried out for 20 minutes at 121 ℃, and the potato soaked powder is cooled and stored for standby.
Meanwhile, if the fermentation liquid (seed liquid) is produced on a large scale: the culture medium with potato paste powder and glucose as raw materials is used for fermentation in a liquid fermentation tank to prepare a large amount of trichoderma fermentation seed liquid.
Apple pomace pretreatment:
taking apple pomace (water content is 80%) after pectin extraction, adding lime with mass fraction of about 0.15% into the apple pomace, physically extruding by using a filter press dehydrator, reducing viscosity, increasing dispersibility, dehydrating to water content of about 70%, drying, grinding, and sieving with 20 mesh sieve for use.
Trichoderma agent:
weighing the pretreated and crushed apple pomace, adding sterile water of potassium dihydrogen phosphate and seed solution, stirring, spreading in a thin layer (within 1 cm) in a sterile culture dish or other sterile container, and spreading 25-31 o Fermenting at C temperature for 2-4 days. Scraping upper spore layer, 36 o And C, drying at low temperature, and crushing to prepare the trichoderma preparation.
The inoculation amount, the addition amount of potassium dihydrogen phosphate, the addition amount of sterile water, the fermentation days and the fermentation temperature in the preparation of the microbial inoculum are 5 main factors, 3 levels are set for each factor, and the total fermentation temperature is L 18 （3 5 ) Orthogonal table orthogonal experiments were performed and the number of trichoderma spores was counted using a hemocytometer, each combination was repeated 3 times, and the average was taken (table 1).
TABLE 1 orthogonal test and extremely bad analysis of Trichoderma preparation
K is the average viable count at different levels for each factor (10 10 Number/g), RFor extreme differences between different levels of factors, the unit is 10 10 Each/g. The inoculation amount and the aseptic water access amount are mass and volume fractions (g/ml) of the pomace, and the addition amount of the monopotassium phosphate and the wheat bran is mass fraction (g/g) of the compost of the pomace.
The extremely poor analysis results in Table 1 show that the addition amount of potassium dihydrogen phosphate and the inoculation amount of seed liquid have the greatest influence on the number of active bacteria of the trichoderma agent, and the fermentation days, the addition amount of sterile water and the fermentation temperature are sequentially carried out; the optimal fermentation combination conditions of the prepared microbial inoculum are that the seed liquid inoculation amount is 10%, 1% of potassium dihydrogen phosphate is added, the fermentation is carried out for 4 days, and 200% of sterile water and 25 ℃ are added. The test is carried out by adopting the combination, and the number of the trichoderma spores reaches 1.5X10 10 Each/g/g. In practical production, the steps of saving raw materials, shortening fermentation period and the like can be taken, wherein the combination of the conditions of No. 1, namely 10 percent of inoculation amount, 0.5 percent of potassium dihydrogen phosphate, 2 days of fermentation, 150 percent of sterile water and 25 percent of fermentation can be adopted o C, trichoderma spore number of 0.24X10 10 The microbial inoculum obtained under the condition can reach the national agricultural microlite microbial inoculum standard (GB 20287-2006), and indexes such as the mixed bacterial rate, the heavy metal content and the like reach the standard, so that the product is qualified (see Table 2).
TABLE 2 parameters of Trichoderma preparation
Example 2
Preparing the apple pomace bio-organic fertilizer:
the apple pomace (water content 80%) after pectin extraction was added with lime having a mass fraction of 0.15%, and was physically pressed by a press dehydrator to be dehydrated to about 70%. Lime can effectively promote the dehydration of apple pomace to reach the water content suitable for composting, but the pH value is raised to above 8, so that the method is not suitable for the growth and propagation of trichoderma. Therefore, after the apple pomace is dehydrated, natural composting is firstly carried out.
Stacking the dehydrated apple pomace into a trapezoid stack shape on an empty field, wherein the height is 1-1.2m, the width is 1.5-2m, the length is set according to the size of the field, and the total volume is ensured to be 1m 3 On top of that, natural hairAfter fermentation for about 3 days, the temperature of the interior of the material is raised to above 50 ℃, composting enters a high-temperature stage and lasts for 10-14 days at 50-60 ℃, then the material is gradually cooled to below 50 ℃, enters a decomposition stage and lasts for about 7 days at 40-50 ℃, and the composting process is completed after the material is cooled to below 40 ℃. In the apple slag composting process, the composting is carried out for 1 time every 3 days, ventilation of materials is promoted, anaerobic fermentation is prevented, and a composting machine or manual composting is adopted according to the volume of the apple slag materials. The temperature in winter is lower, in order to ensure the temperature rise of the composting, the volume of the material can be increased (more than 3m 3 ) And covered with a plastic film or the like. The apple pomace composting process can kill harmful bacteria and weed seeds, the nutrient release can be further promoted in the composting stage, the pH is reduced, the harmlessness and humification of the organic fertilizer are realized, and good conditions are created for the propagation of the trichoderma in the later stage.
