Method for continuously extracting multiple active ingredients from sea buckthorn pomace and application

Abstract

The invention provides a method for continuously extracting multiple active ingredients from sea buckthorn pomace and application, and belongs to the technical field of plant extraction. The extraction method comprises the following steps: performing supercritical CO2 extraction on sea buckthorn pomace powder to obtain sea buckthorn pomace oil, including the sea buckthorn pomace oil with a urea solution, collecting filtrate, and distilling to obtain purified sea buckthorn pomace oil; performing mixed enzymolysis on the extracted sea buckthorn pomace by using cellulase and protease to obtain a first enzymolysis product, performing enzymolysis on the first enzymolysis product by using beta-glucosidase, mixing the obtained crude flavone extract with an ethanol solution to obtain a crude flavone extract solution, and purifying in a chromatographic column to obtain a purified flavone extract; mixing the crude flavone extract with pectinase for enzymolysis, adding absolute ethyl alcohol, and extruding and modifying the collected precipitate to obtain the sea buckthorn pomace dietary fiber. The sea buckthorn pomace oil is high in purity, the flavone aglycone conversion rate of the flavone extract is high, the water-holding capacity of sea buckthorn pomace dietary fibers is high, the sea buckthorn extract can promote proliferation of probiotics, the content of short-chain fatty acid produced by the sea buckthorn extract is high, and intestinal diseases are improved.

Description

Technical Field

[0001] This invention belongs to the field of plant active ingredient extraction technology, and particularly relates to a method and application for continuous extraction of multiple active ingredients from sea buckthorn pomace. Background Technology

[0002] Sea buckthorn, also known as vinegar willow or sour thorn, is a deciduous shrub belonging to the genus Hippophae in the family Elaeagnaceae. It is a plant with both medicinal and edible uses. The main chemical components of sea buckthorn include flavonoids, terpenes, steroids, vitamins, proteins and amino acids, oils and fatty acids, volatile oils, organic acids, sugars, and trace elements. It is a nutritious plant with extremely high medicinal value, possessing biological activities such as killing and inhibiting tumor cells, anticoagulation, lowering blood pressure, preventing vascular embolism, anti-oxidation, anti-fatigue, and enhancing vitality and immunity. However, reports indicate that the sea buckthorn industry generates a large amount of waste fruit residue after producing sea buckthorn products, which not only pollutes the environment but also wastes resources.

[0003] Sea buckthorn pomace is still rich in nutrients such as polyphenols, vitamins, flavonoids, amino acids, organic acids, crude fat, crude protein, crude fiber, and polysaccharides, giving it potential for comprehensive utilization. Extracting nutrients from sea buckthorn pomace can not only increase the added value of deep-processed sea buckthorn products and extend the industrial chain, but also turn waste into treasure, solve environmental pollution problems, and has good economic and social benefits. Currently, the development and utilization of sea buckthorn pomace mainly focuses on the extraction and purification of flavonoids, while components such as dietary fiber and organic acids are not effectively utilized, resulting in a low overall resource recovery rate. There is limited development of sea buckthorn pomace products, and research on extracting multiple active ingredients from sea buckthorn pomace is also lacking. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a method and application for continuous extraction of multiple active ingredients from sea buckthorn pomace.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for continuous extraction of multiple active ingredients from sea buckthorn pomace, comprising the following steps: (1) The sea buckthorn pomace powder was subjected to supercritical CO2 extraction at an extraction pressure of 25~30 MPa, an extraction temperature of 40~45℃, an extraction flow rate of 15~20 L / h, and an extraction time of 120~180 min to collect the sea buckthorn pomace oil and the extracted sea buckthorn pomace. (2) The sea buckthorn pomace oil was encapsulated with urea solution, filtered, and the filtrate was collected to obtain the encapsulated sea buckthorn pomace oil. (3) The sea buckthorn pomace oil after encapsulation is purified by distillation, and the condensed fraction is collected to obtain purified sea buckthorn pomace oil; (4) Mix cellulase and protease, add the extracted sea buckthorn pomace obtained in step (1), perform the first enzymatic hydrolysis to obtain the first enzymatic hydrolysis product, then mix β-glucosidase and the first enzymatic hydrolysis product to perform the second enzymatic hydrolysis, and the obtained second enzymatic hydrolysis product is recorded as crude flavonoid extract. (5) The chromatography column is filled with macroporous adsorption resin to obtain the filled chromatography column; The crude flavonoid extract was mixed with an ethanol solution, and the resulting crude flavonoid extract solution was poured into a packed chromatography column for adsorption. Then, it was eluted sequentially with 15-25% ethanol solution, 45-55% ethanol solution and anhydrous ethanol. The eluent eluted with anhydrous ethanol for 120-160 min was collected to obtain the purified flavonoid extract. (6) Mix the crude flavonoid extract obtained in step (4) with pectinase and hydrolyze it to obtain the hydrolyzed product; (7) Mix the enzymatic hydrolysis product with anhydrous ethanol for alcohol precipitation, filter, and collect the precipitate; subject the precipitate to screw extrusion modification at a temperature of 110~130℃ and a screw speed of 160~200r / min for 2~6min to obtain sea buckthorn fruit residue dietary fiber.

[0006] Preferably, the particle size of the sea buckthorn pomace powder is 40-60 mesh.

[0007] Preferably, in step (2), the mass ratio of the sea buckthorn pomace oil to the urea solution is 2~4:1, the encapsulation time is -6~-2℃, and the encapsulation time is 10~14h; the solvent of the urea solution is an ethanol solution with a volume percentage of 92~97%.

[0008] Preferably, in step (3), the temperature of the distillation purification is 125~165℃.

[0009] Preferably, the distillation purification is carried out in two stages: the first distillation purification is performed at a distillation temperature of 125~135℃ to collect the first condensed fraction, and then the first condensed fraction is subjected to a second distillation purification at 155~165℃ to collect the second condensed fraction.

