CN113336734B - Method for extracting procyanidine from aronia melanocarpa fruits - Google Patents
Method for extracting procyanidine from aronia melanocarpa fruits Download PDFInfo
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- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
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- C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
- C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
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Abstract
The invention provides a method for extracting procyanidine from aronia melanocarpa fruits. The method comprises the following steps: A) mashing Aronia melanocarpa fruit, and filtering to obtain fruit juice and fruit residue; B) fermenting the pomace by adopting aspergillus niger, trichoderma viride and bacillus licheniformis to obtain pomace fermentation liquor; C) mixing the fruit juice and the pomace fermentation liquor to obtain a mixed liquor, and carrying out enzymolysis on the mixed liquor by adopting pectinase and papain to obtain an enzymolysis liquid; D) separating and purifying the enzymolysis liquid to obtain the procyanidin extract. The method can degrade the cell wall of the pomace to be beneficial to dissolving out the procyanidine, so that the procyanidine in the pomace is extracted to the maximum extent, the whole process condition is mild, the required equipment is simple, and large-scale and industrialized production can be realized; meanwhile, the method can utilize the waste of the aronia melanocarpa residues, promote the development of the deep processing industry of the aronia melanocarpa and improve the additional value of the aronia melanocarpa.
Description
Technical Field
The invention relates to the technical field of extraction of plant active ingredients, in particular to a method for extracting procyanidine from aronia melanocarpa fruits.
Background
Procyanidins (OPC) are a class of polyphenolic compounds that occur widely in nature. Researches show that the procyanidine has strong antioxidation, can eliminate free radicals, and has multiple effects of preventing and treating cardiovascular diseases, resisting radiation, regulating immunity and the like in recent years. At present, procyanidins are widely used in the fields of foods, medicines, cosmetics and the like as nutrition enhancers, natural antioxidants, natural preservatives and the like.
The aronia melanocarpa is also named as cherokee rose and aronia melanocarpa, is native to North America, is planted in provinces and markets such as northeast, inner Mongolia, Gansu and the like of China at present, and is rosaceous deciduous shrub integrating edible, medicinal, garden and ecological values. The aronia melanocarpa fruit is rich in flavone, anthocyanin, procyanidine, polyphenol and other substances, and is announced by Weijian in 2018 as a new food raw material. The related data show that the anthocyanin and the flavone (the content of the fresh fruit is as high as 0.25-0.35 percent) in the aronia melanocarpa fruit are the highest in the polyphenol in the known plants, and the content of the procyanidine is 2.5 times of that of the blueberry. The aronia melanocarpa is a preferred raw material for extracting procyanidine, the pomace obtained after juice is squeezed is still rich in procyanidine, the waste materials are utilized, the problems of pollution caused by waste residue discharge and low utilization rate can be effectively solved, the development of the deep processing industry of the aronia melanocarpa is promoted, and the additional value of the aronia melanocarpa is increased.
The existing procyanidin extraction technology can be divided into water extraction method, organic solvent extraction method, microwave extraction method, ultrasonic extraction method, and supercritical CO extraction method2Extraction and extraction methods, etc. The water extraction method has high extraction temperature and long extraction time, and is easy to cause the loss of the procyanidine; the organic solvent extraction has the defects of large extraction solvent consumption, environmental pollution, residual toxicity of the solvent and the like; microwave-assisted extraction method, ultrasonic extraction method and supercritical CO2The extraction method is still in a laboratory stage, and industrial popularization and use are difficult.
CN106674185A discloses a method for extracting procyanidine from aronia melanocarpa fruits or pomace, which comprises soaking the aronia melanocarpa fruits or pomace in an organic solvent, performing flash extraction by using a flash extraction device, and performing suction filtration or centrifugation to obtain an aronia melanocarpa procyanidine extract solution; concentrating the proanthocyanidin extract solution of the aronia melanocarpa by using a negative pressure rotary evaporation device to obtain an aronia melanocarpa proanthocyanidin extract; pre-freezing the extract of the procyanidin of the aronia melanocarpa in an ultra-low temperature refrigerator, and freeze-drying the extract by using a freeze-drying device to obtain freeze-dried powder of the procyanidin of the aronia melanocarpa. However, this method can only extract 89.71mg procyanidins from 5g of aronia melanocarpa fruit, the extraction rate is 1.8%, the extraction rate is low, and procyanidins in aronia melanocarpa fruit cannot be released to the maximum extent.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for extracting procyanidine from aronia melanocarpa fruits, which can degrade cell walls of pomace to facilitate dissolution of procyanidine, so that procyanidine in the pomace is extracted to the maximum degree, and the extraction rate of procyanidine is high.
