CN111676168A - Nano-selenium detoxicant and preparation method thereof, detoxified biological protein selenium and preparation method and application thereof - Google Patents
Nano-selenium detoxicant and preparation method thereof, detoxified biological protein selenium and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a nano-selenium detoxicant, a preparation method thereof, detoxified biological protein selenium, a preparation method and application thereof. The nano-selenium detoxifying agent is prepared by extracting 10-20 parts of schisandra chinensis, 8-15 parts of raw cacumen biotae, 8-15 parts of mirabilite and 5-8 parts of garden burnet with water. The preparation method of the detoxified biological protein selenium comprises the following steps: inoculating bacillus subtilis into a culture medium containing nano-selenium to perform primary fermentation and secondary fermentation, and then inoculating the obtained bacillus subtilis completely adapting to the nano-selenium environment into a third culture medium containing nano-selenium and having the nano-selenium detoxicant to perform tertiary fermentation. The traditional Chinese medicine extracting solution extracted from the schisandra chinensis, the raw cacumen biotae, the mirabilite and the garden burnet is used as the nano-selenium detoxifying agent, a large amount of toxins of the nano-selenium can be removed in the fermentation process to obtain detoxified biological protein selenium, the detoxified biological protein selenium is further fermented to prepare the protein selenium plant nutrient microbial inoculum, no side effect is generated on crops, and the selenium-enriched crops can be produced in a large scale.
Description
Technical Field
The invention relates to the technical field of biofertilizers, and particularly relates to a nano-selenium detoxifying agent and a preparation method thereof, and detoxified biological protein selenium and a preparation method and application thereof.
Background
Biological plant nutrient inocula are increasingly widely applied to plant cultivation, generally contain a plurality of plant beneficial components such as nitrogen, phosphorus, potassium protein and the like which are necessary for plants, but generally lack selenium element. Selenium-rich vegetables and fruits are lacked in the market at present, and if selenium can be obtained from a plant microbial inoculum to increase the selenium content of the fruits and the vegetables and supplement the selenium required by a human body, the selenium-rich vegetables and fruits are a good choice.
At present, nano-selenium exists in the market, but the nano-selenium has heavier toxicity, so that a great amount of probiotics die in the fermentation process of the plant fungicide, the viable count of the bacterial liquid cannot meet the requirement of the fungicide, and the quality of the fungicide is influenced.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a nano-selenium detoxicant, a preparation method thereof, detoxified biological protein selenium, a preparation method and application thereof, so as to improve the problems.
The invention is realized by the following steps:
in a first aspect, the embodiment of the invention provides a nano-selenium detoxifying agent, which is obtained by extracting traditional Chinese medicine raw materials with water, wherein the traditional Chinese medicine raw materials comprise the following components in parts by weight: 10-20 parts of schisandra chinensis, 8-15 parts of raw cacumen biotae, 8-15 parts of mirabilite and 5-8 parts of garden burnet.
In a second aspect, the embodiment of the present invention further provides a preparation method of a nano-selenium detoxifying agent, which includes: extracting the raw materials with water.
Optionally, soaking the traditional Chinese medicine raw materials in water for 20-28 hours, boiling for 3-5 hours, and separating to remove solids, wherein the weight ratio of the water used for soaking to the schisandra chinensis in the traditional Chinese medicine raw materials is 5000-9000: 10 to 20.
In a third aspect, an embodiment of the present invention further provides a preparation method of detoxified bio-protein selenium, including: and inoculating the bacillus subtilis into a first culture medium for primary fermentation, and then inoculating the bacillus subtilis obtained through the primary fermentation into a second culture medium for secondary fermentation to obtain the bacillus subtilis completely adapting to the nano-selenium environment. Wherein the first culture medium and the second culture medium both contain nano selenium.
And inoculating the bacillus subtilis obtained after the secondary fermentation into a third culture medium with nano-selenium for tertiary fermentation, wherein the third culture medium also contains the nano-selenium detoxifying agent.
In a fourth aspect, the embodiment of the invention also provides detoxified biological protein selenium, which is prepared by the preparation method of the detoxified biological protein selenium.
In a fifth aspect, an embodiment of the present invention further provides a method for preparing a selenium protein plant nutrient microbial inoculum, which includes: the detoxified biological protein selenium is used as one of the main raw materials to prepare the protein selenium plant nutrient microbial inoculum by fermentation.
Optionally, the main raw materials comprise detoxified biological selenium protein, yeast powder, peptone and white granulated sugar.
In a sixth aspect, the embodiment of the invention also provides an application of the detoxified biological protein selenium in preparation of a protein selenium plant nutrient microbial inoculum.
One of the above embodiments of the present invention has the following beneficial effects: the liquid organic detoxified biological protein selenium is obtained by taking traditional Chinese medicine extracting solution extracted by schisandra chinensis, raw cacumen biotae, mirabilite and sanguisorba officinalis according to a certain proportion as a nano-selenium detoxicant, and under the action of the nano-selenium detoxicant, a large amount of toxins possessed by nano-selenium can be eliminated in the fermentation process. The protein selenium plant nutrient microbial inoculum prepared by further fermenting the detoxified biological protein selenium does not produce side effect on crops, so that the selenium-enriched crops can be produced in a large scale.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The nano-selenium detoxication agent and the preparation method thereof, the detoxified biological protein selenium and the preparation method and the application thereof provided by the invention are specifically explained below.
Some embodiments of the present invention provide a nano-selenium detoxifying agent, which is obtained by extracting traditional Chinese medicine raw materials with water, wherein the traditional Chinese medicine raw materials comprise, by weight: 10-20 parts of schisandra chinensis, 8-15 parts of raw cacumen biotae, 8-15 parts of mirabilite and 5-8 parts of garden burnet.
