Animal feed additive with anti-inflammatory and anti-oxidation functions

Abstract

The invention provides an animal feed additive with anti-inflammatory and anti-oxidation functions, namely a traditional Chinese medicine type feed additive with anti-biological efficacy, and a preparation method of the animal feed additive comprises the following steps: mixing a dried olive pomace powder sample with a subcritical extraction agent, and carrying out subcritical extraction to obtain an extraction system; removing the subcritical extraction agent of the extraction system to obtain a pomace powder system without pomace oil; mixing the pomace powder system with ethanol, extracting, and carrying out reduced pressure distillation on the obtained extracting solution to obtain an extract; and mixing the extract system with petroleum ether, standing for layering, taking a petroleum ether phase, and filtering the obtained petroleum ether phase. The extracted maslinic acid and oleanolic acid have anti-inflammatory and anti-oxidation effects, animal experiments prove that the maslinic acid and oleanolic acid can enhance intestinal health in a short time, after the polypeptide is added, side effects caused by long-term use of the maslinic acid and oleanolic acid are improved, and the maslinic acid and oleanolic acid can be popularized and used as novel animal feed additives.

Description

Technical Field

[0001] The 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 that are obtained through microbial culture or chemical synthesis and have the ability to specifically kill microorganisms. In 1928, British scientist Alexander Fleming first discovered penicillin, which marked the beginning of the antibiotic era for mankind. Subsequently, in 1946, American scientist Moore and others first discovered that antibiotics have a growth-promoting effect in broiler feed. This discovery led the global animal husbandry industry into a new era of antibiotic growth promotion. Since then, antibiotics have been widely used as feed additives in the breeding of various animals such as pigs, poultry, cattle, sheep, and aquatic animals. Adding antibiotics below the therapeutic dose to feed can promote animal growth, improve feed conversion rate, and reduce the morbidity and mortality of livestock and poultry, which has played an important role in promoting the development of the breeding industry. However, the long-term addition and use of antibiotics has also brought about a series of problems such as bacterial resistance, environmental pollution, drug residues in animal-derived foods, and public health and safety hazards.

[0003] The ban on the use of feed antibiotics in animal production has become a consensus and irreversible trend among countries around the world. The European Union has 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 from January 1, 2020, all types of drug feed additives with growth-promoting functions, except traditional Chinese medicine, will be discontinued, marking that my country's animal husbandry industry has officially entered a new era of antibiotic-free feed.

[0004] As the global ban on feed antibiotics is advancing, finding safe and effective alternatives has become a global research hotspot, and the market has an urgent need for safe and efficient feed antibiotic alternatives. Feed plants and their additives are considered one of the ideal substitutes for feed antibiotics in animal production because of their rich resources, natural sources, comprehensive functions, high safety, low toxicity and side effects, low resistance, and no (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, namely, a Chinese medicine-type feed additive with anti-biological effects.

[0006] The animal feed additive provided by the present invention comprises the following steps:

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

[0008] Removing the subcritical extractant of the extraction system to obtain a pomace powder system after the pomace oil is 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 taken out, and 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%; 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 has a pressure of 0.45-0.8 MPa, a temperature of 35-45° C., and a time of 40-45 min.

[0014] Preferably, the method of removing the subcritical extractant of 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, the speed of the centrifugation is 6000-20000 r / min, and the time is 5-20 min.

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

[0018] The polypeptide is extracted from Malus multiflora;

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

[0020] The maslinic acid and oleanolic acid extracted by the present invention have anti-inflammatory and antioxidant effects, and are confirmed by animal experiments to be able to enhance intestinal health in a short 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 maslinic acid and oleanolic acid can be promoted and used as a new animal feed additive. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : The scavenging rate of maslinic acid and oleanolic acid on DPPH free radicals;

[0022] Figure 2 :Maslinic acid and oleanolic acid on 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 : Secondary mass 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 :Effects of maslinic acid and oleanolic acid on oxidative indices in colitis mice. DETAILED DESCRIPTION

[0029] Studies have found that natural organic substances such as hawthorn acid, oleanolic acid and some pentacyclic triterpene acids contained in food processing byproducts that can be used as feed, such as olive pomace, have good anti-inflammatory and antioxidant effects and enhance intestinal health. Therefore, the research and development of processing byproducts containing functional substances such as anti-inflammatory, antioxidant and intestinal health enhancement using olive pomace as raw materials can be used as animal feed additives.