The invention provides a method for extracting procyanidine from aronia melanocarpa fruits, which comprises the following steps:
A) mashing Aronia melanocarpa fruit, and filtering to obtain fruit juice and fruit residue;
B) fermenting the pomace by adopting aspergillus niger, trichoderma viride and bacillus licheniformis to obtain pomace fermentation liquor;
C) mixing the fruit juice and the pomace fermentation liquor to obtain a mixed liquor, and carrying out enzymolysis on the mixed liquor by adopting pectinase and papain to obtain an enzymolysis liquid;
D) separating and purifying the enzymolysis liquid to obtain the procyanidin extract.
According to the method, enzyme systems such as cellulase and the like generated by combined fermentation of aspergillus niger, trichoderma viride and bacillus licheniformis are adopted to degrade the cell wall of the pomace so as to be beneficial to dissolving out procyanidine, and a mixed culture system constructed by the fungi and the bacteria can realize enzyme system complementation, function complementation and growth mutual promotion; meanwhile, the components such as pectin and protein in the pomace after secondary enzymolysis and fermentation are combined with exogenous pectinase and papain, so that the procyanidine remained in the pomace can be extracted to the maximum extent. The two-stage enzymolysis process overcomes the defects of high temperature, large dosage of organic reagents and the like in the traditional procyanidine extraction, and the two-stage enzymolysis is mutually matched and synergistic, so that the extraction rate of procyanidine in pomace is remarkably improved.
In step a) of the present invention, mashing may be performed using equipment commonly used in the art of food processing, such as a crusher, a beater, etc.; the filtration after the mashing is carried out by sieving with a 100-sand 200-mesh sieve, the filtration effect of the juice is influenced by the mesh size, if the mesh size is less than 100 meshes, fine fruit residue particles cannot be removed, and the filtration effect cannot be achieved; if the grain size is larger than 200 meshes, the pulp filtering time is too long, and the production efficiency is influenced.
Step B) of the present invention may comprise:
adding the pomace into a liquid culture medium, and then inoculating aspergillus niger, trichoderma viride and bacillus licheniformis for fermentation; wherein, the composition of the liquid culture medium can be as follows: peptone 0.2-10g/L, sodium chloride 0.2-10g/L, magnesium sulfate 0.2-10g/L and dipotassium hydrogen phosphate 0.2-10 g/L; the adding amount of the pomace can be 200-500 g/L. The adding amount of the pomace influences the extraction rate of the procyanidine, if the adding amount is less than 200g/L, the material-liquid ratio is too large, and the production cost is increased; if the addition amount is more than 500g/L, the material-liquid ratio is too small, the pomace cannot be fully contacted with the solution, and the extraction rate of the procyanidin is reduced.
In the step B), the inoculation amount of Aspergillus niger can be 0.1-0.5% of the pomace, the inoculation amount of Trichoderma viride can be 0.1-0.5% of the pomace, and the inoculation amount of Bacillus licheniformis can be 0.2-1% of the pomace; the fermentation conditions include: the fermentation temperature is 28-32 ℃, and the ventilation volume is 1: (1.0-1.2), the pressure is 0.04-0.06MPa, the rotating speed is 100-200rpm, and the fermentation time is 20-30 h.
Because the pomace of the aronia melanocarpa still contains a large amount of procyanidine, how to destroy the cell wall of the pomace makes the release of procyanidine become a key for extraction. The research finds that: the enzyme system and the enzyme production capability of a single microorganism or similar microorganisms have certain limitations, so that the cellulose in the pomace is difficult to completely degrade, and the degradation effect is not ideal enough; by adopting the enzyme systems such as cellulase and the like generated by combined fermentation of the aspergillus niger, the trichoderma viride and the bacillus licheniformis, macromolecular fibers in cell walls of peel and pulp fibers can be degraded into micromolecular polysaccharides, so that procyanidine remained in pomace can be effectively dissolved out, and the extraction rate of procyanidine is further improved.
The aspergillus niger, the trichoderma viride and the bacillus licheniformis used in the invention are all products which are conventionally sold in the market at present.
