CN114736314A - Camellia oleifera shell polysaccharide and preparation method thereof - Google Patents
Camellia oleifera shell polysaccharide and preparation method thereof Download PDFInfo
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- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 1
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- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
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Images
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
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- Sustainable Development (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to the field of natural component extraction, and discloses camellia oleifera shell polysaccharide and a preparation method thereof. The preparation method provided by the invention is based on the combination of an enzymolysis method and an alcohol extraction method, can obtain higher yield of the camellia oleifera shell polysaccharide by adopting lower extraction temperature, reduces the loss of the bioactivity of the camellia oleifera shell polysaccharide caused by overhigh extraction temperature, and ensures the quality of the camellia oleifera shell polysaccharide.
Description
Technical Field
The invention relates to the field of extraction and application of camellia oleifera shell active polysaccharide, and particularly relates to camellia oleifera shell polysaccharide and a preparation method thereof.
Background
Camellia oleifera (Camellia oleifera Abel.) is a high-quality plant oil source, and is a evergreen shrub or a small tree in Theaceae. The oil-removed tea seed can be used for extracting oil, and the fruit shell can also be used for extracting tannin extract, furfural, xylitol and the like. In recent years, researchers at home and abroad discover that polysaccharides can be extracted by using camellia oleifera shells as raw materials, and the polysaccharides have the performances of oxidation resistance, moisture retention, hygroscopicity and the like.
At present, the extraction method of camellia oleifera shell polysaccharide mainly comprises a method based on an alcohol extraction method and combining means such as ultrasound, water extraction and extraction. For example, in the article "influence of different extraction methods on the content of camellia oleifera shell polysaccharide", zhugendan et al (2019) detected the polysaccharide yield when camellia oleifera shell polysaccharide is extracted by using a water extraction and alcohol precipitation method, an ultrasonic-ethanol extraction method and other methods, and specific conditions in the extract are optimized, and it is found that the polysaccharide yield can be correspondingly increased when the conditions such as alcohol concentration, extraction temperature and the like are increased. For another example, Chenjing Si et al (2019) introduced a method of mixing and soaking oil tea fruit shell powder with 30 times of 60% ethanol for 1 hour, and then heating and refluxing at 100 ℃ for extraction in the article "optimization of ethanol extraction of oil tea fruit shell polysaccharide and moisture absorption and retention property". Shenjianfu (2010) introduces a method of using an alcohol precipitation method, sequentially using absolute ethyl alcohol, acetone and absolute ethyl ether to wash, and performing vacuum drying to obtain the camellia oleifera shell polysaccharide in the text of research on the extraction and antioxidant effects of the camellia oleifera shell polysaccharide.
However, the current extraction method usually adopts a high-temperature extraction mode, and the high temperature can destroy the biological activity of the camellia oleifera shell polysaccharide and reduce the quality of the camellia oleifera shell polysaccharide. Therefore, it is highly desirable to develop a method for extracting camellia oleifera shell polysaccharide at a low temperature to solve the above problems.
Disclosure of Invention
The invention aims to overcome the problem that the biological activity of camellia oleifera fruit shell polysaccharide is easily damaged under a high temperature condition in an extraction method in the prior art, and provides camellia oleifera fruit shell polysaccharide and a preparation method thereof.
In order to achieve the above object, the present invention provides a camellia oleifera shell polysaccharide, which comprises structural units provided by the following monosaccharides: 5-10 wt% of mannose, 15-20 wt% of fucose, 15-20 wt% of galactose, 5-10 wt% of rhamnose, 1-5 wt% of arabinose and 30-50 wt% of glucose.
A second aspect of the present invention provides a method for preparing camellia oleifera shell polysaccharide, comprising the steps of:
(1) enzymolysis: mixing oil tea fruit shells, water and a compound enzyme under an enzymolysis condition for enzymolysis, and carrying out first solid-liquid separation on the obtained mixture;
(2) alcohol extraction: and contacting the liquid phase product obtained by the first solid-liquid separation with alcohol for alcohol precipitation to obtain an alcohol extraction product.
The third aspect of the invention provides the camellia oleifera shell polysaccharide obtained by the method.
Through the technical scheme, the invention can obtain the following beneficial effects:
(1) according to the method provided by the invention, the camellia oleifera shell polysaccharide is extracted under a low-temperature condition, so that the reduction of the bioactivity of the camellia oleifera shell polysaccharide caused by overhigh extraction temperature can be reduced, and the quality of the camellia oleifera shell polysaccharide is improved.
(2) According to the method provided by the invention, the oil-tea camellia shell raw material is treated by an enzymolysis method, the conditions are mild, the treatment efficiency is high, and the damage of the use of chemical reagents to the bioactivity of oil-tea camellia shell polysaccharide is reduced.
(3) The camellia oleifera shell polysaccharide provided by the invention has good oxidation resistance, and the clearance rate of free radicals such as ABTS, DPPH, OH and the like can reach more than 65% at 30 mug/mL.
Drawings
FIG. 1 is a graph showing the relationship between the enzymolysis temperature and the yield of oil-tea camellia husk polysaccharide obtained in test example 1.
Fig. 2 is a graph comparing the yields of camellia oleifera husk polysaccharide in the examples of the present invention and the comparative examples.
FIGS. 3-5 are graphs comparing the clearance of tea fruit chitin and tea polysaccharide to ABTS free radicals, DPPH free radicals and OH free radicals, respectively, in test example 3.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The first aspect of the invention provides camellia oleifera shell polysaccharide, which comprises structural units provided by the following monosaccharides: 5-10 wt% of mannose, 15-20 wt% of fucose, 15-20 wt% of galactose, 5-10 wt% of rhamnose, 1-5 wt% of arabinose and 30-50 wt% of glucose. The weight percentage numerical value represents the content of the structural unit provided by the corresponding monosaccharide in the camellia oleifera shell polysaccharide provided by the invention.
