A seabuckthorn leaf extract, a feed additive, and a preparation method and application thereof

Abstract

The application discloses a seabuckthorn leaf extract, a feed additive and a preparation method and application thereof, and belongs to the technical field of plant extract preparation. The method takes fresh seabuckthorn leaves collected in a harvesting period as raw materials, and prepares basic seabuckthorn leaf powder through pretreatment, fixation and drying; flavone active components in the seabuckthorn leaves are directionally enriched through ethanol reflux extraction combined with AB-8 macroporous resin fractional purification; the active enrichment is uniformly mixed with a whey protein isolate-malt dextrin Maillard reaction compound, and a special feed additive is prepared through low-temperature spray drying. The active flavone prepared by the application has a purity of more than 40%, and the active substance has high stability and excellent palatability; animal experiments show that the additive can significantly reduce the diarrhea rate of weaned piglets, improve the body antioxidant level, enhance the immune function and relieve weaning stress. The application realizes high-value utilization of seabuckthorn leaf forestry byproduct resources, is suitable for large-scale green pig breeding, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of plant extract preparation technology, and in particular to a sea buckthorn leaf extract, a feed additive, its preparation method and application. Background Technology

[0002] Sea buckthorn ( Hippophae rhamnoides Sea buckthorn (L.) is a plant with both edible and medicinal uses. Its leaves are rich in flavonoids, polyphenols, and other active ingredients, possessing antioxidant and anti-inflammatory physiological functions. In pig production, weaned piglets are prone to diarrhea and stunted growth due to their underdeveloped digestive systems and weak stress response, severely impacting breeding efficiency. Simultaneously, there is an urgent market demand for natural feed additives for piglets, and the application of sea buckthorn leaves, as an easily accessible and low-cost byproduct, in piglet farming has not yet been fully explored.

[0003] Current technologies for treating diarrhea and growth retardation in piglets largely rely on antibiotics or chemical additives. While these can alleviate symptoms to some extent, long-term use can lead to gut microbiota imbalance, drug residues, and potential risks such as drug resistance and residue buildup. Furthermore, they fail to fundamentally improve the piglets' physiological functions. Research on the application of sea buckthorn leaf extract is primarily focused on human health, with limited application in piglet farming. Its specific regulatory effects on piglet gut health and growth performance remain unclear, and mature application protocols have not been developed. Moreover, existing technologies lack systematic research on combining sea buckthorn leaf extract with piglet gut health and growth performance in a piglet farming setting, failing to meet the demand for natural and safe feed additives in large-scale farming.

[0004] Therefore, there is an urgent need to develop a piglet breeding application solution based on sea buckthorn leaf extract. Summary of the Invention

[0005] Existing technologies for preventing and treating diarrhea and improving growth retardation in piglets often rely on antibiotics or chemical additives. These methods are not only costly, prone to drug resistance and residues, and disrupt the intestinal microecology, thus contradicting the concept of green farming, but also cumbersome, time-consuming, and labor-intensive in production and use. This invention provides a sea buckthorn leaf extract, a feed additive, its preparation method, and its application. Based on sea buckthorn leaf extract, this invention utilizes its active ingredients to improve intestinal function in piglets, reduce the incidence of diarrhea, and promote growth, achieving high-value utilization of sea buckthorn leaf by-products while solving the drawbacks of existing chemical additives in piglet farming.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for preparing sea buckthorn leaf extract, comprising the following steps: (1) Raw material pretreatment: Select fresh sea buckthorn leaves that are free from mold and pests during the harvest period, spray them with clean water to thoroughly remove surface mud, impurities and residual dirt, drain the surface water, and place them at room temperature to wither until the moisture content of the sea buckthorn leaves drops to 18% to 22%, they feel soft and have no obvious water stains, and are ready for use. (2) Preparation of basic sea buckthorn leaf powder by blanching and drying: Fresh sea buckthorn leaves after withering are sent to a hot air blanching equipment for blanching treatment. After blanching, they are quickly taken out and lightly kneaded to loosen and shape them to avoid clumping. Then they are transferred to a constant temperature drying equipment and dried at a constant temperature until the moisture content of the material is ≤7%. After being taken out and cooled to room temperature, they are crushed, sieved, and coarse residue is removed to obtain sea buckthorn leaf powder. It is then sealed and stored for later use. (3) Preparation of crude extract: Ethanol reflux extraction or ultrasonic-assisted extraction is performed. After extraction, the extract is filtered through a multi-layer filter cloth and the supernatant is collected to obtain the crude extract of sea buckthorn leaves for later use. (4) Purification: The crude extract of sea buckthorn leaves was sent to a vacuum concentration device for concentration to remove ethanol from the system and obtain an alcohol-free crude extract concentrate. The concentrate was diluted with purified water to adjust the concentration to 1.0-1.5 mg / mL and slowly loaded onto an AB-8 macroporous adsorption resin column. After loading, the column was eluted with purified water at a rate of 3-4 BV / h to remove impurities until the eluent was colorless and transparent. Then, a gradient elution of 20%-80% ethanol solution was used for fractional elution, and 40%-80% of the target eluent was collected. The target eluent was concentrated again under vacuum to remove ethanol and obtain the sea buckthorn leaf extract.

