Pellet feed for improving animal immunity and preparation method thereof

Abstract

The active oligopeptide TDGLTSLD and the astragalus polysaccharide which can improve the resistance or the immunity or the growth capacity of animals are separated from the astragalus seedlings serving as a natural source, the feed containing the oligopeptide and the polysaccharide can effectively improve the immunity of the animals, the growth capacity such as the feed conversion ratio of the animals can be improved, and the feed has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to a pelleted feed for improving animal immunity and its preparation method. Background Technology

[0002] Against the backdrop of rapid development in intensive and large-scale animal husbandry, animals often face multiple challenges, including high stocking densities, environmental stress, and pathogenic microbial invasion, leading to immense pressure on their immune systems, decreased productivity, and frequent disease outbreaks. For a long time, antibiotics have played a crucial role as feed additives in disease prevention and growth promotion; however, their overuse has resulted in increasingly serious problems such as bacterial resistance, drug residues, and environmental pollution, becoming a major concern in global public health. Therefore, developing safe, efficient, and residue-free natural immune enhancers to replace or reduce antibiotic use is a core issue that urgently needs to be addressed by the animal nutrition and feed industry.

[0003] Traditional Chinese medicine (TCM) herbs, due to their natural properties, multi-target effects, and low residues, are considered a potential preferred alternative to antibiotics. Astragalus membranaceus (Huangqi), a traditional Qi-tonifying herb, has had its immune-enhancing effects confirmed by numerous modern pharmacological studies. However, in conventional applications, its dried roots are primarily used, resulting in limited resources and high costs. The astragalus stalks (the above-ground stems and leaves) are often disposed of as agricultural waste after the medicinal harvest, leading to significant resource waste. Research indicates that astragalus stalks and roots contain similar types of active ingredients, such as polysaccharides, flavonoids, saponins, and various amino acids, suggesting their potential for development into immune enhancers.

[0004] Currently, although there are reports of using Astragalus membranaceus or its extracts in animal feed, the following technical limitations exist: First, directly adding whole Astragalus membranaceus or ordinary water extracts results in unstable content of active ingredients, low bioavailability, and potential impact on palatability. Second, research on the high-value development of waste Astragalus membranaceus seedlings, focusing on their protein resources and preparing specific immune-enhancing peptides using modern enzymatic hydrolysis technology, is still insufficient. Third, how to stably and uniformly integrate such liquid or hygroscopic peptide extracts into pelleted feed that is resistant to storage, easy to transport and feed, and maximize the protection of its bioactivity during this processing is a key technical bottleneck for its industrial application.

[0005] Therefore, there is an urgent need in this field for an innovative technological solution that can efficiently transform the waste resource of Astragalus membranaceus seedlings, extract peptides with a clear function of enhancing animal immunity through optimized enzymatic hydrolysis, and further design a scientific method for preparing pelleted feed. Ultimately, this will produce a functional feed product that effectively improves animal health and conforms to the concept of antibiotic-free farming. This will not only reduce feed costs and realize the recycling of agricultural by-products, but also have significant practical implications for promoting the sustainable development of animal husbandry. Summary of the Invention

[0006] To address the lack of technical expertise in existing animal feed preparation methods and their poor nutritional effects, this paper provides a pelleted feed for improving animal immunity and its preparation method.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: The first aspect of this invention provides a method for preparing Astragalus membranaceus oligopeptides and polysaccharides, the preparation method comprising the following steps: 1) Dry the Astragalus membranaceus seedlings, pulverize them to 80-120 mesh and sieve them, add distilled water, stir well, and then sonicate for 1-3 minutes; 2) Heat the ultrasonically treated Astragalus membranaceus seedling aqueous solution in step 1) to 65~100℃, stir and react for 30~60min, then filter and collect the filter cake and filtrate. Add ethanol to the filtrate, let it stand, and Astragalus membranaceus seedling polysaccharide will precipitate. 3) Add the filter cake collected in step 2) to distilled water, stir, adjust the pH of the solution to 8-10 with sodium hydroxide, add alkaline protease to hydrolyze for 8-12 hours, boil for 3-10 minutes to terminate the enzymatic hydrolysis reaction, filter, and then pass through 10 kDa and 3 kDa ultrafiltration membranes in sequence for ultrafiltration. Collect the filtrate with a molecular weight less than 3 kDa, and further separate and purify it using a Sephadex G-25 gel column to obtain P1, P2, and P3.

