Application of ferritin in feed additive
By recombinant Bacillus subtilis expressing deacetylated ferritin as a feed additive, the problem of iron deficiency in piglets is solved, the iron binding amount and health rate are improved, piglet growth is promoted, the feed-to-meat ratio is reduced, and the breeding benefits are improved.
Patent Information
- Application Number
- CN202510589584.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-08
- Publication Date
- 2025-07-08
AI Technical Summary
In modern intensive breeding, iron deficiency in piglets leads to anemia, decreased immunity and high mortality. The existing iron supplementation methods have safety and cost problems, especially the low absorption rate of organic iron and high manufacturing costs.
Deacetylated sarcoferrin expressed by recombinant Bacillus subtilis was used as feed additives. By mutation of the amino acid site of sarcoferrin, iron binding capacity was improved and powdered feed additives were prepared.
The iron binding amount and serum iron content of piglets are increased, the health rate and growth performance are improved, the feed-to-meat ratio is reduced, and the breeding benefits are increased.
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Figure CN120266937A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of feed preparation, and particularly relates to the application of ferritin in feed additives. Background Art
[0002] Iron plays a crucial role in animals. Iron is a core component of hemoglobin, responsible for binding oxygen in red blood cells and transporting it from the lungs to all tissues of the body. In muscle cells, iron constitutes myoglobin, which stores oxygen for use during muscle contraction. Iron is a component of cytochromes (such as cytochrome c oxidase) and assists in ATP production in mitochondria. Multiple enzymes (such as catalase, peroxidase) rely on iron to catalyze reactions, scavenge free radicals, and protect cells from oxidative damage. Iron affects the growth and differentiation of immune cells, and iron deficiency may lead to a decline in immune function. In antioxidant defense, iron-containing enzymes (such as superoxide dismutase) help neutralize reactive oxygen molecules. In the body, ribonucleotide reductase relies on iron, and this enzyme plays a key role in DNA synthesis, affecting cell division and tissue repair. Iron is also involved in the production of neurotransmitters such as dopamine and serotonin, affecting nerve signal conduction.
[0003] After piglets are born, they grow rapidly and their blood volume increases rapidly, requiring a large amount of iron to synthesize hemoglobin. The iron reserve in newborn piglets is only about 50 mg, while the daily iron requirement is about 7 - 10 mg. The iron content in breast milk is low (about 1 - 2 mg / L), which cannot meet the demand. In modern intensive farming, piglets have less chance to contact the soil (a natural source of iron), further increasing the risk of iron deficiency. Iron deficiency can cause anemia in piglets, with pale skin, difficulty breathing, reduced vitality, and slow growth; a decline in immunity, making them prone to diarrhea and respiratory diseases; and iron deficiency is associated with an increased mortality rate in piglets, and severe anemia may lead to sudden death.
[0004] Currently, the common iron supplementation methods in pig farms are as follows: One is injection iron supplementation. Piglets are intramuscularly or subcutaneously injected with iron dextran injection at a dose of 150 - 200 mg / head within 1 - 3 days after birth, and repeated injection is carried out at 2 - 3 weeks of age. When injecting iron, there are individual piglets with poor iron tolerance, and excessive iron can cause poisoning, leading to vomiting, diarrhea, and even death. Moreover, in large-scale and high-density pig farms, injection iron supplementation is time-consuming and laborious, with high labor costs. The other is oral iron supplementation, with ferrous sulfate, iron glycinate, iron methionine, etc. added to feed or drinking water. Organic iron such as iron glycinate and iron methionine has a higher absorption rate than inorganic iron (such as ferrous sulfate), but organic iron is a complex formed by combining inorganic iron (such as ferrous sulfate, ferrous chloride, etc.) with organic ligands (such as amino acids, small peptides, organic acids, etc.), and the manufacturing cost is relatively high, and the absorption rate of piglets is still relatively low.
[0005] Nereis has a relatively high iron content and belongs to a group with relatively rich iron content among marine benthic organisms. Nereis mostly lives in the coastal intertidal zone or estuarine bottom mud, and the sediments in these areas are rich in iron minerals (such as hematite, pyrite) and organically complexed iron input from land sources. Nereis can store excessive iron in the form of ferritin in the coelom or specific tissues. Expressing Nereis ferritin through microorganisms and using it as an organic iron feed additive for iron supplementation in piglets and pregnant and lactating sows can meet the iron demand for the rapid growth of piglets in the initial stage of birth, promote the healthy growth of piglets, and improve the breeding efficiency. Summary of the Invention
[0006] The purpose of the present invention is to provide an application of ferritin in feed additives, belonging to the technical field of feed preparation.