In the step C), the total dosage of the pectinase and the papain (which are collectively called complex enzyme) can be 0.05-0.1% of the weight of the mixed solution, and the weight ratio of the pectinase to the papain can be (1-2): 1, the enzyme activity of the pectinase is (0.5-1.5) multiplied by 104U/g, enzyme activity of papain is (15-25) multiplied by 104U/g。
Preferably, the compound enzyme can be composed of 60 parts by weight of pectinase and 40 parts by weight of papain, and the enzyme activity of the pectinase is 1 x 104U/g, papain 20X 104U/g。
The fruit juice obtained in the step A) is filtered, so that particles such as peel and pomace can be removed, but macromolecular proteins, pectin and other substances in the fruit juice are flocculated along with the prolonging of time, and are difficult to completely remove by conventional centrifugation and other modes; the addition of pectinase and papain can degrade these macromolecular substances into water-soluble small molecular components, thereby improving the clarity of the juice and improving the purity and water solubility of the procyanidin product.
Meanwhile, although cellulase and other enzyme systems generated by fermentation of aspergillus niger, trichoderma viride and bacillus licheniformis can soften and decompose cellulose in cell walls, macromolecular pectin, protein and other components in the pomace can still wrap part of procyanidine, pectinase and papain are adopted to carry out enzymolysis on the pomace continuously, and the extraction rate of procyanidine in the pomace can be further improved.
In the invention, when the weight of the compound enzyme is more than 0.1 percent of the weight of the mixed solution, the dissolution speed of the procyanidine is slow, so that the enzyme dosage is wasted; when the weight of the compound enzyme is less than 0.05 percent of the weight of the mixed solution, the procyanidine cannot be fully dissolved out, and the extraction rate is reduced; therefore, the weight of the complex enzyme is suitably controlled to be 0.05-0.1% of the weight of the mixed solution. In addition, the use of pectinase and papain in amounts less than or greater than the above ranges results in a decrease in the dissolution rate of the nutrient components, and therefore the enzyme amounts of pectinase and papain are suitably in the above ranges.
In the present invention, the enzymatic conditions include: the enzymolysis temperature is 45-55 ℃, and the enzymolysis time is 4-10 h.
During enzymolysis, if the enzymolysis reaction temperature is lower than 45 ℃, the enzymolysis reaction is incomplete, and the dissolution speed of the procyanidine is slow; if the temperature is higher than 55 ℃, the enzymolysis reaction is not complete, and the activity of the enzyme is inhibited; therefore, it is appropriate to control the temperature of the enzymatic hydrolysis at 45 to 55 ℃. Meanwhile, under the condition that the enzymolysis temperature is 45-55 ℃, if the enzymolysis reaction time is less than 4 hours, the enzymolysis reaction is not complete, and the procyanidine can not be completely dissolved out; if the enzymolysis reaction time exceeds 10 hours, the enzymolysis reaction tends to be smooth, and energy waste is caused; therefore, it is appropriate to control the time for the enzymatic hydrolysis to be 4 to 10 hours.
In addition, as the fermentation conditions of aspergillus niger, trichoderma viride and bacillus licheniformis tend to be neutral, and the pH value of the enzymolysis reaction is also in a neutral range, the fermentation liquid after the fermentation is finished can meet the optimal working conditions of pectinase and papain without adjusting the pH value.
The enzymes used in the invention are all products which are conventionally sold in the current market; for example, pectinase sold by Souchehan (Weifang) bioengineering Co., Ltd, papain sold by Nanning Pobo bioengineering Co., Ltd, and the like.
In step D) of the present invention, the separation and purification may include crude extraction; wherein the crude extraction may comprise: filtering the enzymolysis liquid to obtain filtrate; concentrating and drying the filtrate to obtain the crude procyanidin extract.
Specifically, centrifugal filtration can be adopted for filtering the enzymatic hydrolysate, and equipment used for centrifugal filtration can adopt a high-speed centrifuge which is conventionally sold in the current market, such as a disc centrifuge, a tubular centrifuge and the like; the conditions for centrifugation may be those described in the instructions for the operation of the centrifuge used.
The equipment used for concentrating the filtrate may be currently commercially available concentration equipment such as a three-way concentrator, a scraper concentrator, a ball concentrator, etc. Stopping concentrating when the solid content of the concentrated solution reaches above 20%, and drying by spray drying or other drying methods to obtain procyanidin crude extract with procyanidin content of not less than 35%; the detection of the content of the procyanidine is performed according to the method in the technical guideline of physical and chemical health index inspection and evaluation of health food (2020 edition).