[0008] 4) The purified P1 fraction was further separated and purified by RP-HPLC using a C18 column. The RP-HPLC separation conditions were: isocratic elution; mobile phase A was deionized water containing 0.05% TFA; mobile phase B was acetonitrile containing 0.05% TFA; flow rate was 1 mL / min; injection volume was 10 μL; sample concentration was 10 mg / mL; column temperature was 20℃; and the elution peak was detected at 214 nm.

[0009] The RP-HPLC separation chromatogram showed four absorption peaks collected during the elution process, such as... Figure 1 As shown, they are sequentially labeled F1, F2, F3, and F4, and each component is freeze-dried and stored.

[0010] The amino acid sequence of component F2 was analyzed by LC-MS / MS, and the astragalus oligopeptide was identified, with the amino acid sequence SEQ ID NO 1: TDGLTSLD. Figure 2 This is its secondary mass spectrum.

[0011] In a preferred embodiment, the mass-to-volume ratio of Astragalus membranaceus seedlings to distilled water in step 1) is 1:8~25, with units of g / mL.

[0012] In a preferred embodiment, the ultrasonic frequency of the ultrasonic treatment in step 1) is 1000~1800W, and the sieving is through a 100-mesh sieve.

[0013] In a preferred embodiment, step 2) involves heating to 80°C and stirring the reaction for 45 minutes.

[0014] In a preferred embodiment, in step 3), the amount of alkaline protease added is 4000~6000U per gram of filter cake, and the enzymatic hydrolysis conditions are enzymatic hydrolysis in a 50°C shaking water bath for 10 hours, with the pH value of the enzyme reaction controlled at 8.0.

[0015] In a preferred embodiment, in step 3), alkaline protease is hydrolyzed for 10 hours and then boiled for 5 minutes.

[0016] A second aspect of the present invention provides an astragalus oligopeptide, wherein the astragalus oligopeptide is prepared by the above-described preparation method and its amino acid sequence is as follows.

[0017] Preferably, the polypeptide is prepared by recombinant expression and chemical synthesis.

[0018] In a preferred embodiment of the present invention, a polynucleotide is provided that encodes the astragalus oligopeptide.

[0019] In a preferred embodiment, the present invention provides an expression vector comprising a polynucleotide encoding an astragalus oligopeptide.

[0020] In a preferred embodiment, the present invention provides a cell comprising a polynucleotide or expression vector encoding astragalus oligopeptide.

[0021] A third aspect of the invention provides pelleted feed to enhance animal immunity, comprising Astragalus membranaceus oligopeptides and polysaccharides, as well as concentrated feed.

[0022] The oligopeptides and polysaccharides were prepared by the above method.

[0023] The composition of the concentrated feed is as follows: 23-26 parts corn, 5-6 parts wheat bran, 6-10 parts soybean meal, 5-6 parts hemp meal, 0.5-1 part calcium stone powder, and 0.5-1 part salt.

[0024] The present invention also provides the application of the above-mentioned pelleted feed in the preparation of animal immune-enhancing feed.

[0025] Preferably, the enhanced immunity is specifically targeted at ovine pleuropneumonia.

