Active peptide and application thereof as poultry feed additive

Abstract

The invention provides an active peptide and application of the active peptide as a poultry feed additive, namely the active peptide which can be added into poultry feed and has antibacterial and immunity-enhancing effects. The provided active peptide is separated and prepared from the spleen of a chicken, and the amino acid sequence of the active peptide is SEQ ID NO: 1. The active peptide is extracted from the spleen of a chicken and has a good antibacterial effect, and a white feather broiler test proves that the active peptide can improve the immunity of an animal body by promoting immune cell proliferation, regulating cell factor activity and the like, and can be popularized and applied as a poultry feed additive.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of functional active substances, and in particular relates to an active peptide and application thereof as a poultry feed additive. Background Art

[0002] In recent years, with the continuous increase in consumer demand for poultry products, the adjustment of livestock product consumption structure and the rapid development of deep processing industries of livestock products such as pre-prepared meals, poultry farming has become increasingly important in my country's agriculture. According to the National Bureau of Statistics, in the first quarter of 2024, the number of poultry slaughtered nationwide reached nearly 3.711 billion, an increase of 0.1% year-on-year, and the output of poultry meat was 6.1 million tons, an increase of 350,000 tons year-on-year, an increase of 6.1%, the highest in the past five years, and exceeded 6 million tons; the output of poultry eggs was 8.67 million tons, an increase of 120,000 tons year-on-year, an increase of 1.5%, also a record high in the past five years. These data show that my country's poultry farming industry has maintained steady growth in production scale, output and market supply.

[0003] Poultry feed additives are widely used in poultry feeds. These additives play an important role in improving feed utilization efficiency, promoting poultry growth, enhancing immunity and improving product quality. However, their use also has certain disadvantages. For example, improper use may lead to damage to animal health, increased food safety risks and environmental pollution. In particular, the abuse of certain additives such as antibiotics and hormones may have potential toxic effects on poultry, and the residues may also be passed to humans through the food chain, posing a threat to human health.

[0004] Bioactive peptides are small molecule polypeptide compounds with specific biological activities, which are composed of 20 natural amino acids in different compositions and arrangements. These peptide compounds play important physiological functions in organisms, such as promoting gastrointestinal motility, enhancing immunity, regulating metabolism, enhancing memory, and promoting bone development. They are widely present in animals, plants and microorganisms in nature, and can also be extracted, separated, purified or synthesized through modern biotechnology. Due to their unique biological activity and broad application prospects, bioactive peptides have received extensive attention and in-depth research in many fields such as medicine, food, health products, and feed.

[0005] The application of bioactive peptides in poultry additives is quite extensive and has broad prospects. As a safe and efficient feed additive, bioactive peptides are widely used in poultry farming due to their unique nutritional effects, immune enhancement effects and antioxidant properties. It can be used as a nutrient directly to synthesize body protein and chelate and transport minerals to improve the absorption and utilization rate of minerals. At the same time, immune peptides can stimulate the proliferation of lymphocytes in poultry and enhance the phagocytic ability of macrophages, thereby improving the immunity of poultry and reducing the occurrence of diseases. In addition, bioactive peptides also have antioxidant effects, which can inhibit lipid oxidation, extend the shelf life of feed, and improve the growth performance and meat and egg quality of poultry. Therefore, as a poultry additive, bioactive peptides can not only improve the health of poultry and promote growth and development, but also improve product quality and increase breeding efficiency. Summary of the invention

[0006] The purpose of the present invention is to provide an active peptide and its application as a poultry feed additive, that is, an active peptide that can be added to poultry feed and has antibacterial and immunity-enhancing effects.

[0007] The present invention firstly provides an active peptide with antibacterial ability, wherein the active peptide is separated and prepared from the spleen of chicken.

[0008] Furthermore, the amino acid sequence of the active peptide is KKFEDCHLAR (SEQ ID NO: 1).

[0009] The present invention also provides an application of the active peptide, which can be added to poultry feed as an additive;

[0010] Preferably, the amount of active peptide added is 200 mg / kg.

