Process for producing enzyme selenoprotein through liquid compound fermentation of compound strains and application of enzyme selenoprotein

Abstract

The invention discloses a process for producing enzyme selenoprotein by liquid compound fermentation of compound strains, and relates to the technical field of biological fermentation, enterococcus faecalis, aspergillus niger and bacillus mucilaginosus are adopted to obtain a mixed strain according to a specific proportion, the mixed strain is added into a breeding culture medium, sodium selenite is added after amplification, and the enzyme selenoprotein is obtained. Sodium selenite is absorbed by mixed strains and converted into enzyme selenoprotein, repeated fermentation is adopted, the mixed strains and sodium selenite are supplemented, and along with increase of the number of times of fermentation, the metabolic capacity of the strains is gradually enhanced, the content of metabolites is increased, and the content of selenoprotein is gradually increased and tends to be stable; according to the present invention, the stopping of the fermentation process can be timely determined according to the requirement on the product enzyme selenoprotein, after the repeated fermentation, the molecular weight of the enzyme selenoprotein is from 3000 Dalton to 18000 Dalton, the fermentation frequency can be selected according to the requirement, the larger the molecular weight of the enzyme selenoprotein is, the better the absorption is, and the wide application prospect is provided.

Description

Technical Field

[0001] The present invention relates to the field of biological fermentation technology, and specifically relates to a process for producing enzyme selenium protein by liquid compound fermentation of composite strains and its application. Background Art

[0002] Selenium is an important element for animal growth, immunity, reproduction and hormone metabolism. It participates in the metabolism of the animal body by synthesizing selenium proteins. There are more than 30 kinds of proteins synthesized by selenium, which are essential components of the active centers of various enzymes in mammals, such as glutathione peroxidase, deiodinase, etc. Adding an appropriate amount of selenium to animal diets can not only promote animal growth, but also affect meat quality. Selenium can be added to animal feed in the form of inorganic selenium or organic selenium. Inorganic selenium (such as selenite) has low utilization rate, high toxicity and potential environmental pollution during the addition process. There is a risk of poisoning when animals ingest a small amount of inorganic selenium. Now many countries have prohibited the addition of inorganic selenium to animal feed. In contrast, organic selenium has high biological utilization rate, low toxicity and good palatability, and can significantly increase the selenium level in animal tissues, milk and eggs, and improve the production performance of animals.

[0003] Lactic acid bacteria are commonly used probiotic strains, which can convert inorganic selenium into organic selenium through bacterial metabolism, making it more conducive to the absorption and utilization of the animal body. And compared with the existing organic selenium yeast, lactic acid bacteria have unique advantages in antibiotic substitution, but there is less research on the existing organic selenium lactic acid bacteria. Therefore, studying composite strains with high selenium tolerance and producing high-molecular-weight organic selenium proteins has broad application prospects. Summary of the Invention

[0004] To solve the above problems, the purpose of the present invention is to provide a process for producing enzyme selenium protein by liquid compound fermentation of composite strains and its application.

[0005] To achieve the above object, the present invention is realized through the following technical solutions:

[0006] A process for producing enzyme selenium protein by liquid compound fermentation of composite strains, comprising the following steps:

[0007] ① The first fermentation: Add the first breeding medium into the reaction kettle, control the temperature of the reaction kettle at 35 - 38°C, add the mixed strains into it, and ferment at 35 - 38°C for 70 - 75 hours to obtain the first fermentation broth;

[0008] The first breeding medium is composed of glucose, methionine, amino acids and water according to the mass ratio of 95 - 105:8 - 12:95 - 105:95 - 105;

[0009] The mixed strains are obtained by mixing Enterococcus faecalis, Aspergillus niger and Bacillus mucilaginosus according to the volume ratio of 2 - 3:2 - 3:2 - 3;

[0010] The volume ratio of the mixed bacterial strains to the first breeding medium is 6-9:100;

[0011] The viable cell contents of Enterococcus faecalis, Aspergillus niger, and Bacillus mucilaginosus are all 10 8 cfu / g or more;

[0012] ② Second fermentation: Sodium selenite is added to the first fermentation broth obtained in the previous step, and after stirring and mixing evenly, the pH value of the first fermentation broth is measured, and the pH value is adjusted to 6.0-6.5 with sodium bicarbonate. The same volume of mixed bacterial strains as in step ① is added to the reaction kettle, and fermentation is carried out at 35-38 °C for 70-75 hours to obtain a second fermentation broth;

[0013] The mass ratio of the sodium selenite to the first breeding medium is 6-7:100;

[0014] ③ nth fermentation: Repeat the operation steps of step ② 1-5 times, the difference being that the mass of sodium selenite added each time is adjusted;

[0015] The n is any positive integer from 3 to 8.

[0016] Preferably, in step ③, as the number of fermentation times increases, the mass of sodium selenite added does not increase until it becomes 0.

