Feed additive for synergistically improving oxidative stress resistance of broiler chickens based on oleuropein-luteoloside-maslinic acid ternary combination and application of feed additive

Abstract

The invention discloses a feed additive for synergistically improving oxidative stress resistance of broiler chickens based on oleuropein-luteoloside-maslinic acid ternary combination and application of the feed additive, the feed additive is the olea europaea leaf extract, the total polyphenol content of the feed additive is about 52%, the total flavone content is 7%, and the total flavone content is 5%. The feed is mainly composed of oleuropein, luteolin-7-beta-glucoside and maslinic acid, the oleuropein, the luteolin-7-beta-glucoside and the maslinic acid are sequentially added in a molar ratio of 6: 1: 1 to generate a synergistic anti-oxidative stress effect, and when the feed is applied to breeding on the basis of corn-soybean daily ration of broilers, the daily gain of the broilers can be obviously increased, the feed conversion ratio is reduced, and the feed conversion rate is increased. The intestinal micro-ecological health, the anti-oxidative stress effect and the organism immunity are improved, and the infection rate and the death rate of intestinal diseases such as diarrhea of broilers are reduced. The product has the advantages of being simple and convenient to prepare and use, free of toxic and side effects, beneficial to broiler chicken growth and human health, free of the problems of drug resistance, drug residues and the like, and capable of promoting chicken meat quality improvement and the like.

Description

Technical Field

[0001] The present invention relates to the field of animal feed additive design and formulation, and specifically to a feed additive based on an olive leaf extract comprising a ternary combination of oleuropein, luteolin, and maslinic acid, for synergistically enhancing the antioxidant stress resistance of broiler chickens, and its application. Background Art

[0002] The widespread use of antibiotics, resulting in drug residues, increased drug resistance, and potential safety risks to humans, is driving the scientific community to seek natural, safe, and effective alternatives to traditional antibiotics. The abundance of plant resources provides ample opportunities for the screening of natural feed additives. Among these, olive leaves, a representative example of forest-derived resources, are a key source of these natural feed additives. This is because olive leaves are rich in potent antibacterial compounds, exhibiting excellent antioxidant and antibacterial properties, while also being biosafe and low-cost.

[0003] Olive leaves ( Olea europaea Olive leaves (Olea europaea L.) are a valuable agricultural byproduct produced annually during the harvesting of fruit from pruned and swinging olive trees, amounting to approximately 300,000 to 400,000 tons. Most of this is directly crushed, scattered across fields, or burned, with detrimental economic and environmental impacts. One promising avenue for the high-value utilization of olive leaves is their potential as a nutrient for high-quality meat, driven by the high demand for green and healthy livestock farming using natural polyphenols. Olive leaves are a rich source of bioactive phenols, a more significant source than olive oil and fruit. However, olive leaves are rich in crude fiber, which, due to its difficulty in digestion and palatability, may not be suitable for direct feeding to non-ruminant animals such as chickens.

[0004] Oleuropein, a secoiridoid terpenoid (such as oleuropein) and polyphenol (such as hydroxytyrosol), is primarily found in olive leaves, accounting for 8–14% of the dry matter. It also contains flavonoids (such as luteolin, celery glycoside, rutin, and kaempferol) and verbascoside. Studies have shown that oleuropein in olive leaves exhibits pharmacological activities beneficial to human health, including antioxidant, antimicrobial, anticancer, and antidiabetic properties. Furthermore, the content of oleuropein in olive leaves is higher than that in olive oil and also higher than that in other parts of the same olive tree (olive fruit, buds, and bark). While olive leaf extract has a stronger antioxidant capacity than vitamin C and vitamin E, individual phenolic compounds exhibit higher antioxidant capacity in vitro, while mixed phenolic compounds exhibit antioxidant capacities similar to or higher than those of individual phenolic compounds. This is due to a synergistic effect between oleuropein and other bioactive compounds. Olive leaves are also rich in triterpenoids such as hawthorn acid, which can reduce oxidative stress and inflammatory responses in livestock and poultry, thereby improving their health by scavenging free radicals and inhibiting the expression of inflammatory factors (such as TNF-α and IL-6). They also work synergistically with other active ingredients in olive leaves (such as oleuropein and polyphenols) to enhance intestinal barrier function and nutrient absorption efficiency. As a powerful antioxidant, olive leaf extract has been approved by the European Food and Drug Administration for its safety in feed for all animal species.