[0010] Preferably, the amount of cellulase used is 800~1200 U / g, and the amount of protease used is 1200~1700 U / g; the temperature of the first enzymatic hydrolysis is 45~55℃, the time is 1~3h, and the pH of the second enzymatic hydrolysis is 4.5~5.0. The amount of β-glucosidase used is 600~1000 U / g; the temperature of the second enzymatic hydrolysis is 40~50℃, the time is 2~4h, and the pH of the first enzymatic hydrolysis is 7.0~8.0.

[0011] Preferably, in step (5), the macroporous adsorption resin is of type AB-8, and the volume-to-mass ratio of the crude flavonoid extract solution to the amount of macroporous adsorption resin is 140 mL: 5~10 g. The mass-to-volume ratio of the crude flavonoid extract to the ethanol solution is 1 g: (35~45) mL, and the volume percentage of the ethanol solution used to obtain the crude flavonoid extract solution is 15~25%. The adsorption time is 0.5~1.5h; When eluting with 15-25% ethanol solution, the volume used is 0.5 column volumes; when eluting with 45-55% ethanol solution (v / v), the volume used is 1.5 column volumes; when eluting with anhydrous ethanol, the volume used is 4 column volumes. The elution flow rate is 2 column volumes / h; In step (6), the amount of pectinase used is 1800~2200 U / g; the temperature of enzymatic hydrolysis is 45~55℃, the time is 2~4h, and the pH of enzymatic hydrolysis is 3.0~4.0. In step (7), the mass-to-volume ratio of the enzymatically hydrolyzed product to the ethanol solution is 1g:8~12mL; the alcohol precipitation time is 40~50min; the alcohol precipitation temperature is 55~65℃; and the ultrasonic alcohol precipitation power is 150~250W.

[0012] This invention provides a sea buckthorn extract with probiotic effects, wherein the sea buckthorn extract is one or more of the above-mentioned purified sea buckthorn pomace oil, purified flavonoid extract, and sea buckthorn pomace dietary fiber.

[0013] Preferably, the sea buckthorn extract is the purified sea buckthorn pomace oil, the purified flavonoid extract, and the sea buckthorn pomace dietary fiber; the mass ratio of the purified sea buckthorn pomace oil, the purified flavonoid extract, and the sea buckthorn pomace dietary fiber is 1:(1.5~2.5):(6~8).

[0014] This invention provides an application of the above-mentioned sea buckthorn extract in any one or more of the following a to c. a. Preparation of drugs to improve intestinal diseases; b. Preparation of drugs to promote the proliferation of probiotics; c. Increase the content of short-chain fatty acids produced by probiotics.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method and application for the continuous extraction of multiple active ingredients from sea buckthorn pomace. The invention separates and purifies various active ingredients from sea buckthorn pomace, such as sea buckthorn pomace oil, flavonoid extracts, and sea buckthorn pomace dietary fiber. The sea buckthorn pomace oil prepared by this invention has high purity, the flavonoid extract has a high flavonoid aglycone conversion rate, and the sea buckthorn pomace dietary fiber has strong water-holding capacity. Furthermore, the sea buckthorn extract can promote the proliferation of probiotics and produce high levels of short-chain fatty acids, thus improving intestinal diseases. This invention separates and purifies multiple active ingredients from sea buckthorn pomace, improves the resource utilization rate of sea buckthorn, and develops the utilization value of sea buckthorn pomace, which has great economic significance. Detailed Implementation

[0016] This invention provides a method for continuous extraction of multiple active ingredients from sea buckthorn pomace, comprising the following steps: (1) The sea buckthorn pomace powder was subjected to supercritical CO2 extraction at an extraction pressure of 25~30 MPa, an extraction temperature of 40~45℃, an extraction flow rate of 15~20 L / h, and an extraction time of 120~180 min to collect the sea buckthorn pomace oil and the extracted sea buckthorn pomace. (2) The sea buckthorn pomace oil was encapsulated with urea solution, filtered, and the filtrate was collected to obtain the encapsulated sea buckthorn pomace oil. (3) The sea buckthorn pomace oil after encapsulation is purified by distillation, and the condensed fraction is collected to obtain purified sea buckthorn pomace oil; (4) Mix cellulase and protease, add the extracted sea buckthorn pomace obtained in step (1), perform the first enzymatic hydrolysis to obtain the first enzymatic hydrolysis product, then mix β-glucosidase and the first enzymatic hydrolysis product to perform the second enzymatic hydrolysis, and the obtained second enzymatic hydrolysis product is recorded as crude flavonoid extract. (5) The chromatography column is filled with macroporous adsorption resin to obtain the filled chromatography column; The crude flavonoid extract was mixed with an ethanol solution, and the resulting crude flavonoid extract solution was poured into a packed chromatography column for adsorption. Then, it was eluted sequentially with 15-25% ethanol solution, 45-55% ethanol solution and anhydrous ethanol. The eluent eluted with anhydrous ethanol for 120-160 min was collected to obtain the purified flavonoid extract. (6) Mix the crude flavonoid extract obtained in step (4) with pectinase and hydrolyze it to obtain the hydrolyzed product; (7) Mix the enzymatic hydrolysis product with anhydrous ethanol for alcohol precipitation, filter, and collect the precipitate; subject the precipitate to screw extrusion modification at a temperature of 110~130℃ and a screw speed of 160~200r / min for 2~6min to obtain sea buckthorn fruit residue dietary fiber.

[0017] In this invention, sea buckthorn pomace powder is subjected to supercritical CO2 extraction at an extraction pressure of 25-30 MPa, an extraction temperature of 40-45°C, an extraction flow rate of 15-20 L / h, and an extraction time of 120-180 min to collect sea buckthorn pomace oil and extracted sea buckthorn pomace. The particle size of the sea buckthorn pomace powder is preferably 40-60 mesh, more preferably 45-55 mesh, and even more preferably 50 mesh. The sea buckthorn pomace powder is obtained by crushing and sieving sea buckthorn pomace. In a preferred embodiment, sea buckthorn pomace powder is subjected to supercritical CO2 extraction at an extraction pressure of 27-39 MPa, an extraction temperature of 41-44°C, an extraction flow rate of 16-19 L / h, and an extraction time of 130-170 min to collect sea buckthorn pomace oil and extracted sea buckthorn pomace. More preferably, sea buckthorn pomace powder is subjected to supercritical CO2 extraction at an extraction pressure of 28 MPa, an extraction temperature of 43°C, an extraction flow rate of 18 L / h, and an extraction time of 150 min to collect sea buckthorn pomace oil and extracted sea buckthorn pomace. In the sea buckthorn pomace oil obtained by supercritical CO2 extraction of this invention, palmitic acid accounts for 30-35% of the mass percentage of the sea buckthorn pomace oil.