[0007] Furthermore, in step (2), during the blanching process, the blanching temperature is controlled at 180-240℃ and the blanching is continued for 1-2 minutes.

[0008] Furthermore, in step (2), the sieving is performed through a 40-60 mesh sieve.

[0009] Furthermore, in step (3), The ethanol reflux extraction involves adding sea buckthorn leaf powder to an ethanol-water solution at a ratio of 1g:15~25mL to prepare a 50%~70% ethanol extraction system. After stirring and mixing, the mixture is transferred to a reflux extraction device, and the extraction temperature is controlled at 65~75℃ for constant temperature reflux extraction for 1.5~2.5 h. It also includes repeatedly extracting the filter residue 1-2 times, combining the supernatants from the repeated extractions to obtain a crude extract of sea buckthorn leaves.

[0010] Further, in step (3), ultrasonic-assisted extraction includes adding sea buckthorn leaf powder to an ethanol aqueous solution at a material-to-liquid ratio of 1:20~40mL, and ultrasonicating at 60~80℃ for 60~120 minutes, with an ultrasonic frequency of 30~50 kHz and a power of 200~400 W.

[0011] Furthermore, it also includes repeatedly extracting the filter residue 1-2 times, combining the supernatants from the repeated extractions to obtain a crude extract of sea buckthorn leaves.

[0012] Furthermore, in step (4), the loading speed is controlled at 2-3 BV / h; fractional elution is performed at a speed of 2-3 BV / h.

[0013] In a second aspect, the present invention provides a method for preparing a feed additive, comprising the following steps: S1. Preparation of whey protein isolate-maltodextrin complex: Mix whey protein isolate and maltodextrin at a mass ratio of 1:1~5 evenly, place in a constant temperature drying device, control the reaction temperature at 60~80℃ and the reaction time at 12~24h to prepare whey protein isolate-maltodextrin complex. S2. Mixing and blending: Using the whey protein isolate-maltodextrin complex obtained in step S1 as a carrier stabilizer, the complex and sea buckthorn leaf extract are mixed evenly at a mass ratio of 1:20-40. Purified water is added, and the mixture is placed in a constant temperature water bath at a temperature of 45-55°C. The mixture is stirred and blended to prepare a natural composite stable carrier solution. The sea buckthorn leaf extract is any of the sea buckthorn leaf extracts prepared by the above methods. S3. Spray drying: The carrier solution obtained in step S2 is sent to a spray drying device for spray drying. After drying, the powder at the bottom of the tower is collected, stirred and mixed evenly to obtain a special feed additive for sea buckthorn leaf extract.

[0014] Furthermore, in step S3, during the spray drying process, the inlet air temperature is controlled at 160–180°C, the outlet air temperature at 75–85°C, the atomization pressure at 0.2–0.4 MPa, and the feed rate of the liquid at 5–8 mL / min.

[0015] In a third aspect, the present invention provides a feed additive prepared by any of the above methods.

[0016] In a fourth aspect, the present invention provides the use of sea buckthorn leaf extract or feed additive prepared by any of the methods described above in the preparation of drugs for the prevention and / or treatment of diarrhea in livestock or in the preparation of products for improving the health of livestock.

[0017] Furthermore, the animals being raised are weaned piglets.

[0018] Furthermore, the amount of the feed additive added is 0.06% to 0.12% of the daily ration weight.