Fructus Schisandrae is dried mature fruit of Magnoliaceae plant fructus Schisandrae chinensis or fructus Schisandrae Sphenantherae, picked in autumn when the fruit is mature, sun-dried or steamed, sun-dried, and removed fruit stalks and impurities. The folium Platycladi is dry branch tip and leaf of Platycladus orientalis of Cupressaceae. Collected in summer and autumn mostly and dried in the shade. Multiple branches, flat small branches. The leaves are fine and scaly, alternately opposite, attached to the branches, dark green or yellow green. Is brittle and easy to break. Fragrant smell, bitter and astringent taste and slight pungent taste. Mirabilite, also known as Mirabilite, Sal Nitri and Mirabilite, is a crude product of Natrii sulfas, and the crystal obtained by processing and refining is Natrii sulfas. It is called Xuanming powder after dehydration. Mirabilitum, Natrii sulfas, and Natrii sulfas have the same effects. The radix Sangusorbae is perennial herb of Ulmus of Rosaceae, and has spindle-shaped thick and strong heel, petiole lobule, and mauve petal, and fruit is enclosed in calyx tube. Also named as Huangzhuyang, Yuzhang, Yufermented soya beans or Haematitum.
Through a large amount of research and practice, the inventor creatively discovers that the schisandra chinensis, the raw cacumen biotae, mirabilite and the garden burnet are prepared according to the proportion, the extracting solution obtained after the water extraction of the active ingredients of the medicine can be used as a detoxifying agent of nano-selenium, the toxin of the nano-selenium can be removed in a culture medium containing the nano-selenium through the fermentation of bacillus subtilis, and then the detoxified biological protein selenium harmless to plants is obtained. In the fermentation process of preparing the selenium-containing plant nutrient microbial inoculum, the detoxified bioprotein selenium does not harm active microbial flora in the plant nutrient microbial inoculum, so that the plant nutrient microbial inoculum containing a large amount of selenium can be conveniently obtained, and the large-scale production of selenium-rich crops is promoted.
Specifically, some embodiments of the present invention also provide a method for preparing the nano-selenium detoxifying agent, which comprises: extracting the above Chinese medicinal materials with water. In some embodiments, the Chinese medicinal raw materials are soaked in water for 20-28 hours, then boiled for 3-5 hours, and then separated to remove solids, wherein the weight ratio of the water used for soaking to the schisandra chinensis in the Chinese medicinal raw materials is 5000-9000: 10 to 20. The active pharmaceutical ingredients in the traditional Chinese medicine can be fully extracted by a water extraction method of soaking and boiling, so that the detoxification effect is better.
More specifically, the screened traditional Chinese medicines are discarded weeds and impurities, the selected traditional Chinese medicines are washed by clean water, soaked for 24 hours, then the soaked traditional Chinese medicines are boiled for 4 hours, and cooled. And then filtering the cooled solid-liquid mixture, and taking the filtrate as the nano-selenium detoxifying agent.
Some embodiments of the present invention also provide a method for preparing detoxified bio-protein selenium, comprising:
s1, inoculating the bacillus subtilis into the first culture medium for primary fermentation.
Specifically, in some embodiments, the first medium comprises the following components: 3-7 g/L of nano selenium, 0.5-1 g/L of composite trace elements, 2-5 g/L of yeast powder, 2-5 g/L of peptone, 10-50 g/L of glucose, 0.5-1 g/L of sodium chloride and 3000-6000 kg of water.
Through primary fermentation in a first culture medium, a large amount of bacillus subtilis is poisoned and killed in a nano-selenium environment, the method is essentially a mutagenesis process, only a few bacillus subtilis with better tolerance and toxicity survive in the primary fermentation process, and the survived mutagenic bacteria are preliminarily adapted to the environment with nano-selenium.
In some embodiments, the first medium has a pH of 5-6 and is sterilized at 118-120 ℃ for 20-30 min. Specifically, raw materials of the first culture medium are stirred and mixed by a container, the pH of the mixed solution is 5-6, the mixed raw materials are injected into a primary culture tank after dissolution, the first liquid culture medium is sterilized by a wet-heat method, the temperature is 121 ℃, the temperature is kept for 20-30 minutes, and the temperature is reduced to 34-37 ℃.
Further, in some embodiments, the fermentation temperature of the primary fermentation is 36-38 ℃, and the fermentation time is 45-52 hours.
In some embodiments, the primary culture tank is a seeding tank, and the primary fermentation is performed in the seeding tank, wherein the tank pressure of the seeding tank is 0.01-0.03 MPa, preferably 0.02MPa during the fermentation. In some embodiments, the first medium is stirred during the primary fermentation at a speed of 150 to 200rmp, preferably 180 rmp.
Specifically, in the primary fermentation, bacillus subtilis is inoculated into a first culture medium in a suspension form according to the volume ratio of 1-5%.
And S2, inoculating the bacillus subtilis obtained through the primary fermentation into a second culture medium for secondary fermentation to obtain the bacillus subtilis completely adapting to the nano-selenium environment.
Specifically, in some embodiments, the second medium comprises the following components: 3-7 g/L of nano selenium, 0.5-1 g/L of composite trace elements, 2-5 g/L of yeast powder, 2-5 g/L of peptone, 10-50 g/L of glucose, 0.5-1 g/L of sodium chloride and 6000-8000 kg of water.
The bacillus subtilis with better nano-selenium tolerance obtained by primary fermentation is propagated and expanded through secondary fermentation, a part of bacillus subtilis is poisoned and died in the secondary fermentation process, only part of bacillus subtilis with better selenium mutagenesis tolerance grows and propagates in the secondary fermentation, the tolerant bacteria completely adapt to the environment of nano-selenium through the secondary fermentation, and then the bacillus subtilis obtained after the secondary fermentation can be subjected to detoxification culture in a culture medium with nano-selenium.
In some embodiments, the second medium has a pH of 5-6 and is sterilized at 118-120 ℃ for 20-30 min. Specifically, the raw materials are stirred and mixed by a container, the pH value is 5.0-6.0 after dissolution, the raw pulp is thrown into a propagation tank, high-temperature sterilization is carried out for 121 ℃, the temperature is 20-30 minutes, then cooling is carried out, and the temperature is reduced to 34-37 ℃.
In some embodiments, the fermentation temperature of the secondary fermentation is 36-38 ℃ and the fermentation time is 45-52 hours. In a preferred embodiment, the secondary fermentation is carried out by using a propagation tank, wherein the tank pressure of the propagation tank is 0.01-0.03 MPa, preferably 0.02 MPa. In some embodiments, the second medium is stirred during the secondary fermentation at a speed of 150 to 200rmp, preferably 180 rmp.