[0017] Preferably, when n = 3, the mass ratio of the sodium selenite added to the first breeding medium is 3-4:100;

[0018] When n = 4-6, the mass ratio of the sodium selenite added to the first breeding medium is 1-2:100;

[0019] When n = 7-8, the mass of sodium selenite added is 0.

[0020] Preferably, in steps ② and ③, the pH value of the fermentation broth is controlled at 6.0-6.5 during fermentation, and the pH value of the fermentation broth is controlled by adding sodium bicarbonate.

[0021] Preferably, the viable cell content of Enterococcus faecalis is 5-10×10 9 cfu / g, the viable cell content of Aspergillus niger is 5-8×10 9 cfu / g, and the viable cell content of Bacillus mucilaginosus is 8-12×10 9 cfu / g.

[0022] Preferably, the mixed bacterial strains are obtained by mixing Enterococcus faecalis, Aspergillus niger, and Bacillus mucilaginosus according to a volume ratio of 1:1:1.

[0023] Preferably, the first breeding medium is composed of glucose, methionine, amino acids and water in a mass ratio of 10:1:10:10.

[0024] The present invention has the following advantages compared with the prior art:

[0025] In the process of producing enzyme selenium protein by liquid compound fermentation of the composite strain of the present invention, a mixed strain is obtained by using Enterococcus faecalis, Aspergillus niger and Bacillus mucilaginosus in a specific ratio, added to the breeding medium, and after amplification, sodium selenite is added. The mixed strain absorbs sodium selenite and converts it into enzyme selenium protein. Repeated fermentation is adopted, and the mixed strain and sodium selenite are supplemented. As the number of fermentation times increases, the metabolic ability of the strain gradually increases, the content of metabolites increases, and the content of selenium protein gradually increases and tends to be stable.

[0026] The process of liquid compound fermentation of the composite strain of the present invention for producing enzyme selenium protein is uniquely characterized in that it can decide to stop the fermentation process in a timely manner according to the need for the product enzyme selenium protein. After repeated fermentation, the molecular weight of the enzyme selenium protein can range from 3,000 daltons to 18,000 daltons. The number of fermentation times can be selected according to the need. After only two times of selenium-enriched fermentation, the obtained fermentation broth can be used in agriculture, such as for the cultivation of selenium-enriched wheat, selenium-enriched corn and selenium-enriched grains. The fermentation broth obtained after 3 to 4 times of fermentation can be used in animal husbandry, for beef cattle breeding, reducing the diseases of beef cattle, improving immunity and the selenium content in beef; the fermentation broth with high molecular weight in the later several times can be used in the pharmaceutical industry. And the larger the molecular weight of the enzyme selenium protein, the better the absorption, and it has broad application prospects. Description of the Drawings

[0027] Figure 1 It is a state diagram of the eighth fermentation broth. Detailed Embodiments

[0028] The object of the present invention is to provide a process for producing enzyme selenium protein by liquid compound fermentation of a composite strain and its application, which is realized through the following technical solutions:

[0029] The strains of Enterococcus faecalis, Aspergillus niger and Bacillus mucilaginosus of the present invention should be free from contamination by miscellaneous bacteria, have high activity, have genetic stability and storage stability, be non-pathogenic, and have reliable sources and complete certifications.

[0030] The optimal pH for the mixed fermentation of Enterococcus faecalis, Aspergillus niger and Bacillus mucilaginosus for selenium enrichment is 6.0 - 6.5. This range can balance the growth requirements of the three strains, maximize the conversion and enrichment efficiency of selenium, and ensure the stability of the fermentation process at the same time.

[0031] In the present invention, Enterococcus faecalis, Aspergillus niger, and Bacillus mucilaginosus are mixed and fermented in a specific ratio to achieve selenium enrichment, mainly based on the biological characteristics of these three strains and their synergistic effects in the process of selenium transformation and enrichment. Specifically:

[0032] In terms of selenium enrichment and storage:

[0033] These two strains, Enterococcus faecalis and Bacillus mucilaginosus, can efficiently absorb selenium and store it inside the cells to form selenium-enriched cells. Enterococcus faecalis mainly absorbs selenium through transport proteins on the cell wall and cell membrane, while Bacillus mucilaginosus adsorbs selenium by secreting extracellular polysaccharides. The mycelium of Aspergillus niger has a large surface area, which can adsorb and enrich a large amount of selenium. At the same time, its metabolites (such as polysaccharides and proteins) can also combine with selenium to form stable organic selenium complexes. `

[0034] Synergistic effect:

[0035] The reduction effect of Enterococcus faecalis provides Aspergillus niger with a more easily absorbable organic selenium form, and the metabolic activities of Aspergillus niger further promote the transformation and enrichment of selenium. The ability of Bacillus mucilaginosus to decompose phosphorus and release trace elements provides a richer nutrient environment for Enterococcus faecalis and Aspergillus niger. At the same time, the extracellular polysaccharides secreted by it contribute to the adsorption and stabilization of selenium.

