Animal feed additive with anti-inflammatory and antioxidant functions

Abstract

The present invention provides an animal feed additive with anti-inflammatory and antioxidant functions, namely, a traditional Chinese medicine-type feed additive with anti-biotic efficacy. The preparation method comprises the following steps: mixing a dried olive pomace powder sample with a subcritical extractant, performing subcritical extraction, and obtaining an extraction system; removing the subcritical extractant from the extraction system to obtain a pomace powder system from which pomace oil has been removed; mixing the pomace powder system with ethanol, performing leaching, and subjecting the obtained extract to vacuum distillation to obtain an extract; mixing the extract system with petroleum ether, allowing the petroleum ether phase to stand for stratification, and filtering the obtained petroleum ether phase to obtain an extract. The maslinic acid and oleanolic acid extracted by the present invention have anti-inflammatory and antioxidant effects, and animal experiments have confirmed that they can enhance intestinal health in a short period of time. The addition of a polypeptide improves the side effects of the maslinic acid and oleanolic acid caused by their long-term use. The present invention can be promoted and used as a new animal feed additive.

Description

Technical Field

[0001] The present invention relates to the technical field of biological products, and in particular to an animal feed additive with anti-inflammatory and antioxidant functions. Background Art

[0002] Antibiotics are substances obtained through microbial culture or chemical synthesis that specifically kill microorganisms. In 1928, British scientist Alexander Fleming discovered penicillin, ushering in the antibiotic era. Subsequently, in 1946, American scientists Moore et al. first discovered that antibiotics in broiler feed promoted growth, ushering in a new era of antibiotic growth promotion in the global livestock industry. Since then, antibiotics have been widely used as feed additives in a variety of animal species, including pigs, poultry, cattle, sheep, and aquatic animals. Adding subtherapeutic doses of antibiotics to feed can promote animal growth, improve feed conversion rates, and reduce morbidity and mortality in livestock and poultry, playing a significant role in promoting the development of the livestock industry. However, the long-term use of antibiotics has also led to a series of problems, including bacterial resistance, environmental pollution, drug residues in animal-derived foods, and public health and safety risks.

[0003] Banning the use of feed antibiotics in animal production has become a global consensus and an irreversible trend. The European Union completely banned the use of antibiotics as feed additives in 2006. my country's Ministry of Agriculture and Rural Affairs also issued Announcement No. 194 in September 2019, announcing that, effective January 1, 2020, all growth-promoting feed additives, with the exception of traditional Chinese medicines, would be discontinued. This marked the official entry of my country's livestock industry into a new era of antibiotic-free feed.

[0004] As the global ban on feed antibiotics deepens, the search for safe and effective alternatives has become a global research hotspot, with the market urgently demanding safe and effective alternatives to feed antibiotics. Forage plants and their additives are considered an ideal alternative to feed antibiotics in animal production due to their multiple advantages, including abundant resources, natural origins, comprehensive functions, high safety, low toxicity and side effects, resistance to drug resistance, and zero (or low) residues. Summary of the Invention

[0005] The object of the present invention is to provide an animal feed additive with anti-inflammatory and antioxidant functions, that is, a traditional Chinese medicine-type feed additive with anti-biological efficacy.

[0006] The animal feed additive provided by the present invention has a preparation method comprising the following steps:

[0007] The dried olive pomace powder sample and a subcritical extractant are mixed and subjected to subcritical extraction to obtain an extraction system;

[0008] removing the subcritical extractant from the extraction system to obtain a pomace powder system after the pomace oil has been removed;

[0009] The pomace powder system is mixed with ethanol for extraction, and the obtained extract is subjected to reduced pressure distillation to obtain an extract;

[0010] The extract system is mixed with petroleum ether, stirred, allowed to stand for stratification, and the petroleum ether phase is collected. The obtained petroleum ether phase is filtered to obtain a filtrate.

[0011] Preferably, the olive pomace powder sample is pre-treated before being mixed with the subcritical extractant; the pre-treatment comprises the following steps: drying and crushing the olive pomace; the moisture content of the dried material is ≤5%; and the particle size of the crushed material is 20 to 40 meshes.