[0018] In this invention, sea buckthorn pomace oil is encapsulated with a urea solution, filtered, and the filtrate is collected to obtain encapsulated sea buckthorn pomace oil. The mass ratio of sea buckthorn pomace oil to urea solution is 2-4:1, more preferably 2.5-3.5:1, and even more preferably 3:1. The encapsulation time is preferably -6 to -2°C, more preferably -5 to -3°C, and even more preferably -4°C. The encapsulation time is preferably 10-14 hours, more preferably 11-13 hours, and even more preferably 12 hours. The solvent of the urea solution is an ethanol solution with a volume percentage of 92-97%, such as a 95% ethanol solution. The urea solution is prepared by mixing urea and solvent at a volume ratio of 1:2. This invention achieves preliminary separation of saturated and unsaturated fatty acids through urea encapsulation, thereby further increasing the palmitic acid content in the encapsulated sea buckthorn pomace oil, with palmitic acid accounting for more than 45% of the mass percentage of the encapsulated sea buckthorn pomace oil.

[0019] In this invention, the encapsulated sea buckthorn pomace oil is purified by distillation, and the condensed fraction is collected to obtain purified sea buckthorn pomace oil. The distillation purification temperature is 125~165℃, more preferably 130~160℃, such as 130℃ or 160℃; the distillation purification adopts two distillation purification: the first distillation purification is performed at a distillation temperature of 125~135℃, and the first condensed fraction is collected; then the first condensed fraction is subjected to a second distillation purification at 155~165℃, and the second condensed fraction is collected. The distillation purification is performed under ultravacuum, and the ultravacuum pressure is 0.1 Pa. This invention uses two distillation purification, which further increases the palmitic acid content in the purified sea buckthorn pomace oil, and the palmitic acid accounts for more than 70% of the mass percentage of the purified sea buckthorn pomace oil.

[0020] In this invention, cellulase and protease are mixed and added to the extracted sea buckthorn pomace obtained in step (1) for the first enzymatic hydrolysis to obtain the first enzymatic hydrolysis product. Then, β-glucosidase and the first enzymatic hydrolysis product are mixed for the second enzymatic hydrolysis to obtain the second enzymatic hydrolysis product, which is denoted as crude flavonoid extract. The amount of cellulase used is 800-1200 U / g, more preferably 900-1100 U / g, and even more preferably 1000 U / g. In this invention, the unit U / g of the amount of cellulase used means the enzyme activity of cellulase added per gram of extracted sea buckthorn pomace. The amount of protease used is preferably 1200-1700 U / g, more preferably 1300-1600 U / g, and even more preferably 1500 U / g. The unit U / g of the amount of protease used means the enzyme activity of protease added per gram of extracted sea buckthorn pomace. The temperature of the first enzymatic hydrolysis is preferably 45-55℃, more preferably 46-52℃, and even more preferably 50℃. The time of the first enzymatic hydrolysis is preferably 1-3 h, more preferably 1.5-2.5 h, and even more preferably 2 h. The pH of the first enzymatic hydrolysis is preferably 4.5-5.0, more preferably 4.6-4.9, and even more preferably 4.8. The preferred amount of β-glucosidase is 600-1000 U / g, more preferably 700-900 U / g, and even more preferably 800 U / g. The unit U / g of the β-glucosidase amount means the enzyme activity of β-glucosidase added per gram of the first enzymatic hydrolysis product. The preferred temperature for the second enzymatic hydrolysis is 40-50℃, more preferably 42-48℃, and even more preferably 45℃. The preferred time for the second enzymatic hydrolysis is 2-4 h, more preferably 2.5-3.5 h, and even more preferably 3 h. The preferred pH for the second enzymatic hydrolysis is 7.0-8.0, more preferably 7.2-7.8, and even more preferably 7.5.

[0021] In this invention, a macroporous adsorption resin is packed into a chromatography column to obtain a packed chromatography column; the crude flavonoid extract is mixed with an ethanol solution, and the resulting crude flavonoid extract solution is poured into the packed chromatography column for adsorption. Then, it is eluted sequentially with a 15-25% (v / v) ethanol solution, a 45-55% (v / v) ethanol solution, and anhydrous ethanol. The eluent obtained by eluting with anhydrous ethanol for 120-160 min is collected to obtain the purified flavonoid extract. The macroporous adsorption resin is of type AB-8. The volume-to-mass ratio of the crude flavonoid extract solution to the macroporous adsorption resin is 140 mL: 5~10 g, such as 140 mL: 5 g, 140 mL: 6 g, 140 mL: 7 g, 140 mL: 8 g, 140 mL: 9 g, or 140 mL: 10 g. The mass-to-volume ratio of the crude flavonoid extract to the ethanol solution is 1 g: (35~45) mL, more preferably 1 g: (37~42) mL, and even more preferably 1 g: 40 mL. The volume percentage of ethanol solution used to extract the crude flavonoid extract is 15-25%, such as 20%, and the content of the crude flavonoid extract solution poured into the packed chromatography column is 140 mL. The adsorption time is preferably 0.5-1.5 h, more preferably 1 h. The elution volume with 15-25% ethanol solution is 0.5 column volumes; the elution volume with 45-55% ethanol solution is 1.5 column volumes; the elution volume with anhydrous ethanol is 4 column volumes; and the elution flow rate is 2 column volumes / h. The conversion rate of flavonoid aglycones in the purified flavonoid extract prepared by this invention is high, reaching over 80%.