[0036] Therefore, compared with the existing single yeast fermentation, these three strains, Enterococcus faecalis, Aspergillus niger, and Bacillus mucilaginosus, promote each other during the mixed fermentation process, forming an efficient selenium transformation and enrichment system, significantly improving the selenium enrichment efficiency, having a good targeting effect on selenium elements, and the advantages are fast fermentation and small molecular weight.

[0037] After adding sodium selenite to the fermentation broth, with the increase in the number of fermentation times, the ability of the strains to absorb and transform selenium is gradually optimized, resulting in an upward trend in the selenium content in the enzyme selenium protein and then tending to be stable; in different fermentation batches, the forms of selenium (the ratio of organic selenium and inorganic selenium) are different. In the first two fermentations, inorganic selenium is the main component, and in the remaining fermentations, the synergistic effect of Enterococcus faecalis and Aspergillus niger significantly increases the proportion of organic selenium (such as selenocysteine and selenomethionine); with the increase in the number of fermentation times, the metabolic ability of the strains gradually increases, resulting in an increase in the content of metabolites, which in turn affects the properties of the enzyme selenium protein;

[0038] In summary, the first two fermentations are in the optimization stage, and the properties of the enzyme selenium protein (such as selenium content and protein structure) fluctuate greatly. The fermentation conditions in the remaining fermentations tend to be stable, and the properties of the enzyme selenium protein are more consistent, showing high repeatability and stability.

[0039] The process of eight fermentations is as follows:

[0040] A process for producing enzyme selenium protein by liquid compound fermentation of compound strains, comprising the following steps:

[0041] ① First fermentation: Add the first breeding medium into the reaction kettle, control the temperature of the reaction kettle at 35-38 °C, add the mixed strains into it, and ferment at 35-38 °C for 70-75 hours to obtain the first fermentation broth;

[0042] The first breeding medium is composed of glucose, methionine, amino acids and water according to the mass ratio of 95-105:8-12:95-105:95-105;

[0043] The mixed strains are obtained by mixing Enterococcus faecalis, Aspergillus niger and Bacillus mucilaginosus according to the volume ratio of 2-3:2-3:2-3;

[0044] The volume ratio of the mixed strains to the first breeding medium is 6-9:100;

[0045] The viable bacteria content of Enterococcus faecalis, Aspergillus niger and Bacillus mucilaginosus is all 10 8 cfu / g or more;

[0046] ② Second fermentation: Add the first sodium selenite into the first fermentation broth obtained in the previous step, stir and mix evenly, then detect the pH value of the first fermentation broth, adjust the pH value to 6.0-6.5 with sodium bicarbonate, add the mixed strains with the same volume as that in step ① into the reaction kettle, and ferment at 35-38 °C for 70-75 hours to obtain the second fermentation broth;

[0047] The mass ratio of the first sodium selenite to the first breeding medium is 6-7:100;

[0048] After fermentation in this step, the molecular weight of the enzyme selenium protein in the second fermentation broth is about 3100; the selenium protein content is about 2×10 -2 g / kg;

[0049] ③ Third fermentation: Add the second sodium selenite into the second fermentation broth obtained in step ②, stir and mix evenly, then detect the pH value of the second fermentation broth, adjust the pH value to 6.0-6.5 with sodium bicarbonate, add the mixed strains with the same volume as that in step ① into the reaction kettle, and ferment at 35-38 °C for 70-75 hours to obtain the third fermentation broth;

[0050] The mass ratio of the second sodium selenite to the first breeding medium is 3-4:100;

[0051] After fermentation in this step, the molecular weight of the enzyme selenium protein in the third fermentation broth is about 5600; the selenium protein content is about 3×10 -2 g / kg;

[0052] ④ Fourth fermentation: Add sodium selenite (III) to the third fermentation broth obtained in step ③, stir and mix evenly, then measure the pH value of the third fermentation broth, adjust the pH value to 6.0 - 6.5 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 35 - 38 °C for 70 - 75 hours to obtain the fourth fermentation broth;

[0053] The mass ratio of the sodium selenite (III) to the first breeding medium is 1 - 2:100;

[0054] After fermentation in this step, the molecular weight of the enzyme selenium protein in the fourth fermentation broth is about 8100; the selenium protein content is about 3.5×10 -2 g / kg;

[0055] ⑤ Fifth fermentation: Add sodium selenite (IV) to the fourth fermentation broth obtained in step ④, stir and mix evenly, then measure the pH value of the fourth fermentation broth, adjust the pH value to 6.0 - 6.5 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 35 - 38 °C for 70 - 75 hours to obtain the fifth fermentation broth;

[0056] The mass ratio of the sodium selenite (IV) to the first breeding medium is 1 - 2:100;

[0057] After fermentation in this step, the molecular weight of the enzyme selenium protein in the fifth fermentation broth is about 10600; the selenium protein content is about 4×10 -2 g / kg;

