Selenomethionine feed additive and method for its preparation

Abstract

The application relates to the technical field of feed additives, and particularly discloses a selenium-methionine feed additive and a preparation method thereof. The preparation method of the feed additive is as follows: adding wheat powder, a synthetic precursor, KH2PO4 and MgSO4 into navel orange residues, uniformly mixing, obtaining a fermentation substrate, controlling the moisture content of the fermentation substrate, inoculating a complex strain producing glutathione reductase at pH 4-8, uniformly mixing, controlling the fermentation temperature, fermenting for 10-20 days, inoculating bacillus producing methionine synthetase again, fermenting for 10-15 days, drying, crushing, and obtaining the selenium-methionine feed additive. The prepared feed additive has high organic selenium content, selenium-methionine accounts for more than 50% of the total selenium content, and the highest content reaches 73.11%, which meets the standard of the feed additive, and the preparation method is environment-friendly and has low requirements on production environment conditions.

Description

Technical Field

[0001] This application relates to the field of feed additive technology, and more specifically, to a selenomethionine feed additive and its preparation method. Background Technology

[0002] Navel orange pomace is a major byproduct of navel orange juicing. It is high in sugar and water, but because fresh pomace is prone to mold and difficult to preserve, has poor palatability, low protein content, and low digestibility, most of it is disposed of as waste, leading to environmental problems such as soil acidification and eutrophication of water bodies. Although some regions have tried using fresh pomace as ruminant feed, its resource utilization rate is extremely low due to its nutritional deficiencies.

[0003] Selenium is an essential trace element for animal growth and development, and its deficiency can severely affect animal growth, development, and the quality of livestock products. However, traditional inorganic selenium sources (sodium selenite) have significant drawbacks such as high toxicity and low absorption rate. Inorganic selenium sources have a narrow safety threshold, are prone to causing poisoning, and require strict control of mixing uniformity to avoid localized overdose. While organic selenium (such as yeast selenium) has reduced toxicity and improved absorption rate, its production cost is high, and it relies on imported strains or processes, making it difficult to meet large-scale demand. Therefore, there is an urgent need to develop a low-toxicity, high-absorption, and low-cost selenomethionine-containing feed additive and its preparation method, and to construct an efficient bioconversion system for navel orange pomace resources. Summary of the Invention

[0004] To address the technical problems mentioned in the background section, this application provides a selenomethionine feed additive and its preparation method.

[0005] A method for preparing a selenomethionine feed additive includes the following preparation steps:

[0006] After crushing and sieving navel orange residue, wheat flour, synthetic precursors, KH2PO4, and MgSO4 are added and mixed evenly to obtain the fermentation substrate. The moisture content of the fermentation substrate is controlled at 40-65%, and a compound strain producing glutathione reductase is inoculated at a pH of 4-8. The mixture is mixed evenly, and the fermentation temperature is controlled at 20-32℃ for 10-20 days. Then, Bacillus spp. producing methionine synthase is inoculated and fermented for 10-15 days. The mixture is dried and crushed at a temperature of 40-65℃ to obtain the selenomethionine feed additive.

[0007] Preferably, the mass ratio of the navel orange residue, wheat flour, synthetic precursor, KH2PO4 and MgSO4 is 10-80:15-85:0.1-5:0.01-0.5:0.01-0.5.

[0008] Preferably, the synthetic precursor is composed of sodium selenite and cysteine ​​in a mass ratio of 0.6-0.8:1.

[0009] Preferably, the synthetic precursor is composed of sodium selenite and cysteine ​​in a mass ratio of 0.7:1.

[0010] Preferably, the glutathione reductase-producing complex strain consists of Pichia pastoris, Streptomyces and Lactobacillus plantarum with an effective viable count of 10-12:2-4:1-3.

[0011] Preferably, the Bacillus that produces methionine synthase is Bacillus licheniformis and / or Bacillus subtilis.

[0012] Preferably, the inoculation amount of the glutathione reductase-producing complex strain is 5-15% of the fermentation substrate.

[0013] Preferably, the inoculum amount of the Bacillus thuringiensis producing methionine synthase is 8-15% of the fermentation substrate.

