A preparation method of elderberry extract

Abstract

The invention discloses a method for preparing an elderberry extract, and relates to the technical field of plant extraction; the method comprises the following steps: S1, extracting an elderberry raw material by countercurrent extraction to obtain an extract; S2, concentrating the extract to obtain a concentrated solution; S3, mixing the concentrated solution, a glycosyl donor and an enzyme, and then glycosylating to obtain a glycosylated product; S4, passing the glycosylated product through a macroporous resin column, eluting with water and an ethanol aqueous solution with a volume fraction of 50% to 70% in sequence, and collecting the ethanol aqueous solution eluate; S5, microfiltration and nanofiltration of the ethanol aqueous solution eluate in sequence, collecting the filtrate; and concentrating the filtrate to dryness. The preparation method of the invention has simple steps and low cost; and an anthocyanin extract with a mass content of ≥65% is also prepared.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extraction, and specifically is a method for preparing an elderberry extract. Background Art

[0002] Elderberry (scientific name: Sambucus williamsii Hance) is a plant of the genus Sambucus in the family Caprifoliaceae. It is a traditional Chinese medicinal material. The fruit of elderberry is blue-purple-black in color, oval or nearly round in shape, and the fruiting period is from September to October. Elderberry grows on hillsides, bushes or roadsides. Its fruit contains a large amount of anthocyanidins and anthocyanins, which are active ingredients with antioxidant properties. Anthocyanins are water-soluble plant pigments that are widely present in the fruits, flowers, leaves, stems, roots and other organs of plants. They belong to the flavonoids and can appear red, purple-red and blue under different pH or metal ion conditions, thus giving plant tissues and organs colorful colors. Anthocyanins are unstable in nature in vitro and are easily degraded by temperature, pH, oxidants and metal ions. Elderberries contain a large amount of anthocyanins and anthocyanidins. The aglycones of anthocyanins are mostly cyanidins, and the fresh weight content of anthocyanins is 200mg~1000mg / 100g.

[0003] Flavonoid components in plants generally exist in the form of glycosides. Glycosylation is a reaction of late modification of natural products in the biosynthetic process. Enzyme-catalyzed biosynthesis has advantages such as high selectivity and high efficiency. In addition, complex reaction steps such as cumbersome protection and deprotection can be avoided. Moreover, the reaction conditions are mild and environmentally friendly. The obtained product has high purity and high yield, and is used more. At present, the most commonly used enzymes for enzyme-catalyzed biosynthesis of glycosides have two kinds: glycosyltransferase and glycoside synthetase. By adding specific glycosyltransferase and glycosyl donor to the extract containing aglycon to carry out glycosylation reaction, aglycon therein can be converted into glycoside, and the recovery rate of glycoside can be improved to a certain extent.

[0004] Elderberry pomace is the solid waste after the elderberry is squeezed to extract juice or oil. It contains cellulose, anthocyanins and other substances. It can be used as a stable, extensive, cheap and easy-to-obtain raw material for anthocyanin extraction. At present, the pomace has been used to prepare agricultural feed and extract anthocyanins. Studies have found that the content of anthocyanins in the pomace is 75% to 98% of the whole fruit content. Anthocyanin extraction includes solvent method, ultrasonic method, microwave method, high-voltage pulsed electric field method, negative pressure cavitation method, low eutectic solvent method, enzymatic method, supercritical CO2 method, etc.

[0005] CN113774100A discloses a method for extracting anthocyanins from plants, specifically disclosing that a liquid anthocyanin pigment product is prepared into a powdered product through acid-alcohol extraction, reduced pressure concentration, glycosylation reaction, two filtrations and then spraying. This preparation method can improve the solubility of the anthocyanin pigment product and increase the yield of the product.

[0006] CN113876821A discloses a method for extracting anthocyanins from roselle, specifically disclosing that after extracting anthocyanins with acid and alcohol, fat-soluble components such as volatile oil are removed by extraction, and a roselle anthocyanin-enriched extract is obtained by macroporous resin and MCX cationic double column chromatography.

[0007] CN113336813A discloses a method for preparing natural anthocyanins based on Nitraria tangutorum fruit extract, specifically discloses that enzymatic hydrolysis-acidified ethanol ultrasonic-assisted extraction can improve the cell wall breakage rate of Nitraria tangutorum fruit, release more anthocyanins, and improve the pigment extraction rate; resin adsorption and desorption can remove impurities such as metal ions and particulate matter in the solution; microfiltration, ultrafiltration and nanofiltration are used to concentrate and purify the extract, avoiding the damage to the quality of anthocyanins by concentration methods such as hot air drying; the concentration rate of the extract concentrated and purified by this method reaches more than 68.0%, and the color value of Nitraria tangutorum anthocyanins is increased by 35.2%.