And S3, inoculating the bacillus subtilis obtained after the secondary fermentation into a third culture medium with nano-selenium for tertiary fermentation, wherein the third culture medium contains the nano-selenium detoxifying agent in the embodiment.
Specifically, in some embodiments, the weight ratio of the nano-selenium detoxicant to the nano-selenium in the third culture medium is 6000-8000: 20 to 40.
Further, in some embodiments, the third medium comprises the following components: 7-9 g/L of nano-selenium, 0.5-1 g/L of composite trace elements, 2-5 g/L of yeast powder, 2-5 g/L of peptone, 10-50 g/L of glucose, 1-5 g/L of sodium chloride, 6000-8000 kg of nano-selenium detoxication agents and 30000-50000kg of water.
In some embodiments, the composite trace elements comprise: manganese sulfate, potassium dihydrogen phosphate, ferrous sulfate and magnesium sulfate, wherein the mass ratio of various trace element substances can be 1:1: 1: 1. in other embodiments of the present invention, the trace elements are compounded in the above-described material ratios.
By adding the nano-selenium detoxicant of the traditional Chinese medicine extract into the culture medium, 60-80% of selenium is found to be combined with protein when the fermentation is about ten days, 90% of selenium is combined with protein when the fermentation is terminated, and a large amount of toxin is removed under the action of the traditional Chinese medicine. Through ten to twenty days of detoxification and fermentation, stable liquid organic protein selenium, namely detoxified biological protein selenium is produced.
In some embodiments, the third medium has a pH of 5-6 and is sterilized at 118-120 ℃ for 20-30 min. Specifically, the raw materials of the third culture medium are sequentially poured into a stirring tank, the raw materials are stirred and mixed, the nano-selenium detoxicant is poured into the stirring tank after being dissolved, the mixture is stirred with the raw materials, the pH value is 5-6, the mixture is poured into a third-stage fermentation tank, the mixture is sterilized at high temperature of 121 ℃, the temperature is kept for 20-30 minutes, and the temperature is cooled to 34-37 ℃.
In some embodiments, the fermentation temperature of the three-stage fermentation is 36-38 ℃, and the fermentation time is 10-20 days. And (3) performing three-stage fermentation by using a fermentation tank, wherein the tank pressure of the fermentation tank is 0.01-0.03 MPa, preferably 0.02MPa, and the third culture medium is stirred in the three-stage fermentation process, and the stirring speed is 150-200 rmp, preferably 180 rmp.
Some embodiments of the invention also provide detoxified biological protein selenium prepared by the preparation method of the detoxified biological protein selenium.
Some embodiments of the present invention also provide a method for preparing a selenium protein plant nutrient microbial inoculum, which comprises: the protein selenium plant nutrient microbial inoculum is prepared by taking the detoxified biological protein selenium as one of main raw materials through fermentation. Optionally, the main raw materials comprise detoxified biological selenium protein, yeast powder, peptone and white granulated sugar.
Specifically, the preparation method of the selenium protein plant nutrient microbial inoculum comprises the following steps:
s1, fermenting a plurality of strains in the seed liquid culture medium, wherein the plurality of strains are a mixture of a plurality of probiotics.
In some embodiments, the plurality of species comprises: the ratio of the bacillus subtilis to the yeast to the actinomycetes to the fluorescent bacteria is 2:1:1: 1.
In some embodiments, the seed liquid fermentation medium comprises the following components in parts by weight: 50-90 g/L of white granulated sugar, 3-5 g/L of yeast powder, 3-5 g/L of peptone, 0.5-1 g/L of composite trace elements, 1-2 g/L of sodium chloride and 600-1000 kg of water. In some embodiments, the pH of the seed liquid culture medium is 5-6, and the seed liquid culture medium is sterilized at 118-120 ℃ for 20-30 min. Specifically, the raw materials are stirred and mixed by a container, the pH value of the mixed solution is 5.0-6.0, the raw materials are thrown into a first-level seeding tank after being dissolved, the temperature is 121 ℃, the temperature is kept for 20-30 minutes, and the temperature is reduced to 34-37 ℃.
In some embodiments, the fermentation temperature is 34-37 ℃ and the fermentation time is 48-56 hours.
It should be noted that the bacillus subtilis is original bacillus subtilis and has not been cultured in an environment with nano-selenium.
Seed liquid of various probiotics can be obtained through the seed liquid culture medium, and the seed liquid has fermentation activity to a certain extent.
S2, inoculating the seed liquid containing various strains obtained by seed fermentation into a fermentation liquid culture medium for fermentation. The fermentation liquid culture medium comprises the following components in proportion: 3-5 g/L of detoxified biological protein selenium, 0.5-1 g/L of composite trace elements, 2-5 g/L of yeast powder, 2-5 g/L of peptone, 20-80 g/L of glucose, 1-3 g/L of sodium chloride, 6000-8000 kg of nano-selenium detoxifier and 90000kg of water 50000-.
The selenium-rich protein plant nutrient microbial inoculum contains quantitative selenium and a large number of biological bacteria, and can improve the physical and chemical properties of soil and increase the content of organic matters in the soil through the activities of trace elements and the biological bacteria, and has the function of loosening the soil. After the biological protein selenium biological agent is applied to soil, the biological bacteria quickly proliferate to form group dominance to decompose nitrogen, phosphorus and potassium which are fixed in the soil and can not be absorbed and utilized by plants, and fix free nitrogen in the air for the plants to absorb and utilize. In addition, because the selenium source adopted in the fermentation process is the detoxified biological protein selenium, the detoxified biological protein selenium does not produce side effect on crops, does not cause harm to various probiotics in the fermentation process, and is beneficial to the large-scale production of the protein selenium plant nutrient microbial inoculum.
In some embodiments, the pH of the fermentation liquid medium is 5-6, and the fermentation liquid medium is sterilized at 118-120 ℃ for 20-30 min; preferably, the fermentation temperature is 34-37 ℃, and the fermentation time is 18-22 days. Specifically, the temperature of the fermentation tank is 34-37 ℃, the pressure of the fermentation tank is 0.02MPa, and the fermentation is stirred for 180 rmp.