[0014] A selenomethionine feed additive is prepared by the above method.

[0015] In summary, this application has the following beneficial effects:

[0016] In the process of synthesizing selenomethionine feed additives, this application achieves efficient conversion of sodium selenite into selenomethionine by inoculating a compound strain of bacteria that produce glutathione reductase and Bacillus that produces methionine synthase in stages. In the first stage, a complex strain of bacteria producing glutathione reductase was inoculated. Among them, Pichia pastoris can efficiently secrete glutathione reductase, which can reduce high-valence inorganic selenium (such as sodium selenite) to low-valence (such as hydrogen selenide), providing a key intermediate for the subsequent synthesis of organic selenium. At the same time, its vigorous sugar metabolism can utilize the carbohydrates in wheat flour, providing energy for the entire fermentation system. Streptomycin can not only secrete a variety of extracellular enzymes (such as proteases and amylases) to degrade complex components such as cellulose and pectin in navel orange pomace, but also release small molecule carbon and nitrogen sources (such as glucose and amino acids), optimizing the availability of fermentation substrates. Its secondary metabolites can inhibit contamination by other microorganisms and maintain the stability of the fermentation environment. Lactobacillus plantarum maintains the system by producing acid (such as lactic acid), which on the one hand matches the optimal growth environment of Pichia pastoris, and on the other hand inhibits the reproduction of harmful microorganisms, ensuring the specificity of fermentation direction. The short-chain fatty acids accumulated during its metabolism can promote the subsequent colonization and enzyme expression of Bacillus licheniformis. The synergistic fermentation of three microbial strains not only achieves efficient conversion of sodium selenite to selenocysteine ​​with a significantly improved selenium conversion rate, but also reduces sterilization costs through pH self-regulation. In the second stage, *Bacillus licheniformis* producing methionine synthase is inoculated. This *Bacillus licheniformis* secretes highly active methionine synthase, directionally synthesizing selenocysteine ​​into selenomethionine. Through staged fermentation, in the first stage, glutathione reductase-producing bacteria preferentially complete selenium reduction. In the second stage, the methionine synthase-producing *Bacillus licheniformis* focuses on methionine synthesis, avoiding enzyme activity inhibition, thus achieving efficient conversion of inorganic selenium to organic selenium.

[0017] In this application, the efficient conversion of sodium selenite to selenomethionine is achieved by adjusting the feed ratio of sodium selenite and cysteine. Sodium selenite, as an inorganic selenium source, provides selenium while avoiding excessive toxicity. An appropriate ratio satisfies the substrate requirements of the glutathione reductase-catalyzed reduction reaction, while preventing the inhibition of microbial growth by high concentrations of sodium selenite. Cysteine, as a precursor for methionine synthesis, can provide thiol groups and carbon skeletons, and combine with selenium ions to form selenocysteine ​​intermediates, which directly participate in the methionine synthase-catalyzed reaction of Bacillus licheniformis, shortening the metabolic pathway and increasing the yield of the target product. Attached Figure Description

[0018] Figure 1 The conversion rate of selenomethionine after fermentation is shown in the figure for different feed ratios of cysteine ​​and sodium selenite.

[0019] Figure 2 This is a schematic diagram of the biological process of selenomethionine feed additive synthesis. Detailed Implementation

[0020] The present application will be further described in detail below with reference to the embodiments. The wheat flour (food grade) used in the embodiments and comparative examples of this application was purchased from Hebei Chuangzhiyuan Biotechnology Co., Ltd.; sodium selenite (feed grade) was purchased from Guangdong Mingtong Biotechnology Co., Ltd.; cysteine ​​(food grade) was purchased from Shandong Xinxiong Biotechnology Co., Ltd.; Pichia manshurica (product name: Pichia manshurica, product number: B336087), Streptomyces sp. (product name: Streptomyces sp., product number: BMZ126915), Lactobacillus plantarum (product name: Lactobacillus plantarum, product number: B94687); and Bacillus licheniformis (product name: B66977) were all purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0021] Examples 1-3 provide a selenomethionine feed additive and its preparation method.