Strain activation: picking up trichoderma asperellum preserved in agar inclined plane at 4 DEG CTrichoderma asperellum) Inoculating to potato glucose agar (PDA) plate center, culturing at 28deg.C in darkness for 3-4 days, picking edge mycelium or scraping green spore, transferring mycelium or spore into potato glucose liquid culture medium (PD), and culturing at 28deg.C under shaking (rotation speed 150 rpm) for 4-5 days to form a large amount of white flocculent mycelium to obtain Trichoderma fermentation broth (seed solution).
Preparation of PDA culture medium: 7.5g potato soaked powder is weighed, 1000ml of water is added, 20g glucose and 18g agar are added, sterilization is carried out for 20 minutes at 121 ℃, and the potato soaked powder is packaged in a culture dish for cooling and then stored for standby. Preparation of PD culture solution: 7.5g of potato soaked powder is weighed, 1000ml of water is added, 20g of glucose is added, sterilization is carried out for 20 minutes at 121 ℃, and the potato soaked powder is cooled and stored for standby.
Meanwhile, if the fermentation liquid (seed liquid) is produced on a large scale: the culture medium with potato paste powder and glucose as raw materials is used for fermentation in a liquid fermentation tank to prepare a large amount of trichoderma fermentation seed liquid.
Preparing a biological organic fertilizer:
and uniformly spraying the obtained trichoderma fermentation seed liquid to the spread apple pomace compost by using a sprayer, wherein the volume-mass ratio of the inoculation amount of the trichoderma liquid is controlled to be 5-20%. And stacking the materials into a trapezoid stack, turning the stack once every 3 days at room temperature, completing the fermentation of the trichoderma in 7 to 14 days, spreading and airing until the water content is 30%, and crushing to obtain the trichoderma bio-organic fertilizer.
Wheat bran is added into the apple pomace compost before the trichoderma fermentation seed liquid is sprayed, so that the material carbon nitrogen ratio can be optimized, the trichoderma fermentation is facilitated, and the trichoderma bio-organic fertilizer with higher viable bacteria number is obtained.
In the preparation process of the bio-organic fertilizer, the inoculation amount, the wheat bran addition amount and the fermentation days are 3 main factors, 3 levels are set for each factor, and L is used 18 （3 3 ) Orthogonal table orthogonal experiments were performed and each combination was repeated 3 times, taking the average (see table 3). The number of viable trichoderma was calculated by dilution plating using a special purpose separation plate for trichoderma (table 1). Preparation of a special separation culture medium for trichoderma: 7.5g potato soaked powder is weighed, 0.033g of Bengalia, 0.5g of sodium propionate, 20g of glucose and 18g of agar are added, 1000ml of water is added, sterilization is carried out for 20 minutes at 121 ℃, and 0.03g of streptomycin and 0.02g of nystatin are added before pouring.
TABLE 3 orthogonal test and extremely bad analysis of Trichoderma fertilizers
K is the average viable count (hundred million/g) at different levels for each factor, and R is the extremum difference (hundred million/g) between different levels for each factor. The inoculation amount is the mass and volume fraction (g/ml) of the fruit dreg compost, and the wheat bran addition amount is the mass fraction (g/g) of the fruit dreg compost.
The extremely poor analysis results in Table 3 show that the order of the influence of each factor on the number of the viable bacteria of the trichoderma bio-organic fertilizer is fermentation time, the inoculation amount of trichoderma fermentation seed liquid and the addition amount of wheat bran, and the optimal fermentation combination conditions are fermentation for 14 days, the inoculation amount of the trichoderma fermentation seed liquid is 20%, and the addition amount of the wheat bran is 5%. By using the combination for fermentation, the effective viable count of Trichoderma reaches 9.24 hundred million/g (Table 3). In actual production, the links of saving raw materials, shortening the fermentation period and the like are considered, and the combination condition of No. 1, namely, the inoculation amount of trichoderma fermentation seed liquid is 5%, wheat bran is not added, and the trichoderma is fermented for 7 days, wherein the number of active bacteria of the effective active bacteria of the trichoderma reaches 1.31 hundred million/g, the obtained organic fertilizer under the condition reaches the agricultural industry standard (NY 884-2012), and various indexes such as the organic matter content, the heavy metal content and the like of the trichoderma bio-organic fertilizer also reach the standard, so that the product is qualified (Table 4).