[0058] ⑥ Sixth fermentation: Add sodium selenite (V) to the fifth fermentation broth obtained in step ⑤, stir and mix evenly, then measure the pH value of the fifth fermentation broth, adjust the pH value to 6.0 - 6.5 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 35 - 38 °C for 70 - 75 hours to obtain the sixth fermentation broth;

[0059] The mass ratio of the sodium selenite (V) to the first breeding medium is 1 - 2:100;

[0060] After fermentation in this step, the molecular weight of the enzyme selenium protein in the sixth fermentation broth is about 13100; the selenium protein content is about 4×10 -2 g / kg;

[0061] ⑦ Seventh fermentation: Measure the pH value of the sixth fermentation broth, adjust the pH value to 6.0 - 6.5 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 35 - 38 °C for 70 - 75 hours to obtain the seventh fermentation broth; After fermentation in this step, the molecular weight of the enzyme selenium protein in the seventh fermentation broth is about 15600; the selenium protein content is about 4×10 -2 g / kg;

[0062] ⑧ Eighth fermentation: Detect the pH value of the seventh fermentation broth, adjust the pH value to 6.0 - 6.5 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 35 - 38 °C for 70 - 75 hours to obtain the eighth fermentation broth; the state is as Figure 1 shown. After fermentation in this step, the molecular weight of the enzyme selenium protein in the eighth fermentation broth is about 18,100; the selenium protein content is about 4×10 -2 g / kg.

[0063] The characteristics of the present invention are that the number of fermentations can be selected according to needs, and two to eight fermentations can be selected. After only two selenium-enriched fermentations (steps ② and ③), the obtained fermentation broth can be used in agriculture, such as for the cultivation of selenium-enriched wheat, selenium-enriched corn and selenium-enriched grains. The fermentation broth obtained from 3 - 4 fermentations (steps ④ and ⑤) can be used in animal husbandry, for beef cattle breeding, reducing the diseases of beef cattle, improving immunity and the selenium content in beef; and the fermentation broth with high molecular weight in the subsequent fermentations (steps ⑥, ⑦ and ⑧) can be used in the pharmaceutical industry. And the larger the molecular weight of the enzyme selenium protein, the better the absorption.

[0064] The detection standard of the fermentation broth after fermentation in the present invention is T / DLAS 0002 - 2020.

[0065] The detection process of the molecular weight of organic selenium in the present invention is as follows:

[0066] The detection of the molecular weight of organic selenium usually involves the processes of its extraction, separation and quantitative analysis. The following are the main steps for detecting the molecular weight of organic selenium based on the high performance liquid chromatography - inductively coupled plasma mass spectrometry (HPLC - ICP - MS) method.

[0067] Sample preparation: Accurately pipette 1.0 mL - 5 mL of the liquid sample into a 50 mL conical flask, add 20 mL of 0.05 mol / L MES - TRIS buffer solution, add 100 μL of 50 mg / mL protease solution, seal with tin foil and mix well.

[0068] Enzymatic hydrolysis: Control the sample temperature at 60 °C ± 1 °C and continuously carry out enzymatic hydrolysis for 2 h. After taking it out and cooling, transfer it to a 50 mL centrifuge tube.

[0069] Centrifugation: Centrifuge at 5000 r / min for 10 min, take the supernatant, and filter it through a 0.45 μm organic filter membrane to obtain the solution to be measured;

[0070] Liquid chromatography separation: Separate the components of the supernatant through the liquid chromatography system. The chromatographic conditions are a C18 column (250 mm x 4.6 mm, 5 μm), the mobile phase is 98% trifluoroacetic acid aqueous solution (0.2% v / v) - 2% methanol, and the column temperature is set at 30 °C.

[0071] Mass spectrometry: After separation of the components, they are measured by an inductively coupled plasma mass spectrometer, and the retention time and mass-to-charge ratio are recorded.

[0072] Data analysis: Quantitative analysis is performed by the external standard method of peak area to calculate the molecular weight of organic selenium.

[0073] The fermented broth of the present invention can be filtered by a vacuum filtration procedure. During filtration, a filter membrane with a pore size such as 0.22 μm or 0.45 μm can be used for filtration. This filter membrane can effectively retain the microorganisms present in the fermented broth while allowing other components to pass through.

[0074] The following further describes the present invention in conjunction with specific embodiments.