[0012] Preferably, the subcritical extractant includes one or more of butane, propane, isobutane or dimethyl ether.

[0013] Preferably, the subcritical extraction pressure is 0.45-0.8 MPa, the temperature is 35-45° C., and the time is 40-45 min.

[0014] Preferably, the method of removing the subcritical extractant from the extraction system is vacuum distillation; the temperature of the vacuum distillation is 45-50° C., the vacuum degree is ≤0.5 MPa, and the time is 25-30 min.

[0015] Preferably, the volume ratio of the extract system to petroleum ether is 1:1 to 1:20.

[0016] Preferably, the purification is carried out under centrifugal conditions, with a centrifugal speed of 6000 to 20000 r / min and a time of 5 to 20 min.

[0017] Furthermore, the animal feed additive also contains a polypeptide for preventing and treating enteritis;

[0018] The polypeptide is extracted from Crabapple Tree;

[0019] Furthermore, the sequence of the polypeptide is DKQGEP (SEQ ID NO: 1).

[0020] The maslinic acid and oleanolic acid extracted by the present invention have anti-inflammatory and antioxidant effects. Animal experiments have confirmed that they can enhance intestinal health in a short period of time. After the addition of polypeptides, the side effects caused by the long-term use of maslinic acid and oleanolic acid are improved, and the present invention can be promoted and used as a new animal feed additive. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Scavenging rate of DPPH free radicals by maslinic acid and oleanolic acid;

[0022] Figure 2 :Maslinic acid and oleanolic acid to ABTS + Free radical scavenging ability;

[0023] Figure 3 : Effects of maslinic acid and oleanolic acid on NO release;

[0024] Figure 4 : Reversed-phase high performance liquid chromatography chromatogram;

[0025] Figure 5 : MS / MS spectrum of the selected peptide;

[0026] Figure 6 :H&E pathological observation of mouse colon;

[0027] Figure 7 : Levels of IL-4, IL-1β, TNF-α, and IFN-γ in the serum of mice in each group;

[0028] Figure 8 :The effects of maslinic acid and oleanolic acid on oxidative indices in colitis mice. DETAILED DESCRIPTION

[0029] Research has found that natural organic compounds such as maslinic acid, oleanolic acid, and some pentacyclic triterpenic acids, derived from food processing byproducts used as feed, such as olive pomace, exhibit promising anti-inflammatory, antioxidant, and gut health-enhancing properties. Therefore, developing and preparing processing byproducts containing functional substances such as anti-inflammatory, antioxidant, and gut health-enhancing substances using olive pomace as a raw material could be used as animal feed additives.

[0030] The present invention is described in detail below with reference to specific embodiments and accompanying drawings.

[0031] Example 1: Extraction of Maslinic Acid and Oleanolic Acid

[0032] Olive pomace is taken and dried to a moisture content of no more than 5%, crushed in a crusher, sieved through 40 mesh and 20 mesh in sequence, and olive pomace fragments with a particle size of 20-40 mesh are taken. A subcritical extractant is used at 0.45-0.8 MPa, the temperature is maintained at 35-45 DEG C, and extraction is performed for 40-45 minutes. The subcritical extractant is removed to obtain pomace powder, the pomace powder is mixed with ethanol, extracted, and the extract is subjected to reduced pressure distillation to obtain an extract, the obtained system is mixed and stirred with petroleum ether in a volume ratio of 1:1-1:20, and after standing and stratification, the petroleum ether phase is taken, the petroleum ether phase is filtered, and the filtrate obtained by centrifugation at a rotation speed of 6000-20000 r / min is maslinic acid and oleanolic acid.

[0033] Example 2: Determination of the anti-inflammatory and antioxidant effects of maslinic acid and oleanolic acid

[0034] 1. Determination of DPPH free radical scavenging rate

[0035] Measure 0.1mL maslinic acid and oleanolic acid sample solution, add in 10mL volumetric flask, be diluted to near 1mL scale mark with distilled water, then add 3.5mL concentration and be the DPPH ethanolic solution of 0.2mmol / L, continue to be settled to the volumetric flask scale mark with ethanol, guarantee that solution mixes.Mixed solution is placed in lucifuge and reacts 30min.Afterwards, under 517nm wavelength, measure the absorbance of this solution.Simultaneously, prepare respectively the DPPH solution that does not contain sample and the sample solution that does not contain DPPH, measure absorbance separately under same wavelength, with as blank control and sample background correction.In addition, same concentration vitamin C (VC) solution is set as positive control, carries out same treatment and measures its absorbance.Finally, according to gained data calculation sample to the scavenging rate of DPPH free radical.