[0007] To achieve the above purpose, the present invention provides the following technical solutions:
[0008] First, the present invention provides an application of ferritin in feed additives, and the feed additive is a feed additive prepared from deacetylated Nereis ferritin expressed by recombinant Bacillus subtilis.
[0009] Furthermore, the amino acid sequence of the deacetylated Nereis ferritin is SEQ ID NO.2.
[0010] Furthermore, the preparation method of the feed additive is as follows:
[0011] (1) The recombinant Bacillus subtilis expressing deacetylated Nereis ferritin was inoculated into LB culture medium containing 40 μg / mL chloramphenicol at a ratio of 1% by volume, and cultured on a shaker at 37 °C and 250 r / min for 12 h to prepare a primary seed solution;
[0012] (2) According to the fermentation volume, the primary seed solution was transferred to prepare a secondary seed solution and multiple-stage seed solutions;
[0013] (3) The multiple-stage seed solutions were inoculated into the ferritin fermentation medium at a ratio of 1% by volume, and fermented and cultured in a fermenter for 16 h to express deacetylated Nereis ferritin, and the cultured bacterial liquid was collected;
[0014] (4) The bacterial liquid was broken by an ultrasonic crusher and dried by a spray dryer into a powder form, and then packed in plastic bags to make a deacetylated Nereis ferritin feed additive.
[0015] Furthermore, the ferritin fermentation medium is LB medium with the pH value adjusted to 6.0, and then 20 mg / L ferric citrate is added.
[0016] Second, the present invention provides a deacetylated Nereis ferritin, and the amino acid sequence of the deacetylated Nereis ferritin is SEQ ID NO.2.
[0017] Thirdly, the present invention provides a gene for expressing deacetylated nereis ferritin, and the nucleotide sequence of the gene is SEQ ID NO.4.
[0018] The beneficial effects of the present invention are as follows: by analyzing the structure of nereis ferritin and mutating its amino acid sites, the adverse effects of potential acetylation sites on the binding of ferritin to iron atoms are eliminated. A recombinant Bacillus subtilis expressing deacetylated nereis ferritin is constructed to express deacetylated nereis ferritin. The cells are lysed by ultrasonic treatment, and the lysate is spray-dried to prepare a feed additive. The amount of iron bound by deacetylated nereis ferritin is increased by 44.62% compared with that of unmutated nereis ferritin. The serum iron content of piglets fed with the deacetylated nereis ferritin feed additive is 2.51 μg / mL, and the health rate is 98%, both of which are higher than those of the glycine iron and unmutated nereis ferritin feed additive groups. The feed conversion ratio is 1.70:1, which is lower than that of the glycine iron and unmutated nereis ferritin feed additive groups. The deacetylated nereis ferritin feed additive has a good iron supplement effect on piglets, can improve the health status of piglets, promote the growth of piglets, and effectively increase the breeding efficiency. Description of the Drawings
[0019] Figure 1 : Detection chart of the iron-binding content of nereis ferritin.
[0020] Figure 2 : Detection chart of the serum iron content of piglets. Detailed Embodiments
[0021] Example 1: Design of Deacetylated Nereis Ferritin
[0022] The GenBank accession number of the amino acid sequence of nereis ferritin is: AII80418.1, and the amino acid sequence is SEQ ID NO.1. The 68th K and the 108th K are potential acetylation sites. The acetylation site weakens the ability of ferritin to bind iron atoms. To eliminate the acetylation site, the 68th K and the 108th K are mutated to H, and the amino acid sequence of deacetylated nereis ferritin is designed as SEQ ID NO.2.
[0023] Example 2: Construction of Recombinant Bacillus subtilis Expressing Nereis Ferritin
[0024] 1.1 Gene Synthesis
[0025] (1) According to the amino acid sequence SEQ ID NO.1 of nereis ferritin, the DNA sequence SEQ ID NO.3 of nereis ferritin is synthesized, and BamHI and EcoRV restriction enzyme sites are added to the upstream and downstream respectively. It is entrusted to Sangon Biotech (Shanghai) Co., Ltd. for synthesis and cloned onto the pUC57 plasmid to obtain the pUC57-Ferritin plasmid.
[0026] (2) Synthesize the DNA sequence of deacetylated nereis ferritin SEQ ID NO.4 according to the amino acid sequence of SEQ ID NO.2. Add BamHI and EcoRV restriction enzyme sites to the upstream and downstream respectively, entrust Sangon Biotech (Shanghai) Co., Ltd. to synthesize, and clone it onto the pUC57 plasmid to obtain the pUC57-Ferritin-Mut plasmid.