[0019] The beneficial effects of this invention include at least the following: (1) This invention realizes the efficient and high-value utilization of sea buckthorn leaf resources, breaking through the limitations of existing technologies that focus on the development of sea buckthorn resources and the low utilization rate of sea buckthorn fruit and leaves. Through scientific extraction and purification processes, the application value of active ingredients such as flavonoids in sea buckthorn leaves is fully explored, and the previously underutilized sea buckthorn leaves are transformed into efficient green feed additives. This not only expands the application field of sea buckthorn leaves and improves the comprehensive economic benefits of the sea buckthorn industry, but also realizes the recycling of resources, which is in line with the concept of green agricultural development.

[0020] (2) The whey protein isolate-maltodextrin compound prepared by the present invention has the natural nutritional properties of whey protein isolate and the processing compatibility of maltodextrin. It contains no harmful components, is highly compatible with the physiological characteristics of weaned piglets, and is easily digested and absorbed by piglets. It can not only play the role of carrier in encapsulation and slow release, but also form a synergistic effect with sea buckthorn leaf extract, which can help improve the intestinal environment of piglets, enhance the nutritional value of feed, further optimize the growth performance of piglets, and reduce the diarrhea rate. Compared with existing feed additives, there is no need to add additional antibiotics and other chemical agents, which is more in line with the development trend of green feed.

[0021] (3) The feed additive of the present invention is adapted to the physiological needs of weaned piglets. After addition, it can significantly optimize the growth performance of piglets and reduce the diarrhea rate. Moreover, it does not require the addition of antibiotics or other chemical agents. It not only solves the problem that existing piglet feed is prone to diarrhea and slow growth, but also conforms to the development trend of green feed, reduces feed feeding costs, and ensures the safety of piglet products. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0024] The following specific embodiments illustrate the solution proposed in this invention: Example 1 A method for preparing a feed additive includes the following steps: Raw material pretreatment: Select fresh sea buckthorn leaves that are free from mold and pests during the harvest season, spray them with clean water to thoroughly remove surface mud, impurities and residual dirt, drain the surface water, and then spread them out at room temperature to wither until the moisture content of the sea buckthorn leaves drops to 18% to 22%, they feel soft to the touch and have no obvious water stains, and are ready for use.

[0025] Preparation of basic sea buckthorn leaf powder by blanching and drying: Fresh sea buckthorn leaves after withering are sent to a hot air blanching device, the blanching temperature is controlled at 210℃, and blanching is continued for 1.5 minutes. After blanching, the leaves are quickly taken out and lightly kneaded to loosen and shape them to avoid clumping. Then, they are transferred to a constant temperature drying device, the drying temperature is controlled at 55℃, and the material is dried at a constant temperature until the moisture content is ≤7%. After being taken out and cooled to room temperature, the leaves are crushed and passed through a 50-mesh sieve to remove coarse residue, thus obtaining sea buckthorn leaf powder, which is then sealed and stored for later use.

[0026] Preparation of crude extract by ethanol reflux extraction: Take sea buckthorn leaf powder and add ethanol aqueous solution at a material-to-liquid ratio of 1:20 (m / v) to prepare a 60% ethanol extraction system. After stirring and mixing, transfer to a reflux extraction device, control the extraction temperature at 70℃, and reflux extract at a constant temperature for 2 hours. After extraction, filter with multi-layer filter cloth, collect the supernatant, and the filter residue can be extracted once more. Combine the supernatants from the two extractions to obtain the crude extract of sea buckthorn leaves for later use.

[0027] Macroporous resin fractionation purification and targeted enrichment of active components: The crude extract of sea buckthorn leaves was fed into a vacuum concentration device and concentrated under reduced pressure at 60℃ and 0.07MPa to remove ethanol, yielding an alcohol-free crude extract concentrate. The concentrate was diluted with purified water to a concentration of 1.2 mg / mL and slowly loaded onto an AB-8 macroporous adsorption resin column at a loading rate of 2 BV / h. After loading, the column was eluted with purified water at a rate of 3 BV / h until the eluent was colorless and transparent. Then, a gradient elution of 20%–80% ethanol solutions was performed at a rate of 2 BV / h, collecting 40%–80% of the target eluent. The target eluent was concentrated again under reduced pressure to remove ethanol, yielding an active enriched concentrate mainly composed of isorhamnetin glycosides and total polyphenolic flavonoids. The purity of the active flavonoids was controlled to ≥40%, and the concentrate was refrigerated to obtain the sea buckthorn leaf extract. According to the test results, the total flavonoid content of the sea buckthorn leaf extract in this embodiment was 42.36%.