[0022] In this invention, the crude flavonoid extract obtained in step (4) is mixed with pectinase and enzymatically hydrolyzed to obtain the hydrolyzed product. The amount of pectinase used is preferably 1800~2200 U / g, more preferably 1900~2100 U / g, and even more preferably 2000 U / g. The unit U / g of the amount of pectinase used means the enzyme activity of pectinase added per gram of crude flavonoid extract. The temperature of the enzymatic hydrolysis is preferably 45~55℃, more preferably 48~52℃, and even more preferably 50℃. The time is preferably 2~4h, more preferably 2.5~3.5h, and even more preferably 3h. The pH of the enzymatic hydrolysis is preferably 3.0~4.0, more preferably 3.2~3.8, and even more preferably 3.5.

[0023] In this invention, the enzymatically hydrolyzed product is mixed with anhydrous ethanol for alcohol precipitation, filtered, and the precipitate is collected. The precipitate is then subjected to screw extrusion modification at a temperature of 110-130℃ and a screw speed of 160-200 r / min for 2-6 min to obtain sea buckthorn fruit residue dietary fiber. The mass-to-volume ratio of the enzymatically hydrolyzed product to the ethanol solution is 1g:8-12mL, more preferably 1g:9-11mL, and even more preferably 1g:10mL; the alcohol precipitation time is preferably 40-50 min, more preferably 42-48 min, and even more preferably 45 min; the alcohol precipitation temperature is preferably 55-65℃, more preferably 58-63℃, and even more preferably 60℃; the ultrasonic alcohol precipitation power is preferably 150-250W, more preferably 170-220W, and even more preferably 200W. In a preferred embodiment, the precipitate is subjected to screw extrusion modification at a temperature of 115-125℃ and a screw speed of 170-190 r / min for 3-5 minutes to obtain sea buckthorn fruit residue dietary fiber. The sea buckthorn fruit residue dietary fiber prepared by this invention has a water holding capacity of 9.15 g / g.

[0024] This invention provides a sea buckthorn extract with probiotic effects, wherein the sea buckthorn extract is one or more of the above-mentioned purified sea buckthorn pomace oil, purified flavonoid extract, and sea buckthorn pomace dietary fiber.

[0025] In a preferred embodiment, the sea buckthorn extract comprises the purified sea buckthorn pomace oil, purified flavonoid extract, and sea buckthorn pomace dietary fiber described above; the mass ratio of the purified sea buckthorn pomace oil, purified flavonoid extract, and sea buckthorn pomace dietary fiber is 1:(1.5~2.5):(6~8). More preferably, the sea buckthorn extract comprises the purified sea buckthorn pomace oil, purified flavonoid extract, and sea buckthorn pomace dietary fiber described above; the mass ratio of the purified sea buckthorn pomace oil, purified flavonoid extract, and sea buckthorn pomace dietary fiber is 1:2:7.

[0026] This invention provides an application of the above-mentioned sea buckthorn extract in any one or more of the following a to c. a. Preparation of drugs to improve intestinal diseases; b. Preparation of drugs to promote the proliferation of probiotics; c. Increase the content of short-chain fatty acids produced by probiotics.

[0027] In this invention, when preparing a drug to promote the proliferation of probiotics, the probiotics include lactic acid bacteria and / or bifidobacteria; the short-chain fatty acid is one or more of acetic acid, propionic acid and butyric acid.

[0028] This invention separates and purifies multiple active ingredients from sea buckthorn pomace, improving the resource utilization rate of sea buckthorn and developing the utilization value of sea buckthorn pomace, which has great economic significance.

[0029] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] In the following embodiments, the acidic protease was purchased from the Xiasheng Food Flagship Store on the Taobao platform. The manufacturer was Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., and the product name was protease (acidic 537).

[0032] The cellulase was purchased from Xiasheng Enzyme Preparation Boutique on Taobao. The manufacturer is Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., and the product name is Cellulase (Plant Extract Special SPE-017).

[0033] The pectinase was purchased from the Xiasheng Food Flagship Store on Taobao. The manufacturer is Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., and the product name is pectinase.

[0034] The β-glucosidase was purchased from Shanghai Yuanye Biotechnology Co., Ltd., CAS No. 9001-22-3, LOT:D141S234825.

[0035] Example 1 A method for continuous extraction of multiple active ingredients from sea buckthorn pomace, comprising the following steps: (1) Extraction of palmitic acid from sea buckthorn pomace (S1) Crush the sea buckthorn pomace and pass it through a 50-mesh sieve to obtain sea buckthorn pomace powder; (S2) Take 2000g of sea buckthorn pomace powder and put it into a 5000mL extraction vessel. Under the conditions of extraction pressure of 28MPa, extraction temperature of 43℃, extraction flow rate of 18L / h and extraction time of 150min, supercritical CO2 extraction is carried out to collect sea buckthorn pomace oil and extracted sea buckthorn pomace. (S3) Mix urea with a 95% ethanol solution at a volume ratio of 1:2 to obtain a urea solution. Add the sea buckthorn pomace oil obtained in step (S2) to the urea solution, wherein the mass ratio of urea solution to sea buckthorn pomace oil is 3:1. Incorporate at -4℃ for 12 hours, filter and collect the filtrate to obtain the encapsulated sea buckthorn pomace oil. (S4) The encapsulated sea buckthorn pomace oil was purified by first distillation at a vacuum pressure of 0.1 Pa and a distillation temperature of 130 °C. The first condensed fraction was collected and purified by second distillation at a vacuum pressure of 0.1 Pa and a distillation temperature of 160 °C. The second condensed fraction was collected to obtain the purified sea buckthorn pomace oil.

[0036] In this embodiment, the palmitic acid content in sea buckthorn pomace oil, encapsulated sea buckthorn pomace oil, and purified sea buckthorn pomace oil was determined using the GB 5009.168 method.