[0027] 1.2 Construction of recombinant expression vectors
[0028] (1) Use the restriction enzymes BamHI and EcoRV to perform double digestion on the pUC57-Ferriti and pUC57-Ferritin-Mut plasmids, and recover the Ferriti and Ferritin-Mut gene fragments respectively;
[0029] (2) Use the restriction enzymes BamHI and EcoRV to perform double digestion on the expression vector p7257, and recover the gene fragment;
[0030] (3) Ligate the Ferriti and p7257 nucleic acid fragments with T4 DNA ligase, and ligate the Ferritin-Mut and p7257 nucleic acid fragments with T4 DNA ligase. Transform them into Escherichia coli E.coli DH5α competent cells respectively, extract the plasmids, and identify them by double digestion with BamHI and EcoRV to obtain the recombinant expression plasmids p7257-Ferriti and p7257-Ferritin-Mut.
[0031] 1.3 Construction of recombinant Bacillus subtilis strains expressing nereis ferritin and deacetylated nereis ferritin
[0032] (1) After the recombinant plasmid p7257-Ferriti is identified as correct by enzyme digestion and sequencing, transform it into Bacillus subtilis WB800 competent cells, spread it on a 40 μg / mL chloramphenicol plate, pick a single colony into 5 mL of LB culture medium containing 40 μg / mL chloramphenicol, and culture it at 37 °C and 250 r / min on a shaker for 12 h as the seed solution, named WB800-Ferriti.
[0033] (2) After the recombinant plasmid p7257-Ferritin-Mut is identified as correct by enzyme digestion and sequencing, transform it into Bacillus subtilis WB800 competent cells, spread it on a 40 μg / mL chloramphenicol plate, pick a single colony into 5 mL of LB culture medium containing 40 μg / mL chloramphenicol, and culture it at 37 °C and 250 r / min on a shaker for 12 h as the seed solution, named WB800-Ferritin-Mut.
[0034] 1.4 Fermentation expression of nereis ferritin and deacetylated nereis ferritin
[0035] (1) Transfer 0.1 ml of the seed liquid to 10 ml of LB culture medium containing 40 μg / mL chloramphenicol at a ratio of 1% by volume, and culture it in a shaker at 37°C and 250 r / min for 12 h as the primary seed liquid.
[0036] (2) Transfer 10 ml of the primary seed liquid to 1000 ml of LB culture medium containing 40 μg / mL chloramphenicol at a ratio of 1% by volume, and culture it in a shaker at 37°C and 250 r / min for 12 h as the secondary seed liquid.
[0037] (3) Calibrate the pH electrode and dissolved oxygen electrode of the fermenter, and calibrate the flow rate of the peristaltic pump.
[0038] (4) Prepare 70 L of LB medium with a pH value of 6.0, containing 20 mg / L ferric citrate, add it to a 100 L fermenter, and sterilize the medium, fermenter and pipeline at 121°C for 30 min.
[0039] (5) When the culture medium in the fermenter cools down to 37°C, add 700 ml of the secondary seed liquid to the fermenter and start fermenter culture. The fermenter parameters are set as follows: stirring speed 500 - 800 r / min, tank pressure 9 psi, temperature 37°C, and the set DO value (dissolved oxygen) is above 20%.
[0040] (6) Ferment and culture for 16 h, take samples of the bacterial liquid for detection of the iron-binding content, ultrasonically disrupt the bacterial liquid, dry it with a spray dryer to form a powder, and pack it in plastic bags to make the non-mutated nereis ferritin feed additive and deacetylated nereis ferritin feed additive.
[0041] 1.5 Detection of iron-binding content of non-mutated nereis ferritin and deacetylated nereis ferritin
[0042] Centrifuge 10 ml of the sampled fermented bacterial liquid at 5000 rmp for 10 minutes to collect the bacterial cell precipitate. Resuspend the bacterial cell precipitate with 50 ml of ultrapure water, centrifuge at 5000 rmp for 10 minutes, collect the bacterial cell precipitate, and repeat the washing of the bacterial cells 3 times. Resuspend the bacterial cell precipitate with 10 ml of ultrapure water and ultrasonically disrupt it for 10 minutes. Centrifuge at 12000 rmp for 10 minutes, take the supernatant and detect the iron content using the iron release detection method for ferritin.
[0043] (1) Sample pretreatment: Dilute the ferritin sample with PBS and use PBS as the blank control.
[0044] (2) Iron release: Take 100 μL of the sample + 100 μL of 0.5 mol / L HCl, mix well and incubate at 37°C for 30 minutes.
[0045] (3) Reduction reaction: Add 20 μL of 10% ascorbic acid and let it stand at room temperature for 10 minutes to reduce Fe 3+ to Fe 2+ .
[0046] (4) Color reaction: Add 50 μL of 5 mmol / L phenanthroline solution, mix well and react for 10 minutes in the dark.