[0008] CN112336665A discloses a method for preparing an elderberry extract, specifically disclosing that after acid-alcohol stirring extraction, the extract is filtered through a macroporous resin adsorption column, and the 55% ethanol eluate is collected, concentrated, and then dried to obtain an extract with anthocyanin content ≥36%.

[0009] CNCN105294631A discloses an industrial method for preparing anthocyanidins from elderberry fruits, specifically disclosing the use of high-voltage pulse electric field extraction equipment to extract anthocyanidins, and then using membrane equipment and macroporous adsorption resin to purify anthocyanidins to obtain a high-purity anthocyanidin product. This method can produce high-purity anthocyanidins, but requires high-voltage pulse electric field extraction equipment, which requires the purchase of new equipment and is costly.

[0010] It can be seen from the above records that the products prepared in the related technology are mostly concentrated elderberry juice, which is mostly obtained by directly squeezing the fresh fruit and concentrating the filtrate after collection. The anthocyanin content is low and the application range is limited.

[0011] Therefore, the present invention provides a method for preparing an elderberry extract to solve the problems raised in the above background technology. Summary of the invention

[0012] The object of the present invention is to provide a method for preparing an elderberry extract to solve at least one aspect of the problems and defects raised in the above-mentioned background technology.

[0013] Specifically, the present invention provides a method for preparing an elderberry extract, comprising the following steps:

[0014] S1, extracting elderberry residue by countercurrent extraction method to obtain an extract;

[0015] S2, concentrating the extract to obtain a concentrated solution;

[0016] S3, mixing the concentrated solution, the glycosyl donor and the enzyme and performing glycosylation to obtain a glycosylated product;

[0017] S4, passing the glycosylation product through a macroporous resin column, eluting with water and 50% to 70% ethanol aqueous solution by volume, and collecting the ethanol aqueous solution eluate;

[0018] S5, sequentially subjecting the ethanol aqueous solution eluate to microfiltration and nanofiltration, and collecting the filtrate;

[0019] Concentrating the filtrate to dryness;

[0020] The countercurrent extraction method in step S1 uses an acidic ethanol solution as an extractant;

[0021] The pH of the acidic ethanol solution is 3.5-5.

[0022] According to one technical solution of the preparation method technical solution of the present invention, at least the following beneficial effects are achieved:

[0023] The present invention adopts continuous countercurrent extraction, which greatly reduces the amount of extractant used and the processing time of the extractant, thereby shortening the extraction time and saving production costs.

[0024] The present invention converts anthocyanins into anthocyanins by enzymes and glycosyl donors, which simplifies the subsequent purification process by eluting all anthocyanin substances with similar properties with only one gradient, and greatly improves the yield of anthocyanins by 10% to 20%.

[0025] The present invention also uses macroporous resin, microfiltration and fine filtration to purify anthocyanin components, thereby improving the separation effect of flavonoid components; and uses a method combining microfiltration and ultrafiltration to remove impurities such as sugars and proteins, thereby further improving the purity of anthocyanins.

[0026] The preparation method of the invention has simple steps and low cost, and can also prepare anthocyanin extracts with a mass content of ≥65%.

[0027] According to some embodiments of the invention, the elderberry raw material is elderberry pomace.

[0028] According to some embodiments of the present invention, the method for preparing the elderberry residue comprises the following steps:

[0029] After squeezing elderberry juice, the solid and liquid are separated, and the solid phase is collected; and the solid phase is dried to obtain the product.

[0030] According to some embodiments of the invention, the elderberry is fresh elderberry.

[0031] According to some embodiments of the present invention, the drying temperature is 45°C to 55°C.

[0032] The invention uses elderberry residue after juicing as a raw material and adopts a low-temperature drying method to prepare the elderberry residue, which is convenient for storage and transportation, and can prevent anthocyanins from being decomposed by heat, thereby improving the extraction rate of anthocyanins.

[0033] According to some embodiments of the present invention, the drying time is 8 hours to 24 hours.

[0034] According to some embodiments of the present invention, the drying is forced air drying.

[0035] According to some embodiments of the present invention, the wind speed of the blast drying is 0.5 m / s to 2 m / s.

[0036] According to some embodiments of the present invention, the drying is hot air drying or heat pump drying.

[0037] According to some embodiments of the present invention, the solid phase is sieved after drying.

[0038] According to some embodiments of the present invention, the sieving is through a 10-30 mesh sieve.

[0039] According to some embodiments of the present invention, the average particle size of the elderberry raw material is 0.6 mm to 2 mm.