[0037] In the seabuckthorn pomace oil obtained in step (S2), palmitic acid accounts for approximately 30-35% of the mass of the seabuckthorn pomace oil. In the encapsulated seabuckthorn pomace oil obtained in step (S3), palmitic acid accounts for approximately 45% of the mass of the encapsulated seabuckthorn pomace oil. In the purified seabuckthorn pomace oil obtained in step (S4), palmitic acid accounts for approximately 70% of the mass of the purified seabuckthorn pomace oil.

[0038] (2) Extraction of flavonoid aglycones from sea buckthorn pomace (A1) Mix 10000U of cellulase and 15000U of acidic protease, then add 10g of the extracted sea buckthorn pomace obtained in step (S2) and mix evenly. Adjust the pH to 4.8 and enzymatically hydrolyze at 50℃ for 2h to obtain the first enzymatic hydrolysis product. (A2) Mix 8000U of β-glucosidase and 10g of the first enzymatic hydrolysis product evenly, adjust the pH to 7.5, and enzymatically hydrolyze at 45℃ for 3h to obtain the second enzymatic hydrolysis product, which is the crude flavonoid extract. (A3) Mix the crude flavonoid extract with a 20% ethanol solution at a mass-volume ratio of 1g:40mL to obtain a crude flavonoid extract solution; 5g of AB-8 macroporous adsorption resin was packed into the chromatography column to obtain the packed chromatography column. 140 mL of crude flavonoid extract solution was poured into a packed chromatography column lined with a thin layer of absorbent cotton using a glass rod. After adsorption for 1 hour, the column was eluted with 0.5 column volumes of 20% ethanol solution, followed by 1.5 column volumes of 50% ethanol solution to remove impurities. Finally, it was eluted with 4 column volumes of anhydrous ethanol. The eluent obtained after elution with anhydrous ethanol for 60-120 minutes was collected to obtain the purified flavonoid extract. The flow rate for elution with 20% ethanol solution, 50% ethanol solution, and anhydrous ethanol was 2 column volumes / h.

[0039] High performance liquid chromatography (HPLC) was used to detect flavonoid aglycones in the purified flavonoid extract (refer to Wang Jianwei, Li Dailong, and Sun Junshe, “Study on the Enzymatic Conversion of Flavonoid Aglycones in Ginkgo Leaf Extract”, Food Industry Technology, 2008(4):3), and the conversion rate of flavonoid aglycones was calculated using the following formula.

[0040] The conversion rate of flavonoid aglycones = total content of flavonoid aglycones after conversion ÷ (content of flavonoid glycosides before conversion + content of flavonoid aglycones before conversion) × 100%.

[0041] The calculated conversion rate of flavonoid aglycones was 80%.

[0042] (3) Extraction of dietary fiber from sea buckthorn pomace (B1) Add 20,000 U of pectinase to 10 g of the crude flavonoid extract obtained in step (A2), adjust the pH to 3.5, and enzymatically hydrolyze at 50°C for 3 h to obtain the enzymatically hydrolyzed product. (B2) Add 50 mL of anhydrous ethanol to 5 g of the enzymatically hydrolyzed product, and precipitate the product by ultrasonication at 60 °C and 200 W for 45 min. Filter the product and collect the precipitate. The precipitate obtained is pectin polysaccharide. The pectin polysaccharide content was determined by the phenol-sulfuric acid method (calculated as galacturonic acid), and the pectin polysaccharide yield was calculated as: mass of dried pectin precipitate ÷ mass of product after enzymatic hydrolysis × 100%.

[0043] Calculations showed that the yield of pectin polysaccharides increased to 9.2%.

[0044] (B3) Extrusion modification process: The screw speed is 180 r / min, the temperature gradient is 80℃ (feeding zone) → 120℃ (extrusion zone), and the extrusion time is 4 min, so that the moisture content of the pectin polysaccharide material is 15%. Then it is passed through a 60 mesh sieve to obtain sea buckthorn fruit residue dietary fiber.

[0045] The water-holding capacity of sea buckthorn fruit residue dietary fiber was determined by room temperature static centrifugation and weighing method (Bai Xiaozhou, Zhang Yifei. "Influence of steam explosion on the properties of soluble dietary fiber in sea buckthorn fruit residue". Food Research and Development, (2019).). The water-holding capacity (WHC, unit g / g) was calculated by using the formula (W1-W)÷W, where W1 represents the mass of the precipitate after the sample is dried by centrifugation and W is the initial mass of the sea buckthorn fruit residue dietary fiber sample.

[0046] The sea buckthorn pomace prepared in this embodiment has a water-holding capacity of 9.15 g / g.

[0047] Example 2 A method for continuous extraction of multiple active ingredients from sea buckthorn pomace, comprising the following steps: (1) Extraction of palmitic acid from sea buckthorn pomace (S1) Crush the sea buckthorn pomace and pass it through a 40-mesh sieve to obtain sea buckthorn pomace powder; (S2) Take 2000g of sea buckthorn pomace powder and put it into a 5000mL extraction vessel. Under the conditions of extraction pressure of 30MPa, extraction temperature of 45℃, extraction flow rate of 15L / h and extraction time of 120min, supercritical CO2 extraction is carried out to collect sea buckthorn pomace oil and extracted sea buckthorn pomace. (S3) Mix urea with a 97% ethanol solution at a volume ratio of 1:2 to obtain a urea solution. Add the sea buckthorn pomace oil obtained in step (S2) to the urea solution, wherein the mass ratio of urea solution to sea buckthorn pomace oil is 4:1. Incorporate at -2℃ for 10 hours, filter and collect the filtrate to obtain the encapsulated sea buckthorn pomace oil. (S4) The encapsulated sea buckthorn pomace oil was purified by first distillation at a vacuum pressure of 0.1 Pa and a distillation temperature of 125 °C. The first condensed fraction was collected and purified by second distillation at a vacuum pressure of 0.1 Pa and a distillation temperature of 165 °C. The second condensed fraction was collected to obtain the purified sea buckthorn pomace oil.