Further, in the step D), the separation and purification can also comprise fine extraction after crude extraction; wherein, the essence includes: performing dynamic adsorption by using X-5 type macroporous resin, desorbing with 45-55% ethanol solution, eluting until the column water is colorless, collecting desorption solution, concentrating, and drying to obtain procyanidin refined extract; wherein, the adsorption rate can be controlled to be 2-4BV/h, the desorption rate is 2-4BV/h, and the adsorption and desorption rates are all preferably 3 BV/h.
The X-5 type macroporous resin is adopted to selectively adsorb and purify the procyanidin, so that a procyanidin refined extract with higher content can be obtained, and the content of the procyanidin in the procyanidin refined extract is not less than 92%.
The procyanidin products with different purities can be respectively prepared in the step D), when the required procyanidin product has lower purity, the crude procyanidin extract can be directly prepared by adopting the crude extraction step and concentrating and drying, so that the step of purifying macroporous resin is omitted, and the cost is saved; when the purity of the required procyanidin product is higher, the required procyanidin product can be further purified by adopting a fine extraction step, and then concentrated and dried, so that the high-purity procyanidin fine extract is prepared.
Compared with the prior art, the invention has the beneficial effects that:
according to the method for extracting the procyanidine from the aronia melanocarpa fruits, enzyme systems such as cellulase and the like generated by combined fermentation of aspergillus niger, trichoderma viride and bacillus licheniformis are adopted to degrade the cell walls of the pomace so as to be beneficial to dissolution of the procyanidine, the mixed fermentation can realize enzyme system complementation and mutualistic symbiosis, and the defects of weak enzyme production capability, poor enzymolysis effect and the like of a single strain are overcome; in addition, substances such as pectin and protein in the pomace are subjected to secondary enzymolysis by utilizing exogenous pectinase and papain, so that the procyanidine remained in the pomace can be extracted to the maximum degree, and the extraction rate of the procyanidine in the pomace is improved. The method can utilize the waste of the pomace, improves the economic benefit of enterprises, and is beneficial to prolonging the industrial chain of the aronia melanocarpa; the two-stage enzymolysis process consisting of multi-strain mixed fermentation and compound enzyme enzymolysis overcomes the defects of high temperature and large using amount of organic reagents in the traditional procyanidine extraction, has mild conditions and simple required equipment, can realize large-scale and industrialized production, promotes the development of the deep processing industry of the aronia melanocarpa, and improves the additional value of the aronia melanocarpa.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a flow chart of the process for extracting procyanidins from Aronia melanocarpa fruit of example 1.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an", and "the" include plural forms as well, unless the context clearly indicates otherwise, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, devices, components, and/or combinations thereof.
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The raw materials adopted by the embodiments of the invention are as follows:
aspergillus niger: purchased from Shandong Changtai Biotech, Inc.;
and (3) trichoderma viride: purchased from Shandong Changtai Biotech, Inc.;
bacillus licheniformis; purchased from underwriter valley biotechnology limited;
and (3) pectinase: purchased from secukino (Weifang) bioengineering, Inc.;
papain: purchased from south china pompon bioengineering, inc.
Example 1
Referring to fig. 1, the method for extracting procyanidins from aronia melanocarpa fruits in the embodiment comprises the following steps:
1) raw material crushing and pulping
Mechanically mashing Aronia melanocarpa fruit into pulp, and sieving with 100 mesh sieve to obtain fruit juice and fruit residue.
2) Microbial fermentation of pomace
Deionized water was added to the fermentor, a liquid medium was prepared from 0.2g/L peptone, 2g/L sodium chloride, 10g/L magnesium sulfate and 5g/L dipotassium hydrogen phosphate, and the above-mentioned pomace was added to the liquid medium at a rate of 200 g/L.
Inoculating aspergillus niger, trichoderma viride and bacillus licheniformis into the liquid culture medium added with the pomace, wherein the inoculation amounts of the aspergillus niger, the trichoderma viride and the bacillus licheniformis are 0.5%, 0.2% and 0.2% of the pomace respectively; stirring and fermenting at constant temperature of 30 deg.C, ventilation amount of 1:1.0, pressure of 0.05MPa, and rotation speed of 120rpm for 20 hr to obtain Aronia melanocarpa residue fermentation liquid.
3) Enzymolysis
Adding the fruit juice obtained in the step 1) into the pomace fermentation liquor obtained in the step 2) to obtain a mixed liquor.
And adding a complex enzyme into the mixed solution for enzymolysis, wherein the addition amount of the complex enzyme is 0.05 percent of the weight of the mixed solution, and the weight ratio of the pectinase to the papain is 1:1, the enzyme activity of the pectinase is 0.5 multiplied by 104U/g, papainThe enzyme activity of (A) is 20X 104U/g, and carrying out enzymolysis for 4h at the enzymolysis temperature of 50 ℃ to obtain the aronia melanocarpa enzymatic hydrolysate.