[0075] Example 1

[0076] A process for the liquid compound fermentation of a composite strain to produce enzyme selenium protein, comprising the following steps:

[0077] ① First fermentation: 95 kg of glucose, 8 kg of methionine, 95 kg of amino acids and 95 kg of water are mixed evenly in a batching tank, added to a reaction kettle, the temperature of the reaction kettle is controlled at 35 °C, a mixed strain is added thereto, and fermentation is carried out at 35 °C for 75 hours to obtain a first fermentation broth;

[0078] The mixed strain is obtained by mixing Enterococcus faecalis, Aspergillus niger and Bacillus mucilaginosus in a volume ratio of 1:1:1;

[0079] The volume ratio of the mixed strain to the first breeding medium is 6:100;

[0080] The viable counts of Enterococcus faecalis, Aspergillus niger and Bacillus mucilaginosus are all 10 8 cfu / g or more;

[0081] ② Second fermentation: 17.58 kg of sodium selenite is added to the first fermentation broth obtained in step ①, stirred and mixed evenly, the pH value of the first fermentation broth is detected, the pH value is adjusted to 6.0 with sodium bicarbonate, a mixed strain with the same volume as that in step ① is added to the reaction kettle, and fermentation is carried out at 35 °C for 75 hours to obtain a second fermentation broth;

[0082] ③ Third fermentation: 8.79 kg of sodium selenite is added to the second fermentation broth obtained in step ②, stirred and mixed evenly, the pH value of the first fermentation broth is detected, the pH value is adjusted to 6.0 with sodium bicarbonate, a mixed strain with the same volume as that in step ① is added to the reaction kettle, and fermentation is carried out at 35 °C for 75 hours to obtain a third fermentation broth, which is the enzyme selenium protein.

[0083] Example 2

[0084] A process for producing enzyme selenium protein by liquid compound fermentation of a composite strain, comprising the following steps:

[0085] ① First fermentation: Mix 105 kg of glucose, 12 kg of methionine, 105 kg of amino acids and 105 kg of water evenly in a batching tank, add them to a reaction kettle, control the temperature of the reaction kettle at 38 °C, add the mixed strain to it, and ferment at 38 °C for 70 hours to obtain the first fermentation broth;

[0086] The mixed strain is obtained by mixing Enterococcus faecalis, Aspergillus niger and Bacillus mucilaginosus in a volume ratio of 2:2:3;

[0087] The volume ratio of the mixed strain to the first breeding medium is 9:100;

[0088] The viable bacteria content of Enterococcus faecalis, Aspergillus niger and Bacillus mucilaginosus is all above 108 cfu / g;

[0089] ② Second fermentation: Add 22.89 kg of sodium selenite to the first fermentation broth obtained in step ①, stir and mix evenly, then detect the pH value of the first fermentation broth, adjust the pH value to 6.5 with sodium bicarbonate, add the same volume of mixed strain as in step ① to the reaction kettle, and ferment at 38 °C for 70 hours to obtain the second fermentation broth;

[0090] ③ Third fermentation: Add 13.08 kg of sodium selenite to the second fermentation broth obtained in step ②, stir and mix evenly, then detect the pH value of the first fermentation broth, adjust the pH value to 6.5 with sodium bicarbonate, add the same volume of mixed strain as in step ① to the reaction kettle, and ferment at 38 °C for 70 hours to obtain the third fermentation broth, which is the enzyme selenium protein.

[0091] Example 3

[0092] A process for producing enzyme selenium protein by liquid compound fermentation of a composite strain, comprising the following steps:

[0093] ① First fermentation: Mix 100 kg of glucose, 10 kg of methionine, 100 kg of amino acids and 100 kg of water evenly in a batching tank, add them to a reaction kettle, control the temperature of the reaction kettle at 37 °C, add the mixed strain to it, and ferment at 37 °C for 72 hours to obtain the first fermentation broth;

[0094] The mixed strain is obtained by mixing Enterococcus faecalis, Aspergillus niger and Bacillus mucilaginosus in a volume ratio of 2:3:2;

[0095] The volume ratio of the mixed strain to the first breeding medium is 8:100;

[0096] The viable bacteria content of Enterococcus faecalis, Aspergillus niger and Bacillus mucilaginosus is all 10 8 cfu / g or more;

[0097] ② Second fermentation: Add 20.15 kg of sodium selenite to the first fermentation broth obtained in step ①, stir and mix evenly, then measure the pH value of the first fermentation broth, adjust the pH value to 6.2 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 37 °C for 72 hours to obtain the second fermentation broth;

[0098] The mass ratio of the sodium selenite to the first breeding medium is 6.5:100;

[0099] ③ Third fermentation: Add 10.85 kg of sodium selenite to the second fermentation broth obtained in step ②, stir and mix evenly, then measure the pH value of the first fermentation broth, adjust the pH value to 6.2 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 37 °C for 72 hours to obtain the third fermentation broth, which is the enzyme selenium protein.

[0100] The enzyme selenium protein obtained in Examples 1 to 3 can be directly used in agriculture, such as for the cultivation of selenium-rich wheat, selenium-rich corn and selenium-rich grains, and can be used as foliar fertilizer or base fertilizer.