[0028] Preparation of whey protein isolate-maltodextrin complex: whey protein isolate and maltodextrin were mixed evenly at a mass ratio of 1:2 and placed in a constant temperature drying device. The reaction temperature was controlled at 70℃ and the reaction time was 24h to prepare the whey protein isolate-maltodextrin complex.

[0029] Compound mixing: whey protein isolate-maltodextrin compound was selected as the carrier stabilizer. The compound and sea buckthorn leaf extract were mixed evenly at a mass ratio of 1:30. Purified water was added, and the mixture was placed in a constant temperature water bath at 50°C. The mixture was stirred at low speed to prepare a natural composite stable carrier solution.

[0030] The finished product is obtained by spray drying: The above-mentioned stable carrier solution is fed into a spray drying equipment and a low-temperature spray drying process is adopted. The inlet air temperature is controlled at 170℃, the outlet air temperature is controlled at 80℃, the atomization pressure is 0.3MPa, and the feed rate of the liquid is 6 mL / min. After drying, the powder at the bottom of the tower is collected, stirred and mixed evenly to obtain a special feed additive for sea buckthorn leaf extract. The additive has an active flavonoid purity of ≥18%, uniform particles, and excellent palatability.

[0031] Packaging and storage: Seal the finished powder (feed additive) into moisture-proof and light-proof food-grade packaging bags and store it in a cool, dry and ventilated environment at room temperature.

[0032] Example 2 A method for preparing a feed additive includes the following steps: Raw material pretreatment: Select fresh sea buckthorn leaves that are free from mold and pests during the harvest season, spray them with clean water to thoroughly remove surface mud, impurities and residual dirt, drain the surface water, and then spread them out at room temperature to wither until the moisture content of the sea buckthorn leaves drops to 18% to 22%, they feel soft to the touch and have no obvious water stains, and are ready for use.

[0033] Preparation of basic sea buckthorn leaf powder by blanching and drying: Fresh sea buckthorn leaves after withering are sent to a hot air blanching device, the blanching temperature is controlled at 180℃, and blanching is continued for 2 minutes. After blanching, the leaves are quickly taken out and lightly kneaded to loosen and shape them to avoid clumping. Then, they are transferred to a constant temperature drying device, the drying temperature is controlled at 50℃, and the material is dried at a constant temperature until the moisture content is ≤7%. After being taken out and cooled to room temperature, they are crushed and passed through a 40-mesh sieve to remove coarse residue, thus obtaining sea buckthorn leaf powder, which is sealed and stored for later use.

[0034] Preparation of crude extract by ethanol reflux extraction: Take sea buckthorn leaf powder and add ethanol aqueous solution at a material-to-liquid ratio of 1:30 (m / v). Sonicate at 70 ℃ for 100 minutes with an ultrasonic frequency of 40 kHz and a power of 300 W. After extraction, filter with multi-layer filter cloth and collect the supernatant. The filter residue can be extracted once more. Combine the supernatants from the two extractions to obtain the crude extract of sea buckthorn leaves for later use.

[0035] Macroporous resin fractionation purification and targeted enrichment of active components: The crude extract of sea buckthorn leaves was fed into a vacuum concentration device and concentrated under reduced pressure at 55℃ and a vacuum degree of 0.06MPa to remove ethanol from the system, obtaining an alcohol-free crude extract concentrate. The concentrate was diluted with purified water to adjust the concentration to 1 mg / mL and slowly loaded onto an AB-8 type macroporous adsorption resin column at a loading rate of 2 BV / h. After loading, the column was eluted with purified water at a rate of 3 BV / h until the eluent was colorless and transparent. Then, a gradient elution of 20%–80% ethanol solutions was performed at a rate of 2 BV / h, collecting 40%–80% of the target eluent. The target eluent was concentrated again under reduced pressure to remove ethanol, yielding an active enriched concentrate mainly composed of isorhamnetin glycosides and total polyphenolic flavonoids. The purity of the active flavonoids was controlled to ≥40%, and the concentrate was refrigerated to obtain the sea buckthorn leaf extract. According to the test results, the total flavonoid content of the sea buckthorn leaf extract in this embodiment was 41.2%.