[0005] With increasing attention to promoting human health, high-quality chicken meat is in high demand worldwide. For safety and efficacy reasons, research on poultry meat-enhancing feed additives has primarily focused on natural plant extracts. Olive leaf extract can be used as an effective additive in the poultry industry to improve growth performance and antioxidant properties. Furthermore, the addition of olive leaf extract to chicken meat offers consumers the benefits of high-quality meat and nutritional enhancement within an integrated agriculture-food-nutrition chain. For example, broiler chickens fed a diet supplemented with 200 mg oleuropein / kg and an olive leaf extract containing 20% ​​oleuropein and 10% GAE total polyphenols for 42 days showed no significant differences in feed intake, mortality, or slaughter parameters (small intestine weight and breast muscle yield). Broilers were fed a diet supplemented with 0, 5, 10, and 15 mL / L of water (4.4 mg / mL of oleuropein in the extract). The results showed that treatment with 15 mL of olive leaf extract significantly increased daily feed intake, total feed intake, final weight, total weight gain, and feed conversion rate (FCR). p<0.05) (Oke et al., 2017). Olive leaf extract, as a natural feed additive, has the potential to enhance broiler growth and improve meat quality. However, a comprehensive understanding of the relationship between milk and meat quality, antioxidant activity, and cecal microbiome following appropriate olive leaf extract feeding remains lacking. Currently, the effect of olive leaf extract supplementation on improving meat quality in broiler corn-soybean diets remains limited. Therefore, the application of synergistic natural plant extracts as animal feed additives holds great promise and feasibility. Summary of the Invention

[0006] The present invention provides a ternary synergistic antioxidant olive leaf extract feed additive. The preparation method is simple, easy to operate, and easy to industrialize. The present invention also provides a feed additive containing olive leaf extract at a specific dosage, which has the effects of regulating the microecological balance of broiler chickens, inhibiting the growth of Escherichia coli, and promoting the proliferation of probiotics such as lactic acid bacteria and bifidobacteria.

[0007] 1. A feed additive for broiler chickens that synergistically enhances the antioxidant stress resistance of olive leaf extract based on the ternary combination of oleuropein, luteolin and maslinic acid, and its application, characterized in that: Chinese olive leaves are used as raw materials, subjected to ethanol-water solvent hot extraction, accelerated mass transfer release under high-speed mechanical stirring, and efficiently purified and enriched by macroporous resin. The collected liquid is further enriched by three-stage filtration of a physical membrane S, and the ethanol is recovered by rotary evaporation and then spray-dried. After oleuropein, luteolin and maslinic acid are measured by high-performance liquid phase, the olive leaf extract is compounded according to an appropriate ratio of oleuropein, luteolin and maslinic acid. The olive leaf extract obtained has the following active ingredients: oleuropein, luteolin and maslinic acid. In an intestinal cell HCT-116 model subjected to H2O2 oxidative stress, the olive leaf extract significantly reduces the ROS level. When the prepared olive leaf extract is added to the diet of broiler chickens as a feed additive, the olive leaf extract can significantly enhance the antioxidant stress resistance, regulate the intestinal microecology of broiler chickens, reduce the mortality rate, and thus improve the quality of broiler chickens.

[0008] 2. The method according to claim 1, wherein the olive leaves are produced in Longnan, Gansu and Guangyuan, Sichuan, and have a water content of less than 5%.

[0009] 3. according to claim 1, it is characterized in that, in described ethanol-water solvent, the volume proportion of ethanol is 70-90%.

[0010] 4. The method according to claim 1, wherein the mechanical stirrer is made of stainless steel and has a stirring rate of 15,000-20,000 r / min; the hot extraction temperature ranges from 90°C to 100°C, the liquid-to-solid ratio L / kg ranges from 10 to 15, and the extraction time is 4-5 h.