[0101] Example 4

[0102] A process for producing enzyme selenium protein by liquid compound fermentation of a composite strain, comprising the following steps:

[0103] ① First fermentation: Mix 95 kg of glucose, 8 kg of methionine, 95 kg of amino acids and 95 kg of water evenly in a batching tank, add them to the reaction kettle, control the temperature of the reaction kettle at 35 °C, add the mixed strains to it, and ferment at 35 °C for 75 hours to obtain the first fermentation broth;

[0104] The mixed strains are obtained by mixing Enterococcus faecalis, Aspergillus niger and Bacillus mucilaginosus in a volume ratio of 1:1:1;

[0105] The volume ratio of the mixed strains to the first breeding medium is 6:100;

[0106] Among them, the viable count of Enterococcus faecalis is 5×10 9 cfu / g, the viable count of Aspergillus niger is 5×10 9 cfu / g, and the viable count of Bacillus mucilaginosus is 8×10 9 cfu / g;

[0107] ② Second fermentation: Add 17.58 kg of sodium selenite to the first fermentation broth obtained in step ①, stir and mix evenly, then measure the pH value of the first fermentation broth, adjust the pH value to 6.0 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 35 °C for 75 hours to obtain the second fermentation broth;

[0108] ③Third fermentation: Add 8.79 kg of sodium selenite to the second fermentation broth obtained in step ②, stir and mix evenly, then measure the pH value of the second fermentation broth, adjust the pH value to 6.0 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 35 °C for 75 hours to obtain the third fermentation broth;

[0109] ④Fourth fermentation: Add 2.93 kg of sodium selenite to the third fermentation broth obtained in step ③, stir and mix evenly, then measure the pH value of the third fermentation broth, adjust the pH value to 6.0 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 35 °C for 75 hours to obtain the fourth fermentation broth;

[0110] ⑤Fifth fermentation: Add 2.93 kg of sodium selenite to the fourth fermentation broth obtained in step ③, stir and mix evenly, then measure the pH value of the fourth fermentation broth, adjust the pH value to 6.0 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 35 °C for 75 hours to obtain the fifth fermentation broth;

[0111] ⑥Sixth fermentation: Add 2.93 kg of sodium selenite to the fifth fermentation broth obtained in step ③, stir and mix evenly, then measure the pH value of the fifth fermentation broth, adjust the pH value to 6.0 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 35 °C for 75 hours to obtain the sixth fermentation broth;

[0112] ⑦Seventh fermentation: Measure the pH value of the sixth fermentation broth, adjust the pH value to 6.0 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 35 °C for 75 hours to obtain the seventh fermentation broth;

[0113] ⑧Eighth fermentation: Measure the pH value of the seventh fermentation broth, adjust the pH value to 6.0 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 35 °C for 75 hours to obtain the eighth fermentation broth.

[0114] In Example 4, the product fermentation broths in steps ③ - ⑧ are all enzyme selenium proteins. The fermentation processes in ③ - ⑧ can be stopped as needed. For example, if the fermentation broth is used in agriculture, the subsequent fermentations do not need to be carried out after the third fermentation; if the fermentation broth is used in animal husbandry, the fermentation can be stopped at step ④ or step ⑤. If the fermentation broth is used in the pharmaceutical industry and a larger molecular weight of enzyme selenium protein is required, the fermentation processes in steps ⑥ - ⑧ can be continued.

[0115] Example 5

[0116] A process for producing enzyme selenium protein by liquid compound fermentation with a composite strain, comprising the following steps:

[0117] ① First fermentation: Mix 105 kg of glucose, 12 kg of methionine, 105 kg of amino acids and 105 kg of water evenly in a batching tank, add them to a reaction kettle, control the temperature of the reaction kettle at 38 °C, add the mixed strains to it, and ferment at 38 °C for 70 hours to obtain the first fermentation broth;

[0118] The mixed strains are obtained by mixing Enterococcus faecalis, Aspergillus niger and Bacillus mucilaginosus in a volume ratio of 1:2:3;

[0119] The volume ratio of the mixed strains to the first breeding medium is 9:100;

[0120] Among them, the viable count of Enterococcus faecalis is 10×10 9 cfu / g, the viable count of Aspergillus niger is 8×10 9 cfu / g, and the viable count of Bacillus mucilaginosus is 12×10 9 cfu / g;

[0121] ② Second fermentation: Add 22.89 kg of sodium selenite to the first fermentation broth obtained in step ①, stir and mix evenly, then detect the pH value of the first fermentation broth, adjust the pH value to 6.5 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 38 °C for 70 hours to obtain the second fermentation broth;

[0122] ③ Third fermentation: Add 13.08 kg of sodium selenite to the second fermentation broth obtained in step ②, stir and mix evenly, then detect the pH value of the first fermentation broth, adjust the pH value to 6.5 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 38 °C for 70 hours to obtain the third fermentation broth;

[0123] ④ Fourth fermentation: Add 6.54 kg of sodium selenite to the third fermentation broth obtained in step ③, stir and mix evenly, then detect the pH value of the third fermentation broth, adjust the pH value to 6.5 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 38 °C for 70 hours to obtain the fourth fermentation broth;

[0124] ⑤ Fifth fermentation: Add 6.00 kg of sodium selenite to the fourth fermentation broth obtained in step ③, stir and mix evenly, then detect the pH value of the fourth fermentation broth, adjust the pH value to 6.5 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 38 °C for 70 hours to obtain the fifth fermentation broth;