[0048] (2) Extraction of flavonoid aglycones from sea buckthorn pomace (A1) Mix 8000U of cellulase and 12000U of acidic protease, then add 10g of the extracted sea buckthorn pomace obtained in step (S2) and mix evenly. Adjust the pH to 4.5 and enzymatically hydrolyze at 50℃ for 3h to obtain the first enzymatic hydrolysis product. (A2) Mix 6000U of β-glucosidase and 10g of the first enzymatic hydrolysis product evenly, adjust the pH to 7.5, and enzymatically hydrolyze at 40℃ for 4h to obtain the second enzymatic hydrolysis product, which is the crude flavonoid extract. (A3) Mix the crude flavonoid extract with a 20% ethanol solution at a mass-volume ratio of 1g:45mL to obtain a crude flavonoid extract solution; 10g of AB-8 macroporous adsorption resin was packed into the chromatography column to obtain the packed chromatography column. 140 mL of crude flavonoid extract solution was poured into a packed chromatography column lined with a thin layer of absorbent cotton using a glass rod. After adsorption for 1 h, the column was eluted with 0.5 column volumes of 15% ethanol solution, followed by 1.5 column volumes of 45% ethanol solution to remove impurities. Finally, it was eluted with 4 column volumes of anhydrous ethanol. The eluent obtained after elution with anhydrous ethanol for 60–120 min was collected to obtain the purified flavonoid extract. The flow rate for elution with 15% ethanol solution, 45% ethanol solution, and anhydrous ethanol was 2 column volumes / h.

[0049] (3) Extraction of dietary fiber from sea buckthorn pomace (B1) Add 18000U of pectinase to 10g of the crude flavonoid extract obtained in step (A2), adjust the pH to 4.0, and enzymatically hydrolyze at 55℃ for 4h to obtain the enzymatically hydrolyzed product. (B2) Add 40 mL of anhydrous ethanol to 5 g of the enzymatically hydrolyzed product, and sonicate at 65 °C and 250 W for 45 min. Filter and collect the precipitate, which is pectin polysaccharide.

[0050] (B3) Extrusion modification process: The screw speed is 180 r / min, the temperature gradient is 80℃ (feeding zone) → 120℃ (extrusion zone), the extrusion time is 2 min, and then it is passed through a 60 mesh sieve to obtain sea buckthorn fruit residue dietary fiber.

[0051] Example 3 A method for continuous extraction of multiple active ingredients from sea buckthorn pomace, comprising the following steps: (1) Extraction of palmitic acid from sea buckthorn pomace (S1) Crush the sea buckthorn pomace and pass it through a 60-mesh sieve to obtain sea buckthorn pomace powder; (S2) Take 2000g of sea buckthorn pomace powder and put it into a 5000mL extraction vessel. Under the conditions of extraction pressure of 25MPa, extraction temperature of 40℃, extraction flow rate of 20L / h and extraction time of 180min, supercritical CO2 extraction is carried out to collect sea buckthorn pomace oil and extracted sea buckthorn pomace. (S3) Mix urea with a 92% ethanol solution at a volume ratio of 1:2 to obtain a urea solution. Add the sea buckthorn pomace oil obtained in step (S2) to the urea solution, wherein the mass ratio of urea solution to sea buckthorn pomace oil is 2:1. Incorporate at -6℃ for 14 hours, filter and collect the filtrate to obtain the encapsulated sea buckthorn pomace oil. (S4) The encapsulated sea buckthorn pomace oil was purified by first distillation at a vacuum pressure of 0.1 Pa and a distillation temperature of 135 °C. The first condensed fraction was collected and purified by second distillation at a vacuum pressure of 0.1 Pa and a distillation temperature of 155 °C. The second condensed fraction was collected to obtain the purified sea buckthorn pomace oil.

[0052] (2) Extraction of flavonoid aglycones from sea buckthorn pomace (A1) Mix 12000U of cellulase and 17000U of acidic protease, then add 10g of the extracted sea buckthorn pomace obtained in step (S2) and mix evenly. Adjust the pH to 4.0 and enzymatically hydrolyze at 55℃ for 1h to obtain the first enzymatic hydrolysis product. (A2) Mix 10000U of β-glucosidase and 10g of the first enzymatic hydrolysis product evenly, adjust the pH to 8.0, and enzymatically hydrolyze at 50℃ for 2h to obtain the second enzymatic hydrolysis product, which is the crude flavonoid extract. (A3) Mix the crude flavonoid extract with a 15% ethanol solution at a mass-volume ratio of 1g:45mL to obtain a crude flavonoid extract solution; 8g of AB-8 macroporous adsorption resin was packed into the chromatography column to obtain the packed chromatography column. 140 mL of crude flavonoid extract solution was poured into a packed chromatography column lined with a thin layer of absorbent cotton using a glass rod. After adsorption for 1 h, the column was eluted with 0.5 column volumes of 25% ethanol solution, followed by 1.5 column volumes of 55% ethanol solution to remove impurities. Finally, it was eluted with 4 column volumes of anhydrous ethanol. The eluent obtained after elution with anhydrous ethanol for 60–120 min was collected to obtain the purified flavonoid extract. The flow rate for elution with 25% ethanol solution, 55% ethanol solution, and anhydrous ethanol was 2 column volumes / h.

[0053] (3) Extraction of dietary fiber from sea buckthorn pomace (B1) Add 22000U of pectinase to 10g of the crude flavonoid extract obtained in step (A2), adjust the pH to 3.0, and enzymatically hydrolyze at 45℃ for 2h to obtain the enzymatically hydrolyzed product. (B2) Add 60 mL of anhydrous ethanol to 5 g of the enzymatically hydrolyzed product, and sonicate at 55 °C and 150 W for 50 min. Filter and collect the precipitate, which is pectin polysaccharide.

[0054] (B3) Extrusion modification process: The screw speed is 180 r / min, the temperature gradient is 80℃ (feeding zone) → 120℃ (extrusion zone), the extrusion time is 6 min, and then it is passed through a 60 mesh sieve to obtain sea buckthorn fruit residue dietary fiber.