Table 4 parameters of trichoderma fertilizers
Application example 1
Test of growth promoting effect of trichoderma preparation on matrimony vine and honeysuckle in coastal saline-alkali soil
And carrying out field cell tests in the coastal saline soil region of the yellow river delta. And 2021, selecting a medium-light salinized land block (with the salt content of 0.3%), and planting Chinese wolfberry and honeysuckle bare root seedlings. The row spacing of Chinese wolfberry plants is 1.5 meters, the cell length is 5.5 meters, and the cell width is 4 meters; the row spacing of honeysuckle plants is 1 meter, and the length of a cell is 5 meters and the width of the cell is 4 meters. At the beginning of four months, 1g of the trichoderma preparation obtained in example 1 was applied around the roots of the plants in the treated cells, and no trichoderma preparation was applied in the control cells. The treatment and control set 4 duplicate cells, respectively. Growing season for 4-11 months, measuring leaf photosynthetic rate, collecting plant for 11 months, cleaning, oven drying, and measuring plant dry weight (see figure 1).
The result of figure 1 shows that the application of trichoderma agent can raise photosynthetic rate of wolfberry and honeysuckle in saline-alkali soil, increase biomass accumulation and promote plant growth, and the trichoderma in the microbial inoculum can raise salt tolerance of plant and can be suitable for saline-alkali soil environment.
Application example 2
Growth promotion test and partial nitrogen fertilizer replacement test of trichoderma koningii bio-organic fertilizer in coastal saline-alkali soil
And carrying out field cell tests in the coastal saline soil region of the yellow river delta. Selecting a moderately and slightly salinized land block (with the salt content of 0.3%) in the year of 4 months in 2020, planting two-year-old bare root seedlings of Chinese wolfberry, and performing conventional field management in growing seasons, wherein the plant row spacing is 1.5 meters, the cell length is 5.5 meters and the width is 4 meters. In the test, the trichoderma bio-organic fertilizer is abbreviated as trichoderma bacterial fertilizer. The experiment was divided into 6 treatments, each containing 4 duplicate cells. Treatment 1 is notFertilizer application control, treatment 2 was application of Trichoderma fertilizer sterilization (500 g/m 2 ) Treatment 3 is the application of Trichoderma viride fertilizer (500 g/m 2 ) Treatment 4 is to apply Trichoderma fertilizer and 50% fertilizer (Trichoderma bio-organic fertilizer 500 g/m 2 ，43 g/m 2 Urea), treatment 5 is to apply the trichoderma bio-organic fertilizer and 75% fertilizer (trichoderma bio-organic fertilizer 500 g/m 2 ，64.5 g/m 2 Urea), treatment 6 is conventional full fertilizer application (86 g/m) 2 Urea). At the end of 10 months, a plant is randomly selected in each district, fruits and plants are collected, dried and weighed (see figure 2). Trial treatment and sample collection were repeated 2021 (see fig. 3).
The trichoderma fertilizer sterilizing material is prepared by sterilizing the trichoderma bio-organic fertilizer prepared in the example 2 at high temperature and high pressure (sterilizing trichoderma to become organic fertilizer without beneficial bacteria).
As can be seen from fig. 2, the trichoderma fertilizer is inactivated by high temperature and high pressure, but the nutrient contents of organic matters, nitrogen, phosphorus, potassium and the like are not obviously changed, and the sterilizing matters of the trichoderma fertilizer are equivalent to the organic composting of fruit residues. The application of trichoderma fertilizer sterilizing substance increases the yield and biomass of dried fruits of Chinese wolfberry, and has remarkable effect of increasing 2021. Simultaneously applying trichoderma fertilizer
The yield and biomass of the dried fruits of the Chinese wolfberry are obviously increased, and the increase amplitude is higher than that of the application of sterilizing substances, which proves that the trichoderma fertilizer can more effectively promote the growth and yield formation of the Chinese wolfberry in the saline-alkali soil relative to the fruit residue organic fertilizer.
As can be seen from fig. 3, the biomass and dry fruit yield of medlar in the areas where the trichoderma fertilizer and the fertilizer are applied by 50% and the fertilizer is applied by 100% are significantly increased, and the difference between the two is not obvious. The biomass and the yield of medlar in 75% of cells where the trichoderma bacterial manure and the chemical fertilizer are applied are also obviously increased and are obviously higher than those in the cells where the conventional chemical fertilizer is applied, which shows that the Shi Mumei bacterial manure can realize the fertilizer application reduction and promote the growth and the yield improvement of medlar.