Further, the efficacy and balance of the probiotic flora in the selenium protein plant nutrient inoculum are further enriched according to the needs. The preparation method can also comprise the following steps:
and S3, after the fermentation in the fermentation liquid culture medium is finished, inoculating the mixed culture solution of the cultured mature lactic acid bacteria and acetic acid bacteria into the fermentation liquid culture medium for continuous fermentation. Optionally, the fermentation time is 20-28 hours. It should be noted that the fermentation conditions are the same as those described above.
Specifically, in some embodiments, the ratio of lactic acid bacteria to acetic acid bacteria is 2:1, and the ratio of the amount of lactic acid bacteria to the amount of bacillus subtilis in the plurality of bacterial species is 1: 1.
in some embodiments, the mixed solution of lactic acid bacteria and acetic acid is cultured in a reseeding tank. The culture medium in the reseeding tank is: 2-5 g/L yeast powder, 2-5 g/L peptone, 20-50 g/L glucose, 0.5-1 g/L composite trace elements, 0.5-1 g/L sodium chloride and 3000-6000 kg purified water. Sterilizing the culture medium at the high temperature of 121 ℃, preserving the heat for 20-30 minutes, and cooling to 34-37 ℃. The culture conditions are that the temperature of the tank is 34-37 ℃, the stirring is carried out for 180rmp under the pressure of the tank being 0.02Mpa, and the culture period is 48-54 hours.
It should be noted that the water used in the embodiment of the present invention is pure water.
In some embodiments, the microbial inoculum fermented by the mixed bacterial liquid of lactic acid bacteria and acetic acid bacteria is directly poured into a storage tank, and is subjected to after-ripening at the temperature of 5-15 ℃, wherein the after-ripening time is 20-60 days.
Some embodiments of the invention also provide application of the detoxified biological selenium protein in preparation of a selenium protein plant nutrient microbial inoculum.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The implementation provides a preparation method of a nano-selenium detoxifying agent, which comprises the following steps:
the raw material ratio is as follows: 12 parts of schisandra chinensis, 8 parts of raw cacumen biotae, 8 parts of mirabilite, 5 parts of garden burnet and 5000 parts of purified water.
Removing weed and impurities from the selected Chinese medicinal materials, cleaning the selected Chinese medicinal materials with clear water, soaking for 24 hr, boiling for 4 hr, and cooling. And then filtering the cooled solid-liquid mixture, and taking the filtrate as a nano-selenium detoxifying agent for later use.
Example 2
The implementation provides a preparation method of a nano-selenium detoxifying agent, which comprises the following steps:
the raw material ratio is as follows: 20 parts of schisandra chinensis, 15 parts of raw cacumen biotae, 15 parts of mirabilite, 8 parts of garden burnet and 9000 parts of purified water.
Removing weed and impurities from the selected Chinese medicinal materials, cleaning the selected Chinese medicinal materials with clear water, soaking for 24 hr, boiling for 4 hr, and cooling. And then filtering the cooled solid-liquid mixture, and taking the filtrate as a nano-selenium detoxifying agent for later use.
Example 3
The implementation provides a preparation method of a nano-selenium detoxifying agent, which specifically comprises the following steps:
the raw material ratio is as follows: 15 parts of schisandra chinensis, 12 parts of raw cacumen biotae, 12 parts of mirabilite, 6 parts of garden burnet and 7000 parts of purified water.
Removing weed and impurities from the selected Chinese medicinal materials, cleaning the selected Chinese medicinal materials with clear water, soaking for 24 hr, boiling for 4 hr, and cooling. And then filtering the cooled solid-liquid mixture, and taking the filtrate as a nano-selenium detoxifying agent for later use.
Example 4
The embodiment provides a preparation method of detoxified biological protein selenium, which specifically comprises the following steps:
s1, inoculating the bacillus subtilis into the first culture medium for primary fermentation.
The first culture medium comprises the following components in proportion: 3g/L of nano selenium, 0.5g/L of composite trace elements, 2g/L of yeast powder, 2g/L of peptone, 10g/L of glucose, 0.5g/L of sodium chloride and 3000kg of water.
Stirring and mixing the raw materials of the first culture medium by a container, dissolving, pumping the mixed raw materials into a primary culture tank, sterilizing the liquid first culture medium by a damp-heat method, keeping the temperature at 121 ℃ for 20 minutes, and cooling to 34 ℃.
Bacillus subtilis was inoculated into the first medium in the form of a suspension at 1%. Performing primary fermentation in a seeding tank under 0.01 MPa. And stirring the first culture medium in the primary fermentation process, wherein the stirring speed is 150rmp, and the fermentation time is 45 hours.
And S2, inoculating the bacillus subtilis obtained through the primary fermentation into a second culture medium for secondary fermentation to obtain the bacillus subtilis completely adapting to the nano-selenium environment.
The second culture medium comprises the following components in proportion: 3g/L of nano-selenium, 0.5g/L of composite trace element, 2g/L of yeast powder, 2g/L of peptone, 10g/L of glucose, 0.5g/L of sodium chloride and 6000kg of water.
Stirring and mixing the raw materials by a container, pumping the raw stock into a propagation tank, sterilizing at high temperature for 121 ℃, cooling for 30 minutes, and cooling to 34 ℃.
The fermentation temperature of the secondary fermentation is 36 ℃, and the fermentation time is 45 hours. The pot pressure of the propagation pot is 0.01 MPa. And stirring the second culture medium in the secondary fermentation process, wherein the stirring speed is 150 rmp.
S3, inoculating the bacillus subtilis obtained after the secondary fermentation into a third culture medium with nano-selenium for tertiary fermentation.
The third culture medium comprises the following components in proportion: 7g/L of nano-selenium, 0.5g/L of composite trace elements, 2g/L of yeast powder, 2g/L of peptone, 10g/L of glucose, 1g/L of sodium chloride, 6000kg of nano-selenium detoxication agent and 30000kg of water. Wherein the nano-selenium detoxifying agent is the nano-selenium detoxifying agent prepared in example 3.
The composite trace elements comprise manganese sulfate, monopotassium phosphate, ferrous sulfate and magnesium sulfate in equal mass ratio.