[0011] 5. The method according to claim 1, wherein the macroporous resin type comprises one of D101, AB-8, XAD-5, XAD-2 and HP20.

[0012] 6. The method according to claim 1, wherein the separation conditions of the macroporous resin are as follows: an elution solvent concentration range of 60% to 90% ethanol-water and a flow rate range of 2.0 to 10.0 mL / min.

[0013] 7. The method according to claim 1, wherein the collected liquid is further enriched by three-stage filtration using a physical membrane, wherein the membranes used are microfiltration membrane, ultrafiltration membrane, and nanofiltration membrane, respectively, and the materials thereof are ceramic membrane and polyethersulfone; the membrane pore sizes are approximately 0.1-10 μm, 0.001-0.1 μm, and 0.001-0.01 μm, respectively; and the final nanofiltration molecular weight cut-off is 200-1000 Da.

[0014] 8. The method of claim 1, wherein the HPLC contents of the main active ingredients of the olive leaf extract are oleuropein, luteolin, and maslinic acid, respectively, at 52%, 7%, and 6%, respectively, with a synergistic molar ratio of 6:1:1. The method of claim 1, wherein the mass ratio of the olive leaf extract added to the corn-soybean diet for broiler chickens is in the range of 0.01% to 1%.

[0015] 9. The method according to claim 1, characterized in that, compared with a group not supplemented with olive leaf extract, the activities of total superoxide dismutase (T-SOD) and catalase (CAT) in the serum of chickens increased by more than 40%, and the serum malondialdehyde content decreased by more than 30%; the activity of total superoxide dismutase (T-SOD) in chicken meat increased by more than 50%, the content of the antioxidant glutathione (GSH) increased by more than 80%, and the malondialdehyde (MDA) content decreased by more than 40%; and the intestinal microecological health of broiler chickens was regulated, compared with a group not supplemented with olive leaf extract, the harmful Escherichia coli was reduced by 12%, and the probiotics such as lactic acid bacteria and bifidobacteria increased by 20%; the shear force of chicken meat was reduced by more than 35%, and the mortality rate was significantly reduced.

[0016] Beneficial effects The preparation method of the olive leaf extract feed additive provided by the present invention is simple, efficient, and easy to operate. It is a purely physical process and does not require expensive equipment or high energy consumption. In addition, by accurately compounding the olive leaf extract rich in the ternary active ingredients of oleuropein, luteolin, and maslinic acid as a feed additive and applying it to the corn-soybean diet of broilers, the oxidative stress of intestinal cells can be significantly reduced. Not only can the GSH content, the antioxidant activity of total TOC enzyme and CAT enzyme in broiler serum and chicken be significantly increased, and the MDA content in serum and chicken be significantly reduced, but the health of intestinal microecology can also be improved, that is, the number of Escherichia coli can be reduced and the number of probiotics such as lactic acid bacteria and bifidobacteria can be increased. The shear force of chicken is reduced by more than 35%, and the broiler feed additive can be used to improve meat quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 HPLC spectrum of 80% ethanol-water olive leaf extract.

[0018] Figure 2 Effects of different addition amounts of olive leaf extract on total superoxide dismutase (T-SOD) activity, total oxidative activity (T-AOC), reduced glutathione (GSH) and malondialdehyde (MDA) content in chicken breast.

[0019] Figure 3 Effects of different addition amounts of olive leaf extract on total superoxide dismutase (T-SOD) activity, total oxidative activity (T-AOC), catalase (CAT) activity and malondialdehyde (MDA) content in broiler serum.

[0020] Figure 4 Effects of different olive leaf extract supplementation levels on the cecal intestinal microecological health (proliferation of total bacteria, lactic acid bacteria, bifidobacteria and Escherichia coli) in broiler chickens fed for 35 days.

[0021] Figure 5 Effects of different olive leaf extract dosages on the mortality rate of broiler chickens after feeding for 35 days.

[0022] Figure 6 Effects of different olive leaf extract supplementation levels on daily gain (ADG), daily intake (ADFI) and feed conversion ratio (FCR) of broiler chickens after 35 days of feeding.