Claims (9)
1. The application of the apple pomace as the resource is characterized in that: the application of apple pomace in preparing trichoderma preparation or trichoderma bio-organic fertilizer as fermentation materials.
2. The use of apple pomace according to claim 1 for recycling, wherein: the fermentation material is obtained by dehydrating apple pomace after pectin extraction to water content of about 70%, and drying.
3. The use of apple pomace according to claim 1 for recycling, wherein: the trichoderma preparation is obtained by inoculating trichoderma liquid into fermentation materials and fermenting;
the trichoderma bio-organic fertilizer is obtained by spraying trichoderma liquid into a fermented material after composting and fermenting.
4. A trichoderma preparation using apple pomace as a raw material is characterized in that: inoculating trichoderma liquid into a raw material taking apple pomace as a ferment, and fermenting to obtain the trichoderma liquid.
5. A trichoderma preparation using apple pomace as a raw material according to claim 4, wherein: the trichoderma spinosum is addedTrichoderma asperellum) Inoculating the bacterial liquid of (a) into a raw material taking apple pomace as a ferment for fermentation treatment at a constant temperature; wherein, potassium dihydrogen phosphate is added into the raw materials to adjust the pH value to be neutral.
6. A method for preparing trichoderma preparation using apple pomace as raw material according to claim 4, which is characterized in that: the trichoderma spinosum is addedTrichoderma asperellum) Inoculating the bacterial liquid of (a) into a raw material taking apple pomace as a ferment for fermentation treatment at a constant temperature; wherein, potassium dihydrogen phosphate is added into the raw materials to adjust the pH value to be neutral.
7. A trichoderma bio-organic fertilizer using apple pomace as a raw material is characterized in that: the apple pomace is used as a raw material of a fermented product, and is obtained by composting, spraying trichoderma liquid to the apple pomace, and then fermenting.
8. The trichoderma bio-organic fertilizer using apple pomace as a raw material according to claim 7, wherein: the trichoderma spinosum is addedTrichoderma asperellum) Spraying the bacterial liquid of (2) into the raw material of the fermented product of apple pomace after composting treatment, and then fermenting at room temperature;
or, the trichoderma asperellum containingTrichoderma asperellum) Spraying the bacterial liquid of (2) into the raw material of the fermented product of apple pomace after composting treatment, and then fermenting at room temperature; wherein, the raw material of the fermented product is the apple pomace after composting treatment, and wheat bran is added.
9. A method for preparing the trichoderma bio-organic fertilizer by using apple pomace as a raw material, which is characterized in that: the trichoderma spinosum is addedTrichoderma asperellum) Spraying the bacterial liquid of the formula (I) into the raw material of the composted apple pomace serving as a ferment, and then fermenting at room temperature;
or, the trichoderma asperellum containingTrichoderma asperellum) Spraying the bacterial liquid of the formula (I) into the raw material of the composted apple pomace serving as a ferment, and then fermenting at room temperature; wherein, the raw material of the fermented product is the apple pomace after composting treatment, and wheat bran is added.
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| CN202310486784.2A CN116179371A (en) | 2023-05-04 | 2023-05-04 | Application of apple pomace as resource |
| CN202310547875.2A CN116693334A (en) | 2023-05-04 | 2023-05-16 | Application of apple pomace as resource |
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| CN202310486784.2A CN116179371A (en) | 2023-05-04 | 2023-05-04 | Application of apple pomace as resource |
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| CN202310547875.2A Pending CN116693334A (en) | 2023-05-04 | 2023-05-16 | Application of apple pomace as resource |
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| CN100353858C (en) * | 2005-03-18 | 2007-12-12 | 西北大学 | Method for manufacturing feedstuff protein by solid state fermentation of apple dregs liquid |
| CN106348888B (en) * | 2016-08-31 | 2019-09-10 | 山东省科学院生态研究所 | A method of trichoderma as biological organic fertilizer is produced using shallot Folium Allii fistulosi waste |
| CN106754426B (en) * | 2017-01-17 | 2020-05-08 | 中国科学院烟台海岸带研究所 | Trichoderma asperellum and application thereof |
| CN115651846A (en) * | 2022-09-14 | 2023-01-31 | 山东省科学院生物研究所 | Trichoderma fungicide, biological organic fertilizer, preparation method and application thereof |
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