[0036]

[0037] Where A1 is the absorbance value of the sample solution after reaction; A2 is the absorbance value of the solution without adding DPPH; A0 is the absorbance value of the solution without adding sample.

[0038] Figure 1 It can be seen that the scavenging effect of maslinic acid and oleanolic acid solutions on DPPH free radicals is close to that of VC, indicating that they have good antioxidant properties.

[0039] 2. ABTS + Determination of free radical scavenging ability

[0040] Measure 0.5 mL of sample solution into two 10 mL volumetric flasks and add 2.5 mL of ABTS to each + The working solution was then accurately diluted to the mark with distilled water to ensure uniform mixing. After standing for 10 minutes in the dark at room temperature, the absorbance of each solution was measured at a wavelength of 734 nm using a spectrophotometer. At the same time, two control groups were set up: one group contained only ABTS + The working solution (without sample) was used to measure the background absorbance, and the other group was the sample solution (without ABTS) + The sample's absorbance was determined using a VC solution (the working solution) at the same wavelength. Finally, a VC solution with the same concentration level was used as a positive control, and the free radical scavenging rate of the sample was calculated based on the data obtained.

[0041]

[0042] Where A1 is the absorbance value of the sample solution after reaction; A2 is the absorbance value without adding ABTS + A0 is the absorbance value of the working solution; A0 is the absorbance value of the solution without adding sample.

[0043] Figure 2 It can be seen that hawthorn acid and oleanolic acid have a significant effect on ABTS + It has a good scavenging effect, with a scavenging rate of over 70%.

[0044] 3. NO release detection

[0045] The experimental design included three groups: a blank control group (containing only DMEM culture medium, no cells), an LPS model group (LPS concentration of 1.0 μg / mL), and an experimental group (LPS concentration of 1.0 μg / mL combined with 50 μg / mL each of maslinic acid and oleanolic acid). Each sample was added to the culture wells at a volume of 100 μL and then incubated in a 37°C, CO2-containing incubator for 24 hours. A nitrite (NaNO2) standard was diluted in culture medium, and 50 μL of cell culture supernatant was collected from each well. After standing at room temperature for 5 minutes, equal volumes of Griess reagent I (50 μL) and Griess reagent II (50 μL) were added sequentially. The optical density (OD) at a wavelength of 540 nm was measured and combined with a NaNO2 standard curve to accurately calculate the amount of NO generated in each group.

[0046] As a key regulatory factor, NO plays an important role in various physiological functions and inflammatory processes, especially in the LPS-induced RAW264.7 macrophage acute inflammation model. Figure 3 The effects of hawthorn acid and oleanolic acid on NO secretion in this model: after LPS (1.0 μg / mL) treatment of cells for 24 hours, the NO level in the model group was significantly increased compared with the control group (P < 0.01), verifying the effectiveness of the inflammation model; and after the addition of hawthorn acid and oleanolic acid, the NO content was significantly decreased compared with the model group (P < 0.05), indicating that hawthorn acid and oleanolic acid have good anti-inflammatory effects.

[0047] Maslinic acid and oleanolic acid were used as feed additives in mouse breeding. The results showed that after two months of continuous feeding of the mice with the feed additives containing maslinic acid and oleanolic acid, enteritis was found in the fed group of mice. It is speculated that continuous feeding of the feed additives containing maslinic acid and oleanolic acid will cause enteritis in the fed animals. Therefore, feed prepared with maslinic acid and oleanolic acid as feed additives is more suitable for short-term treatment.