And sequentially pouring the raw materials of the third culture medium into a stirring tank, stirring and mixing the raw materials, pouring the nano-selenium detoxicant into the stirring tank after dissolving, mixing and stirring the nano-selenium detoxicant with the raw materials, pouring the mixture into a third-stage fermentation tank, sterilizing at high temperature of 121 ℃, preserving heat for 20 minutes, and cooling to 34 ℃.
And inoculating the bacillus subtilis obtained after the secondary fermentation into a third culture medium for fermentation, wherein the fermentation temperature of the tertiary fermentation is 36 ℃, and the fermentation time is 10 days. The pressure of the fermentation tank is 0.01Mpa, the third culture medium is stirred in the three-stage fermentation process, and the stirring speed is 150. After the third-stage fermentation, stable liquid organic protein selenium, namely detoxified biological protein selenium is obtained.
Example 5
S1, inoculating the bacillus subtilis into the first culture medium for primary fermentation.
The first culture medium comprises the following components in proportion: 7g/L of nano-selenium, 1g/L of composite trace element, 5g/L of yeast powder, 5g/L of peptone, 50g/L of glucose, 1g/L of sodium chloride and 6000kg of water.
Stirring and mixing the raw materials of the first culture medium by a container, dissolving, pumping the mixed raw materials into a primary culture tank, sterilizing the liquid first culture medium by a damp-heat method, keeping the temperature at 121 ℃ for 30 minutes, and cooling to 37 ℃.
Bacillus subtilis is inoculated into a first culture medium in a suspension form in a proportion of 5%. Performing primary fermentation in a seeding tank, wherein the tank pressure of the seeding tank is 0.03Mpa during fermentation. The first culture medium is stirred in the primary fermentation process, the stirring speed is 200rmp, and the fermentation time is 52 hours.
And S2, inoculating the bacillus subtilis obtained through the primary fermentation into a second culture medium for secondary fermentation to obtain the bacillus subtilis completely adapting to the nano-selenium environment.
The second culture medium comprises the following components in proportion: 7g/L of nano-selenium, 1g/L of composite trace element, 5g/L of yeast powder, 5g/L of peptone, 50g/L of glucose, 1g/L of sodium chloride and 8000kg of water.
Stirring and mixing the raw materials by a container, pumping the raw stock into a propagation tank, sterilizing at the high temperature of 121 ℃, preserving the heat for 30 minutes, then cooling, and reducing the temperature to 37 ℃.
The fermentation temperature of the secondary fermentation is 38 ℃, and the fermentation time is 52 hours. The pot pressure of the propagation pot is 0.03 MPa. And stirring the second culture medium in the secondary fermentation process, wherein the stirring speed is 200 rmp.
S3, inoculating the bacillus subtilis obtained after the secondary fermentation into a third culture medium with nano-selenium for tertiary fermentation.
The third culture medium comprises the following components in proportion: 9g/L of nano-selenium, 1g/L of composite trace element, 5g/L of yeast powder, 5g/L of peptone, 50g/L of glucose, 5g/L of sodium chloride, 8000kg of nano-selenium detoxicant and 50000kg of water. Wherein the nano-selenium detoxifying agent is the nano-selenium detoxifying agent prepared in example 3.
The composite trace elements comprise manganese sulfate, monopotassium phosphate, ferrous sulfate and magnesium sulfate in equal mass ratio.
And sequentially pouring the raw materials of the third culture medium into a stirring tank, stirring and mixing the raw materials, pouring the nano-selenium detoxicant into the stirring tank after dissolving, mixing and stirring the nano-selenium detoxicant with the raw materials, pouring the mixture into a third-stage fermentation tank, sterilizing at high temperature of 121 ℃, preserving heat for 30 minutes, and cooling to 37 ℃.
And inoculating the bacillus subtilis obtained after the secondary fermentation into a third culture medium for fermentation, wherein the fermentation temperature of the tertiary fermentation is 38 ℃, and the fermentation time is 20 days. The pressure of the fermentation tank is 0.03Mpa, and the third culture medium is stirred in the three-stage fermentation process, wherein the stirring speed is 200 rmp. After the third-stage fermentation, stable liquid organic protein selenium, namely detoxified biological protein selenium is obtained.
Example 6
S1, inoculating the bacillus subtilis into the first culture medium for primary fermentation.
The first culture medium comprises the following components in proportion: 5g/L of nano-selenium, 0.7g/L of composite trace elements, 4g/L of yeast powder, 3g/L of peptone, 30g/L of glucose, 0.8g/L of sodium chloride and 4500kg of water.
Stirring and mixing the raw materials of the first culture medium by a container, dissolving, pumping the mixed raw materials into a primary culture tank, sterilizing the liquid first culture medium by a damp-heat method, keeping the temperature at 121 ℃ for 25 minutes, and cooling to 37 ℃.
Bacillus subtilis was inoculated into the first medium in the form of a suspension at 3%. Performing primary fermentation in a seeding tank, wherein the tank pressure of the seeding tank is 0.02Mpa during fermentation. The first culture medium is stirred in the primary fermentation process, the stirring speed is 180rmp, and the fermentation time is 48 hours.
And S2, inoculating the bacillus subtilis obtained through the primary fermentation into a second culture medium for secondary fermentation to obtain the bacillus subtilis completely adapting to the nano-selenium environment.
The second culture medium comprises the following components in proportion: 5g/L of nano selenium, 0.8g/L of composite trace element, 3g/L of yeast powder, 4g/L of peptone, 30g/L of glucose, 0.8g/L of sodium chloride and 7000kg of water.
Stirring and mixing the raw materials by a container, pumping the raw stock into a propagation tank, sterilizing at the high temperature of 121 ℃, preserving the heat for 25 minutes, then cooling, and reducing the temperature to 35 ℃.
The fermentation temperature of the secondary fermentation is 37 ℃, and the fermentation time is 48 hours. The pot pressure of the propagation pot is 0.02 MPa. And stirring the second culture medium in the secondary fermentation process, wherein the stirring speed is 180 rmp.
S3, inoculating the bacillus subtilis obtained after the secondary fermentation into a third culture medium with nano-selenium for tertiary fermentation.
The third culture medium comprises the following components in proportion: 8g/L of nano-selenium, 0.8g/L of composite trace elements, 4g/L of yeast powder, 4g/L of peptone, 30g/L of glucose, 3g/L of sodium chloride, 7000kg of nano-selenium detoxicant and 40000kg of water. Wherein the nano-selenium detoxifying agent is the nano-selenium detoxifying agent prepared in example 3.