[0023] Figure 7 Effects of different olive leaf extract addition amounts on shear force (tenderness) of chicken meat after feeding for 35 days. DETAILED DESCRIPTION

[0024] The present invention is further described below through specific implementation cases, but is not intended to limit the present invention.

[0025] A feed additive for synergistically enhancing broiler chickens' resistance to oxidative stress based on an olive leaf extract containing a ternary combination of oleuropein, luteolin, and maslinic acid, and its application, comprising the following steps: Dried olive leaves from Longnan, Gansu Province, were used as raw material. Rapid extraction was performed using an ethanol-water mixture (ethanol content ranged from 50% to 95% by volume) under mechanical stirring conditions of 15,000 to 20,000 r / min, extraction time of 4 to 5 h, and a liquid-to-solid ratio (kg / L) of 10 to 20. The extract was filtered while hot, concentrated (diluted with water to a concentration of 10% oleuropein), and purified by passing through a macroporous resin. After passing through microfiltration / ultrafiltration / nanofiltration membranes, the extract was concentrated by rotary evaporation, stirred with an appropriate amount of water, and spray-dried. The active ingredients, including oleuropein, luteolin, and maslinic acid, were determined. The prepared olive leaf extract was added to broiler diets at a mass ratio of 0.01 to 1%, and its effects on blood quality, meat quality, and intestinal microecological environment were evaluated.

[0026] The detection adopted by the present invention: The main active ingredients of olive leaf extract were chromatographed on an Agilent Technologies Poroshell 120, EC-C18 (150 mm × 3.0 mm ID, 2.7 μm particle size) column at 26°C. The mobile phase consisted of acetonitrile (A), water (pH = 3.2), and formic acid (B) at a flow rate of 0.4 mL min. −1 The injection volume ranged from 5 to 10 μL. Detection wavelengths were 210 and 280 nm, respectively. A started at 5% at 0.1 min, increased from 5% to 40% at 40 min, remained at 40% until 45 min, then increased to 70% at 50 min and remained at 70% until 60 min. It then increased to 100% at 65 min, remained at 100% until 68 min, and finally returned to 5% at 70 min. Example

[0027] (1) Preparation of the ternary component extract of olive leaves 11 kg of olive leaf powder was weighed and added to 110 L of 85% ethanol-water in an extraction kettle. The mixture was mechanically stirred at 90°C (at a rate of 15,000 r / min). The filtrate was collected and concentrated by rotary evaporation, and then passed through XAD-5 macroporous resin (elution solvent concentration range: 70% ethanol-water, flow rate: 3.0 mL / min). The collected liquid was sequentially passed through PTF microfiltration membrane, PURO-UF1060 ultrafiltration membrane and NF nanofiltration membrane, and then concentrated by rotary evaporation. An appropriate amount of water was added and the mixture was spray-dried to obtain 0.32 kg of olive leaf extract (oleuropein content: 53%, luteolin content: 7.2%, and maslinic acid content: 6.9%).

[0028] (2) Evaluation of olive leaf extract in broiler feeding The prepared olive leaf extract was premixed with broiler diets. One-day-old Avi White-feathered chicks were fed a conventional diet for 7 days. Afterward, 0.2% olive leaf extract was added to the broiler diet for 35 days. A conventional broiler diet (see Table 1) served as a blank control. Following slaughter according to national standards, antioxidant levels of chicken breast meat, such as glutathione (GSH), thiocyanate (T-SOD), and maltodextrin (MDA), were measured. A total of 120 chicks were randomly divided into two groups (60 chicks per group, three replicates, 10 chicks per replicate): Control (fed with a complete diet) and F2 (f2) (f2) (f2) (f2). All chicks were fed a commercial feed supplemented with olive leaf extract. The daily diet formula is shown in Table 1. A complete commercial diet without olive leaf extract was used as a blank control. At the end of the experiment when broilers were fed olive leaf extract for 35 days, 5 broilers were randomly selected from each group, slaughtered, serum collected, and chicken breast and cecum obtained. The compounds in the meat were tested, and the test parameters included: glutathione (GSH), malondialdehyde (MDA), total superoxide dismutase (T-SOD), etc.; the microorganisms in the cecum were tested by metagenomics, and the test parameters included total bacterial count, Lactobacillus, Bifidobacterium and Escherichia coli.