Preferably, the camellia oleifera shell polysaccharide comprises structural units provided by the following monosaccharides: 6.5-7.5 wt% of mannose, 18-20 wt% of fucose, 15-17 wt% of galactose, 5-7 wt% of rhamnose, 1-3 wt% of arabinose and 40-50 wt% of glucose.
Preferably, the camellia oleifera shell polysaccharide comprises structural units provided by the following monosaccharides: 6.8-7.5 wt% of mannose, 18.5-19.5 wt% of fucose, 15-16.5 wt% of galactose, 6-7 wt% of rhamnose, 2-3 wt% of arabinose and 45-50 wt% of glucose.
According to a preferred embodiment of the present invention, wherein the weight average molecular weight of the camellia oleifera shell polysaccharide is 50000-70000. In addition, the inventor also finds that the camellia oleifera husk polysaccharide is brown powder, has the molecular weight distribution of 3000-250000, and has the characteristics of being easily soluble in water or aqueous solutions (such as aqueous solutions of acid, alkali and salt) and not being soluble in organic solvents (such as alcohol, ketone, ether and the like).
At present, the method for preparing the camellia oleifera shell polysaccharide in the field is mainly based on an alcohol extraction method supplemented with other means, wherein a higher extraction temperature (for example, 100 ℃) is usually adopted in the extraction process to improve the yield of the camellia oleifera shell polysaccharide. However, high temperature can increase the yield and easily destroy the bioactivity of camellia oleifera shell polysaccharide. The inventor of the invention skillfully discovers that the oil-tea camellia husk polysaccharide can be extracted at higher yield at lower extraction temperature by combining an enzymolysis method and an alcohol extraction method and matching with the preferable complex enzyme and extraction conditions of the invention. Meanwhile, the method can also retain higher bioactivity of the camellia oleifera shell polysaccharide and improve the quality of the camellia oleifera shell polysaccharide.
In a second aspect, the present invention provides a method for preparing camellia oleifera shell polysaccharide, comprising the steps of:
(1) enzymolysis: mixing oil tea fruit shells, water and a compound enzyme under an enzymolysis condition for enzymolysis, and carrying out first solid-liquid separation on an obtained enzymolysis product;
(2) alcohol extraction: and contacting the liquid phase product obtained by the first solid-liquid separation with alcohol for alcohol precipitation to obtain an alcohol extraction product.
The camellia oleifera shells are byproducts generated in the process of squeezing the camellia oleifera oil by the camellia oleifera oil processing enterprises, and in consideration of equipment abrasion, oil quality, oil yield and the like, the camellia oleifera oil processing enterprises generally firstly peel the camellia oleifera fruits (namely, peel the camellia oleifera fruits and separate the kernels from the shells), and then extract oil from the kernels. Any camellia oleifera shell used for preparing camellia oleifera shell polysaccharide in the prior art can be subjected to polysaccharide extraction by adopting the method provided by the invention. According to a preferred embodiment of the present invention, in the step (1), the camellia oleifera shells are generated when camellia oleifera fruits are subjected to a hulling treatment before the camellia oleifera oil is squeezed.
In order to enable the oil tea fruit shell to be in full contact with the complex enzyme and improve the enzymolysis efficiency and effect, preferably, the oil tea fruit shell exists in a powder form. The oil tea fruit shell powder can be a related product obtained commercially, and can also be prepared into powder by the oil tea fruit shell.
The method provided by the invention has no special limitation on the particle size of the camellia oleifera shell powder, and only needs to achieve the purpose of fully contacting with the complex enzyme in an enzymolysis system. More preferably, the particle size of the camellia oleifera shell powder is 150-450 μm. That is, the camellia oleifera shell powder can pass through at least 40 mesh sieve and at most 100 mesh sieve.
Any enzyme which is available in the field and can be used for enzymolysis of the oil-tea camellia husk (such as main components, cellulose, starch, protein and the like) can be used as a component in the compound enzyme provided by the invention. In view of cost, enzymolysis efficiency and effect, the compound enzyme comprises the following components: at least one of cellulase, amylase and protease.
Preferably, the cellulase is selected from at least one of a C1 enzyme, a CX enzyme, and a glucosidase enzyme.
Preferably, the amylase is selected from at least one of alpha-amylase, beta-amylase and gamma-amylase.
Preferably, the protease is selected from at least one of papain, trypsin, cathepsin and subtilisin. More preferably papain.
The enzyme used in the method provided by the invention can be a related product which is commercially obtained and contains the enzyme of the type described above, and can also be the enzyme which is obtained by self-preparation.
In the method provided by the invention, the dosage of the camellia oleifera shells, water and the complex enzyme in the step (1) is not particularly limited as long as the camellia oleifera shells (powder) can be subjected to sufficient enzymolysis. The amount of the oil-tea camellia shell, water and the complex enzyme can be adjusted by a person skilled in the art according to the actual conditions such as the components and the characteristics of the complex enzyme. According to a preferred embodiment of the present invention, wherein, in the step (1), the weight ratio of the camellia oleifera shell to the water is 1: 10-30.
According to a preferred embodiment of the present invention, wherein the cellulase is used in an amount of 500-1500U relative to 1g of the oil tea camellia husk.
Preferably, the amount of the cellulase is 700-1500U relative to 1g of the oil tea fruit shell.
According to a preferred embodiment of the present invention, wherein the amount of the amylase is 400-1200U relative to 1g of the oil tea camellia husk.