The composite trace elements comprise manganese sulfate, monopotassium phosphate, ferrous sulfate and magnesium sulfate in equal mass ratio.
And sequentially pouring the raw materials of the third culture medium into a stirring tank, stirring and mixing the raw materials, pouring the nano-selenium detoxicant into the stirring tank after dissolving, mixing and stirring the nano-selenium detoxicant with the raw materials, pouring the mixture into a third-stage fermentation tank, sterilizing at high temperature of 121 ℃, preserving heat for 25 minutes, and cooling to 37 ℃.
And inoculating the bacillus subtilis obtained after the secondary fermentation into a third culture medium for fermentation, wherein the fermentation temperature of the tertiary fermentation is 37 ℃, and the fermentation time is 18 days. The pressure of the fermentation tank is 0.02MPa, and the third culture medium is stirred in the three-stage fermentation process, wherein the stirring speed is 180 rmp. After the third-stage fermentation, stable liquid organic protein selenium, namely detoxified biological protein selenium is obtained.
Example 7
The invention provides a preparation method of a selenium protein plant microbial inoculum, which comprises the following steps:
s1, fermenting the mixed bacterial colony of bacillus subtilis, yeast, actinomycetes and fluorescent bacteria in the seed liquid fermentation culture medium.
The seed liquid fermentation culture medium comprises the following components in parts by weight: 50g/L of white granulated sugar, 3g/L of yeast powder, 3g/L of peptone, 0.5g/L of composite trace elements, 1g/L of sodium chloride and 600kg of water.
Stirring and mixing the raw materials by a container, pouring the raw materials into a first-level seeding tank after dissolving, keeping the temperature for 20 minutes at the high temperature of 121 ℃, and cooling to 34 ℃.
The fermentation process is carried out in a seeding tank, the fermentation temperature is 37 ℃, and the fermentation time is 48 hours.
The bacillus subtilis is original bacillus subtilis and is not cultured in an environment with nano-selenium, and the proportion of bacillus subtilis, yeast, actinomycetes and fluorescent bacteria is 2:1:1: 1.
s2, inoculating the seed liquid containing various strains obtained by seed fermentation into a fermentation liquid culture medium for fermentation. The fermentation liquid culture medium comprises the following components in proportion: 3g/L of detoxified biological protein selenium, 0.5g/L of composite trace element, 2g/L of yeast powder, 2g/L of peptone, 20g/L of glucose, 1g/L of sodium chloride, 6000kg of nano-selenium detoxicant and 50000kg of water. Wherein the nano-selenium detoxifying agent is the nano-selenium detoxifying agent prepared in example 3. The detoxified protein selenium is the product prepared in example 6.
Sterilizing the fermentation liquid culture medium at 118 ℃ for 20-30 min, and inoculating the seed liquid; the temperature of the fermentation tank is 34 ℃, the pressure of the fermentation tank is 0.02MPa, and the fermentation tank is stirred for 180 rmp.
Example 8
The invention provides a preparation method of a selenium protein plant microbial inoculum, which comprises the following steps:
s1, fermenting the mixed bacterial colony of bacillus subtilis, yeast, actinomycetes and fluorescent bacteria in the seed liquid fermentation culture medium.
The seed liquid fermentation culture medium comprises the following components in parts by weight: 90g/L of white granulated sugar, 5g/L of yeast powder, 5g/L of peptone, 0.8g/L of composite trace elements, 2g/L of sodium chloride and 1000kg of purified water.
Stirring and mixing the raw materials by a container, pouring the raw materials into a first-level seeding tank after dissolution, keeping the temperature at 121 ℃ for 30 minutes, and cooling to 37 ℃.
The fermentation process is carried out in a seeding tank, the fermentation temperature is 37 ℃, and the fermentation time is 56 hours.
The bacillus subtilis is original bacillus subtilis and is not cultured in an environment with nano-selenium, and the proportion of bacillus subtilis, yeast, actinomycetes and fluorescent bacteria is 2:1:1: 1.
s2, inoculating the seed liquid containing various strains obtained by seed fermentation into a fermentation liquid culture medium for fermentation. The fermentation liquid culture medium comprises the following components in proportion: 5g/L of detoxified biological protein selenium, 1g/L of composite trace element, 5g/L of yeast powder, 5g/L of peptone, 80g/L of glucose, 3g/L of sodium chloride, 8000kg of nano-selenium detoxifier and 90000kg of water. Wherein the nano-selenium detoxifying agent is the nano-selenium detoxifying agent prepared in example 3. The detoxified protein selenium is the product prepared in example 6.
Sterilizing the fermentation liquid culture medium at 118 deg.C for 29min, and inoculating seed liquid; the temperature of the fermentation tank is 37 ℃, the pressure of the fermentation tank is 0.02MPa, and the fermentation tank is stirred for 180 rmp.
Example 9
The invention provides a preparation method of a selenium protein plant microbial inoculum, which comprises the following steps:
s1, fermenting the mixed bacterial colony of bacillus subtilis, yeast, actinomycetes and fluorescent bacteria in the seed liquid fermentation culture medium.
The seed liquid fermentation culture medium comprises the following components in parts by weight: 70g/L of white granulated sugar, 3g/L of yeast powder, 3g/L of peptone, 0.8g/L of composite trace elements, 2g/L of sodium chloride and 800kg of purified water.
Stirring and mixing the raw materials by a container, pouring the raw materials into a first-level seeding tank after dissolution, keeping the temperature at 121 ℃ for 25 minutes, and cooling to 37 ℃.
The fermentation process is carried out in a seeding tank, the fermentation temperature is 37 ℃, and the fermentation time is 50 hours.
The bacillus subtilis is original bacillus subtilis and is not cultured in an environment with nano-selenium, and the proportion of bacillus subtilis, yeast, actinomycetes and fluorescent bacteria is 2:1:1: 1.
s2, inoculating the seed liquid containing various strains obtained by seed fermentation into a fermentation liquid culture medium for fermentation. The fermentation liquid culture medium comprises the following components in proportion: 4g/L of detoxified biological protein selenium, 0.8g/L of composite trace element, 4g/L of yeast powder, 4g/L of peptone, 50g/L of glucose, 2g/L of sodium chloride, 7000kg of nano selenium detoxificant and 70000kg of water. Wherein the nano-selenium detoxifying agent is the nano-selenium detoxifying agent prepared in example 3. The detoxified protein selenium is the product prepared in example 6.