4) Separation, crude extraction
And (3) centrifugally filtering the enzymatic hydrolysate of the aronia melanocarpa to remove impurities, collecting filtrate, concentrating the filtrate, and performing spray drying to obtain the procyanidin crude extract. The detection shows that the content of procyanidine in the procyanidine crude extract is 36.4%.
Example 2
The method for extracting procyanidine from aronia melanocarpa fruits comprises the following steps:
1) raw material crushing and pulping
Mechanically mashing Aronia melanocarpa fruit into pulp, and sieving with 200 mesh sieve to obtain fruit juice and fruit residue.
2) Microbial fermentation of pomace
Adding deionized water into a fermentation tank, preparing a liquid culture medium according to 10g/L of peptone, 0.2g/L of sodium chloride, 0.2g/L of magnesium sulfate and 10g/L of dipotassium hydrogen phosphate, and then adding the pomace obtained in the step 1) into the liquid culture medium according to the adding amount of 500 g/L.
Inoculating aspergillus niger, trichoderma viride and bacillus licheniformis into a liquid culture medium added with the pomace, wherein the inoculation amounts of the aspergillus niger, the trichoderma viride and the bacillus licheniformis are 0.1%, 0.1% and 1% of the pomace respectively; stirring and fermenting at 28 deg.C and ventilation rate of 1:1.2 under the condition of pressure of 0.04MPa and rotation speed of 100rpm for 30h to obtain Aronia melanocarpa residue fermentation liquid.
3) Enzymolysis
Adding the fruit juice obtained in the step 1) into the pomace fermentation liquor obtained in the step 2) to obtain a mixed liquor.
And adding a complex enzyme into the mixed solution for enzymolysis, wherein the addition amount of the complex enzyme is 0.1 percent of the weight of the mixed solution, and the weight ratio of the pectinase to the papain is 2: 1, the enzyme activity of the pectinase is 1 x 104U/g, papain 15X 104U/g, and carrying out enzymolysis for 10h at the enzymolysis temperature of 45 ℃ to obtain the aronia melanocarpa enzymatic hydrolysate.
4) Separating, crude extracting and fine extracting
Centrifuging the enzymatic hydrolysate of Aronia melanocarpa, filtering to remove impurities, and collecting filtrate.
And (3) dynamically adsorbing the filtrate by using X-5 type macroporous resin, desorbing by using 45% ethanol at the adsorption and desorption rate of 3BV/h, eluting until the lower column is colorless, collecting the desorption solution, concentrating, and performing spray drying to obtain the procyanidin refined extract. The detection shows that the content of procyanidine in the procyanidine refined extract is 93.8%.
Example 3
The method for extracting procyanidine from aronia melanocarpa fruits comprises the following steps:
1) raw material crushing and pulping
Mechanically mashing Aronia melanocarpa fruit into pulp, and sieving with 120 mesh sieve to obtain fruit juice and fruit residue.
2) Microbial fermentation of pomace
Deionized water was added to the fermenter, and a liquid medium was prepared from 2g/L peptone, 10g/L sodium chloride, 5g/L magnesium sulfate and 0.2g/L dipotassium hydrogen phosphate, and then the marc of step 1) was added to the liquid medium at a rate of 300 g/L.
Inoculating aspergillus niger, trichoderma viride and bacillus licheniformis into the liquid culture medium added with the pomace, wherein the inoculation amounts of the aspergillus niger, the trichoderma viride and the bacillus licheniformis are 0.3%, 0.5% and 0.5% of the pomace respectively; stirring and fermenting at the fermentation temperature of 32 deg.C, ventilation rate of 1:1.1, pressure of 0.06MPa, and rotation speed of 200rpm for 22h to obtain Aronia melanocarpa residue fermentation liquid.
3) Enzymolysis
Adding the fruit juice obtained in the step 1) into the pomace fermentation liquor obtained in the step 2) to obtain a mixed liquor.
And adding a complex enzyme into the mixed solution for enzymolysis, wherein the addition amount of the complex enzyme is 0.06 percent of the weight of the mixed solution, and the weight ratio of the pectinase to the papain is 1.5: 1, the enzyme activity of the pectinase is 1.5 multiplied by 104U/g, enzyme activity of papain is 25 multiplied by 104U/g, and carrying out enzymolysis for 9h at the enzymolysis temperature of 55 ℃ to obtain the aronia melanocarpa enzymatic hydrolysate.