Preferably, the amount of the amylase is 450-1000U relative to 1g of the oil tea fruit shell.
According to a preferred embodiment of the present invention, wherein the protease is used in an amount of 3000-8000U relative to 1g of the oil tea camellia husk.
Preferably, the protease is used in an amount of 3500-7500U relative to 1g of the oil tea fruit shell.
According to a preferred embodiment of the present invention, wherein the cellulase, amylase and protease are used in a ratio of 1: 0.8-2.5: 2-6, preferably 1: 0.9-2: 3-5.
In the method provided by the invention, the enzymolysis conditions in the step (1) can be adjusted according to actual conditions. According to a preferred embodiment of the present invention, in step (1), the conditions of the enzymatic hydrolysis include: the temperature is 30-60 ℃, the stirring speed is 500-.
In order to make the enzymolysis system heated uniformly and facilitate observing the enzymolysis degree, preferably, the enzymolysis temperature is controlled by heating in a water bath.
In the process provided by the present invention, the first solid-liquid separation in step (1) may be carried out in any manner known in the art. According to a preferred embodiment of the present invention, wherein the first solid-liquid separation mode is selected from centrifugal separation and/or natural sedimentation separation.
In view of efficiency, effect and the like of solid-liquid separation, preferably, the first solid-liquid separation mode is centrifugal separation, and conditions include: the centrifugal speed is 2000-5000rpm, the centrifugal time is 10-30min, the temperature is 30-60 ℃, and the pH value is 5-6. The temperature and pH values refer to the (initial) temperature and pH value of the material in the first solid-liquid separation system.
Any alcohol existing in the field and capable of being used for extracting the camellia oleifera shell polysaccharide can be applied to the method provided by the invention. According to a preferred embodiment of the present invention, wherein, in the step (2), the alcohol is selected from the group consisting of C1-C5 alcohols.
From the viewpoint of cost, extraction efficiency, and the like, it is preferable that the alcohol is selected from at least one of methanol and/or ethanol and an aqueous solution thereof.
In view of the safety of the product, it is more preferable to contact the liquid-phase product obtained by the first solid-liquid separation with (anhydrous) ethanol or an aqueous solution thereof (preferably wherein the volume fraction of ethanol is 90% or more).
In the method provided by the present invention, the amount of the alcohol is not particularly limited, and may be adjusted according to the actual situation. In view of energy saving, consumption reduction and reduction of polysaccharide loss during alcohol precipitation, it is preferable that the alcohol is used in an amount of 3 to 25mL, preferably 5 to 20mL, relative to 1g of the liquid-phase product on a dry basis. The dry basis is a solid substance obtained by drying the liquid-phase product obtained by the first solid-liquid separation.
In the method provided by the invention, the alcohol precipitation condition is not particularly limited, and any condition for extracting the camellia oleifera shell polysaccharide by alcohol precipitation in the field can be applied to the method provided by the invention. In order to further improve the yield of the camellia oleifera shell polysaccharide and reduce the loss of the polysaccharide during alcohol precipitation, according to a preferred embodiment of the present invention, the alcohol precipitation conditions include: the temperature is 3-5 ℃, the time is 12-24h, and the volume concentration of alcohol in the alcohol precipitation system is 70-85%.
In order to improve the efficiency of alcohol precipitation while reducing the amount of alcohol used, according to a preferred embodiment of the present invention, the method further comprises a step of concentrating the liquid-phase product obtained by the first solid-liquid separation before the step (2).
Any means of concentration known in the art may be suitable for use in the methods provided herein. In order to better control the temperature of the concentration while ensuring the efficiency and effect of the concentration, the concentration is preferably selected from the group consisting of rotary evaporation concentration and/or water bath evaporation concentration.
In the method of the present invention, the concentration step may be performed so as to reduce the volume of the liquid phase obtained by the first solid-liquid separation in step (1). For the purposes of saving reagents, reducing subsequent work load, reducing polysaccharide loss, etc., according to a preferred embodiment of the present invention, wherein the volume after concentration is 1/5-1/3 before concentration.
In the method of the present invention, the concentration conditions are not particularly limited as long as the above-mentioned object can be achieved and the above-mentioned effects can be achieved. In order to improve the concentration efficiency while retaining the activity of the camellia oleifera shell polysaccharide as much as possible, preferably, the concentration is performed by a method selected from the group consisting of rotary evaporation concentration, with the conditions including: the temperature is less than or equal to 50 ℃. More preferably at a temperature of 40-50 ℃ for a period of 120- > 180 min.
According to a preferred embodiment of the present invention, wherein the method further comprises a step of performing a second solid-liquid separation on the alcohol extract after the step (2).
Any solid-liquid separation method known in the art may be suitable for use in the present invention. Preferably, the second solid-liquid separation mode is at least one selected from centrifugal separation and/or natural sedimentation.
In view of treatment efficiency and effect, more preferably, the second solid-liquid separation mode is centrifugal separation, and the conditions include: the centrifugal speed is 5000-.
According to a preferred embodiment of the present invention, wherein the method further comprises a step of drying the obtained solid phase after the second solid-liquid separation.
Any drying means known in the art may be suitable for use in the methods provided herein. In order to ensure good drying effect and simultaneously retain the biological activity of the camellia oleifera shell polysaccharide as much as possible, the drying mode is preferably selected from freeze drying and/or vacuum drying.
More preferably, the drying is by freeze drying under conditions comprising: the temperature is between-30 ℃ and-20 ℃, and the time is 480 and 600 min.
The third aspect of the present invention provides the camellia oleifera shell polysaccharide obtained by the method as described above.