[0036] Preparation of whey protein isolate-maltodextrin complex: whey protein isolate and maltodextrin were mixed evenly at a mass ratio of 1:1 and placed in a constant temperature drying device. The reaction temperature was controlled at 60℃ and the reaction time was 24h to prepare the whey protein isolate-maltodextrin complex.

[0037] Compound mixing: whey protein isolate-maltodextrin compound was selected as the carrier stabilizer. The compound and sea buckthorn leaf extract were mixed evenly at a mass ratio of 1:20. Purified water was added, and the mixture was placed in a constant temperature water bath at 45°C. The mixture was stirred at low speed to prepare a natural composite stable carrier solution.

[0038] The finished product is obtained by spray drying: The above-mentioned stable carrier solution is fed into a spray drying equipment and a low-temperature spray drying process is adopted. The inlet air temperature is controlled at 160℃, the outlet air temperature is controlled at 75℃, the atomization pressure is 0.2MPa, and the feed rate of the liquid is 5 mL / min. After drying, the powder at the bottom of the tower is collected, stirred and mixed evenly to obtain a special feed additive for sea buckthorn leaf extract. The additive has an active flavonoid purity of ≥18%, uniform particles, excellent palatability, and high stability of active substances.

[0039] Packaging and storage: Seal the finished powder (feed additive) into moisture-proof and light-proof food-grade packaging bags and store it in a cool, dry and ventilated environment at room temperature.

[0040] Example 3 A method for preparing a feed additive includes the following steps: Raw material pretreatment: Select fresh sea buckthorn leaves that are free from mold and pests during the harvest season, spray them with clean water to thoroughly remove surface mud, impurities and residual dirt, drain the surface water, and then spread them out at room temperature to wither until the moisture content of the sea buckthorn leaves drops to 18% to 22%, they feel soft to the touch and have no obvious water stains, and are ready for use.

[0041] Preparation of basic sea buckthorn leaf powder by blanching and drying: Fresh sea buckthorn leaves after withering are sent to a hot air blanching device, the blanching temperature is controlled at 240℃, and blanching is continued for 1 minute. After blanching, the leaves are quickly taken out and lightly kneaded to loosen and shape them to avoid clumping. Then, they are transferred to a constant temperature drying device, the drying temperature is controlled at 50℃, and the material is dried at a constant temperature until the moisture content is ≤7%. After being taken out and cooled to room temperature, they are crushed and passed through a 60-mesh sieve to remove coarse residue, thus obtaining sea buckthorn leaf powder, which is sealed and stored for later use.

[0042] Preparation of crude extract by ethanol reflux extraction: Take sea buckthorn leaf powder and add ethanol aqueous solution at a material-to-liquid ratio of 1:25 (m / v) to prepare a 60% ethanol extraction system. After stirring and mixing, transfer to a reflux extraction device, control the extraction temperature at 75℃, and reflux extract at a constant temperature for 1.5h. After extraction, filter with multi-layer filter cloth, collect the supernatant, and the filter residue can be extracted once more. Combine the supernatants from the two extractions to obtain the crude extract of sea buckthorn leaves for later use.

[0043] Macroporous resin fractionation purification and targeted enrichment of active components: The crude extract of sea buckthorn leaves was fed into a vacuum concentration device and concentrated under reduced pressure at 65℃ and a vacuum degree of 0.08MPa to remove ethanol from the system, obtaining an alcohol-free crude extract concentrate. The concentrate was diluted with purified water to adjust the concentration to 1.5 mg / mL, and then slowly loaded onto an AB-8 type macroporous adsorption resin column at a loading rate of 3 BV / h. After loading, the column was first eluted with purified water at a rate of 4 BV / h until the eluent was colorless and transparent. Then, a gradient elution of 20%–80% ethanol solutions was performed at a rate of 3 BV / h, collecting 40%–80% of the target eluent. The target eluent was concentrated again under reduced pressure to remove ethanol, yielding an active enriched concentrate mainly composed of isorhamnetin glycosides and total polyphenolic flavonoids. The purity of the active flavonoids was controlled to be ≥40%, and the concentrate was refrigerated to obtain the sea buckthorn leaf extract. According to the test results, the total flavonoid content of the sea buckthorn leaf extract in this embodiment was 44.5%.