Sterilizing the fermentation liquid culture medium at 118 deg.C for 25min, and inoculating seed liquid; the temperature of the fermentation tank is 37 ℃, the pressure of the fermentation tank is 0.02MPa, and the fermentation tank is stirred for 180 rmp.
And S3, after the fermentation in the fermentation liquid culture medium is finished, inoculating the mixed culture solution of the mature cultured lactic acid bacteria and acetic acid bacteria into the fermentation liquid culture medium for further fermentation for 24 hours. It should be noted that the fermentation conditions are the same as those described above.
The mixed solution of the lactic acid bacteria and the acetic acid is obtained by culturing in a reseeding tank, and the ratio of the lactic acid bacteria to the acetic acid bacteria is 2: 1. The culture medium in the reseeding tank is: 3g/L of yeast powder, 3g/L of peptone, 40g/L of glucose, 0.8g/L of composite trace elements, 0.8g/L of sodium chloride and 5000kg of purified water. Sterilizing the culture medium at 121 deg.C, maintaining the temperature for 25min, cooling to 37 deg.C. The culture conditions are that the temperature of the tank is 37 ℃, the pressure of the tank is 0.02Mpa, the stirring is carried out for 180rmp, and the culture period is 48 hours.
Directly pouring the microbial inoculum fermented by the mixed bacterial liquid of lactic acid bacteria and acetic acid bacteria into a storage tank, and after-ripening at the temperature of 8 ℃ for 40 days. After being cultured, the selenium-protein plant nutrient microbial inoculum can be subpackaged into 10 kg kegs, and then the kegs are screwed, covered and put in storage.
Comparative example 1
The general liquid microbial inoculum purchased in the market is purchased from the research center of microorganisms of Ministry of agriculture.
Comparative example 2
The preparation process is different from that of example 9 only in that nano-selenium is used to replace detoxified bio-protein selenium in step 2 of the method of example 9, and purified water is used to replace nano-selenium detoxificant.
Test example 1
The liquid inocula in comparative example 1 and comparative example 2 were compared with the proteinic selenium plant nutrient inocula obtained in example 9 by live count comparison. The results are shown in Table 1.
TABLE 1
As can be seen from Table 1, the liveness count of the selenium protein plant nutrient microbial inoculum adopted in the embodiment of the invention is higher than that of common nano-selenium, the selenium protein plant nutrient microbial inoculum reaches the standard that the number of probiotics in the microbial fertilizer of Ministry of agriculture is more than 2 hundred million/ml, and the nano-selenium is far lower than the national standard. Therefore, the biological protein selenium is formed by detoxification, the biological protein selenium is superior to nano selenium, and the nano selenium microbial inoculum cannot reach the national standard, so that the detoxification protein selenium is preferably used for producing the protein selenium plant nutrient microbial inoculum.
Test example 2
The ingredients of the selenium protein plant nutrient inoculum of example 9 were tested as shown in table 2.
TABLE 2
In conclusion, the selenium protein plant nutrient microbial inoculum provided by the embodiment of the invention contains quantitative selenium and a large number of biological bacteria, and can improve the physical and chemical properties of soil and increase the content of soil organic matters through the activities of trace elements and biological bacteria, and has the effect of loosening the soil. After the biological protein selenium biological agent is applied to soil, the biological bacteria quickly proliferate to form group dominance to decompose nitrogen, phosphorus and potassium which are fixed in the soil and can not be absorbed and utilized by plants, and fix free nitrogen in the air for the plants to absorb and utilize. The selenium-protein plant nutrient microbial inoculum can not generate side effect on crops, the antibiotic bacteria in the selenium-protein plant microbial inoculum can kill fruit flies, insects and leukoderma in the proliferation and metabolism process of soil, the microbial inoculum is endogenous hormones such as other active substances, can self regulate the physiological process, and can not generate side effect on the crops.
In addition, the protein selenium plant nutrient microbial inoculum can promote the growth of crops, make the roots of the crops developed, quickly emerge, and the branches and leaves green, mature in advance, improve the stress resistance of the crops, and show the functions of disease resistance, drought resistance and lodging resistance. The selenium-protein plant nutrient microbial inoculum is nontoxic and residue-free, can decompose residues of chemical fertilizers and pesticides applied for a long time in soil, dissolves heavy metal residues after sewage irrigation, purifies the environment, and is a preferred fertilizer in the production of green agricultural products. Furthermore, the selenium protein plant nutrient microbial inoculum can also take biogas slurry as a carrier, wherein the biogas slurry contains a large amount of trace elements, nitrogen, phosphorus, potassium and organic proteins which are necessary for plants.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The nano-selenium detoxifying agent is characterized by being prepared by extracting traditional Chinese medicine raw materials with water, wherein the traditional Chinese medicine raw materials comprise the following components in parts by weight: 10-20 parts of schisandra chinensis, 8-15 parts of raw cacumen biotae, 8-15 parts of mirabilite and 5-8 parts of garden burnet.
2. The method of preparing a nano-selenium detoxifying agent of claim 1, comprising: extracting the traditional Chinese medicine raw materials with water;
preferably, the traditional Chinese medicine raw materials are soaked in water for 20-28 hours, then boiled for 3-5 hours, and then separated to remove solids, wherein the weight ratio of the water used for soaking to the schisandra chinensis in the traditional Chinese medicine raw materials is 5000-9000: 10 to 20.
3. A preparation method of detoxified biological protein selenium is characterized by comprising the following steps:
inoculating bacillus subtilis into a first culture medium for primary fermentation, and then inoculating the bacillus subtilis obtained through the primary fermentation into a second culture medium for secondary fermentation to obtain the bacillus subtilis completely adapting to a nano-selenium environment; wherein the first culture medium and the second culture medium both contain nano-selenium;
inoculating the bacillus subtilis obtained after the secondary fermentation into a third culture medium with nano-selenium for tertiary fermentation, wherein the third culture medium also has the nano-selenium detoxifying agent as claimed in claim 1.