The present invention will be described in detail below by way of examples. It should be understood that the following examples are only intended to further illustrate and explain the contents of the present invention by way of example, and are not intended to limit the present invention.
In the following embodiment, the camellia oleifera shell that adopts squeezes the workshop for anhui dong xu Dabieshan agricultural science and technology limited company and squeezes the production process in the tea-seed oil, carries out the shelling processing to the camellia oleifera fruit, the camellia oleifera shell that produces when obtaining the camellia seed that is used for extracting oil. Cellulase (mainly containing enzyme components such as C1 enzyme, CX enzyme and glucosidase) and papain are purchased from Huzhou Li Biotechnology limited, wherein the activity of the cellulase is 1.5 ten thousand U/g, and the activity of the papain is 40 ten thousand U/g. Amylase (mainly containing enzyme components such as alpha-amylase, beta-amylase, gamma-amylase and the like) is purchased from Yiwu Yilong bioscience, Inc., and the enzyme activity is 10 ten thousand U/g. Other reagents, which are referred to in the following examples, were purchased from a normal chemical/biological reagent supplier without specific description, and were analytically pure.
In the following examples, the preparation method of the camellia oleifera shell powder used was as follows: grinding the oil tea fruit shells subjected to impurity removal and cleaning to the particle size of about 300 mu m.
In the following examples, ethanol extract precipitates were lyophilized using a vacuum freeze dryer of type YRD-30.2A, manufactured by Shanghai Yucheng freezing facility, Inc.
Example 1
Enzymolysis: accurately weighing 10g of oil tea fruit shell powder, and mixing the oil tea fruit shell powder with 100g of water and complex enzyme.
The compound enzyme formula is as follows: 7000U cellulase, 4500U amylase, 35000U papain.
The obtained mixture was heated in a water bath at 60 ℃ for 4h while stirring at 500 rpm. Keeping the temperature of the material at 60 ℃, adjusting the pH value to 6, and then carrying out first solid-liquid separation: centrifuging at 2000rpm for 30min, and taking supernatant as a first solid-liquid separation product.
Concentration: and concentrating the supernatant to 1/3 of the original volume by rotary evaporation at 50 ℃ to obtain a concentrated first solid-liquid separation product.
Alcohol extraction: mixing the concentrated first solid-liquid separation product with absolute ethyl alcohol according to a volume ratio of 1:4, standing in a refrigerator at 4 ℃ overnight (about 12h), and obtaining an alcohol extraction product.
Second solid-liquid separation: and centrifuging the alcohol extraction product at 5000rpm for 20min to obtain alcohol extraction precipitate.
And (3) drying: freeze-drying the alcohol extraction precipitate (temperature-30 deg.C, time 480 min). The camellia oleifera shell polysaccharide A1 is obtained.
Example 2
Enzymolysis: accurately weighing 10g of oil tea fruit shell powder, and mixing the oil tea fruit shell powder with 200g of water and complex enzyme.
The compound enzyme formula is as follows: 11000U cellulase, 7250U amylase, 55000U papain.
The obtained mixture was heated in a water bath at 45 ℃ for 5h while stirring at 1000 rpm. Keeping the temperature of the materials at 45 ℃, adjusting the pH value to 5.5, and then carrying out first solid-liquid separation: centrifuging at 3000rpm for 20min, and taking supernatant as a first solid-liquid separation product.
And (3) concentrating: and concentrating the supernatant to 1/4 of the original volume by rotary evaporation at 40 ℃ to obtain a concentrated first solid-liquid separation product.
Alcohol extraction: mixing the concentrated first solid-liquid separation product with anhydrous ethanol at a volume ratio of 1:5, standing in a refrigerator at 4 deg.C overnight (about 18h), to obtain ethanol extract.
Second solid-liquid separation: and centrifuging the alcohol extraction product at 12000rpm for 15min to obtain alcohol extraction precipitate.
And (3) drying: freeze-drying the alcohol extraction precipitate (temperature-25 deg.C, time 540 min). The camellia oleifera shell polysaccharide A2 is obtained.
Example 3
Enzymolysis: accurately weighing 10g of camellia oleifera shell powder, and mixing the camellia oleifera shell powder with 300g of water and complex enzyme.
The compound enzyme formula is as follows: 15000U cellulase, 10000U amylase, 75000U papain.
The obtained mixture was heated in a water bath at 30 ℃ for 8h while stirring at 2000 rpm. Keeping the temperature of the material at 30 ℃, adjusting the pH value to 5, and then carrying out first solid-liquid separation: centrifuging at 5000rpm for 10min, and taking supernatant as a first solid-liquid separation product.
Concentration: and concentrating the supernatant to 1/5 of the original volume by rotary evaporation at 50 ℃ to obtain a concentrated first solid-liquid separation product.
Alcohol extraction: mixing the concentrated first solid-liquid separation product with absolute ethyl alcohol according to the volume ratio of 1:4.5, standing in a refrigerator at 4 ℃ for about 24 hours to obtain an alcohol extraction product.
Second solid-liquid separation: and centrifuging the alcohol extraction product at 12000rpm for 10min to obtain alcohol extraction precipitate.
And (3) drying: freeze-drying the alcohol extraction precipitate (temperature-20 deg.C, time 600 min). The camellia oleifera shell polysaccharide A3 is obtained.
Example 4
The procedure of example 1 was followed except that the papain enzyme was replaced with 35000U of the protease (purchased from Huzhou, Lily Biotech Ltd., mainly containing trypsin, having an enzyme activity of 10 ten thousand U/g). The camellia oleifera shell polysaccharide A4 is obtained.