[0029] pass Figure 1-Figure 7 Experimental results show that, compared with a blank control group without olive leaf extract, the olive leaf extract feed additive of the present invention not only significantly increased the total superoxide dismutase (T-SOD) content (by up to 52%) and glutathione (GSH) content (by up to 91%), and significantly reduced the MDA content (by up to 43%) in broiler chicken serum and chicken meat, indicating enhanced antioxidant capacity, but also significantly improved the cecal microecology of broiler chickens (both lactic acid bacteria and bifidobacteria increased by more than 15%, and the number of Escherichia coli decreased by more than 12%). Furthermore, it significantly reduced the mortality rate of broiler chickens, reduced the shear force of chicken meat (by more than 35%), and had a certain growth-promoting effect. Example

[0030] (1) Preparation of the ternary component extract of olive leaves 9.6 kg of olive leaf powder was weighed and added into an extraction kettle with 97 L of 85% ethanol-water. The mixture was mechanically stirred at 95°C (at a rate of 18,000 r / min) and filtered while hot. The collected filtrate was concentrated by rotary evaporation and diluted with water to a concentration of 12% oleuropein. The concentrate was passed through AB-8 macroporous resin (elution solvent concentration range: 85% ethanol-water, flow rate: 8 mL / min). The collected liquid was sequentially passed through PTF microfiltration membrane, PURO-UF1060 ultrafiltration membrane and NF nanofiltration membrane. The extract was concentrated by rotary evaporation again and spray-dried with an appropriate amount of water to obtain 0.35 kg of olive leaf extract (oleuropein content: 50%, luteolin content: 7%, and maslinic acid content: 7.2%).

[0031] (2) Experiment on feeding broiler chickens with olive leaf extract The prepared olive leaf extract was premixed with broiler diets. One-day-old Avi White-feathered chicks were fed a conventional diet for 7 days. Afterward, 0.3% olive leaf extract was added to the broiler diet for 35 days. A conventional broiler diet (see Table 1) served as a blank control. Following slaughter according to national standards, antioxidant levels of chicken breast meat, such as glutathione (GSH), thiothreonine oxide (T-SOD), and methylated amino acid (MDA), were measured. A total of 120 chicks were randomly divided into two groups (60 chicks per group, three replicates, 10 chicks per replicate): G1 (fed with a complete diet) and G2 (fed with 0.3% olive leaf extract). All chicks were fed a commercial feed supplemented with olive leaf extract. The daily diet formula is shown in Table 1. A complete commercial diet without olive leaf extract was used as a blank control. At the end of the experiment when broilers were fed olive leaf extract for 35 days, 5 broilers were randomly selected from each group, slaughtered, and serum was collected. Chicken breast meat and cecum were obtained for testing compounds in the meat. The test parameters included: glutathione (GSH), malondialdehyde (MDA), and total superoxide dismutase (T-SOD); the microorganisms in the cecum were tested. The test parameters included total bacterial count, Lactobacillus, Bifidobacterium and Escherichia coli.

[0032] Compared to the blank control, the olive leaf extract feed additive not only significantly increased total superoxide dismutase (T-SOD) levels (up to 49%) and glutathione (GSH) levels (up to 87%), and significantly reduced MDA levels (up to 38%), indicating enhanced antioxidant capacity, but also significantly improved the cecal microecology of broiler chickens (both lactic acid bacteria and bifidobacteria increased by over 15%, and the number of Escherichia coli decreased by over 13%). Furthermore, it significantly reduced the mortality rate of broiler chickens and lowered the shear force on the chicken meat, demonstrating a certain growth-promoting effect. Example

[0033] (1) Preparation of ternary components of olive leaf extract 10 kg of olive leaf powder was weighed and mechanically stirred at room temperature (speed of 20000 r / min). The powder was filtered while hot, and the filtrate was collected and concentrated by rotary evaporation. The concentrate was diluted with water to 12% oleuropein, and then passed through AB-8 macroporous resin (elution solvent concentration range: 91% ethanol-water, flow rate of 10.0 mL / min). The collected liquid was sequentially passed through PTF microfiltration membrane, PURO-UF1060 ultrafiltration membrane and NF nanofiltration membrane, and then concentrated by rotary evaporation. An appropriate amount of water was added and the powder was spray-dried to obtain 0.29 kg of olive leaf extract with a solid content of 52.36% oleuropein, 6.32% luteolin and 6.13% maslinic acid.