4) Separating, crude extracting and fine extracting
Centrifuging the enzymatic hydrolysate of Aronia melanocarpa, filtering to remove impurities, and collecting filtrate.
And (3) dynamically adsorbing the filtrate by using X-5 type macroporous resin, desorbing by using 55% ethanol at the adsorption and desorption rate of 4BV/h, eluting until the lower column is colorless, collecting the desorption solution, concentrating, and performing spray drying to obtain the procyanidin refined extract. The content of procyanidine in the procyanidine refined extract is 92.6% by detection.
Example 4
The method for extracting procyanidine from aronia melanocarpa fruits comprises the following steps:
1) raw material crushing and pulping
Mechanically mashing Aronia melanocarpa fruit into pulp, and sieving with 150 mesh sieve to obtain fruit juice and fruit residue.
2) Microbial fermentation of pomace
Adding deionized water into a fermentation tank, preparing a liquid culture medium according to 5g/L peptone, 2g/L sodium chloride, 1g/L magnesium sulfate and 5g/L dipotassium hydrogen phosphate, and then putting the pomace obtained in the step 1) into the liquid culture medium according to 300 g/L.
Inoculating aspergillus niger, trichoderma viride and bacillus licheniformis into the liquid culture medium added with the pomace, wherein the inoculation amounts of the aspergillus niger, the trichoderma viride and the bacillus licheniformis are 0.2%, 0.2% and 0.5% of the pomace respectively; stirring and fermenting at 29 deg.C and ventilation 1:1.0 under constant temperature and pressure 0.05MPa and rotation speed 150rpm for 24h to obtain Aronia melanocarpa residue fermentation liquid.
3) Enzymolysis
Adding the fruit juice obtained in the step 1) into the pomace fermentation liquor obtained in the step 2) to obtain a mixed liquor.
And adding a complex enzyme into the mixed solution for enzymolysis, wherein the addition amount of the complex enzyme is 0.06 percent of the weight of the mixed solution, and the weight ratio of the pectinase to the papain is 2: 1, the enzyme activity of the pectinase is 1 x 104U/g, enzyme activity of papain is 20 multiplied by 104U/g, and carrying out enzymolysis for 6h at the enzymolysis temperature of 48 ℃ to obtain the aronia melanocarpa enzymatic hydrolysate.
4) Separation, crude extraction
Centrifuging and filtering the enzymatic hydrolysate of the aronia melanocarpa to remove impurities, collecting filtrate, concentrating the filtrate, and performing spray drying to obtain the crude procyanidine extract. Detection shows that the content of procyanidine in the procyanidine crude extract is 40.3%.
Example 5
The method for extracting procyanidine from aronia melanocarpa fruits comprises the following steps:
1) raw material crushing and pulping
Mechanically mashing Aronia melanocarpa fruit into pulp, and sieving with 120 mesh sieve to obtain fruit juice and fruit residue.
2) Microbial fermentation of pomace
Deionized water was added to the fermentor, a liquid medium was prepared from 4g/L peptone, 2g/L sodium chloride, 0.5g/L magnesium sulfate and 5g/L dipotassium hydrogen phosphate, and then the pomace of step 1) was added to the liquid medium at 400 g/L.
Inoculating aspergillus niger, trichoderma viride and bacillus licheniformis into the liquid culture medium added with the pomace, wherein the inoculation amounts of the aspergillus niger, the trichoderma viride and the bacillus licheniformis are 0.4%, 0.4% and 0.6% of the pomace respectively; stirring and fermenting at 31 deg.C, ventilation rate of 1:1.1, pressure of 0.05MPa, and rotation speed of 180rpm for 28h to obtain Aronia melanocarpa residue fermentation liquid.
3) Enzymolysis
Adding the fruit juice obtained in the step 1) into the pomace fermentation liquor obtained in the step 2) to obtain a mixed liquor.
And adding a complex enzyme into the mixed solution for enzymolysis, wherein the addition amount of the complex enzyme is 0.08 percent of the weight of the mixed solution, and the weight ratio of the pectinase to the papain is 1.5: 1, the enzyme activity of the pectinase is 1 x 104U/g, enzyme activity of papain is 20 multiplied by 104U/g, and carrying out enzymolysis for 8h at the enzymolysis temperature of 52 ℃ to obtain the aronia melanocarpa enzymatic hydrolysate.