Example 5
The procedure of example 1 was followed except that the temperature of the water bath in the enzymatic step was raised to 62 ℃. The camellia oleifera shell polysaccharide A5 is obtained.
Example 6
According to the method in example 1, except that the complex enzyme formula is as follows: 5000U cellulase, 4000U amylase, 30000U papain. The camellia oleifera shell polysaccharide A6 is obtained.
Example 7
According to the method in example 1, except that the complex enzyme formula is as follows: 15000U cellulase, 12000U amylase, 80000U papain. The camellia oleifera shell polysaccharide A7 is obtained.
Example 8
The process in example 1 was followed except that the camellia oleifera shell polysaccharide preparation was carried out using unground camellia oleifera shells. The camellia oleifera shell polysaccharide A8 is obtained.
Comparative example 1
The camellia oleifera shell polysaccharide is extracted by adopting the following water extraction-alcohol precipitation method:
10g of camellia oleifera shell powder was accurately weighed and mixed with 100g of water.
Water extraction: the obtained mixture was heated in a water bath at 60 ℃ for 4h while stirring at 500 rpm. Then, first solid-liquid separation is carried out, centrifugation is carried out for 30min under the condition of 2000rpm, and supernate is taken as a first solid-liquid separation product.
Concentration: and (3) carrying out rotary evaporation concentration on the supernatant at 50 ℃ to obtain 1/3 with the original volume, thereby obtaining a concentrated first solid-liquid separation product.
Alcohol extraction: the concentrated first solid-liquid separation product was mixed with ethanol at a volume ratio of 1:4, and left to stand in a refrigerator at 4 ℃ overnight (about 12 hours) to obtain an alcohol extract.
Second solid-liquid separation: centrifuging the alcohol extract at 5000rpm for 20min to obtain alcohol extraction precipitate.
And (3) drying: freeze-drying the alcohol extraction precipitate (temperature-30 deg.C, time 480 min). The camellia oleifera shell polysaccharide D1 is obtained.
Test example 1
Yield and properties of oil tea fruit polysaccharide
The camellia oleifera shell polysaccharide obtained in the above examples and comparative examples was weighed, and the camellia oleifera shell polysaccharide yield thereof was calculated according to the following formula. The results are detailed in table 1 and fig. 2.
The color of the camellia oleifera husk polysaccharide obtained in the above examples and comparative examples was observed at a camellia oleifera husk polysaccharide mass (g)/camellia oleifera husk raw material mass (g) × 100%, and the weight average molecular weight and molecular weight distribution of the camellia oleifera husk polysaccharide were measured by gel chromatography using a high performance liquid chromatograph manufactured by shimadzu corporation of japan and having a model of prominenceLC-20A, and the results are detailed in table 1.
TABLE 1 Camellia oleifera shell polysaccharide yield and weight average molecular weight
By observation and comparison, the colors of the camellia oleifera husk polysaccharides a1, a4, a5, A6 and a7 are darker than those of a2 and A3, wherein the color of a1 is closest to and only slightly darker than that of a2 and A3, the colors of a4, A6 and a7 are slightly darker than that of a1, and the color of a5 is the darkest among them and is closer to A8 and D1. The oil tea fruit shell polysaccharides A5, A8 and D1 have darker colors, wherein the color of D1 is the darkest, and the color of A8 times is the lightest, and the color of A5 is the lightest.
The solubility of the camellia oleifera shell polysaccharide obtained in the above examples and comparative examples was examined using different solvents. The results show that the above oil tea fruit polysaccharides all have similar solubilities as follows: it is easily dissolved in water, and aqueous solutions of acids (such as hydrochloric acid, sulfuric acid, etc.), bases (such as sodium hydroxide, etc.), salts (such as sodium chloride, etc.), but is not dissolved in organic solvents such as alcohols (such as absolute ethanol, absolute methanol, etc.), ketones (such as acetone, etc.), ethers (such as diethyl ether, etc.).
(II) influence of enzymolysis temperature on yield of oil tea fruit polysaccharide
The oil tea fruit polysaccharide was extracted by setting different enzymatic temperature gradients (5 ℃, 25 ℃, 27 ℃, 30 ℃, 45 ℃, 60 ℃, 62 ℃, 70 ℃) according to the method of example 1 (the conditions and steps except for the enzymatic temperature were the same as those of example 1). Respectively calculating the yield of the oil-tea camellia fruit polysaccharide at different temperatures, and drawing a relation curve of the enzymolysis temperature and the yield of the oil-tea camellia fruit shell polysaccharide (detailed figure 1).
As can be seen from FIG. 1, when the enzymolysis temperature is lower than 45 ℃, the yield of the oil tea fruit polysaccharide is improved along with the increase of the temperature, wherein the yield is the largest along with the increase of the temperature between 25 ℃ and 30 ℃; when the temperature reaches 45 ℃, the yield of the camellia oleifera fruit polysaccharide is highest, and then the yield is slightly reduced along with the temperature rise; when the temperature reaches 60 ℃, the yield of the camellia oleifera fruit polysaccharide is sharply reduced along with the temperature rise.
Test example 2
The main monosaccharide structural units and the contents of the oil tea fruit shell polysaccharides obtained in the above examples and comparative examples were measured by gas chromatography using a gas chromatograph GC-2010Pro manufactured by shimadzu corporation. The results are detailed in table 2.