[0044] Preparation of whey protein isolate-maltodextrin complex: whey protein isolate and maltodextrin were mixed evenly at a mass ratio of 1:5 and placed in a constant temperature drying device. The reaction temperature was controlled at 80℃ and the reaction time was 12h to prepare the whey protein isolate-maltodextrin complex.

[0045] Compound mixing: Whey protein isolate-maltodextrin compound was selected as the carrier stabilizer. The compound and sea buckthorn leaf extract were mixed evenly at a mass ratio of 1:40. Purified water was added, and the mixture was placed in a constant temperature water bath at 55°C. The mixture was stirred at low speed to prepare a natural composite stable carrier solution.

[0046] The finished product is obtained by spray drying: The above-mentioned stable carrier solution is fed into a spray drying equipment and a low-temperature spray drying process is adopted. The inlet air temperature is controlled at 180℃, the outlet air temperature is controlled at 85℃, the atomization pressure is 0.4MPa, and the feed rate of the liquid is 8mL / min. After drying, the powder at the bottom of the tower is collected, stirred and mixed evenly to obtain a special feed additive for sea buckthorn leaf extract. The additive has an active flavonoid purity of ≥18%, uniform particles, excellent palatability, and high stability of active substances.

[0047] Packaging and storage: Seal the finished powder (feed additive) into moisture-proof and light-proof food-grade packaging bags and store it in a cool, dry and ventilated environment at room temperature.

[0048] Comparative Example 1 The only difference between this comparative example and Example 1 is that: The purification step using the AB-8 macroporous adsorption resin column was omitted, and the crude extract was directly concentrated under reduced pressure to obtain sea buckthorn leaf extract.

[0049] The total flavonoid content in the sea buckthorn leaf extract was found to be only 20.14%, which is much lower than the 42.36% in Example 1.

[0050] Note: This comparative example only measures the purity of the flavonoid component of the extract and does not conduct actual piglet feeding experiments. The necessity of the macroporous resin purification step is verified through the purity difference.

[0051] Comparative Example 2 The only difference between this comparative example and Example 1 is that: Replace AB-8 macroporous adsorption resin with D101, and leave everything else unchanged.

[0052] The total flavonoid content in the sea buckthorn leaf extract was found to be only 35.87%.

[0053] Note: This comparative example only measures the purity of the flavonoid component of the extract and does not conduct actual piglet feeding experiments. The necessity of the AB-8 macroporous adsorption resin model is verified through the purity difference.

[0054] Comparative Example 3 The only difference between this comparative example and Example 1 is that: The withering and blanching processes at room temperature are omitted. Fresh sea buckthorn leaves that are free from mold, pests and diseases are directly washed, drained, crushed, sieved and then processed.

[0055] The total flavonoid content in the sea buckthorn leaf extract was found to be only 38.97%.

[0056] Note: This comparative example only measures the purity of the flavonoid components in the extract and does not conduct actual piglet feeding experiments. The necessity of withering and blanching at room temperature is verified through the purity difference.

[0057] Comparative Example 4 The only difference between this comparative example and Example 1 is that: Maltodextrin and sea buckthorn leaf extract were mixed at a mass ratio of 1:30, dissolved in purified water, and stirred at a constant temperature of 50°C at low speed to prepare a composite solution. The solution was then subjected to subsequent spray drying, thus omitting the addition of whey protein isolate.

[0058] Everything else is the same as in Example 1.

[0059] Test case 1. Test conditions Test animals: 28-day-old weaned piglets with good health, normal mental state, no diarrhea or disease were selected and randomly divided into 6 groups of 8 piglets each. There were no significant differences in initial weight and sex ratio among the groups (P>0.05) to ensure the fairness of the test.

[0060] Test feed: The basal diet was a complete compound feed for weaned piglets, which met the requirements of "Nutritional Requirements for Pigs" (GB / T39235-2020). Except for the dosage and purity of sea buckthorn leaf extract, the other components and nutritional levels of the feeds in each group were completely identical; the specific grouping is shown in Table 1 below: Table 1: Feeding conditions for different groups

[0061] Feeding environment: Each group of piglets is raised in the same standardized pig house. The ambient temperature is controlled at 24-26℃, the relative humidity is 60-70%, natural light is combined with artificial light (12 hours of light per day), ventilation is good, disinfection is carried out regularly, and the stocking density is consistent (0.35㎡ per piglet).