[0022] Example 1

[0023] A method for preparing a selenomethionine feed additive includes the following preparation steps:

[0024] Crush the navel orange pulp, pass it through a 10mm sieve, add wheat flour, the synthesis precursors KH₂PO₄ and MgSO₄, mix thoroughly to obtain the fermentation substrate. Control the moisture content of the fermentation substrate to 40%, and at pH 4, inoculate with a glutathione reductase-producing compound strain at an inoculation rate of 5% of the fermentation substrate. Mix thoroughly, control the fermentation temperature at 20℃, and ferment for 10 days. Then, inoculate with methionine synthase-producing Bacillus at an inoculation rate of 8% of the fermentation substrate, and ferment for 10 days at 40℃. At ℃, the mixture is dried and pulverized to obtain selenomethionine feed additive. The mass ratio of navel orange pomace, wheat flour, synthetic precursor, KH2PO4 and MgSO4 is 10:15:0.1:0.01:0.01. The synthetic precursor is composed of sodium selenite and cysteine ​​in a mass ratio of 0.6:1. The compound strain for producing glutathione reductase is composed of Pichia pastoris, Streptomyces and Lactobacillus plantarum with an effective viable count of 10:2:1. The Bacillus licheniformis is the methionine synthase-producing Bacillus licheniformis.

[0025] Example 2

[0026] A method for preparing a selenomethionine feed additive includes the following preparation steps:

[0027] Crush the navel orange pomace, pass it through a 25mm sieve, add wheat flour, the synthesis precursors KH2PO4 and MgSO4, mix thoroughly to obtain the fermentation substrate. Control the moisture content of the fermentation substrate to 50%, and at pH 6, inoculate with a glutathione reductase-producing compound strain at an inoculum of 10% of the fermentation substrate, mix thoroughly, and control the fermentation temperature at 26℃ for 15 days. Then, inoculate with methionine synthase-producing Bacillus at an inoculum of 12% of the fermentation substrate and ferment for 12 days. The selenomethionine feed additive was obtained by drying and pulverizing at 50℃. The mass ratio of navel orange pomace, wheat flour, synthetic precursor, KH2PO4 and MgSO4 was 50:60:3:0.2:0.2. The synthetic precursor was composed of sodium selenite and cysteine ​​in a mass ratio of 0.7:1. The compound strain for producing glutathione reductase was composed of Pichia pastoris, Streptomyces and Lactobacillus plantarum with an effective viable count of 11:3:2. The Bacillus licheniformis was the methionine synthase-producing Bacillus licheniformis.

[0028] Example 3

[0029] A method for preparing a selenomethionine feed additive includes the following preparation steps:

[0030] The navel orange pomace was crushed, passed through a 35mm sieve, and then mixed with wheat flour, the synthesis precursors KH₂PO₄ and MgSO₄. The mixture was thoroughly mixed to obtain the fermentation substrate. The moisture content of the substrate was controlled at 65%. At pH 8, a glutathione reductase-producing compound strain was inoculated at 15% of the substrate. The mixture was thoroughly mixed, and the fermentation temperature was controlled at 32℃ for 20 days. Then, a methionine synthase-producing Bacillus was inoculated at 15% of the substrate and fermented for 15 days. The selenomethionine feed additive was obtained by drying and pulverizing at 65℃. The mass ratio of navel orange pomace, wheat flour, synthetic precursor, KH2PO4 and MgSO4 was 80:85:5:0.5:0.5. The synthetic precursor was composed of sodium selenite and cysteine ​​in a mass ratio of 0.8:1. The compound strain for producing glutathione reductase was composed of Pichia pastoris, Streptomyces and Lactobacillus plantarum with an effective viable count of 12:4:3. The Bacillus licheniformis was the methionine synthase producing bacillus.