[0030] The present invention is described in detail below in conjunction with specific embodiments and 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%, and then crushed by a pulverizer, sieved with 40 mesh and 20 mesh in turn, and olive pomace fragments with a particle size of 20-40 mesh are taken. As for the subcritical extractant, 0.45-0.8MPa is used, the temperature is maintained at 35-45°C, and extraction is performed for 40-45 minutes. The subcritical extractant is removed to obtain pomace powder, and the pomace powder is mixed with ethanol, extracted, and the extract is subjected to reduced pressure distillation to obtain an extract, and the system is mixed and stirred with petroleum ether in a volume ratio of 1:1-1:20, and the petroleum ether phase is taken after standing and stratification, and the petroleum ether phase is filtered, and the filtrate obtained by centrifugation at a rotation speed of 6000-20000r / min is hawthorn 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 hawthorn acid and oleanolic acid sample solution, add in 10mL volumetric flask, dilute to near 1mL mark with distilled water, then add 3.5mL concentration and be 0.2mmol / L DPPH ethanol solution, continue to be fixed to volumetric flask mark with ethanol, ensure that solution is evenly mixed. Mixed solution is placed in luminous environment and reacts 30min. Afterwards, measure the absorbance of this solution under 517nm wavelength. Meanwhile, prepare DPPH solution without sample and sample solution without DPPH respectively, measure absorbance separately under the same wavelength, with as blank control and sample background correction. In addition, set same concentration vitamin C (VC) solution as positive control, carry out same treatment and measure its absorbance. Finally, calculate the scavenging rate of sample to DPPH free radical according to obtained data.

[0036]

[0037] Wherein, 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 hawthorn 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. + Working solution, then accurately diluted to the scale with distilled water to ensure uniform mixing. After standing for 10 minutes at room temperature and protected from light, the absorbance of each solution was measured at a wavelength of 734nm 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) + Working solution) was used to determine the sample's own absorbance, all measured at the same wavelength. Finally, 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 obtained data.

[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 clearing effect, with a clearing 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 1.0 μg / mL), and an experimental group (LPS concentration 1.0 μg / mL combined with 50 μg / mL of hawthorn acid and 50 μg / mL of oleanolic acid). Each group of samples 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. The nitrite (NaNO2) standard was diluted with culture medium, and 50 μL of cell culture supernatant was collected from each well. After standing at room temperature for 5 minutes, equal amounts of Griess reagent I (50 μL) and Griess reagent II (50 μL) were added in turn. The amount of NO generated in each group was accurately calculated by measuring the optical density value (OD) at a wavelength of 540 nm and combining it with the NaNO2 standard curve.

[0046] As a key regulatory factor, NO plays an important role in a variety of physiological functions and inflammatory processes, especially in the LPS-induced RAW264.7 macrophage acute inflammation model. Figure 3 The effect 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; after adding 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 continuous feeding of mice with maslinic acid and oleanolic acid feed additives for 2 months, enteritis was found in the fed group of mice. It is speculated that continuous feeding of maslinic acid and oleanolic acid feed additives 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 having the effect of preventing and treating enteritis

[0049] The tissue of the Malus multiflorus 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°C, and the hydrolysis was performed for 5 hours. After the end, the enzymatic solution was placed in a 100°C water bath to inactivate the enzyme for 15 minutes, and then placed in a centrifuge and centrifuged at 10000r / min for 10 minutes. The supernatant was separated and collected, concentrated and dried to obtain the Malus multiflorus 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, Tris-HCl buffer containing 0-2 mol / L NaCl was used as the eluent, and then purified by HPLC: the hydrolyzate was purified by a reverse 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 to 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, see 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 polypeptide sequences screened were sent to Shanghai Sangon Biotechnology Co., Ltd. for synthesis. RAW264.7 cells were cultured at 37°C and 5% CO2 using complete culture medium containing 10% fetal bovine serum. The cells were passaged every 24 hours. Cells in the logarithmic growth phase were taken and cultured with different polypeptides 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] From the results in Table 1, it can be seen that polypeptide c has the best clearing effect on TNF-α and IL-1β, indicating that it has the best in vitro anti-inflammatory property, and its sequence is DKQGEP (SEQ ID NO: 1).

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

[0054]

[0055] Forty 6-week-old male C57BL / 6 mice were used, with body weights 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 drank 2% maslinic acid and oleanolic acid solutions continuously and freely. At the same time, the mice in the control group were also gavaged with 100 mg / kg of sulfasalazine daily, while the mice in the experimental group were gavaged with maslinic acid and oleanolic acid solutions 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 normal group mice 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 model group mice was severely damaged, the crypt epithelial cell morphology was distorted, and there was infiltration of a large number of inflammatory cells, indicating severe damage to the intestinal barrier function. Although the colon structure of the control group was relatively intact, infiltration of some inflammatory cells and edema of the intestinal wall were still observed. It is worth noting that in the colon tissue sections of the experimental group mice, the crypt structure was clearly visible, with almost no edema or inflammatory cell infiltration. The results confirmed that the addition of polypeptide c to masaic acid and oleanolic acid can effectively reduce the tissue damage of masaic 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 maslinic 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, test group 2). The test group was gavaged with 2% DSS water daily, the test group 1 was also gavaged with 100 mg / kg of 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 mouse intestines were taken to analyze the expression of related genes and cytokines in the colon tissue.