[0040] According to some embodiments of the present invention, the volume fraction of ethanol in the acidic ethanol solution is 30% to 50%.

[0041] According to some embodiments of the present invention, the pH of the acidic ethanol solution is adjusted using acid.

[0042] According to some embodiments of the invention, the acid is citric acid, acetic acid and hydrogen chloride.

[0043] According to some embodiments of the present invention, the countercurrent extraction method is performed for 3 to 5 hours.

[0044] According to some embodiments of the present invention, the temperature of the countercurrent extraction method is 40°C to 55°C.

[0045] According to some embodiments of the present invention, the mass volume ratio of the elderberry raw material to the acidic ethanol solution is 1 g: 8 mL to 12 mL.

[0046] According to some embodiments of the present invention, the concentration temperature in step S2 is 40°C to 55°C.

[0047] According to some embodiments of the present invention, the pH of the concentrated solution is 5-6.

[0048] The present invention adjusts the pH value during the concentration process to prevent the degradation of anthocyanins caused by excessive acidity and the increase of subsequent glycosylation costs.

[0049] According to some embodiments of the present invention, the volume fraction of ethanol in the concentrated solution is below 10%.

[0050] According to some embodiments of the present invention, the pH of the concentrated solution is adjusted using an alkaline substance.

[0051] According to some embodiments of the invention, the alkaline substance comprises ammonia monohydrate, bicarbonate and a strong base.

[0052] According to some embodiments of the invention, the bicarbonate comprises at least one of sodium bicarbonate and potassium bicarbonate.

[0053] According to some embodiments of the invention, the strong base comprises at least one of sodium hydroxide and potassium hydroxide.

[0054] According to some embodiments of the present invention, the glycosyl donor is at least one of dextrin, starch and glucose.

[0055] According to some embodiments of the invention, the enzyme is at least one of cyclodextrin glucose transferase and α-glucoside hydrolase.

[0056] According to some embodiments of the present invention, the glycosylation temperature is 50°C to 60°C.

[0057] According to some embodiments of the present invention, the glycosylation time is 8 h to 12 h.

[0058] According to some embodiments of the present invention, the mass ratio of the enzyme to the elderberry raw material is 0.5 to 1:1000.

[0059] According to some embodiments of the present invention, the mass ratio of the glycosyl donor to the elderberry raw material is 1:100-500.

[0060] According to some embodiments of the present invention, the enzyme is inactivated after glycosylation.

[0061] According to some embodiments of the present invention, the temperature for inactivating the enzyme is 95°C to 100°C.

[0062] According to some embodiments of the present invention, the enzyme inactivation time is 10 min to 20 min.

[0063] According to some embodiments of the present invention, the macroporous resin column is one of a D101 column, a HP-5 column and an AB-8 column.

[0064] According to some embodiments of the present invention, the diameter-to-column ratio of the macroporous resin column is 1:8-10.

[0065] According to some embodiments of the present invention, the loading concentration of the macroporous resin column is 100 mg / mL to 200 mg / mL.

[0066] According to some embodiments of the present invention, the loading volume of the macroporous resin column is 3BV to 5BV.

[0067] According to some embodiments of the present invention, the loading flow rate of the macroporous resin column is 0.8 BV / h to 1.5 BV / h.

[0068] According to some embodiments of the present invention, the amount of water used for elution in step S5 is 1 BV to 3 BV.

[0069] According to some embodiments of the present invention, the water elution flow rate in step S5 is 1 BV / h to 2 BV / h.

[0070] According to some embodiments of the present invention, the amount of the ethanol aqueous solution with a volume fraction of 50% to 70% in step S5 is 2BV to 4BV.

[0071] According to some embodiments of the present invention, the elution flow rate of the ethanol aqueous solution with a volume fraction of 50% to 70% in step S5 is 0.8 BV / h to 2 BV / h.

[0072] According to some embodiments of the present invention, the microfiltration uses a filter membrane with a pore size of 0.3 μm to 0.5 μm.

[0073] According to some embodiments of the present invention, the filter membrane selected for the microfiltration is one of a polysulfone membrane, a polyacrylonitrile membrane and a polyvinyl chloride membrane.

[0074] According to some embodiments of the present invention, the temperature of the microfiltration is 30°C to 45°C.

[0075] According to some embodiments of the present invention, the pressure of the microfiltration is 1 bar to 3 bar.

[0076] According to some embodiments of the present invention, the ultrafiltration uses a filter membrane with a pore size of 2nm to 10nm.

[0077] According to some embodiments of the present invention, the filter membrane selected for ultrafiltration is one of a polyamide membrane, a sulfonated polysulfone membrane and a polysulfone membrane.