[0034] (2) Evaluation of olive leaf extract in broiler feeding The prepared olive leaf extract was premixed with broiler diets. One-day-old Avi White-feathered chicks were fed a conventional diet for 7 days. Afterward, 0.3% olive leaf extract was added to the broiler diet for 35 days. A conventional broiler diet (see Table 1) served as a blank control. Following slaughter according to national standards, antioxidant levels of chicken breast meat, such as glutathione (GSH), thiothreonine oxide (T-SOD), and methylated amino acid (MDA), were measured. A total of 120 chicks were randomly divided into two groups (H1) (fed with a complete diet) and H2 (fed with 0.3% olive leaf extract). All chicks were fed a commercial diet supplemented with olive leaf extract. The daily diet formula is shown in Table 1. A complete commercial diet without olive leaf extract was used as a blank control. At the end of the experiment when broilers were fed olive leaf extract for 35 days, 5 broilers were randomly selected from each group, slaughtered, and serum was collected. Chicken breast meat and cecum were obtained for testing compounds in the meat. The test parameters included: glutathione (GSH), malondialdehyde (MDA), and total superoxide dismutase (T-SOD); the microorganisms in the cecum were tested. The test parameters included total bacterial count, Lactobacillus, Bifidobacterium and Escherichia coli.

[0035] Compared to a blank control group without olive leaf extract, the olive leaf extract feed additive not only significantly increased total superoxide dismutase (T-SOD) levels (by 53%) and glutathione (GSH) levels (by 86%), and significantly reduced MDA levels (by 39%), indicating enhanced antioxidant capacity, but also significantly improved the cecal microecology of broiler chickens (both lactic acid bacteria and bifidobacteria increased by over 18%, and the number of Escherichia coli decreased by over 13%). Furthermore, it significantly reduced the mortality rate of broiler chickens and lowered the shear force on the chicken meat, demonstrating a certain growth-promoting effect.

[0036] HCT-116 cells in the logarithmic growth phase were collected and 3×10 cells were used per dish. 3 6 × 10 cells were seeded in a 6 cm cell culture dish, or 6 × 10 cells were seeded in each well. 3 Cells were seeded in 96-well cell culture plates and incubated for 24 hours in a CO2 incubator at 37°C, 5% CO2, and 100% saturated humidity. The culture medium was then replaced with DMEM containing varying concentrations of H2O2 (0, 5, 50, 100, 200, and 500 µmol / L) for 4 hours. The experiment was repeated three times, and the appropriate H2O2 concentration was selected based on cell viability analysis. In the olive leaf extract protective experiment, HCT-116 cells were incubated in DMEM for 24 hours, followed by DMEM containing varying concentrations of the olive leaf extract (oleuropein / luteolin / maslinic acid) for 24 hours. Finally, the culture medium was replaced with DMEM containing H2O2 for 4 hours. The experiment was repeated three times.

[0037] The experimental results show that different ratios of oleuropein / luteolin / maslinic acid in olive leaf extract have an effect on the oxidative stress of intestinal HCT-116 cells, as shown in the table below. The combination with a molar ratio of 6:1:1 has the highest ROS scavenging efficiency and the strongest synergistic effect.

[0038]

[0039] After feeding broilers with 0.2-0.3% olive leaf extract (oleuropein: oleuropein: hawthorn acid = 6:1:1) in the above-mentioned implementation cases 1-3 for 35 days, compared with the blank group without olive leaf extract, the broilers fed with 0.3% olive leaf extract had an average daily weight gain increased by 3.3%, an average daily feed intake of 2.0%, a feed conversion rate decreased by 1.6%, and a mortality rate decreased; the shear force of the chicken was reduced by more than 35%, which has a growth-promoting effect on broilers and improves the quality of chicken, especially the antioxidant level in serum and chicken meat is significantly improved, the level of resistance to oxidative stress in the broiler intestine is significantly improved, and the intestinal microecological health of broilers is significantly improved.