4) Separating, crude extracting and fine extracting
Centrifuging the enzymatic hydrolysate of Aronia melanocarpa, filtering to remove impurities, and collecting filtrate.
And (3) dynamically adsorbing the filtrate by using X-5 type macroporous resin, desorbing by using 50% ethanol at the adsorption and desorption rate of 2BV/h, eluting until the lower column is colorless, collecting the desorption solution, concentrating, and performing spray drying to obtain the procyanidin refined extract. The content of procyanidine in the procyanidine refined extract is 95.8% by detection.
Comparative example 1
Extracting procyanidine from aronia melanocarpa fruits by adopting an ethanol extraction method, wherein the material-liquid ratio is 1: 20, ethanol concentration of 50%, extraction temperature of 70 deg.C, extraction time of 7h, other steps refer to example 5.
Comparative example 2
Extracting procyanidine from aronia melanocarpa fruits by using a water extraction method, wherein the material-liquid ratio is 1: 20, extraction temperature 90 ℃, extraction time 7h, other steps refer to example 5.
Comparative example 3
Procyanidins were extracted from aronia melanocarpa fruits only using a microbial fermentation method, i.e., fermentation was performed only using aspergillus niger, trichoderma viride, and bacillus licheniformis (excluding the complex enzyme enzymolysis step), and the other steps were referred to example 5.
Comparative example 4
Procyanidins were extracted from aronia melanocarpa fruits only using an enzymatic hydrolysis method, i.e., an enzymatic hydrolysis using only pectinase and papain (excluding a microbial fermentation step), and the other steps were referred to in example 5.
Comparative example 5
The procedure of example 5 was repeated except that only Aspergillus niger and Trichoderma viride were inoculated for fermentation in the microbial fermentation step.
Comparative example 6
Substantially the same as example 5, except that Bacillus subtilis was used in place of Bacillus licheniformis in example 5.
Comparative example 7
Procyanidins were extracted from aronia melanocarpa fruits only using an enzymatic hydrolysis method (excluding a microbial fermentation step), and the mixed enzyme used consisted of cellulase, pectinase and papain, and the other steps were referred to example 5.
Comparative example 8
The procedure was as in example 5 except that bromelain was used in place of papain in example 5.
Comparative example 9
The microbial fermentation adopts a compound bacterium consisting of aspergillus oryzae, lactic acid bacteria and saccharomycetes, the enzymolysis adopts xylanase and amylase, and other steps refer to example 5.
After the procyanidin of aronia melanocarpa is extracted by the methods of example 5 and comparative examples 1 to 9, the extraction rate and the procyanidin purity are measured, and the calculation formula of the extraction rate is as follows:
the extraction rate is equal to the mass of procyanidine in the extract/the mass of aronia melanocarpa fruit multiplied by 100 percent
The results are shown in Table 1.
TABLE 1 procyanidin extraction yield and purity from different extraction methods
| Procyanidin extraction ratio of% | Procyanidin purity% | |
| Example 5 | 2.37 | 95.8 |
| Comparative example 1 | 1.94 | 94.6 |
| Comparative example 2 | 1.42 | 93.7 |
| Comparative example 3 | 1.83 | 92.3 |
| Comparative example 4 | 1.64 | 93.1 |
| Comparative example 5 | 1.91 | 93.5 |
| Comparative example 6 | 1.87 | 92.4 |
| Comparative example 7 | 1.73 | 92.6 |
| Comparative example 8 | 1.93 | 94.2 |
| Comparative example 9 | 1.38 | 91.3 |
From the data analysis in table 1, it can be seen that:
1. the extraction rate of the procyanidin extracted by the traditional ethanol extraction method and the traditional water extraction method is low, and the content is reduced probably because the procyanidin is partially decomposed due to high temperature in the extraction process;
2. compared with the example 5, the extraction rates of the procyanidin in the single microbial fermentation method in the comparative example 3 and the single enzymolysis method in the comparative example 4 are lower, which shows that the cell walls of the pericarp and the pulp fiber of the aronia melanocarpa can be fully degraded by the two-stage enzymolysis process combining microbial fermentation and exogenous enzymolysis, the procyanidin is released to the maximum extent, and the extraction rate of the procyanidin is greatly improved;
3. comparative example 5 only inoculated aspergillus niger and trichoderma viride, comparative example 6 adopted bacillus subtilis, procyanidin extraction rate was lower than that of example 5, which indicates that cell walls in pomace could not be degraded sufficiently by using the same flora or other types of bacillus, and procyanidin extraction rate was greatly reduced;
4. comparative example 7 adopts exogenous cellulase, pectinase and papain for enzymolysis, the effect is far inferior to that of a compound enzyme system generated by microbial fermentation; example 8 the maximum release of procyanidins was also not achieved using bromelain.