[0078] According to some embodiments of the present invention, the ultrafiltration temperature is 30°C to 45°C.

[0079] According to some embodiments of the present invention, the ultrafiltration pressure is 0.1 MPa to 0.8 MPa.

[0080] According to some embodiments of the present invention, the flow rate of the ultrafiltration is 1.5 m / s to 2 m / s.

[0081] According to some embodiments of the present invention, the concentration temperature in step S5 is 40°C to 55°C. DETAILED DESCRIPTION

[0082] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0083] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0084] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0085] The detection method of total anthocyanins in the embodiment of the present invention is as follows:

[0086] SW / T2-2013 Determination of total anthocyanins in cranberry extract from plant extracts.

[0087] In the embodiment of the present invention, the cyclodextrin glucosyltransferase was purchased from Xi'an Longmao Biotechnology Co., Ltd.; model: food grade.

[0088] In the embodiment of the present invention, α-glucoside hydrolase was purchased from Anhui Yinghe Biotechnology Co., Ltd.; model: food grade.

[0089] Example 1

[0090] This embodiment is a method for preparing an elderberry extract, which comprises the following steps:

[0091] S1. Preparation of elderberry raw materials:

[0092] After squeezing fresh elderberry juice, separate the solid and liquid and collect the solid phase;

[0093] The solid phase was dried in a blast dryer (drying temperature was 45° C., drying time was 24 h), then crushed and sieved (10 meshes), to obtain an elderberry raw material (the total anthocyanin mass content was 4.56% as determined by HPLC);

[0094] S2, continuous countercurrent extraction:

[0095] The elderberry raw material (5 kg) obtained in step S1 is subjected to continuous countercurrent extraction in a continuous countercurrent extraction device (the extractant is an acidic ethanol aqueous solution with a volume fraction of 30% (pH is 3.5, acetic acid is selected for pH adjustment); the amount of the extractant is 40 L; the extraction temperature is 55° C., and the extraction time is 3 h), and the extract is collected;

[0096] S3, Concentration:

[0097] The extract obtained in step S2 is concentrated under reduced pressure at 55° C. (the volume after concentration is 1 / 3 of the volume of the extract), and concentrated ammonia water is used to adjust the pH during the concentration process (the pH is controlled between 5 and 6) to obtain a concentrated solution;

[0098] S4, Glycosylation:

[0099] The concentrate, cyclodextrin (cyclodextrin dosage is 10 g, cyclodextrin is heated and dissolved in 50 mL of water and then added) and cyclodextrin glucosyltransferase (5 g) are mixed and glycosylated (glycosylation temperature is 60° C., glycosylation time is 8 h). After glycosylation is completed, the enzyme is inactivated at 95° C. for 20 min, and the glycosylation solution is obtained after cooling.

[0100] S5, elution:

[0101] The glycosylation solution prepared in step S4 was passed through a D101 macroporous resin (the loading flow rate of the glycosylation solution was 0.8 BV / h, the loading amount of the glycosylation solution was 3 BV, and the diameter-to-column ratio of the macroporous resin was 1:6). After the loading was completed, water (the amount of water was 1 BV, the flow rate was 1 BV / h) and ethanol aqueous solution (volume fraction was 50%, the amount was 2 BV, and the flow rate was 1 BV / h) were used for elution in sequence, and the ethanol aqueous solution eluate was collected;

[0102] S6, microfiltration and ultrafiltration:

[0103] The ethanol aqueous solution eluate obtained in step S5 is subjected to microfiltration (temperature 30° C., pressure 3 bar, filter membrane is a polysulfone microfiltration membrane (pore size 0.5 μm)), and the microfiltrate is collected;

[0104] The microfiltrate was ultrafiltered (temperature 30°C, pressure 0.8 MPa, flow rate 1.5 m / s, filter membrane polyamide ultrafiltration membrane (pore size 2 nm)) to obtain an ultrafiltrate;

[0105] The ultrafiltrate was concentrated to dryness (the concentration temperature was 55° C.) to obtain an elderberry extract.

[0106] The weight of the elderberry extract in this example is 348.61 g, and the color is purple-red.

[0107] The mass content of anthocyanins in the extract in this example is 68.98% (measured as cyanidin-3-glucoside by HPLC), the recovery rate is 105.47%, and the improvement is 20.07% (the reason for the improvement is that anthocyanins in the extract are converted into anthocyanins through glycosylation, and the recovery rate of the untransformed anthocyanins in Comparative Example 1 is 85.40%).