[0026] Compared with the prior art, the present invention achieves the following beneficial technical effects: This invention uses Astragalus membranaceus vines as a natural feed source, from which oligopeptides and polysaccharides that can improve animal immunity or growth are isolated. The pelleted feed includes Astragalus membranaceus vine oligopeptides and polysaccharides, as well as concentrated feed. This feed can effectively improve animal immunity and growth, and has broad application prospects. Attached Figure Description

[0027] Figure 1 Ultra-high performance liquid chromatography (UHPLC) separation chromatogram of component P1.

[0028] Figure 2 Secondary mass spectrum of Astragalus oligopeptides.

[0029] Figure 3 The effects of the control group and the pelleted feed group on increasing animal immunoglobulin IgA and IgM. * indicates P < 0.05.

[0030] Figure 4 The effects of the control group and the pelleted feed group on increasing animal immunoglobulin IgG. * indicates P < 0.05.

[0031] Figure 5 The effects of the control group and the pelleted feed group on improving daily weight gain and feed conversion ratio in animals. * indicates P < 0.05. Detailed Implementation

[0032] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0033] Unless otherwise specified, all experimental methods used in the examples are conventional methods. The alkaline protease used was Alcalase® conc BG (enzyme activity 12.5 AU-A / g).

[0034] Example 1: Preparation of Astragalus peptides and polysaccharides The preparation method includes the following steps: 1) After drying the Astragalus membranaceus seedlings, grind them into powder using a grinder and pass them through a 100-mesh sieve. Weigh 300g of Astragalus membranaceus seedling powder into a container, add distilled water at a ratio of 15 times the weight of the Astragalus membranaceus powder, mix thoroughly, and sonicate for 3 minutes at an ultrasonic frequency of 1500W.

[0035] 2) Heat the ultrasonically treated Astragalus membranaceus seedling aqueous solution in step 1) to 100℃, stir and react for 45 min, then filter and collect the filter cake and filtrate. Add ethanol to the filtrate, let it stand, and Astragalus membranaceus seedling polysaccharide will precipitate. 3) Add the filter cake collected in step 2) to distilled water, stir, adjust the pH of the solution to 8 with sodium hydroxide, add 6000 U / g alkaline protease to hydrolyze for 10 h, boil for 8 min to terminate the enzymatic hydrolysis reaction, filter, and after the enzymatic hydrolysate cools, adjust the pH to 7.0 with 1M HCl solution, centrifuge at 8000 rpm for 10 min, and collect the supernatant; 4) The supernatant obtained in step 3) is ultrafiltered sequentially through 10 kDa and 3 kDa ultrafiltration membranes. The filtrate with a molecular weight less than 3 kDa is collected and further separated and purified using a Sephadex G-25 gel column to obtain P1, P2, and P3.

[0036] The components in groups P1-P3 that could significantly improve animal immunity were screened. The results showed that the peptide components in groups P2 and P3 did not have a significant effect on improving animal immunity, but group P1 had a significant effect on improving animal immunity (P<0.05).

[0037] Example 2: Isolation of Astragalus oligopeptides The purified P1 fraction was further separated and purified by RP-HPLC using a C18 column. The RP-HPLC separation conditions were: isocratic elution; mobile phase A was deionized water containing 0.05% TFA; mobile phase B was acetonitrile containing 0.05% TFA; flow rate was 1 mL / min; injection volume was 10 μL; sample concentration was 10 mg / mL; column temperature was 20℃; and the elution peak was detected at 214 nm.

[0038] The RP-HPLC separation chromatogram showed four absorption peaks collected during the elution process, such as... Figure 1 As shown, they are sequentially labeled F1, F2, F3, and F4, and each component is freeze-dried and stored.

[0039] The amino acid sequence of component F2 was analyzed by LC-MS / MS, and the astragalus oligopeptide was identified with the amino acid sequence TDGLTSLD. Its secondary mass spectrum is shown below. Figure 2 As shown.

[0040] Example 3 Characterization of the effect of pelleted feed Natural TDGLTSLD is difficult to obtain, so organic solid-phase synthesis is used to prepare TDGLTSLD in order to reduce production costs.