[0062] Feeding method: Free access to feed and water is adopted. Leftover feed should be cleaned up in a timely manner to ensure that the feed is fresh and free from mold. The drinking water is clean tap water, which is changed twice a day.

[0063] Growth performance indicators: At the beginning of the test (28 days old) and the end of the test (49 days old), each piglet in each group was weighed on an empty stomach, and the total weight and individual weight of each group were recorded. The average daily gain (ADG) and feed conversion ratio (F / G) were calculated.

[0064] Diarrhea rate index: During the formal testing period, the diarrhea status of piglets in each group was observed twice a day, once in the morning and once in the afternoon, and the number of piglets with diarrhea and the number of days with diarrhea were recorded. Diarrhea judgment criteria: the feces are loose and watery, cannot be formed, and are attached to the area around the anus. Diarrhea rate (%) = (number of piglets with diarrhea in each group × number of days with diarrhea) / (total number of piglets in each group × number of days of formal testing) × 100.

[0065] Serum antioxidant and immune indicators: On the day of the test, blood was collected from the anterior vena cava of each group of piglets after fasting. The serum was separated by centrifugation (3000 r / min, 10 min) and stored at -20℃. The activities of GSH-Px and SOD and the content of MDA in the serum (antioxidant indicators) were measured by enzyme-linked immunosorbent assay (ELISA), and the contents of IgG and IgM (immune indicators) were measured. The operation was strictly carried out in accordance with the instructions of the corresponding kit.

[0066] All test data were statistically analyzed using SPSS 26.0 software and expressed as mean ± standard deviation. Differences between groups were analyzed using one-way ANOVA, and P < 0.05 was considered statistically significant.

[0067] 2. Test Results (1) The growth performance results of different groups are shown in Table 2 below: Table 2: Growth Performance Results

[0068] (2) The diarrhea rates of different groups are shown in Table 3 below: Table 3: Diarrhea Rate Results

[0069] (3) Serum antioxidant and immune indicators are shown in Table 4 below; Table 4: Serum antioxidant and immune indicators

[0070] In summary, it can be seen that by adding an appropriate amount of the feed additive prepared in this invention to the diet of weaned piglets, the diarrhea rate of weaned piglets can be significantly reduced, the antioxidant level of the body can be improved, the immune function can be enhanced, the weaning stress of weaned piglets can be effectively alleviated, the health of weaned piglets can be improved, and the high-value utilization of sea buckthorn leaf agricultural and forestry by-products can be realized.

[0071] It should be noted that in this embodiment of the invention, the total flavonoid content is determined by a chemical method (aluminum salt colorimetric method). The specific steps are as follows: accurately weigh an appropriate amount of sample, dissolve it in 60% ethanol and dilute to the mark, shake well and let it stand for filtration, take an appropriate amount of the filtrate, add aluminum salt colorimetric reagent, shake well and place it in the dark for 30 min, and measure the absorbance at a wavelength of 510 nm; use rutin as a reference to draw a standard curve, calculate the total flavonoid content in the sample according to the standard curve, set up 3 parallel experiments for each group, and take the average value as the final determination result, compare the differences in flavonoid content of different samples, and verify the effect of different treatment processes on the enrichment of active ingredients.