[0108] Example 2

[0109] This embodiment is a method for preparing an elderberry extract, which comprises the following steps:

[0110] S1. Preparation of elderberry raw materials:

[0111] After squeezing fresh elderberry juice, separate the solid and liquid and collect the solid phase;

[0112] The solid phase was dried in a blast dryer (drying temperature was 55° C., drying time was 8 h), then crushed and sieved (20 meshes), to obtain an elderberry raw material (the total anthocyanin mass content was 5.07% as determined by HPLC);

[0113] S2, continuous countercurrent extraction:

[0114] The elderberry raw material (100 kg) obtained in step S1 is subjected to continuous countercurrent extraction in a continuous countercurrent extraction device (the extractant is an acidic ethanol aqueous solution with a volume fraction of 50% (pH is 5, citric acid is selected for pH adjustment); the amount of the extractant is 1000 L; the extraction temperature is 40° C., and the extraction time is 5 h), and the extract is collected;

[0115] S3, Concentration:

[0116] The extract obtained in step S2 is concentrated under reduced pressure at 55° C. (the volume after concentration is 1 / 5 of the volume of the extract), and the pH is adjusted with sodium hydroxide solution during the concentration process (the pH is controlled between 5 and 6) to obtain a concentrated solution;

[0117] S4, Glycosylation:

[0118] The concentrate, dextrin (the amount of dextrin is 1 kg, and UPD-glucose dextrin is added after being heated and dissolved in 5 L of water) and cyclodextrin glucosyltransferase (the amount is 50 g) are mixed and glycosylated (the glycosylation temperature is 50° C., and the glycosylation time is 12 h). After the glycosylation is completed, the enzyme is inactivated at 100° C. for 10 min, and the glycosylation solution is obtained after cooling.

[0119] S5, elution:

[0120] The glycosylation solution prepared in step S4 was passed through an HP-5 macroporous resin (the loading flow rate of the glycosylation solution was 1 BV / h, the loading amount of the glycosylation solution was 5 BV, and the diameter-to-column ratio of the macroporous resin was 1:8). After the loading was completed, water (the amount of water was 3 BV, the flow rate was 1 BV / h) and ethanol aqueous solution (volume fraction was 70%, the amount was 4 BV, and the flow rate was 1 BV / h) were used for elution in sequence, and the ethanol aqueous solution eluate was collected;

[0121] S6, microfiltration and ultrafiltration:

[0122] The ethanol aqueous solution eluate obtained in step S5 is subjected to microfiltration (temperature 45° C., pressure 1 bar, filter membrane is a polyvinyl chloride microfiltration membrane (pore size 0.3 μm)), and the microfiltrate is collected;

[0123] The microfiltrate was ultrafiltered (temperature 45°C, pressure 0.1 MPa, flow rate 2 m / s, filter membrane was sulfonated polysulfone ultrafiltration membrane (pore size 10 nm)) to obtain an ultrafiltrate;

[0124] The ultrafiltrate was concentrated to dryness (the concentration temperature was 50° C.) to obtain an elderberry extract.

[0125] The weight of the elderberry extract in this example is 7.62 kg and the color is purple-red.

[0126] The mass content of anthocyanins in the extract of this embodiment is 66.97% (measured as cyanidin-3-glucoside by HPLC), the recovery rate is 100.67%, and the improvement is 15.27%.

[0127] Example 3

[0128] This embodiment is a method for preparing an elderberry extract, which comprises the following steps:

[0129] S1. Preparation of elderberry raw materials:

[0130] After squeezing fresh elderberry juice, separate the solid and liquid and collect the solid phase;

[0131] The solid phase was dried in a blast dryer (drying temperature was 50° C., drying time was 12 h), and then crushed and sieved (30 meshes) to obtain an elderberry raw material (the total anthocyanin mass content was 4.95% as determined by HPLC);

[0132] S2, continuous countercurrent extraction:

[0133] The elderberry raw material (500 kg) obtained in step S1 is subjected to continuous countercurrent extraction in a continuous countercurrent extraction device (the extractant is an acidic ethanol aqueous solution with a volume fraction of 40% (pH is 4, and hydrochloric acid is used for pH adjustment); the amount of the extractant is 6000 L; the extraction temperature is 50° C., and the extraction time is 4 h), and the extract is collected;

[0134] S3, Concentration:

[0135] The extract obtained in step S2 is concentrated under reduced pressure at 50° C. (the volume after concentration is 1 / 4 of the volume of the extract), and the pH is adjusted with a sodium bicarbonate solution during the concentration process (the pH is controlled between 5 and 6) to obtain a concentrated solution;

[0136] S4, Glycosylation:

[0137] The concentrate, glucose (the amount of glucose is 1.67 kg, and the glucose is heated and dissolved in 16.7 L of water and then added) and α-glucosidase (the amount is 400 g) are mixed and then glycosylated (the glycosylation temperature is 55° C., and the glycosylation time is 10 h). After the glycosylation is completed, the enzyme is inactivated at 95° C. for 20 min, and the glycosylation solution is obtained after cooling;

[0138] S5, elution:

[0139] The glycosylation solution prepared in step S4 was passed through an AB-8 macroporous resin (the loading flow rate of the glycosylation solution was 1 BV / h, the loading amount of the glycosylation solution was 4 BV, and the diameter-to-column ratio of the macroporous resin was 1:10). After the loading was completed, water (the amount of water was 1 BV, the flow rate was 1 BV / h) and ethanol aqueous solution (volume fraction was 60%, the amount was 3 BV, and the flow rate was 1 BV / h) were used for elution in sequence, and the ethanol aqueous solution eluate was collected;

[0140] S6, microfiltration and ultrafiltration:

[0141] The ethanol aqueous solution eluate obtained in step S5 is subjected to microfiltration (temperature 40° C., pressure 2 bar, filter membrane is polyacrylonitrile microfiltration membrane (pore size 0.4 μm)), and the microfiltrate is collected;

[0142] The microfiltrate was ultrafiltered (temperature 40°C, pressure 0.5 MPa, flow rate 2 m / s, filter membrane was polyamide ultrafiltration membrane (pore size 8 nm)) to obtain ultrafiltrate;

[0143] The ultrafiltrate was concentrated to dryness (the concentration temperature was 50° C.) to obtain an elderberry extract.

[0144] The weight of the elderberry extract in this example is 33.83 kg, and the color is purple-red.

[0145] The mass content of anthocyanins in the extract of this embodiment is 70.28% (measured by HPLC, calculated as cyanidin-3-glucoside), the recovery rate is 96.05%, and the improvement amount is 10.65%.

[0146] Comparative Example 1

[0147] This comparative example is a method for preparing an elderberry extract, which comprises the following steps:

[0148] S1. Preparation of elderberry raw materials:

[0149] After squeezing fresh elderberry juice, separate the solid and liquid and collect the solid phase;

[0150] The solid phase was dried in a blast dryer (drying temperature was 45° C., drying time was 24 h), then crushed and sieved (10 meshes), to obtain an elderberry raw material (the total anthocyanin mass content was 4.56% as determined by HPLC);

[0151] S2, continuous countercurrent extraction:

[0152] The elderberry raw material (5 kg) obtained in step S1 is subjected to continuous countercurrent extraction in a continuous countercurrent extraction device (the extractant is an acidic ethanol aqueous solution with a volume fraction of 30% (pH is 3.5, acetic acid is selected for pH adjustment); the amount of the extractant is 40 L; the extraction temperature is 55° C., and the extraction time is 3 h), and the extract is collected;

[0153] S3, Concentration:

[0154] The extract obtained in step S2 is concentrated under reduced pressure at 55° C. (the volume after concentration is 1 / 3 of the volume of the extract) to obtain a concentrated solution;

[0155] S4, elution:

[0156] The concentrated solution obtained in step S3 was passed through a D101 macroporous resin (the sample loading flow rate of the concentrated solution was 0.8 BV / h, the sample loading amount of the concentrated solution was 3 BV, and the diameter-to-column ratio of the macroporous resin was 1:6). After the sample loading was completed, water (the amount of water was 1 BV, the flow rate was 1 BV / h) and ethanol aqueous solution (volume fraction was 50%, the amount was 2 BV, and the flow rate was 1 BV / h) were used for elution in sequence, and the ethanol aqueous solution eluate was collected;

[0157] S5, microfiltration and ultrafiltration:

[0158] The ethanol aqueous solution eluate obtained in step S4 is subjected to microfiltration (temperature 30° C., pressure 3 bar, filter membrane is a polysulfone microfiltration membrane (pore size 0.5 μm)), and the microfiltrate is collected;

[0159] The microfiltrate was ultrafiltered (temperature 30°C, pressure 0.8 MPa, flow rate 1.5 m / s, filter membrane polyamide ultrafiltration membrane (pore size 2 nm)) to obtain an ultrafiltrate;

[0160] The ultrafiltrate was concentrated to dryness (the concentration temperature was 55° C.) to obtain an elderberry extract.

[0161] The weight of the elderberry extract in this comparative example is 296.5 g, and the color is purple-red.

[0162] The mass content of anthocyanins in the extract of this comparative example is 65.67% (measured by HPLC, calculated as cyanidin-3-glucoside), and the recovery rate is 85.40%.