[0031] Comparative Example 1

[0032] A method for preparing a selenomethionine feed additive includes the following preparation steps:

[0033] Crush the navel orange pomace, pass it through a 25mm sieve, add wheat flour, the synthesis precursors KH2PO4 and MgSO4, mix thoroughly to obtain the fermentation substrate. Control the moisture content of the fermentation substrate to 50%, and at pH 6, inoculate with a glutathione reductase-producing compound strain at an inoculum of 10% of the fermentation substrate, mix thoroughly, and control the fermentation temperature at 26℃ for 15 days. Then, inoculate with methionine synthase-producing Bacillus at an inoculum of 12% of the fermentation substrate and ferment for 12 days. The selenomethionine feed additive was obtained by drying and pulverizing at 50℃. The mass ratio of navel orange pomace, wheat flour, synthetic precursor, KH2PO4 and MgSO4 was 50:60:3:0.2:0.2. The synthetic precursor was composed of sodium selenite and cysteine ​​in a mass ratio of 0.4:1. The compound strain for producing glutathione reductase was composed of Pichia pastoris, Streptomyces and Lactobacillus plantarum with an effective viable count of 11:3:2. The Bacillus licheniformis was the methionine synthase producing bacillus.

[0034] Comparative Example 2

[0035] A method for preparing a selenomethionine feed additive includes the following preparation steps:

[0036] Crush the navel orange pomace, pass it through a 25mm sieve, add wheat flour, the synthesis precursors KH2PO4 and MgSO4, mix thoroughly to obtain the fermentation substrate. Control the moisture content of the fermentation substrate to 50%, and at pH 6, inoculate with a glutathione reductase-producing compound strain at an inoculum of 10% of the fermentation substrate, mix thoroughly, and control the fermentation temperature at 26℃ for 15 days. Then, inoculate with methionine synthase-producing Bacillus at an inoculum of 12% of the fermentation substrate and ferment for 12 days. The selenomethionine feed additive was obtained by drying and pulverizing at 50℃. The mass ratio of navel orange pomace, wheat flour, synthetic precursor, KH2PO4 and MgSO4 was 50:60:3:0.2:0.2. The synthetic precursor was composed of sodium selenite and cysteine ​​in a mass ratio of 0.9:1. The compound strain for producing glutathione reductase was composed of Pichia pastoris, Streptomyces and Lactobacillus plantarum with an effective viable count of 11:3:2. The Bacillus licheniformis was the methionine synthase producing bacillus.

[0037] Comparative Example 3

[0038] A method for preparing a selenomethionine feed additive includes the following preparation steps:

[0039] The navel orange pomace was crushed, passed through a 25mm sieve, and then mixed with wheat flour, the synthetic precursor KH2PO4, and MgSO4. After thorough mixing, a fermentation substrate was obtained. The moisture content of the fermentation substrate was controlled at 50%. At pH 6, yeast producing glutathione reductase was inoculated at an inoculation rate of 10% of the fermentation substrate. The mixture was thoroughly mixed, and the fermentation temperature was controlled at 26℃ for 15 days. Then, Bacillus thuringiensis producing methionine synthase was inoculated at an inoculation rate of 12% of the fermentation substrate and fermented for 12 days. The mixture was then dried and crushed at 50℃ to obtain the selenomethionine feed additive. The mass ratio of navel orange pomace, wheat flour, synthetic precursor, KH2PO4, and MgSO4 was 50:60:3:0.2:0.2. The synthetic precursor consisted of sodium selenite and cysteine ​​in a mass ratio of 0.7:1. The yeast producing glutathione reductase was Pichia pastoris, and the Bacillus thuringiensis producing methionine synthase was Bacillus licheniformis.

[0040] Comparative Example 4

[0041] A method for preparing a selenomethionine feed additive includes the following preparation steps:

[0042] Crush the navel orange pomace, pass it through a 25mm sieve, add wheat flour, KH₂PO₄ and MgSO₄, mix well to obtain the fermentation substrate. Control the moisture content of the fermentation substrate to 50%, and at pH 6, inoculate with a glutathione reductase-producing compound strain at an inoculum of 10% of the fermentation substrate. Mix well, control the fermentation temperature at 26℃, and ferment for 15 days. Then, inoculate with methionine synthase-producing Bacillus at an inoculum of 12% of the fermentation substrate and ferment for 12 days. The product is dried and pulverized at 50℃ to obtain selenomethionine feed additive. The mass ratio of navel orange residue, wheat flour, synthetic precursor, KH2PO4 and MgSO4 is 50:60:3:0.2:0.2. The synthetic precursor is composed of sodium selenite and cysteine ​​in a mass ratio of 0.7:1. The compound strain for producing glutathione reductase is composed of Pichia pastoris and Streptomyces with an effective viable count of 11:3. The Bacillus for producing methionine synthase is Bacillus licheniformis.