[0011] The present invention extracts an active peptide from the spleen of chicken, which has good antibacterial effect. It has been proved by experiments on white-feathered broilers that it can improve the immune ability of animal bodies by promoting the proliferation of immune cells, regulating the activity of cytokines and the like, and can be promoted and applied as a poultry feed additive. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 :Active peptide reverse phase high performance liquid purification diagram;

[0013] Figure 2 :The content of IgG and IgA in serum of experimental chickens;

[0014] Figure 3 :The content of TNF-α and IL-2 in serum of experimental chickens;

[0015] Figure 4 : Graph showing serum IL-6 and IL-10 levels in experimental chickens. DETAILED DESCRIPTION

[0016] The present invention is described in detail below in conjunction with embodiments and drawings.

[0017] Example 1: Isolation and identification of active peptides

[0018] Healthy white-feathered broilers purchased from a farm in Yantai City were dissected and their spleens were taken out. They were fully crushed and mixed evenly with physiological saline, and then subjected to three repeated freeze-thaw treatments. At 4°C, the mixture was centrifuged at 15,000 r / min for 50 minutes, and the supernatant was taken and the same centrifugation step was repeated. Next, the supernatant was ultrafiltered using a 1000Da ultrafiltration membrane, and the filtrate after ultrafiltration was freeze-dried. The freeze-dried sample was diluted with PBS solution and centrifuged at 15,000 r / min for 15 minutes, then filtered through a 0.22 μm filter membrane and freeze-dried again. Next, the freeze-dried sample was diluted with solution B (containing 2% acetonitrile and 0.065% trifluoroacetic acid), centrifuged at 15,000 r / min for 15 minutes, and then filtered through a 0.22 μm filter membrane again. The filtrate components with antibacterial activity were subjected to reverse HPLC purification analysis, and the active peak ( Figure 1 ). The harvested eluate was subjected to MALDI-TOF-MS analysis, and the results showed that its amino acid sequence was KKFEDCHLAR (SEQ ID NO: 1).

[0019] Example 2: Study on the antibacterial effect of active peptides

[0020] First, the strains of Escherichia coli, Salmonella, Staphylococcus aureus and Edwardsiella tarda were revived, streaked on the surface of the solid culture medium, and cultured overnight at 28°C. Subsequently, a single colony was picked from the culture medium and inoculated into a conical flask containing 25 ml of liquid culture medium, which was placed in a shaker and cultured at 28°C for 12 to 18 hours. After that, the absorbance of the bacterial solution at a wavelength of 600 nanometers was measured, and the concentration of the bacterial solution was adjusted to a range of 0.6 to 0.8 using a liquid culture medium. Next, the bacterial solution was diluted to contain 5×10 5 CFU, and 90 μl was added to each well of a 96-well plate.

[0021] The amino acid sequence KKFEDCHLAR (SEQ ID NO: 1) was sent to a biological company for synthesis and diluted in a liquid culture medium. Take 10 μl of the diluted active peptide and add it to the corresponding wells of the 96-well plate to make the total amount of the reaction system in each well reach 100 μl. After covering the 96-well plate, shake and culture it at 28°C for 24 hours. After that, observe the situation of each well, and use an enzyme reader to measure and record the OD value at a wavelength of 600 nanometers. Among the series of active peptide concentrations obtained by the two-fold dilution method, the minimum concentration that can inhibit bacterial growth is defined as the minimum inhibitory concentration (MIC). At the same time, bacterial solution and liquid culture medium are used as negative and positive controls, respectively, representing 0% and 100% inhibition rates, respectively.

[0022] As shown in Table 1, the screened active peptides have good antibacterial properties against Escherichia coli, Salmonella, Staphylococcus aureus and Edwardsiella tarda, and have better antibacterial effects on Escherichia coli and Salmonella.

[0023] Table 1: Antibacterial effect MIC (μg / mL) of active peptides

[0024]

[0025] Example 3: Animal Experiments of Active Peptides

[0026] 120 healthy white broilers (weight 300g±10g) at 30 days old were purchased from a farm in Yantai City and randomly divided into 4 groups, 10 in each group, and 3 replicates in each group. The control group was fed with a basic diet, and the experimental groups were fed with active peptides added to the diet at low doses (50mg / kg), medium doses (100mg / kg), and high doses (200mg / kg) for 60 days.