[0048] Example 3: Extraction of polypeptides with the effect of preventing and treating enteritis

[0049] The tissue of the Chinese hawthorn was crushed and ground into a homogenate, ddH2O was added and mixed, and then 3000U / mg of trypsin was added, the pH was adjusted to 7.5, the temperature was maintained at 50℃, and hydrolysis was carried out for 5h. After the end, the enzymatic hydrolyzate was placed in a 100℃ water bath to inactivate the enzyme for 15min, and then placed in a centrifuge and centrifuged at 10000r / min for 10min. The supernatant was separated and collected, concentrated and dried to obtain the Chinese hawthorn polypeptide dry powder.

[0050] The enzymatically hydrolyzed Begonia polypeptide was initially purified using an anion exchanger DEAE-52, 20 mM sodium acetate was selected as the buffer, and Tris-HCl buffer containing 0-2 mol / L NaCl was used as the eluent. The hydrolyzate was then purified using a reversed-phase column 300SB-C18 (4.6×250 mm, 5 μm, Agilent). Acetonitrile (ACN) containing 0.1% formic acid (FA) and ACN were used as eluents x and y, respectively. The flow rate was set at 0.5 mL / min, and the elution was performed in the following order: 1-5 min, 100% x; 5-40 min, 5-90% y (linear gradient); 40-50 min, 100% x. The detector wavelength was 280 nm. Figure 4 , using mass spectrometry to determine the peptide sequence ( Figure 5 ), the obtained polypeptide sequences were input into the BIOPEP-UWM bioactive peptide database for activity prediction, and the most active peptides were screened out, whose sequences were: DHTLP (polypeptide a), IDFLEH (polypeptide b) and DKQGEP (polypeptide c).

[0051] The three most active peptide sequences screened were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. RAW264.7 cells were cultured in complete medium containing 10% fetal bovine serum at 37°C and 5% CO2, and passaged every 24 hours. Cells in the logarithmic growth phase were taken and cultured with different peptides for 2 days. 20 μL of MTT solution was added and cultured for another 3 hours. The in vitro anti-inflammatory properties were determined using ELISA.

[0052] As can be seen from the results in Table 1, polypeptide c has the best scavenging effect on TNF-α and IL-1β, indicating that it has the best in vitro anti-inflammatory property. Its sequence is DKQGEP (SEQ ID NO: 1).

[0053] Table 1: In vitro anti-inflammatory activity of three screened Trichosanthes peptides (clearance rate / %)

[0054]

[0055] Forty six-week-old male C57BL / 6 mice were used, with a body weight within the range of (20±3g). One week of adaptive feeding was performed, during which all groups were given normal drinking water and feed. The mice were divided into four groups, with 10 mice in each group, namely the normal group, the model group, the control group and the experimental group. After the adaptive feeding was completed, except for the normal group, the other three groups of mice were allowed to drink 2% maslinic acid and oleanolic acid solution freely. At the same time, the mice in the control group were also gavaged with 100mg / kg of sulfasalazine daily, while the mice in the experimental group were gavaged with maslinic acid and oleanolic acid solution with added polypeptides daily. After 60 days, the mice were fasted, but not watered, for 12 hours. Afterwards, the mice were dissected and blood samples were collected through the orbital vein. During the dissection, blood samples and colon tissues were collected for H&E staining and pathological observation.

[0056] Under the induction of maslinic acid and oleanolic acid, colonic tissue showed obvious damage characteristics, including destruction of intestinal epithelium, loss of goblet cells, strong inflammatory cell infiltration and disappearance of crypts. Figure 6 As shown in the figure, the colon structure of the mice in the normal group remained intact, the crypts were clearly visible, rich in goblet cells, and there were no obvious signs of damage. In contrast, the colon mucosa of the mice in the model group was severely damaged, the crypt epithelial cell morphology was distorted, and there was infiltration of a large number of inflammatory cells, indicating that the intestinal barrier function was severely damaged. Although the colon structure of the control group was relatively intact, some inflammatory cell infiltration and edema of the intestinal wall were still observed. It is worth noting that in the colon tissue sections of the mice in the experimental group, the crypt structure was clearly visible, with almost no edema or inflammatory cell infiltration. The results confirmed that the addition of polypeptide c to maslinic acid and oleanolic acid can effectively reduce the tissue damage of maslinic acid and oleanolic acid to the mouse colon, and its protective effect is more significant than that of SASP.