[0043] Comparative Example 5

[0044] A method for preparing a selenomethionine feed additive includes the following preparation steps:

[0045] The navel orange pulp was crushed and passed through a 25mm sieve. Wheat flour, the synthesis precursors KH₂PO₄ and MgSO₄ were added and mixed thoroughly to obtain the fermentation substrate. The moisture content of the fermentation substrate was controlled at 50%. At pH 6, a glutathione reductase-producing compound strain was inoculated at 10% of the fermentation substrate, mixed thoroughly, and the fermentation temperature was controlled at 26℃ for 15 days. Then, a methionine synthase-producing Bacillus was inoculated at 12% of the fermentation substrate and fermented for 12 days. The selenomethionine feed additive is obtained by drying and pulverizing at 50℃. The mass ratio of navel orange pomace, wheat flour, synthetic precursor, KH2PO4 and MgSO4 is 50:60:3:0.2:0.2. The synthetic precursor is composed of sodium selenite and cysteine ​​in a mass ratio of 0.7:1. The compound strain for producing glutathione reductase is composed of Pichia pastoris and Lactobacillus plantarum with an effective viable count of 11:2. The Bacillus licheniformis is the methionine synthase producing bacillus.

[0046] Comparative Example 6

[0047] A method for preparing a selenomethionine feed additive includes the following preparation steps:

[0048] The navel orange pomace was crushed and passed through a 25mm sieve. Wheat flour, KH₂PO₄, and MgSO₄ precursors were added and mixed thoroughly to obtain the fermentation substrate. The moisture content of the fermentation substrate was controlled at 50%. At pH 6, a glutathione reductase-producing compound strain was inoculated at 10% of the fermentation substrate and mixed thoroughly. Then, a methionine synthase-producing Bacillus was inoculated at 12% of the fermentation substrate. The fermentation temperature was controlled at 26℃ for 27 days. The fermentation was then carried out at 50℃. At ℃, the mixture is dried and pulverized to obtain selenomethionine feed additive. The mass ratio of navel orange residue, wheat flour, synthetic precursor, KH2PO4 and MgSO4 is 50:60:3:0.2:0.2. The synthetic precursor is composed of sodium selenite and cysteine ​​in a mass ratio of 0.7:1. The compound strain for producing glutathione reductase is composed of Pichia pastoris, Streptomyces and Lactobacillus plantarum with an effective viable count of 11:3:2. The Bacillus licheniformis is the methionine synthase-producing Bacillus licheniformis.

[0049] Comparative Example 7

[0050] A method for preparing a selenomethionine feed additive includes the following preparation steps:

[0051] Crush the navel orange pulp, pass it through a 25mm sieve, add wheat flour, the synthesis precursors KH2PO4 and MgSO4, mix thoroughly to obtain the fermentation substrate. Control the moisture content of the fermentation substrate to 50%, maintain a pH of 6, and control the fermentation temperature at 26℃. First, inoculate with Bacillus methionine synthase-producing bacteria at an inoculum of 12% of the fermentation substrate and ferment for 12 days. Then, inoculate with a glutathione reductase-producing compound strain at an inoculum of 10% of the fermentation substrate, mix thoroughly, and ferment for 15 days at a temperature of... The selenomethionine feed additive was obtained by drying and pulverizing at 50℃. The mass ratio of navel orange pomace, wheat flour, synthetic precursor, KH2PO4 and MgSO4 was 50:60:3:0.2:0.2. The synthetic precursor was composed of sodium selenite and cysteine ​​in a mass ratio of 0.7:1. The compound strain for producing glutathione reductase was composed of Pichia pastoris, Streptomyces and Lactobacillus plantarum with an effective viable count of 11:3:2. The Bacillus licheniformis was the methionine synthase-producing Bacillus licheniformis.