[0055] Example 4 A sea buckthorn extract with probiotic effects, wherein the sea buckthorn extract is prepared by mixing the purified sea buckthorn pomace oil prepared in Example 1, the purified flavonoid extract prepared in Example 1, and the sea buckthorn pomace dietary fiber prepared in Example 1 in a mass ratio of 1:2:7.

[0056] Experimental Example 1 The effects of purified sea buckthorn pomace oil, purified flavonoid extract, and sea buckthorn pomace dietary fiber and its complex prepared in Example 1 on the regulation of intestinal probiotics. 1. Experimental Samples Sample 1 is the purified sea buckthorn pomace oil prepared in step (1) of Example 1; Sample 2 is the purified flavonoid extract prepared in step (2) of Example 1; Sample 3 is the sea buckthorn pomace dietary fiber prepared in step (3) of Example 1; Sample 4 is a compound extract (also known as sea buckthorn extract), which is prepared by mixing purified sea buckthorn pomace oil obtained in Example 1, purified flavonoid extract and sea buckthorn pomace dietary fiber in a mass ratio of 1:2:7.

[0057] Positive sample: Inulin (purity ≥98%, conforming to GB / T 41377 standard).

[0058] Fecal donors: 6 healthy adult volunteers (BMI 18.5~23.9, no antibiotics or probiotics taken in the past month). Fresh fecal samples were collected in the early morning, transported under refrigeration at 4℃, and processed within 24 hours. The processing steps are as follows: Weigh 0.5g of fresh fecal sample and add it to a sterile centrifuge tube containing 5mL of sterile PBS to make the fecal concentration 10% (w / v) to obtain the initial bacterial suspension. Vortex at 2500rpm for 4min to obtain a well mixed bacterial suspension. Filter and collect the filtrate to obtain the fecal bacterial suspension.

[0059] Fermentation medium: 5 g / L tryptone, 5 g / L yeast extract, 2 g / L glucose and 0.5 g / L L-cysteine ​​hydrochloride, pH adjusted to 7.0±0.2, solvent is water.

[0060] 2. Experimental Grouping The experiment was randomly divided into 5 groups: blank control group (CK), positive control group (PC), fruit pomace oil treatment group (S1 group), flavonoid extract treatment group (S2 group), fruit pomace dietary fiber treatment group (S3 group), and compound extract treatment group (S4 group). The blank control group consisted of 45 mL of fermentation medium and 5 mL of fecal bacterial suspension, incubated at 37°C for 48 h in an anaerobic incubator; the positive control group consisted of 45 mL of fermentation medium, 5 mL of fecal bacterial suspension, and 0.5 g of inulin, incubated at 37°C for 48 h in an anaerobic incubator; the S1 group consisted of 45 mL of fermentation medium, 5 mL of fecal bacterial suspension, and 0.1 mg of purified sea buckthorn fruit pomace oil, incubated at 37°C for 48 h in an anaerobic incubator; and the S2 group consisted of... Group S3 consisted of a mixture of 45 mL fermentation medium, 5 mL fecal bacterial suspension, and 0.2 mg purified flavonoid extract, which was then incubated at 37°C in an anaerobic incubator for 48 h. Group S4 consisted of a mixture of 45 mL fermentation medium, 5 mL fecal bacterial suspension, and 0.7 mg sea buckthorn fruit residue dietary fiber, which was then incubated at 37°C in an anaerobic incubator for 48 h. After 48 h of incubation, the content of short-chain fatty acids (SCFAs) and the viable count of beneficial bacteria were determined in each group.

[0061] The determination of short-chain fatty acid (SCFA) content (gas chromatography) involves the following steps: (1) Sample pretreatment: Take 1 mL of fermentation broth, centrifuge at 8000 r / min for 10 min, take 0.5 mL of supernatant, add 0.1 mL of 25% metaphosphoric acid solution (Sigma), vortex mix, let stand at 4℃ for 30 min, centrifuge at 12000 r / min for 15 min, filter the supernatant through a 0.22 μm filter membrane to obtain filtrate; (2) Chromatographic conditions: Agilent 7890A gas chromatograph, HP-INNOWAX capillary column (30m×0.32mm×0.25μm), column temperature program: 40℃ for 3min, increase to 150℃ at 5℃ / min, hold for 5min; injection port temperature 200℃, FID detector temperature 250℃; carrier gas is nitrogen (purity 99.999%), flow rate 1mL / min, split ratio 10:1, injection volume 1μL; (3) Quantitative analysis: External standard method, prepare acetic acid, propionic acid and butyric acid standards (Sigma) concentration gradient (0.5, 1, 2, 5, 10 mg / mL), plot the standard curve, and calculate the content of each SCFA in the sample (unit: mg / mL).

[0062] The method for counting viable beneficial bacteria (plate count method) is as follows: Bifidobacterium count: Take 1 mL of fermentation broth and serially dilute with physiological saline to 10⁻⁶. -6 ~10 -8 Take 0.1 mL and spread it on BS medium (containing 50 μg / mL vancomycin), anaerobic culture at 37℃ for 48 h, count the typical red, round, and well-defined colonies, and express the results as log CFU / mL; Lactic acid bacteria count: The diluted solution was spread onto MRS medium (containing 0.05 g / L Bengal red), and anaerobically incubated at 37°C for 48 h. The typical red, pinpoint-shaped colonies were counted, and the results were expressed as log CFU / mL. Proliferation rate calculation: Proliferation rate (%) = (48h viable bacteria count - 0h viable bacteria count) / 0h viable bacteria count × 100%.

[0063] The results of the short-chain fatty acid content determination are shown in Table 1, and the results of the beneficial bacteria live count are shown in Table 2.

[0064] Table 1. Results of short-chain fatty acid content determination after different treatment groups

[0065] The results in Table 1 show that the total SCFAs content in group S4 was 151.9% higher than that in group CK, and the butyric acid content in group S4 was 212.6% higher than that in group CK, which was significantly higher than that in groups S1, S2, S3 and the positive control group, indicating that sea buckthorn extract has a synergistic effect in promoting intestinal bacterial fermentation and acid production.