[0047] (5) Measure absorbance: Measure the absorbance (OD value) at a wavelength of 562 nm.
[0048] (6) Plot the standard curve: Synchronously operate with the FeSO4 solution to make a standard curve (0 - 100 μmol / L).
[0049] (7) Calculate iron content: Iron content (μg Fe / mg sample) = measured iron concentration (μmol / L) × sample dilution factor × 55.85 / ferritin concentration (mg / L)
[0050] From Figure 1 it can be seen that the iron content in the recombinant Bacillus subtilis lysate expressing unmutated nereis ferritin is 30.97 μg Fe / mg protein, and the iron content in the recombinant Bacillus subtilis lysate expressing deacetylated nereis ferritin is 44.79 μg Fe / mg protein. The amount of iron bound by deacetylated nereis ferritin is 44.62% higher than that of unmutated nereis ferritin.
[0051] Example 3: Effect of deacetylated nereis ferritin feed additive on the growth performance of piglets
[0052] Select 300 28-day-old weaned Landrace piglets with a body weight of 8 ± 0.2 kg, randomly divide them into 3 groups with 100 piglets per group, raise them in isolation, and feed the piglets with the basic feed at 8:00 and 16:00 every day, allowing free access to food and water. The first group is the control group, adding 100 mg / kg of ferrous glycinate to the basic feed; the second group adds 100 mg / kg of unmutated nereis ferritin feed additive to the basic feed; the third group adds 100 mg / kg of deacetylated nereis ferritin feed additive to the basic feed. The test period is 30 days. During this period, observe the feeding and mental state of the test pigs every day, and calculate the feed-to-gain ratio (geometric mean) of each group of pigs at the end of the experiment. On the 7th day of the test, randomly select 10 piglets from each group to collect venous blood, separate the serum, and detect the iron content in the serum.
[0053] From Table 1 and Figure 2It can be seen that the serum iron content of piglets in the control group with glycine iron added to the basal diet was low, at 1.12 μg / mL, the health rate was only 91%, and the feed-to-meat ratio was the highest, at 1.84:1; the serum iron content of piglets in the group with the unmutated nereis ferritin feed additive added to the basal diet was 2.05 μg / mL, the health rate was 94%, and the feed-to-meat ratio was 1.77:1. The serum iron content of piglets in the group with the deacetylated nereis ferritin feed additive added to the basal diet was 2.51 μg / mL, the health rate was 98%, both of which were the highest levels among the three groups, and the feed-to-meat ratio was the lowest level among the three groups, 1.70:1. It can be seen that the deacetylated nereis ferritin feed additive has a good effect on iron supplementation in piglets, can improve the health status of piglets, promote the growth of piglets, and effectively increase the breeding efficiency.
[0054] Table 1 Effects of different iron supplementation feed additives on the growth performance of piglets
[0055] Grouping Serum iron content (μg / mL) Health rate Feed conversion ratio Ferrous glycinate 1.12 91％ 1.84:1 Unaltered nereis iron 2.05 94％ 1.77:1 Deacetylated nereis iron 2.51 98％ 1.70:1
Claims
1. Application of ferritin in feed additive, characterized in that, The feed additive is a feed additive made of deacetylated nereis ferritin expressed by recombinant Bacillus subtilis, and the amino acid sequence of the deacetylated nereis ferritin is SEQ ID NO.
2.
2. The application according to claim 1, characterized in that, The preparation method of the feed additive is as follows: (1) The recombinant Bacillus subtilis expressing deacetylated nereis ferritin was inoculated into LB culture medium containing 40 μg / mL chloramphenicol at a ratio of 1% by volume, and cultured in a shaker at 37 °C and 250 r / min for 12 h to prepare a primary seed liquid; (2) According to the fermentation volume, the primary seed liquid was transferred to prepare a secondary seed liquid and multiple-stage seed liquids; (3) The multiple-stage seed liquids were inoculated into the ferritin fermentation medium at a ratio of 1% by volume, and fermented and cultured in a fermenter for 16 h to express deacetylated nereis ferritin, and the cultured bacterial liquid was collected; (4) The bacterial liquid was broken by an ultrasonic crusher, dried by a spray dryer into a powder, and packaged in plastic bags to make a deacetylated nereis ferritin feed additive.
3. The ferritin fermentation medium according to claim 2, wherein, The ferritin fermentation medium is LB medium with the pH value adjusted to 6.0, and then 20 mg / L ferric citrate is added.
4. A deacetylated nereis ferritin, characterized in that, The amino acid sequence of the deacetylated nereis ferritin is SEQ ID NO.
2.
5. A gene for expressing deacetylated nereis ferritin, characterized in that, The nucleotide sequence of the gene is SEQ ID NO.4.