[0052] Performance testing

[0053] Preliminary experimental studies in this application revealed that the proportion of selenomethionine yield to total selenium after fermentation (conversion rate) is related to the pre-fermentation feed ratio of cysteine ​​and sodium selenite. Within a certain range, this correlation with selenomethionine yield was positively correlated. By controlling other fermentation conditions identically, only the feed ratio of cysteine ​​and sodium selenite in the precursor was changed, and the conversion rate of selenomethionine after fermentation was measured. See Table 1 for details. Figure 1 This feed additive containing selenomethionine undergoes a synthetic biological process such as... Figure 2 As shown.

[0054] Speciation of selenium: Speciation and quantification were performed using high performance liquid chromatography-atomic fluorescence spectrometry.

[0055] Total selenium content determination: The test was conducted in accordance with industry standard HJ 694-2014 "Determination of mercury, arsenic, selenium, bismuth and antimony in water by atomic fluorescence spectrometry".

[0056] Table 1. Conversion rate of selenomethionine after fermentation with different feed ratios of cysteine ​​and sodium selenite.

[0057]

[0058] The selenium forms of the selenomethionine feed additives prepared in Examples 1-3 and Comparative Examples 1-7 of this application were tested respectively. The test results are shown in Table 2.

[0059] Table 2. Test results of the selenomethionine feed additives prepared in Examples 1-3 and Comparative Examples 1-7

[0060]

[0061] As shown in Tables 1-2, the method for preparing selenomethionine feed additives provided in this application is not only simple in terms of production equipment and process, but also has low requirements for environmental conditions. Utilizing solid-state fermentation, it does not generate waste gas, wastewater, or waste residue, making it environmentally friendly and energy-saving. The preparation method of this application produces organic selenium with a high content, where selenomethionine accounts for more than 50% of the total selenium content, reaching a maximum of 73.11%, which meets the standards for feed additives (the standard for imported selenomethionine is more than 50%).

[0062] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing a feed additive of selenomethionine, characterized by, The preparation steps include the following: After crushing and sieving navel orange residue, wheat flour, synthetic precursors, KH2PO4 and MgSO4 are added and mixed evenly to obtain fermentation substrate. The moisture content of the fermentation substrate is controlled at 40-65%. At a pH of 4-8, a compound strain of glutathione reductase is inoculated, mixed evenly, and the fermentation temperature is controlled at 20-32℃. Fermentation is carried out for 10-20 days. Then, Bacillus spp. that produces methionine synthase is inoculated and fermented for 10-15 days. At a temperature of 40-65℃, the mixture is dried, crushed, and selenomethionine feed additive is obtained. The synthetic precursor consists of sodium selenite and cysteine ​​in a mass ratio of 0.6-0.8:

1.

2. The method for preparing the selenium-methionine feed additive according to claim 1, characterized by, The mass ratio of the navel orange residue, wheat flour, synthetic precursor, KH2PO4 and MgSO4 is 10-80:15-85:0.1-5:0.01-0.5:0.01-0.

5.

3. The method for preparing selenomethionine feed additive according to claim 1, characterized in that, The synthetic precursor consists of sodium selenite and cysteine ​​in a mass ratio of 0.7:

1.

4. The method for preparing the selenomethionine feed additive according to claim 1, characterized in that, The glutathione reductase-producing complex strain consists of Pichia pastoris, Streptomyces and Lactobacillus plantarum with an effective viable count of 10-12:2-4:1-3.

5. The method for preparing the selenomethionine feed additive according to claim 1, characterized in that, The methionine synthase-producing Bacillus is Bacillus licheniformis and / or Bacillus subtilis.

6. The method for preparing the selenomethionine feed additive according to claim 1, characterized in that, The inoculation amount of the glutathione reductase-producing compound strain is 5-15% of the fermentation substrate.

7. The method for preparing selenomethionine feed additive according to claim 1, characterized in that, The inoculum size of the Bacillus that produces methionine synthase is 8-15% of the fermentation substrate.

8. A selenomethionine feed additive prepared by any one of claims 1-7.

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