[0163] Comparative Example 2

[0164] This comparative example is a method for preparing an elderberry extract, which comprises the following steps:

[0165] S1. Preparation of elderberry raw materials:

[0166] After squeezing fresh elderberry juice, separate the solid and liquid and collect the solid phase;

[0167] The solid phase was dried in a blast dryer (drying temperature was 45° C., drying time was 24 h), then crushed and sieved (10 meshes), to obtain an elderberry raw material (the total anthocyanin mass content was 4.56% as determined by HPLC);

[0168] S2, continuous countercurrent extraction:

[0169] The elderberry raw material (5 kg) obtained in step S1 is subjected to continuous countercurrent extraction in a continuous countercurrent extraction device (the extractant is an acidic ethanol aqueous solution with a volume fraction of 30% (pH is 3.5, acetic acid is selected for pH adjustment); the amount of the extractant is 40 L; the extraction temperature is 55° C., and the extraction time is 3 h), and the extract is collected;

[0170] S3, Concentration:

[0171] The extract obtained in step S2 is concentrated under reduced pressure at 55° C. (the volume after concentration is 1 / 3 of the volume of the extract) to obtain a concentrated solution;

[0172] S4, Glycosylation:

[0173] The concentrate, cyclodextrin (cyclodextrin dosage is 10 g, cyclodextrin is heated and dissolved in 50 mL of water and then added) and cyclodextrin transferase (5 g) are mixed and glycosylated (glycosylation temperature is 60° C., glycosylation time is 8 h). After the glycosylation is completed, the enzyme is inactivated at 95° C. for 20 min, and the glycosylation solution is obtained after cooling.

[0174] S5, elution:

[0175] The glycosylation solution prepared in step S4 was passed through a D101 macroporous resin (the loading flow rate of the glycosylation solution was 0.8 BV / h, the loading amount of the glycosylation solution was 3 BV, and the diameter-to-column ratio of the macroporous resin was 1:6). After the loading was completed, water (the amount of water was 1 BV, the flow rate was 1 BV / h) and ethanol aqueous solution (volume fraction was 50%, the amount was 2 BV, and the flow rate was 1 BV / h) were used for elution in sequence, and the ethanol aqueous solution eluate was collected;

[0176] S6, microfiltration and ultrafiltration:

[0177] The ethanol aqueous solution eluate obtained in step S5 is subjected to microfiltration (temperature 30° C., pressure 3 bar, filter membrane is a polysulfone microfiltration membrane (pore size 0.5 μm)), and the microfiltrate is collected;

[0178] The microfiltrate was ultrafiltered (temperature 30°C, pressure 0.8 MPa, flow rate 1.5 m / s, filter membrane polyamide ultrafiltration membrane (pore size 2 nm)) to obtain an ultrafiltrate;

[0179] The ultrafiltrate was concentrated to dryness (the concentration temperature was 55° C.) to obtain an elderberry extract.

[0180] The weight of the elderberry extract in this comparative example is 321.52 g, and the color is purple-red.

[0181] The mass content of anthocyanin in the extract of this comparative example is 63.47% (measured by HPLC, calculated as cyanidin-3-glucoside), and the recovery rate is 89.50%.

[0182] Comparative Example 3

[0183] This comparative example is a method for preparing an elderberry extract, which comprises the following steps:

[0184] S1. Preparation of elderberry raw materials:

[0185] After squeezing fresh elderberry juice, separate the solid and liquid and collect the solid phase;

[0186] The solid phase was dried in a blast dryer (drying temperature was 45° C., drying time was 24 h), then crushed and sieved (10 meshes), to obtain an elderberry raw material (the total anthocyanin mass content was 4.56% as determined by HPLC);

[0187] S2, continuous countercurrent extraction:

[0188] The elderberry raw material (5 kg) obtained in step S1 is subjected to continuous countercurrent extraction in a continuous countercurrent extraction device (the extractant is an acidic ethanol aqueous solution with a volume fraction of 30% (pH is 3.5, acetic acid is selected for pH adjustment); the amount of the extractant is 40 L; the extraction temperature is 55° C., and the extraction time is 3 h), and the extract is collected;

[0189] S3, Concentration:

[0190] The extract obtained in step S2 is concentrated under reduced pressure at 55° C. (the volume after concentration is 1 / 3 of the volume of the extract), and concentrated ammonia water is used to adjust the pH during the concentration process (the pH is controlled between 5 and 6) to obtain a concentrated solution;

[0191] S4, Glycosylation:

[0192] The concentrate, cyclodextrin (cyclodextrin dosage is 10 g, cyclodextrin is heated and dissolved in 50 mL of water and then added) and cyclodextrin transferase (5 g) are mixed and glycosylated (glycosylation temperature is 60° C., glycosylation time is 8 h). After the glycosylation is completed, the enzyme is inactivated at 95° C. for 20 min, and the glycosylation solution is obtained after cooling.