TABLE 2 Main monosaccharide structural units and content (% by weight) in Camellia oleifera shell polysaccharide
| Numbering | Mannose | Fucose sugar | Galactose | Rhamnose | Arabinose | Glucose |
| A1 | 7.03 | 19.01 | 15.73 | 6.63 | 2.65 | 48.93 |
| A2 | 7.3 | 19.15 | 15.75 | 6.55 | 2.55 | 48.55 |
| A3 | 7.5 | 19.04 | 15.54 | 6.73 | 2.8 | 48.19 |
| A4 | 7.1 | 19.2 | 16.02 | 6.65 | 2.4 | 48.43 |
| A5 | 7.45 | 19.1 | 16.05 | 6.8 | 2.6 | 47.8 |
| A6 | 7.2 | 19.3 | 15.92 | 6.5 | 2.3 | 48.58 |
| A7 | 7.35 | 18.9 | 16.1 | 6.78 | 2.2 | 48.47 |
| A8 | 6.9 | 19.23 | 15.83 | 6.2 | 2.9 | 48.74 |
| D1 | 6.8 | 19.4 | 15.67 | 6.6 | 2.43 | 48.9 |
Test example 3
In this test example, the oxidation resistance of the camellia oleifera shell polysaccharide a3 obtained in example 3 was measured by a radical scavenging experiment.
The preparation methods of the sample solution, the working solution and the test solution used in the following experiments were as follows:
sample solution: diluting the 1mg/mL oil-tea camellia husk polysaccharide aqueous solution into different concentration gradients by using 50 volume percent ethanol aqueous solution according to sample concentrations (35 mu g/mL, 30 mu g/mL, 25 mu g/mL, 20 mu g/mL, 15 mu g/mL, 10 mu g/mL and 5 mu g/mL), thus obtaining the sample solution. The sample concentration is the final concentration of camellia oleifera husk polysaccharide A3 in the free radical scavenging experimental system.
Working fluid: TPTZ (dithiothreitol) aqueous solution (10mmol/L), FeCl3The aqueous solution (20mmol/L) and the acetic acid buffer solution (0.3mol/L) are mixed according to the volume ratio of 1:10, and the working solution is obtained. It is preheated to 37 deg.C before use.
Testing liquid: and (3) uniformly mixing 1mL of sample solution and 3mL of working solution to obtain the test solution. The test solution is mixed and then is stood for 10min in a water bath at 37 ℃ before detection.
In order to better determine the antioxidant capacity level of camellia oleifera fruit shell polysaccharide A3, in the following experiments, tea polysaccharide (purchased from Changsha Sanfu biological company) is used as a positive control, a free radical scavenging experiment is carried out by adopting the same concentration and method as that of camellia oleifera fruit shell polysaccharide, and the free radical scavenging effect level of camellia oleifera fruit polysaccharide A3 is determined by comparing the free radical scavenging rates of the tea camellia oleifera fruit shell polysaccharide and the camellia oleifera fruit shell polysaccharide.
ABTS free radical scavenging experiment
The test principle is as follows: ABTS is a relatively stable free radical, and when added to Phosphate Buffered Saline (PBS), the solution appears blue-green with a strong absorption peak near 734 nm. When a free radical scavenger is present, the single electrons of ABTS are paired to lighten the color, the absorbance at the maximum absorption wavelength becomes small, and the degree of the decrease in absorbance is quantitatively related to the degree of the radical scavenging.
The test method comprises the following steps:
ABTS solution: ABTS (purchased from Hefeibomei Biotech, Inc., purity analytical purity) was prepared into a 7mmol/L solution in blue-green color with Phosphate Buffered Saline (PBS).
Test solutions prepared by using sample solutions with different concentration gradients (1 mL each) and the ABTS solution were mixed according to the ratio of 4: 3, reacting at room temperature for 6min, measuring the absorbance at 734nm, and repeating for 3 times for each test solution.
And observing the color difference between the solution treated by the contrast test solution and the ABTS solution, and finding that the color of the treated solution is lighter than that of the ABTS solution.
A spectrophotometer model 7225 of Shanghai Kemei instrument Co., Ltd is adopted to respectively detect the absorbance of the solution at 734nm after the ABTS solution and the test solution prepared from the sample solutions with different concentration gradients are treated, and the ABTS clearance is calculated according to the following formula. The results are shown in detail in FIG. 2 and Table 3.
ABTS clearance (absorption value of test solution 734 nm/absorption value of ABTS solution 734 nm) x 100%
TABLE 3 comparison of scavenging ability of Camellia oleifera husk polysaccharide and tea polysaccharide on ABTS free radicals
From table 3 and fig. 2, it can be seen that camellia oleifera fruit chitin a3 has a scavenging effect on ABTS free radicals, the concentration of the camellia oleifera fruit chitin a3 is in positive correlation with the scavenging rate, and the scavenging effect level is close to that of camellia oleifera polysaccharide.
(II) DPPH radical scavenging experiment
The test principle is as follows: DPPH is a very stable nitrogen-centered radical, which if removed by the sample indicates that the sample has the effect of reducing the effective concentration of free radicals, alkyl radicals or peroxy radicals and breaking the lipid peroxidation chain reaction. DPPH has a single electron, has a strong absorption peak at 517nm, and the methanol solution of DPPH is dark purple, and when a free radical scavenger exists, DPPH can be paired with the single electron of DPPH to enable the absorption peak to gradually disappear.
The test method comprises the following steps:
DPPH solution: DPPH (purchased from Hefei Bomei Biotech, Ltd., purity of analytical purity) is prepared into a solution of 60 mu mol/L by adopting absolute methanol, and the solution is dark purple.
Test solutions prepared by using sample solutions with different concentration gradients (1 mL each) and a DPPH solution were mixed according to the ratio of 8: 3, shaking uniformly, standing for 30min at room temperature in a dark place, adding into a cuvette, and measuring the absorbance at 517nm, wherein each test solution is repeated for 3 times.