[0072] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0073] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0074] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing sea buckthorn leaf extract, characterized in that, Includes the following steps: (1) Raw material pretreatment: Select fresh sea buckthorn leaves that are free from mold and pests during the harvest period, spray them with clean water to thoroughly remove surface mud, impurities and residual dirt, drain the surface water, and place them at room temperature to wither until the moisture content of the sea buckthorn leaves drops to 18% to 22%, they feel soft and have no obvious water stains, and are ready for use. (2) Preparation of basic sea buckthorn leaf powder by blanching and drying: Fresh sea buckthorn leaves after withering are sent to a hot air blanching equipment for blanching treatment. After blanching, they are quickly taken out and lightly kneaded to loosen and shape them to avoid clumping. Then they are transferred to a constant temperature drying equipment and dried at a constant temperature until the moisture content of the material is ≤7%. After being taken out and cooled to room temperature, they are crushed, sieved, and coarse residue is removed to obtain sea buckthorn leaf powder. It is then sealed and stored for later use. (3) Preparation of crude extract: Take sea buckthorn leaf powder and perform ethanol reflux extraction or ultrasonic-assisted extraction. After extraction, filter with multi-layer filter cloth, collect the supernatant to obtain sea buckthorn leaf crude extract for later use. (4) Purification: The crude extract of sea buckthorn leaves was sent to a vacuum concentration device for concentration to remove ethanol from the system and obtain an alcohol-free crude extract concentrate. The concentrate was diluted with purified water to adjust the concentration to 1.0-1.5 mg / mL and slowly loaded onto an AB-8 macroporous adsorption resin column. After loading, the column was eluted with purified water at a rate of 3-4 BV / h to remove impurities until the eluent was colorless and transparent. Then, a gradient elution of 20%-80% ethanol solution was used for fractional elution, and 40%-80% of the target eluent was collected. The target eluent was concentrated again under vacuum to remove ethanol and obtain the sea buckthorn leaf extract.

2. The method according to claim 1, characterized in that, In step (2), during the fixation process, the fixation temperature is controlled at 180-240℃ and the fixation is continued for 1-2 minutes; And / or, sieve through a 40-60 mesh sieve.

3. The method according to claim 1, characterized in that, In step (3), The ethanol reflux extraction involves adding sea buckthorn leaf powder to an ethanol-water solution at a ratio of 1g:15~25mL to prepare a 50%~70% ethanol extraction system. After stirring and mixing, the mixture is transferred to a reflux extraction device, and the extraction temperature is controlled at 65~75℃ for constant temperature reflux extraction for 1.5~2.5 h. It also includes repeatedly extracting the filter residue 1-2 times, combining the supernatants from the repeated extractions to obtain a crude extract of sea buckthorn leaves.

4. The method according to claim 1, characterized in that, In step (3), Ultrasonic-assisted extraction involves adding sea buckthorn leaf powder to an ethanol-water solution at a material-to-liquid ratio of 1:20-40 mL, and then sonicating at 60-80 ℃ for 60-120 minutes at a frequency of 30-50 kHz and a power of 200-400 W.

5. The method according to claim 1, characterized in that, In step (4), the loading speed is controlled at 2-3 BV / h; fractionation elution is performed at a speed of 2-3 BV / h.

6. A method for preparing a feed additive, characterized in that, Includes the following steps: S1. Preparation of whey protein isolate-maltodextrin complex: Mix whey protein isolate and maltodextrin at a mass ratio of 1:1~5 evenly, place in a constant temperature drying device, control the reaction temperature at 60~80℃ and the reaction time at 12~24h to prepare whey protein isolate-maltodextrin complex. S2. Mixing and blending: Using the whey protein isolate-maltodextrin complex obtained in step S1 as a carrier stabilizer, the complex and sea buckthorn leaf extract are mixed evenly at a mass ratio of 1:20-40. Purified water is added, and the mixture is placed in a constant temperature water bath at a temperature of 45-55°C. The mixture is stirred and blended to prepare a natural composite stable carrier solution. The sea buckthorn leaf extract is the sea buckthorn leaf extract prepared by any of the methods described in claims 1-5. S3. Spray drying: The carrier solution obtained in step S2 is sent to a spray drying device for spray drying. After drying, the powder at the bottom of the tower is collected, stirred and mixed evenly to obtain a special feed additive for sea buckthorn leaf extract.

7. The preparation method according to claim 6, characterized in that, In step S3, during the spray drying process, the inlet air temperature is controlled at 160–180℃, the outlet air temperature at 75–85℃, the atomization pressure at 0.2–0.4 MPa, and the feed rate of the liquid at 5–8 mL / min.

8. The feed additive prepared according to claim 6 or 7.

9. The use of the sea buckthorn leaf extract prepared by any of the methods described in claims 1-6 or the feed additive described in claim 9 in the preparation of drugs for the prevention and / or treatment of diarrhea in livestock or in the preparation of products for improving the health of livestock.

10. The application according to claim 8, characterized in that, The animals being raised are weaned piglets.

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