5. Comparative example 9 adopts composite bacteria consisting of aspergillus oryzae, lactic acid bacteria and yeast to carry out microbial fermentation, and adopts composite enzyme consisting of xylanase and amylase to carry out enzymolysis, the extraction rate of procyanidine is greatly reduced compared with that of example 5, which shows that the composite bacteria and the composite enzyme are not randomly selected, and are preferable combinations with unique advantages for degrading the cell wall of the sorbus pohuashanensis, and the other composite bacteria and the composite enzyme have weaker degradation effect on the cell wall of the sorbus pohuashanensis, and the release effect of procyanidine is greatly limited.
This indicates that: only cellulase and other enzyme systems generated by combined fermentation of aspergillus niger, trichoderma viride and bacillus licheniformis are adopted to degrade the cell wall of the pomace, and substances such as pectin, protein and the like in the pomace are secondarily enzymolyzed by exogenous pectinase and papain, so that the procyanidine remained in the pomace can be extracted to the maximum extent, and the extraction rate of the procyanidine in the pomace is greatly improved.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for extracting procyanidine from Aronia melanocarpa fruit is characterized by comprising the following steps:
A) mashing Aronia melanocarpa fruit, and filtering to obtain fruit juice and fruit residue;
B) fermenting the pomace by adopting aspergillus niger, trichoderma viride and bacillus licheniformis to obtain pomace fermentation liquor;
C) mixing the fruit juice and the pomace fermentation liquor to obtain a mixed liquor, and carrying out enzymolysis on the mixed liquor by adopting pectinase and papain to obtain an enzymolysis liquid;
D) separating and purifying the enzymolysis liquid to obtain the procyanidin extract.
2. The method as claimed in claim 1, wherein in step A), the filtration is 100-mesh and 200-mesh.
3. The method of claim 1, wherein step B) comprises:
adding the pomace into a liquid culture medium, and then inoculating aspergillus niger, trichoderma viride and bacillus licheniformis for fermentation; wherein, the composition of the liquid culture medium is as follows: peptone 0.2-10g/L, sodium chloride 0.2-10g/L, magnesium sulfate 0.2-10g/L and dipotassium hydrogen phosphate 0.2-10 g/L; the adding amount of the pomace is 200-500 g/L.
4. The method according to claim 1 or 3, wherein in step B), the amount of Aspergillus niger is 0.1-0.5% of the amount of pomace, the amount of Trichoderma viride is 0.1-0.5% of the amount of pomace, and the amount of Bacillus licheniformis is 0.2-1% of the amount of pomace.
5. The process according to claim 1 or 3, wherein in step B) the fermentation conditions comprise: the fermentation temperature is 28-32 ℃, and the ventilation volume is 1: 1.0-1: 1.2, the pressure is 0.04-0.06MPa, the rotating speed is 100-200rpm, and the fermentation time is 20-30 h.
6. The method according to claim 1, wherein in step C), the pectinase is used in combination withThe total dosage of the papain is 0.05-0.1% of the weight of the mixed solution, and the weight ratio of the pectinase to the papain is 1: 1-2: 1, the enzyme activity of the pectinase is 0.5 multiplied by 104U/g-1.5×104U/g, enzyme activity of papain is 15 multiplied by 104U/g-25×104U/g。
7. The method according to claim 1 or 6, wherein in step C), the enzymatic conditions comprise: the enzymolysis temperature is 45-55 ℃, and the enzymolysis time is 4-10 h.
8. The method according to claim 1, wherein in step D), the separation and purification comprises crude extraction; the crude extraction comprises the following steps: filtering the enzymolysis liquid to obtain filtrate; concentrating and drying the filtrate to obtain the crude procyanidin extract.
9. The method as claimed in claim 8, wherein in step D), the separation and purification further comprises fine extraction after the crude extraction; the fine extraction comprises the following steps: performing dynamic adsorption by using X-5 type macroporous resin, desorbing with 45-55% ethanol solution, eluting until the column water is colorless, collecting desorption solution, concentrating, and drying to obtain procyanidin refined extract.
10. The method of claim 9, wherein the adsorption rate is controlled to be 2 to 4BV/h and the desorption rate is controlled to be 2 to 4 BV/h.
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