[0027] 1. Test chicken serum immune index

[0028] In each group, experimental chickens of moderate size were selected for venous blood sampling, and serum immune indicators such as immunoglobulin G (IgG), immunoglobulin A (IgA), tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), interleukin-6 (IL-6) and interleukin-10 (IL-10) were detected using kits.

[0029] Compared with the control group, the concentrations of IgG and IgA in the serum of white-feathered broiler chickens in all experimental groups were significantly increased (P<0.05)( Figure 2 ), while the concentrations of TNF-α, IL-2 and IL-6 decreased significantly (P<0.05). At the same time, the concentration of IL-10 in serum also increased significantly (P<0.05) ( Figure 3 and Figure 4). This indicates that supplementing the diet with active peptides (KKFEDCHLAR) can significantly increase the levels of immunoglobulins and anti-inflammatory factors in serum, while effectively reducing the levels of pro-inflammatory factors.

[0030] 2. Number of intestinal microorganisms in experimental chickens

[0031] After blood collection, the experimental chickens were killed by bleeding from the neck, and then 1 g of the contents were collected from their cecum and placed in a sterile centrifuge tube. The lactic acid bacteria, Escherichia coli, and Salmonella in the contents were cultured using MRS, EMB, and SS culture media, respectively, and their numbers were calculated.

[0032] Table 2: Microbial content in the intestinal tract of experimental chickens (lgCFU / g)

[0033]

[0034] From the data in Table 2, it can be seen that with the increase in the amount of active peptides added, the number of Escherichia coli in the cecum of white-feathered broiler chickens in each group showed a significant downward trend, and the difference was significant (P < 0.05). Compared with the control group without the addition of active peptides, the number of Salmonella in the cecum of white-feathered broiler chickens in the high-dose group was significantly reduced (P < 0.05), and the number of Lactobacillus in each dose group was significantly increased (P < 0.05), indicating that active peptides can regulate intestinal bacteria and improve the microbial structure in the intestine of broiler chickens.

[0035] 3. Organ index

[0036] After the experimental chickens were killed, the main immune organs (bursa of Fabricius, thymus, spleen) were removed and weighed, and the organ index was calculated.

[0037] Organ index (%) = organ fresh weight (g) / live weight before slaughter (g) × 100

[0038] Table 3: Effects of active peptides on immune organ indexes of white-feathered broiler chickens (%)

[0039]

[0040] The key immune tissues in poultry include the thymus, spleen and bursa of Fabricius, and the relevant indexes of these immune organs can reflect the immune status of poultry to a certain extent. As shown in Table 3, compared with the control group, the thymus and spleen indexes of the experimental chickens in each dose group were significantly increased (P < 0.05), and the bursa index of the experimental chickens in the high dose group was significantly higher than that in other groups (P < 0.05).

[0041] The chicken-derived active peptide provided by the present invention has significant antibacterial ability, can improve the immune ability of animals by promoting the proliferation of immune cells, improving the anti-infection ability of animals, regulating cytokine activity and inhibiting excessive inflammatory reactions, and can be promoted and applied as a poultry feed additive.

Claims

1. An active peptide with antibacterial ability, characterized in that: The active peptide is separated and prepared from chicken spleen.

2. The active peptide according to claim 1, characterized in that The amino acid sequence of the active peptide is SEQ ID NO:

1.

3. Use of the active peptide according to claim 1 in the preparation of antibacterial products.

4. The use according to claim 3, characterized in that The antibacterial product is a product against Escherichia coli, Salmonella, Staphylococcus aureus or Edwardsiella tarda.

5. Use of the active peptide according to claim 1 as a feed additive.

6. The use according to claim 5, characterized in that The application is as a poultry feed additive.

7. A poultry feed, characterized in that: The active peptide according to claim 1 is added to the poultry feed.

8. The poultry feed according to claim 7, characterized in that The added concentration of the active peptide in the poultry feed is 200 mg / kg.

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