[0125] ⑥ Sixth fermentation: Add 3.27 kg of sodium selenite to the fifth fermentation broth obtained in step ③, stir and mix evenly, then detect the pH value of the fifth fermentation broth, adjust the pH value to 6.5 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 38 °C for 70 hours to obtain the sixth fermentation broth;

[0126] ⑦ Seventh fermentation: Detect the pH value of the sixth fermentation broth, adjust the pH value to 6.5 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 38 °C for 70 hours to obtain the seventh fermentation broth;

[0127] ⑧ Eighth fermentation: Detect the pH value of the seventh fermentation broth, adjust the pH value to 6.5 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 38 °C for 70 hours to obtain the eighth fermentation broth.

[0128] In Example 5, the product fermentation broths in steps ③ to ⑧ are all enzyme selenium proteins. The fermentation processes in ③ to ⑧ can be stopped as needed. For example, if the fermentation broth is used in agriculture, the subsequent fermentations do not need to be carried out after the third fermentation; if the fermentation broth is used in animal husbandry, the fermentation can be stopped at step ④ or step ⑤. If the fermentation broth is used in the pharmaceutical industry and a larger molecular weight of enzyme selenium protein is required, the fermentation processes in steps ⑥ to ⑧ can be continued.

[0129] Example 6

[0130] A process for producing enzyme selenium protein by liquid compound fermentation of a composite strain, comprising the following steps:

[0131] ① First fermentation: Mix 100 kg of glucose, 10 kg of methionine, 100 kg of amino acids and 100 kg of water evenly in a batching tank, add them to the reaction kettle, control the temperature of the reaction kettle at 37 °C, add the mixed strains to it, and ferment at 37 °C for 72 hours to obtain the first fermentation broth;

[0132] The mixed strains are obtained by mixing Enterococcus faecalis, Aspergillus niger and Bacillus mucilaginosus in a volume ratio of 1:1:1;

[0133] The volume ratio of the mixed strains to the first breeding medium is 8:100;

[0134] Among them, the viable count of Enterococcus faecalis is 8×10 9 cfu / g, the viable count of Aspergillus niger is 6×10 9 cfu / g, and the viable count of Bacillus mucilaginosus is 10×10 9 cfu / g;

[0135] ② Second fermentation: Add 20.15 kg of sodium selenite to the first fermentation broth obtained in step ①, stir and mix evenly, then detect the pH value of the first fermentation broth, adjust the pH value to 6.2 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 37 °C for 72 hours to obtain the second fermentation broth;

[0136] The mass ratio of the sodium selenite to the first breeding medium is 6.5:100;

[0137] ③ Third fermentation: Add 10.85 kg of sodium selenite to the second fermentation broth obtained in step ②, stir and mix evenly, then detect the pH value of the first fermentation broth, adjust the pH value to 6.2 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 37 °C for 72 hours to obtain the third fermentation broth;

[0138] ④ Fourth fermentation: Add 6.20 kg of sodium selenite to the third fermentation broth obtained in step ③, stir and mix evenly, then detect the pH value of the third fermentation broth, adjust the pH value to 6.2 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 37 °C for 72 hours to obtain the fourth fermentation broth;

[0139] ⑤ Fifth fermentation: Add 6.00 kg of sodium selenite to the fourth fermentation broth obtained in step ③, stir and mix evenly, then detect the pH value of the fourth fermentation broth, adjust the pH value to 6.2 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 37 °C for 72 hours to obtain the fifth fermentation broth;

[0140] ⑥ Sixth fermentation: Add 4.65 kg of sodium selenite to the fifth fermentation broth obtained in step ③, stir and mix evenly, then detect the pH value of the fifth fermentation broth, adjust the pH value to 6.2 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 37 °C for 72 hours to obtain the sixth fermentation broth;

[0141] ⑦ Seventh fermentation: Detect the pH value of the sixth fermentation broth, adjust the pH value to 6.2 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 37 °C for 72 hours to obtain the seventh fermentation broth;

[0142] ⑧ Eighth fermentation: Detect the pH value of the seventh fermentation broth, adjust the pH value to 6.2 with sodium bicarbonate, add the same volume of mixed strains as in step ① to the reaction kettle, and ferment at 37 °C for 72 hours to obtain the eighth fermentation broth.

[0143] In Example 6, the product fermentation broths in steps ③ to ⑧ are all enzyme selenium proteins. The fermentation processes in ③ to ⑧ can be stopped as needed. For example, if the fermentation broth is used in agriculture, the subsequent fermentation treatments do not need to be carried out after the third fermentation; if the fermentation broth is used in animal husbandry, the fermentation can be stopped at step ④ or step ⑤. If the fermentation broth is used in the pharmaceutical industry and a larger molecular weight of the enzyme selenium protein is required, the fermentation processes in steps ⑥ to ⑧ can be continued.