And observing the color difference between the solution treated by the contrast test solution and the DPPH solution, and finding that the color of the treated solution is lighter than that of the DPPH solution.
A spectrophotometer model 7225 of Shanghai Kemei instruments Co., Ltd is adopted to respectively detect the absorbance of the solution at 517nm after the solution is treated by the test solution prepared from the DPPH solution and the sample solutions with different concentration gradients, and the DPPH clearance rate is calculated according to the following formula. The results are shown in FIG. 3 and Table 4.
DPPH clearance (517 nm absorbance of test solution/517 nm absorbance of DPPH solution) x 100%
TABLE 4 comparison of scavenging ability of oil tea fruit husk polysaccharide and tea polysaccharide for DHHP free radical
| Sample concentration (μ g/mL) | Oil tea fruit shell polysaccharide clearance (%) | Tea polysaccharide clearance (%) |
| 5 | 11.84 | 12.53 |
| 10 | 22.63 | 17.47 |
| 15 | 33.45 | 25.64 |
| 20 | 43.35 | 33.18 |
| 25 | 56.42 | 40.57 |
| 30 | 71.27 | 49.63 |
| 35 | 72.43 | 60.32 |
From table 4 and fig. 3, it can be seen that camellia oleifera fruit chitin a3 has a scavenging effect on DHHP free radicals, the concentration of the camellia oleifera fruit chitin a3 is in positive correlation with the scavenging rate, and the scavenging effect is better than that of camellia oleifera polysaccharide.
(III) OH free radical scavenging experiment
The test principle is as follows: phenanthroline-Fe2+The aqueous solution of (A) has an orange-red color with a maximum absorption peak at 536 nm. When OH radicals are present, phenanthroline-Fe2+Oxidized to phenanthrene-Fe3+Its absorption peak at 536nm disappeared.
The test method comprises the following steps: phenanthroline-Fe2+An oxidation method.
phenanthroline-Fe2+Solution: using deionized water to react with o-phenanthroline-Fe2+(limited chemical reagents from the national pharmaceutical group)The purity was analytically pure) was prepared as a 0.75mmol/L solution, orange red.
Test solutions prepared by adopting sample solutions with different concentration gradients (1 mL each) and o-phenanthroline-Fe2+The solution was prepared according to 4: 1, then 1mL ferrous sulfate solution (0.75mmol/L) and 1mL 0.01% H2O2OH radicals were generated by Fenton reaction, and the reaction system was placed in a water bath at 37 ℃ for 1 hour, and then its absorbance value was measured at 536 nm. Three replicates were done for each test solution.
The color difference of the solution treated by the test solution and the solution treated by the equal volume of deionized water after Fenton oxidation is observed and contrasted, and the color of the solution treated by the test solution is darker.
Respectively detecting the phenanthrene-Fe with a spectrophotometer model 7225 of Shanghai Kemei apparatus Co., Ltd2+After the solution and Fenton oxidation, the absorbance of the solution after the treatment of the test solution prepared from the sample solutions with different concentration gradients and the solution after the treatment of deionized water at 536nm is calculated according to the following formula. The results are shown in detail in FIG. 4 and Table 5.
OH clearance (phenanthroline-Fe)2+536nm absorbance of solution-536 nm absorbance of test solution treatment solution)/(phenanthroline-Fe2+536nm absorbance of the solution-536 nm absorbance of the deionized water treated solution). times.100%
TABLE 5 comparison of OH radical scavenging ability of Camellia oleifera husk polysaccharide and tea polysaccharide
| Sample concentration (μ g/mL) | Oil tea fruit shell polysaccharide clearance (%) | Tea polysaccharide clearance (%) |
| 5 | 21.53 | 19.57 |
| 10 | 31.72 | 27.32 |
| 15 | 48.24 | 31.92 |
| 20 | 56.35 | 46.21 |
| 25 | 70.42 | 60.23 |
| 30 | 75.06 | 69.25 |
| 35 | 73.83 | 71.15 |
From table 5 and fig. 4, it can be seen that camellia oleifera fruit chitin a3 has a scavenging effect on OH free radicals, the concentration thereof is in positive correlation with the scavenging rate, and the scavenging effect is better than that of camellia oleifera polysaccharide.
According to the free radical scavenging experiment results, the camellia oleifera shell polysaccharide extracted by the method has better oxidation resistance, and the oxidation resistance level (free radical scavenging capacity level) of the camellia oleifera shell polysaccharide is equal to or better than that of tea polysaccharide. In addition, the clearance rate of the camellia oleifera shell polysaccharide on free radicals such as ABTS, DPPH, OH and the like can reach more than 65% at 30 mu g/mL, wherein the clearance rate on both ABTS free radicals and DPPH free radicals exceeds 70%.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (10)
1. The camellia oleifera shell polysaccharide is characterized by comprising a structural unit provided by the following monosaccharides: 5-10 wt% of mannose, 15-20 wt% of fucose, 15-20 wt% of galactose, 5-10 wt% of rhamnose, 1-5 wt% of arabinose and 30-50 wt% of glucose.
2. The camellia oleifera shell polysaccharide according to claim 1, wherein the weight average molecular weight of the camellia oleifera shell polysaccharide is 50000-70000.
3. A method for preparing oil-tea camellia husk polysaccharide, which is characterized by comprising the following steps:
(1) enzymolysis: mixing oil tea fruit shells, water and a compound enzyme under an enzymolysis condition for enzymolysis, and carrying out first solid-liquid separation on an obtained enzymolysis product;
(2) alcohol extraction: and contacting the liquid phase product obtained by the first solid-liquid separation with alcohol for alcohol precipitation to obtain an alcohol extraction product.