Claims

1. A feed additive based on an olive leaf extract of a ternary combination of oleuropein, luteolin and maslinic acid for synergistically enhancing the antioxidant stress resistance of broilers and its application, characterized in that: Using Chinese olive leaves as raw materials, they were hot-extracted with ethanol-water solvents, and the mass transfer release was accelerated by high-speed mechanical stirring. They were efficiently purified and enriched by macroporous resins, and the collected liquid was further enriched by three-stage filtration of physical membranes. The ethanol was recovered by rotary evaporation and then spray-dried. After oleuropein, luteolin and hawthorn acid were determined by high-performance liquid phase, they were compounded according to the appropriate ratio of oleuropein-luteolin-hawthorn acid. The olive leaf extract obtained had the active ingredients of oleuropein, luteolin and hawthorn acid. In the intestinal cell HCT-116 model subjected to H2O2 oxidative stress, the olive leaf extract significantly reduced the ROS level. Adding the prepared olive leaf extract to the diet as a feed additive for broilers can significantly improve the antioxidant stress capacity, regulate the intestinal microecology of broilers, reduce the mortality rate, and thus improve the quality of broilers.

2. According to claim 1, it is characterized in that The olive leaves are produced in Longnan, Gansu and Guangyuan, Sichuan, and have a water content of less than 5%.

3. According to claim 1, it is characterized in that In the ethanol-water solvent, the volume proportion of ethanol is 70-90%.

4. According to claim 1, it is characterized in that The mechanical stirrer is made of stainless steel and has a stirring rate of 15,000-20,000 r / min; the hot extraction temperature range is 90-100°C, the liquid-to-solid ratio L / kg ranges from 10-15, and the extraction time is 4-5 h.

5. According to claim 1, it is characterized in that The macroporous resin model includes one of D101, AB-8, XAD-5, XAD-2 and HP20.

6. The method according to claim 1, wherein: The separation conditions of the macroporous resin are as follows: the elution solvent concentration range is 60% to 90% ethanol-water, and the flow rate range is 2.0 to 10.0 mL / min.

7. The method according to claim 1, wherein: The collected liquid is then further enriched through three-stage filtration using physical membranes. The membranes used are microfiltration membrane / ultrafiltration membrane / nanofiltration, respectively, and their materials are ceramic membrane and polyethersulfone; the membrane pore sizes are approximately 0.1-10 μm, 0.001-0.1 μm, and 0.001-0.01 μm, respectively, and the final nanofiltration retention molecular weight is 200-1000 Da.

8. According to claim 1, it is characterized in that The main active ingredients of olive leaf extract are oleuropein, luteolin and maslinic acid, with HPLC contents of 52%, 7% and 6% respectively, and their synergistic molar ratio is 6:1:

1. The method according to claim 1 is characterized in that the mass ratio of olive leaf extract added to the corn-soybean diet of broiler chickens is in the range of 0.01~1%.

9. According to claim 1, it is characterized in that Improving the ability to resist oxidative stress means that compared with the group without adding olive leaf extract, the activities of total superoxide dismutase (T-SOD) and catalase (CAT) in the serum of chickens increased by more than 40%, and the serum malondialdehyde content decreased by more than 30%; the activity of total superoxide dismutase (T-SOD) in chicken meat increased by more than 50%, the content of antioxidant glutathione (GSH) increased by more than 80%, and the malondialdehyde (MDA) content was also reduced by more than 40%; regulating the intestinal microecological health of broilers means that compared with the group without adding olive leaf extract, harmful Escherichia coli decreased by 12%, and probiotics such as lactic acid bacteria and bifidobacteria both proliferated by 20%; the shear force of chicken meat decreased by more than 35%, and the mortality rate was significantly reduced.

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