[0193] S5, elution:

[0194] The glycosylation solution prepared in step S4 was passed through a D101 macroporous resin (the loading flow rate of the glycosylation solution was 0.8 BV / h, the loading amount of the glycosylation solution was 3 BV, and the diameter-to-column ratio of the macroporous resin was 1:6). After the loading was completed, water (the amount of water was 1 BV, the flow rate was 1 BV / h) and ethanol aqueous solution (volume fraction was 50%, the amount was 2 BV, and the flow rate was 1 BV / h) were used for elution in sequence, and the ethanol aqueous solution eluate was collected;

[0195] S6, microfiltration and ultrafiltration:

[0196] The ethanol aqueous solution eluate obtained in step S5 is concentrated to dryness (the concentration temperature is 55° C.) to obtain an elderberry extract.

[0197] The weight of the elderberry extract in this comparative example is 401.51 g, and the color is purple-red.

[0198] The mass content of anthocyanins in the extract of this comparative example is 43.01% (measured as cyanidin-3-glucoside by HPLC), and the recovery rate is 75.74%.

[0199] In summary, the present invention uses elderberry residue after juicing as raw material and adopts low-temperature drying to prepare elderberry residue. On the one hand, it is convenient for storage and transportation, and on the other hand, it can prevent anthocyanins from being decomposed by heat, thereby improving the extraction rate of anthocyanins; the continuous countercurrent extraction is adopted, which greatly reduces the amount of solvent used, reduces the solvent treatment time, thereby shortening the process time and saving costs. The present invention adjusts the pH value during the concentration process of the acid-alcohol extract to prevent excessive acidity from causing the degradation of anthocyanins and causing an increase in subsequent glycosylation costs. The present invention promotes the conversion of anthocyanins into anthocyanins by glycosylation. On the one hand, it simplifies the subsequent purification process, and only one gradient is needed to elute all anthocyanin substances with similar properties. On the other hand, it greatly improves the yield of anthocyanins, and the recovery rate of anthocyanins is increased by 10% to 20%. The present invention adopts macroporous resin and membrane separation method to purify anthocyanin components (the macroporous resin is regenerated and used), has a good separation effect on flavonoid components, and adopts microfiltration and ultrafiltration to remove impurities such as sugars and proteins, further improving the purity of anthocyanin. The steps are simple and the cost is low, and anthocyanin extracts with a total content of ≥65% are prepared.

[0200] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an elderberry extract, characterized in that: The following steps are involved: S1, extracting the elderberry raw material by countercurrent extraction method to obtain an extract; S2, concentrating the extract to obtain a concentrated solution; S3, mixing the concentrated solution, the glycosyl donor and the enzyme and performing glycosylation to obtain a glycosylated product; S4, passing the glycosylation product through a macroporous resin column, eluting with water and 50% to 70% ethanol aqueous solution by volume, and collecting the ethanol aqueous solution eluate; S5, sequentially subjecting the ethanol aqueous solution eluate to microfiltration and ultrafiltration, and collecting the filtrate; Concentrating the filtrate to dryness; The countercurrent extraction method in step S1 uses an acidic ethanol solution as an extractant; The pH of the acidic ethanol solution is 3.5-5; the volume fraction of ethanol in the acidic ethanol solution is 30%-50%; The macroporous resin column is one of a D101 column, an HP-5 column and an AB-8 column; The enzyme is at least one of cyclodextrin glucose transferase and α-glucosidase; The pH of the concentrated solution is 5-6.

2. The method for preparing the elderberry extract according to claim 1, characterized in that: The glycosyl donor is at least one of dextrin, starch and glucose.

3. The method for preparing the elderberry extract according to claim 1, characterized in that: The microfiltration uses a filter membrane with a pore size of 0.3μm~0.5μm.

4. The method for preparing the elderberry extract according to claim 3, characterized in that: The filter membrane selected for microfiltration is one of polysulfone membrane, polyacrylonitrile membrane and polyvinyl chloride membrane.

5. The method for preparing the elderberry extract according to claim 1, characterized in that: The ultrafiltration uses a filter membrane with a pore size of 2nm~10nm.

6. The method for preparing the elderberry extract according to claim 5, characterized in that: The filter membrane selected for ultrafiltration is one of a polyamide membrane, a sulfonated polysulfone membrane and a polysulfone membrane.