4. The method according to claim 3, wherein, in step (1), the camellia oleifera shells are used in powder form;
and/or the compound enzyme comprises: at least one of cellulase, amylase, and protease;
preferably, the particle size of the oil tea shell powder is 150-450 μm;
preferably, the cellulase is selected from at least one of a C1 enzyme, a CX enzyme, and a glucosidase enzyme;
preferably, the amylase is selected from at least one of alpha-amylase, beta-amylase, and gamma-amylase;
preferably, the protease is selected from at least one of papain, trypsin, cathepsin and subtilisin.
5. The method according to claim 3 or 4, wherein in step (1), the weight ratio of the camellia oleifera shell to water is 1: 10-30;
and/or the dosage of the cellulase is 500-1500U relative to 1g of the oil-tea camellia husk;
and/or the dosage of amylase is 400-1200U relative to 1g of the oil-tea camellia husk;
and/or the dosage of the protease is 3000-8000U relative to 1g of the oil-tea camellia husk;
preferably, the dosage of the cellulase is 700-1500U relative to 1g of the oil-tea camellia husk;
preferably, the dosage of the amylase is 450-1000U relative to 1g of the oil-tea camellia husk;
preferably, the amount of protease is 3500-7500U relative to 1g of the oil tea fruit shell;
more preferably, the cellulase, amylase and protease are used in a ratio of 1: 0.8-2.5: 2-6.
6. The method according to any one of claims 3 to 5, wherein in step (1), the conditions of the enzymatic hydrolysis comprise: the temperature is 30-60 ℃, the stirring speed is 500-;
and/or the first solid-liquid separation mode is selected from centrifugal separation and/or natural sedimentation separation;
preferably, the enzymolysis temperature is controlled by adopting a water bath heating mode;
preferably, the first solid-liquid separation mode is centrifugal separation, and the conditions comprise: the centrifugal speed is 2000-5000rpm, the centrifugal time is 10-30min, the temperature is 30-60 ℃, and the pH value is 5-6.
7. The process according to claim 3, wherein, in step (2), the alcohol is selected from C1-C5 alcohols;
preferably, the alcohol is selected from at least one of methanol and/or ethanol and aqueous solutions thereof;
preferably, the alcohol is used in an amount of 3 to 25mL, preferably 5 to 20mL, relative to 1g of the liquid phase product on a dry basis;
more preferably, the conditions of alcohol precipitation include: the temperature is 3-5 ℃, the time is 12-24h, and the volume concentration of alcohol in the alcohol precipitation system is 70-85%.
8. The process according to claim 3, wherein the process further comprises a step of concentrating the liquid-phase product obtained by the first solid-liquid separation, prior to the step (2);
and/or, the method further comprises the step of subjecting the alcohol extract to a second solid-liquid separation after step (2);
preferably, the concentration mode is selected from rotary evaporation concentration and/or water bath evaporation concentration;
preferably, the second solid-liquid separation mode is selected from centrifugal separation and/or natural settling separation;
more preferably, the post-concentration volume is 1/5-1/3 before concentration;
more preferably, the second solid-liquid separation mode is centrifugal separation, and the conditions comprise: the centrifugal speed is 5000-;
further preferably, the concentration is performed by a method selected from rotary evaporation concentration, and the conditions comprise: the temperature is less than or equal to 50 ℃, more preferably the temperature is 40-50 ℃, and the time is 120-180 min.
9. The method according to claim 8, wherein the method further comprises the step of drying the obtained solid phase after the second solid-liquid separation;
preferably, the drying is selected from freeze drying and/or vacuum drying;
more preferably, the drying is by freeze drying under conditions comprising: the temperature is between-30 ℃ and-20 ℃ and the time is 480-.
10. The camellia oleifera shell polysaccharide obtained by the preparation according to any one of claims 3 to 9.
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|---|---|---|---|---|
| US5952023A (en) * | 1996-02-05 | 1999-09-14 | Lipton, Division Of Conopco, Inc. | Enzyme extraction process for tea |
| CN101560265A (en) * | 2009-06-01 | 2009-10-21 | 浙江大学 | Method for preparing oil-tea camellia husk polysaccharide and purifying method |
| CN101560266A (en) * | 2009-06-01 | 2009-10-21 | 浙江大学 | Oil-tea camellia husk polysaccharide and application thereof |
| CN102796203A (en) * | 2011-05-27 | 2012-11-28 | 中南林业科技大学 | Method for preparing anti-oxidization active camellia olefera cake polysaccharide |
| CN105820263A (en) * | 2016-04-15 | 2016-08-03 | 深圳大学 | Process for extracting camellia oleifera shell husk chitin |
| CN109833377A (en) * | 2019-03-27 | 2019-06-04 | 长沙理工大学 | A kind of extract from fruit shell of camellia oleifera abel and its preparation method and application |
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2022
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5952023A (en) * | 1996-02-05 | 1999-09-14 | Lipton, Division Of Conopco, Inc. | Enzyme extraction process for tea |
| CN101560265A (en) * | 2009-06-01 | 2009-10-21 | 浙江大学 | Method for preparing oil-tea camellia husk polysaccharide and purifying method |
| CN101560266A (en) * | 2009-06-01 | 2009-10-21 | 浙江大学 | Oil-tea camellia husk polysaccharide and application thereof |
| CN102796203A (en) * | 2011-05-27 | 2012-11-28 | 中南林业科技大学 | Method for preparing anti-oxidization active camellia olefera cake polysaccharide |
| CN105820263A (en) * | 2016-04-15 | 2016-08-03 | 深圳大学 | Process for extracting camellia oleifera shell husk chitin |
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