[0066] Table 2. Results of beneficial bacteria proliferation after different treatment groups

[0067] The results in Table 2 show that the proliferation rates of Bifidobacterium and Lactobacillus in the S4 group were 584.8% and 507.2% higher than those in the CK group, respectively, and also higher than those in the positive control (PC group), indicating that the sea buckthorn extract of the present invention has the advantage of synergistically promoting the growth of beneficial bacteria.

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for continuous extraction of multiple active ingredients from sea buckthorn pomace, characterized in that, Includes the following steps: (1) The sea buckthorn pomace powder was subjected to supercritical CO2 extraction at an extraction pressure of 25~30 MPa, an extraction temperature of 40~45℃, an extraction flow rate of 15~20 L / h, and an extraction time of 120~180 min to collect the sea buckthorn pomace oil and the extracted sea buckthorn pomace. (2) The sea buckthorn pomace oil was encapsulated with urea solution, filtered, and the filtrate was collected to obtain the encapsulated sea buckthorn pomace oil. (3) The sea buckthorn pomace oil after encapsulation is purified by distillation, and the condensed fraction is collected to obtain purified sea buckthorn pomace oil; (4) Mix cellulase and protease, add the extracted sea buckthorn pomace obtained in step (1), perform the first enzymatic hydrolysis to obtain the first enzymatic hydrolysis product, then mix β-glucosidase and the first enzymatic hydrolysis product to perform the second enzymatic hydrolysis, and the obtained second enzymatic hydrolysis product is recorded as crude flavonoid extract. (5) The chromatography column is filled with macroporous adsorption resin to obtain the filled chromatography column; The crude flavonoid extract was mixed with an ethanol solution, and the resulting crude flavonoid extract solution was poured into a packed chromatography column for adsorption. Then, it was eluted sequentially with 15-25% ethanol solution, 45-55% ethanol solution and anhydrous ethanol. The eluent eluted with anhydrous ethanol for 120-160 min was collected to obtain the purified flavonoid extract. (6) Mix the crude flavonoid extract obtained in step (4) with pectinase and hydrolyze it to obtain the hydrolyzed product; (7) Mix the enzymatic hydrolysis product with anhydrous ethanol for alcohol precipitation, filter, and collect the precipitate; subject the precipitate to screw extrusion modification at a temperature of 110~130℃ and a screw speed of 160~200r / min for 2~6min to obtain sea buckthorn fruit residue dietary fiber.

2. The extraction method according to claim 1, characterized in that, The particle size of the sea buckthorn pomace powder is 40-60 mesh.

3. The extraction method according to claim 1, characterized in that, In step (2), the mass ratio of sea buckthorn pomace oil to urea solution is 2~4:1, the encapsulation time is -6~-2℃, and the encapsulation time is 10~14h; the solvent of the urea solution is an ethanol solution with a volume percentage of 92~97%.

4. The extraction method according to claim 1, characterized in that, In step (3), the temperature for distillation purification is 125~165℃.

5. The extraction method according to claim 1, characterized in that, The distillation purification process involves two distillations: a first distillation purification is performed at a distillation temperature of 125-135℃ to collect the first condensed fraction; then, the first condensed fraction is subjected to a second distillation purification at 155-165℃ to collect the second condensed fraction.

6. The extraction method according to claim 1, characterized in that, The amount of cellulase used is 800~1200 U / g, and the amount of protease used is 1200~1700 U / g; the temperature of the first enzymatic hydrolysis is 45~55℃, the time is 1~3h, and the pH of the first enzymatic hydrolysis is 4.5~5.

0. The amount of β-glucosidase used is 600~1000 U / g; the temperature of the second enzymatic hydrolysis is 40~50℃, the time is 2~4h, and the pH of the second enzymatic hydrolysis is 7.0~8.

0.

7. The extraction method according to claim 1, characterized in that, In step (5), the macroporous adsorption resin is of type AB-8, and the volume-to-mass ratio of the crude flavonoid extract solution to the amount of macroporous adsorption resin is 140 mL: 5~10 g. The mass-to-volume ratio of the crude flavonoid extract to the ethanol solution is 1g:35~45mL, and the volume percentage of the ethanol solution used to obtain the crude flavonoid extract solution is 15~25%. The adsorption time is 0.5~1.5h; When eluting with 15-25% ethanol solution, the volume used is 0.5 column volumes; when eluting with 45-55% ethanol solution (v / v), the volume used is 1.5 column volumes; when eluting with anhydrous ethanol, the volume used is 4 column volumes. The elution flow rate is 2 column volumes / h; In step (6), the amount of pectinase used is 1800~2200 U / g; the temperature of enzymatic hydrolysis is 45~55℃, the time is 2~4h, and the pH of enzymatic hydrolysis is 3.0~4.

0. In step (7), the mass-to-volume ratio of the enzymatically hydrolyzed product to the ethanol solution is 1g:8~12mL; the alcohol precipitation time is 40~50min; the alcohol precipitation temperature is 55~65℃; and the ultrasonic alcohol precipitation power is 150~250W.

8. A sea buckthorn extract with probiotic effects, characterized in that, The sea buckthorn extract is one or more of the following: the purified sea buckthorn pomace oil as described in claim 1, the purified flavonoid extract as described in claim 1, and the sea buckthorn pomace dietary fiber as described in claim 1.

9. The sea buckthorn extract according to claim 8, characterized in that, The seabuckthorn extract is the purified seabuckthorn pomace oil, the purified flavonoid extract, and the seabuckthorn pomace dietary fiber as described in claim 1; the mass ratio of the purified seabuckthorn pomace oil, the purified flavonoid extract, and the seabuckthorn pomace dietary fiber is 1:(1.5~2.5):(6~8).

10. The use of the sea buckthorn extract according to claim 8 or 9 in any one or more of the following a to c, a. Preparation of drugs to improve intestinal diseases; b. Preparation of drugs to promote the proliferation of probiotics; c. Increase the content of short-chain fatty acids produced by probiotics.

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