[0144] In the embodiment of the present invention, during fermentation, as the fermentation progresses, the acidity of the fermentation broth increases. Therefore, the pH value of the fermentation broth is controlled to be 6.0 - 6.5, and the pH value of the fermentation broth is controlled by adding sodium bicarbonate, which is beneficial to the fermentation of Enterococcus faecalis, Aspergillus niger, and Bacillus mucilaginosus.

[0145] The Shandong Academy of Agricultural Sciences used the enzyme selenium protein (molecular weight 3100 - 5600) of the present invention as base fertilizer and foliar fertilizer in Dinglichang Town, Yuncheng County, Shandong Province for planting 3000 mu of organic selenium wheat, and the organic selenium content of the wheat reached 430 μg / kg (detection basis: GB / T5009.93 - 2017). The enzyme selenium protein (molecular weight 3100 - 5600) of the present invention was used to plant rice in the Bai Wenguo Rice Planting Family Farm, Dawu District, Panjin City, Liaoning Province, and the organic selenium content of the rice reached 343 μg / kg (detection basis: GB / T5009.93 - 2017); the enzyme selenium protein (molecular weight 8100 - 10600) of the present invention was used in the Jilin Jimu Company to apply selenium protein to beef cattle, reducing the diseases of beef cattle and improving immunity, and the selenium content of the beef reached 110 μg / kg (detection basis: GB / T5009.93 - 2017), indicating that the enzyme selenium protein of the present invention can be widely applied to industries such as animal husbandry, agriculture, and medicine.

Claims

1. A process for producing enzyme selenium protein by liquid compound fermentation of a compound bacterial strain, characterized in that: It includes the following steps: ① First fermentation: Add the first breeding medium into the reaction kettle, control the temperature of the reaction kettle at 35 - 38 °C, add the mixed strains into it, and ferment at 35 - 38 °C for 70 - 75 hours to obtain the first fermentation broth; The first breeding medium is composed of glucose, methionine, amino acids and water according to the mass ratio of 95 - 105:8 - 12:95 - 105:95 - 105; The mixed strains are obtained by mixing Enterococcus faecalis, Aspergillus niger and Bacillus mucilaginosus according to the volume ratio of 2 - 3:2 - 3:2 - 3; The volume ratio of the mixed strains to the first breeding medium is 6 - 9:100; The viable counts of Enterococcus faecalis, Aspergillus niger, and Bacillus mucilaginosus are all 10 8 cfu / g or more; ② Second fermentation: Add sodium selenite into the first fermentation broth obtained in the previous step, stir and mix evenly, then detect the pH value of the first fermentation broth, adjust the pH value to 6.0 - 6.5 with sodium bicarbonate, add the mixed strains with the same volume as that in step ① into the reaction kettle, and ferment at 35 - 38 °C for 70 - 75 hours to obtain the second fermentation broth; The mass ratio of the sodium selenite to the first breeding medium is 6 - 7:100; ③ The nth fermentation: Repeat the operation steps of step ② for 1 - 5 times, the difference is to adjust the mass of sodium selenite added each time; The n is any positive integer from 3 to 8.

2. The process for producing enzyme selenium protein by liquid compound fermentation with a composite strain according to claim 1, characterized in that: In step ③, with the increase of the fermentation times, the mass of sodium selenite added does not increase until it is 0.

3. The process for producing enzyme selenium protein by liquid compound fermentation with a composite strain according to claim 1, characterized in that: When n = 3, the mass ratio of the sodium selenite added to the first breeding medium is 3 - 4:100; When n = 4 - 6, the mass ratio of the sodium selenite added to the first breeding medium is 1 - 2:100; When n = 7 - 8, the mass of the sodium selenite added is 0.

4. The process for producing enzyme selenium protein by liquid compound fermentation with a composite bacterial strain according to claim 1, characterized in that: In step ② and step ③, control the pH value of the fermentation broth at 6.0 - 6.5 during fermentation, and control the pH value of the fermentation broth by adding sodium bicarbonate.

5. The process for producing enzyme selenium protein by liquid compound fermentation with a composite bacterial strain according to claim 1, characterized in that: The viable count of Enterococcus faecalis is 5 - 10×10 9 cfu / g, the viable count of Aspergillus niger is 5 - 8×10 9 cfu / g, and the viable count of Bacillus mucilaginosus is 8 - 12×10 9 cfu / g.

6. The process for producing enzyme selenium protein by liquid double fermentation with a composite bacterial strain according to claim 1, characterized in that: The mixed strains are obtained by mixing Enterococcus faecalis, Aspergillus niger and Bacillus mucilaginosus according to the volume ratio of 1:1:

1.

7. The process for producing enzyme selenium protein by liquid compound fermentation with a composite strain according to claim 1, characterized in that: The first breeding medium is composed of glucose, methionine, amino acids and water according to the mass ratio of 10:1:10:

10.

8. Use of the enzyme selenium protein obtained in claim 1, characterized in that: It is used in farm fertilizers, feed additives or pharmaceutical additives.

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