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

[0060] The kit was 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 level of IL-4 in the model group decreased significantly (p<0.05), while the level of IL-4 in the experimental group 1 and the experimental group 2 increased significantly. The above studies confirmed that hawthorn acid and oleanolic acid can reduce DSS-induced intestinal inflammation, and the anti-inflammatory effect of hawthorn acid and oleanolic acid solution with the addition of polypeptide C is better than that of hawthorn acid and oleanolic acid alone. After use, the intestinal inflammatory response decreased significantly, and the anti-inflammatory effect was significantly enhanced, thereby achieving the purpose of alleviating intestinal inflammatory response.

[0062] 2. Determination of oxidation indexes in mouse serum

[0063] Enzyme-linked immunosorbent assay (ELISA) was used to determine the content of myeloperoxidase (MPO) in mouse serum; the content of catalase (CAT), glutathione reductase (GR) and malondialdehyde (MDA) in mouse serum was determined by micro-method; the content of superoxide dismutase (SOD) in mouse serum was determined by enzyme labeled method.

[0064] In DSS-induced colitis in mice, oxidative stress in colon tissue is an important indicator. Studies have shown that the occurrence of colitis is often accompanied by an increase in oxidative stress. Therefore, improving oxidative stress in colon tissue is a potential measure to alleviate the occurrence of colitis. Figure 8 The levels of SOD, CAT, GR, MDA and MPO in mouse serum are shown in the figure. Compared with the normal group, the levels of CAT (p<0.05), SOD (P<0.05) and GR (p<0.05) in the model group mice were significantly reduced, while the levels of MDA (p<0.05). The level of MPO (p<0.05) was significantly increased, indicating that DSS caused oxidative adversity in mice and caused oxidative damage to the colon tissue of mice. After intervention with the addition of maslinic acid and oleanolic acid, the oxidation indexes showed an opposite trend to the model group. The levels of CAT and GR in mice were significantly increased (p<0.05), as was the level of SOD, while the levels of MDA (p<0.05) and MPO (p<0.05) were reduced. The intervention effects of maslinic acid and oleanolic acid with the addition of polypeptide C were better, indicating that maslinic acid and oleanolic acid caused oxidative stress in the colon of mice, and the antioxidant effect was 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. An animal feed additive, characterized in that: The method for preparing the animal feed additive comprises the following steps: The dried olive pomace powder sample and a subcritical extractant are mixed and subjected to subcritical extraction to obtain an extraction system; Removing the subcritical extractant of the extraction system to obtain a pomace powder system after the pomace oil is removed; The pomace powder system is mixed with ethanol for extraction, and the obtained extract is subjected to reduced pressure distillation to obtain an extract; The extract system is mixed with petroleum ether, stirred, and after standing for stratification, the petroleum ether phase is taken out, and the obtained petroleum ether phase is filtered to obtain the filtrate as an animal feed additive.

2. The animal feed additive according to claim 1, characterized in that The olive pomace powder sample is pre-treated before being mixed with the subcritical extractant; the pre-treatment comprises the following steps: The olive pomace is dried and crushed; the moisture content of the material after drying is ≤5%; the particle size of the material after crushing is 20-40 meshes.

3. The animal feed additive according to claim 1 or 2, characterized in that The subcritical extractant includes one or more of butane, propane, isobutane or dimethyl ether.

4. The animal feed additive according to claim 1, characterized in that The subcritical extraction has a pressure of 0.45-0.8 MPa, a temperature of 35-45° C., and a time of 40-45 min.

5. The animal feed additive according to claim 1, characterized in that The method of removing the subcritical extractant of the extraction system is vacuum distillation; the temperature of the vacuum distillation is 45-50°C, the vacuum degree is ≤0.5MPa, and the time is 25-30min.

6. The animal feed additive according to claim 1, characterized in that The volume ratio of the extract system to petroleum ether is 1:1 to 1:

20.

7. The animal feed additive according to claim 1, characterized in that The animal feed additive also contains polypeptides for preventing and treating enteritis.

8. The animal feed additive according to claim 7, characterized in that The amino acid sequence of the polypeptide is SEQ ID NO:

1.

9. An animal feed, characterized in that: The animal feed additive according to claim 1 is added to the animal feed.

10. The animal feed according to claim 9, characterized in that The animal feed is also added with a polypeptide having an amino acid sequence of SEQ ID NO: 1.

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