[0057] Example 4: Alleviating effect of a mixture of polypeptide and hawthorn acid and oleanolic acid on colitis in mice

[0058] Mice were screened using the criteria in Example 3 and randomly divided into four groups: a normal group and a test group (model group, test group 1, and test group 2). The test group was gavaged with 2% DSS water daily, the test group 1 was also gavaged with 100 mg / kg maslinic acid and oleanolic acid daily, the test group 2 was gavaged with a mixture of polypeptide and maslinic acid and oleanolic acid, and the normal group was not treated. After 28 days, the mice were killed by cervical dislocation, and the intestines of the mice were removed to analyze the expression of related genes and cytokines in the colon tissue.

[0059] 1. Determination of inflammatory factor indicators in mice

[0060] Kits were used to detect the levels of interleukin-4 (IL-4), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and mouse interferon γ (IFN-γ) in mouse serum.

[0061] Cytokine levels in mice Figure 7 Compared with the model group, the levels of TNF-α, IL-1β, and IFN-γ in the first and second experimental groups were significantly increased (p<0.05), and the data of the second experimental group were closer to the normal group. IL-4 is an important anti-inflammatory cytokine. Figure 7 It can be seen that compared with the normal group, the IL-4 level in the model group was significantly decreased (p<0.05), while the IL-4 levels in the experimental groups 1 and 2 were significantly increased. The above studies confirm that maslinic acid and oleanolic acid can reduce DSS-induced intestinal inflammation. The anti-inflammatory effect of maslinic acid and oleanolic acid solution with the addition of polypeptide C is better than that of maslinic acid and oleanolic acid alone. After use, the intestinal inflammatory response was significantly reduced, and the anti-inflammatory effect was significantly enhanced, thereby achieving the purpose of alleviating intestinal inflammatory response.

[0062] 2. Determination of oxidative markers in mouse serum

[0063] The myeloperoxidase (MPO) content in mouse serum was determined by enzyme-linked immunosorbent assay (ELISA); the catalase (CAT), glutathione reductase (GR) and malondialdehyde (MDA) content in mouse serum was determined by microassay; the superoxide dismutase (SOD) content in mouse serum was determined by enzyme-linked immunosorbent assay (ELISA).

[0064] In DSS-induced colitis in mice, oxidative stress in colonic tissue is an important indicator. Studies have shown that the occurrence of colitis is often accompanied by increased oxidative stress. Therefore, improving oxidative stress in colonic tissue is a potential measure to alleviate the occurrence of colitis. Figure 8 Figures 2 and 3 show the levels of SOD, CAT, GR, MDA, and MPO in mouse serum. Compared with the normal group, the model group showed significantly decreased CAT (p<0.05), SOD (p<0.05), and GR (p<0.05) levels, while MDA (p<0.05) and MPO (p<0.05) levels were significantly increased. This suggests that DSS induces oxidative stress in mice, causing oxidative damage to their colonic tissue. After intervention with maslinic acid and oleanolic acid, oxidative indices showed an opposite trend compared to the model group. CAT and GR levels were significantly increased (p<0.05), as was SOD, while MDA (p<0.05) and MPO (p<0.05) levels were decreased. The intervention with maslinic acid and oleanolic acid, which were supplemented with polypeptide C, showed a more effective effect, indicating that maslinic acid and oleanolic acid induce oxidative stress in the mouse colon, and that the antioxidant effect is stronger after the addition of polypeptide C.

[0065] The hawthorn acid and oleanolic acid extracted by the present invention have anti-inflammatory and antioxidant effects. They are used as short-term treatments, and the addition of polypeptide C improves enteritis caused by long-term use, enhances intestinal health, and can be applied and promoted as natural feed additives.

Claims

1. A polypeptide having the efficacy of preventing and treating enteritis, characterized in that: The amino acid sequence of the polypeptide is SEQ ID NO:

1.

2. Use of the polypeptide according to claim 1 in the preparation of feed additives.

3. An animal feed additive, characterized in that The animal feed additive is added with maslinic acid and oleanolic acid extracts, and the polypeptide according to claim 1.

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