[0041] Astragalus oligopeptides, astragalus polysaccharides, and concentrated feed are mixed in a specific ratio to achieve an astragalus oligopeptide content of approximately 50 mg / kg and an astragalus polysaccharide content of approximately 500 mg / kg. After adding an appropriate amount of water and stirring thoroughly, pelleted feed is obtained. The concentrated feed consists of: 23-26 parts corn, 5-6 parts wheat bran, 6-10 parts soybean meal, 5-6 parts hemp meal, 0.5-1 part calcium limestone powder, and 0.5-1 part salt.

[0042] Animal experiments were conducted using a single-factor comparative design. Sixty healthy 3-month-old lambs were randomly divided into two groups: a pelleted feed group and a control group. The control group was not supplemented with the astragalus oligopeptides and polysaccharides prepared in Example 1 and was fed normally according to conventional methods. Feeding continued for 4 weeks. During the feeding period, lamb mortality was recorded. After the feeding period, blood was collected from the jugular vein of all lambs, serum was separated, and serum biochemical indicators, hormone levels, and serum immune factors were measured. Serum immunoglobulins IgM, IgG, and IgA were measured using an enzyme-linked immunosorbent assay (ELISA) kit (purchased from Jiangsu Enzyme Immunoassay Co., Ltd.).

[0043] Accurately record the amount of feed and leftover feed for the experimental sheep, and calculate the average daily feed intake per sheep per week and the average daily feed intake for the entire period. Weigh the sheep on an empty stomach at 7:30 am at the beginning and end of the experiment, and calculate the average daily weight gain, feed conversion ratio, mortality rate and other indicators for each sheep.

[0044] like Figure 3-4 As shown, compared with the control group, the serum levels of immunoglobulins IgM, IgG, and IgA were significantly increased in the pelleted feed group (P<0.05). This indicates that pelleted feed containing astragalus oligopeptides and astragalus polysaccharides can significantly improve the immunity of animals.

[0045] As shown in Table 1, the survival rate of sheep fed in the control group was 86.7%, while the survival rate of sheep fed with pelleted feed was 100%.

[0046]

[0047] like Figure 5 As shown, sheep fed with pelleted feed had significantly better daily weight gain and feed conversion ratio than the control group (P<0.05). This indicates that pelleted feed containing astragalus oligopeptides and astragalus polysaccharides can significantly improve the growth capacity of goats, which may be closely related to its ability to enhance the goats' immunity and better resist animal diseases.

[0048] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. An astragalus oligopeptide for improving immunity of animals, characterized in that, The amino acid sequence of the oligopeptide is TDGLTSLD.

2. The oligopeptide according to claim 1, characterized in that, The oligopeptide is a variant of TDGLTSLD obtained by 1 amino acid substitution, addition or deletion, and the variant still has the effect of improving animal immunity.

3. The oligopeptide according to claim 1 or 2, characterized in that, The oligopeptide is prepared by genetic engineering expression or chemical synthesis.

4. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of claim 1 or 2. The polynucleotide of claim 4.

5. An expression vector, characterized by, The polynucleotide of claim 4 or the expression vector of claim 5.

6. A recombinant cell, characterized in that, The oligopeptide of any one of claims 1-3, astragalus polysaccharide and concentrate feed.

7. A granular feed for improving immunity of animals, characterized by, 8. The pellet feed of claim 7, wherein the concentrate feed comprises one or more of corn, bran, soybean meal, sesame meal, calcium stone powder and salt.

9. The feed of claim 7, wherein the concentrate feed comprises 23-26 parts of corn, 5-6 parts of bran, 6-10 parts of soybean meal, 5-6 parts of sesame meal, 0.5-1 part of calcium stone powder and an appropriate amount of salt.

10. Use of the oligopeptide of claims 1-3 and / or the pellet feed of any one of claims 7-9 in the preparation of a feed for improving animal immunity or growth ability. ​

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