4. The preparation method of detoxified bio-protein selenium of claim 3, wherein the weight ratio of the nano-selenium detoxifier to the nano-selenium in the third culture medium is 6000-8000: 20-40 parts of;
preferably, the third culture medium comprises the following components in proportion: 7-9 g/L of nano-selenium, 0.5-1 g/L of composite trace element, 2-5 g/L of yeast powder, 2-5 g/L of peptone, 10-50 g/L of glucose, 1-5 g/L of sodium chloride, 6000-8000 kg of nano-selenium detoxifying agent and 30000-50000kg of water;
preferably, the composite trace elements comprise manganese sulfate, monopotassium phosphate, ferrous sulfate and magnesium sulfate in equal parts by weight;
preferably, the pH value of the third culture medium is 5-6, and the third culture medium is sterilized at 118-120 ℃ for 20-30 min;
preferably, the fermentation temperature of the third-stage fermentation is 36-38 ℃, and the fermentation time is 10-20 days;
preferably, a fermentation tank is adopted for three-stage fermentation, the tank pressure of the fermentation tank is 0.01-0.03 MPa, and the third culture medium is stirred during the three-stage fermentation process, wherein the stirring speed is 150-200 rmp.
5. The method of claim 3 or 4, wherein the first culture medium comprises the following components: 3-7 g/L of nano selenium, 0.5-1 g/L of composite trace elements, 2-5 g/L of yeast powder, 2-5 g/L of peptone, 10-50 g/L of glucose, 0.5-1 g/L of sodium chloride and 3000-6000 kg of water;
preferably, the pH value of the first culture medium is 5-6, and the first culture medium is sterilized at 118-120 ℃ for 20-30 min;
preferably, the fermentation temperature of the primary fermentation is 36-38 ℃, and the fermentation time is 45-52 hours;
preferably, a seeding tank is adopted for primary fermentation, the tank pressure of the seeding tank is 0.01-0.03 Mpa, and the first culture medium is stirred in the primary fermentation process, wherein the stirring speed is 150-200 rmp;
preferably, in the primary fermentation, bacillus subtilis is inoculated into the first culture medium in a suspension form in an amount of 1-5%.
6. The method of claim 5, wherein the second medium comprises the following components: 3-7 g/L of nano selenium, 0.5-1 g/L of composite trace elements, 2-5 g/L of yeast powder, 2-5 g/L of peptone, 10-50 g/L of glucose, 0.5-1 g/L of sodium chloride and 6000-8000 kg of water;
preferably, the pH value of the second culture medium is 5-6, and the second culture medium is sterilized at 118-120 ℃ for 20-30 min;
preferably, the fermentation temperature of the secondary fermentation is 36-38 ℃, and the fermentation time is 45-52 hours;
preferably, a propagation tank is adopted for secondary fermentation, the tank pressure of the propagation tank is 0.01-0.03 MPa, and the second culture medium is stirred in the secondary fermentation process, wherein the stirring speed is 150-200 rmp.
7. A detoxified bio-protein selenium, which is produced by the production method according to any one of claims 3 to 6.
8. A method for preparing a selenium protein plant nutrient microbial inoculum is characterized by comprising the following steps: preparing the selenium-proteinate plant nutrient inoculum by fermenting the detoxified biological protein selenium as one of main raw materials as claimed in claim 7;
preferably, the main raw materials comprise detoxified biological protein selenium, carbohydrates and protein substances;
preferably, the main raw materials comprise detoxified biological selenium protein, yeast powder, peptone and white granulated sugar.
9. The method for preparing a selenium protein plant nutrient inoculum according to claim 8, which comprises the following steps: inoculating seed liquid containing various strains obtained by seed fermentation into a fermentation liquid culture medium for fermentation; the fermentation liquid culture medium comprises the following components in parts by weight: 3-5 g/L of detoxified biological protein selenium, 0.5-1 g/L of composite trace element, 2-5 g/L of yeast powder, 2-5 g/L of peptone, 20-80 g/L of glucose, 1-3 g/L of sodium chloride, 6000-8000 kg of nano-selenium detoxifier and 50000-90000kg of water;
preferably, the pH value of the fermentation liquid culture medium is 5-6, and the fermentation liquid culture medium is sterilized at 118-120 ℃ for 20-30 min; preferably, the fermentation temperature is 34-37 ℃, and the fermentation time is 18-22 days;
preferably, the seed liquid is obtained by fermenting a plurality of strains in a seed liquid fermentation culture medium;
preferably, the seed liquid fermentation medium comprises the following components in proportion: 50-90 g/L of white granulated sugar, 3-5 g/L of yeast powder, 3-5 g/L of peptone, 0.5-1 g/L of composite trace elements, 1-2 g/L of sodium chloride and 600-1000 kg of water;
preferably, the plurality of species comprises: bacillus subtilis, saccharomycetes, actinomycetes and fluorescent bacteria; preferably, the strain number ratio of the bacillus subtilis to the yeast to the actinomycetes to the fluorescent bacteria is 2:1:1: 1;
preferably, the pH value of the seed liquid fermentation medium is 5-6, and the seed liquid fermentation medium is sterilized at 118-120 ℃ for 20-30 min; preferably, the fermentation temperature is 34-37 ℃, and the fermentation time is 48-56 hours;
preferably, the preparation method of the selenium protein plant nutrient microbial inoculum further comprises the steps of supplementing a mixed bacterial liquid of mature cultured lactic acid bacteria and acetic acid bacteria into a fermentation liquid culture medium for continuous fermentation after fermentation in the fermentation liquid culture medium is completed; preferably, the fermentation time is 20-28 hours;
preferably, the ratio of the number of the lactic acid bacteria to the number of the acetic acid bacteria is 2: 1;
preferably, the microbial inoculum fermented by the mixed bacterial liquid of the lactic acid bacteria and the acetic acid bacteria is directly poured into a storage tank, and is subjected to after-ripening at the temperature of 5-15 ℃, wherein the after-ripening time is 20-60 days.
10. The use of detoxified bio-protein selenium of claim 7 in the preparation of a selenium-protein plant nutrient inoculant.
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