CN103274979B - Method for separating and purifying astaxanthin from antarctic krill shells - Google Patents
Method for separating and purifying astaxanthin from antarctic krill shells Download PDFInfo
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- Coloring Foods And Improving Nutritive Qualities (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for separating and purifying astaxanthin from antarctic krill shells. The method comprises the following steps of carrying out enzymolysis treatment on antarctic krill shells serving as raw materials through alcalase so as to obtain enzyme-hydrolyzed protein liquid, krill shell residues and pigment precipitates, extracting pigments from the krill shell residues and the pigment precipitates by respectively using acetone and ethanol, combining extracts, saponifying the combined extracts by using NaOH-ethanol so as to obtain saponified liquid, adsorbing free astaxanthin in the saponified liquid by using AB-8 macroporous resin, carrying out further separation and purification on the adsorbed free astaxanthin through a silica gel column, and carrying out freeze-drying so as to obtain high-purity astaxanthin powder. According to the method, the krill shells are processed by using the alcalase, so that the release of the astaxanthin is facilitated, and meanwhile, the protein is protected; and a method that macroporous adsorption resin and silica gel column chromatography are combined is adopted, so that the realization of large-batch production is facilitated.
Description
Technical field
The present invention relates to the method that a seed shrimp shell produces biological products, particularly relate to a kind of method of separation and purification astaxanthin from krill shell.
Background technology
In recent years, increasingly serious along with resource problem, the exploitation of ocean resource become the problem falling over each other to study in various countries.According to estimates, the biomass of krill is 3.79 hundred million tons, and almost spread all over marine site, the whole South Pole, resource is extremely abundant.Various countries pay close attention to the biomass of krill in each marine site and life habit, and amount of fishing increases year by year.Krill has very high development and application values, except containing except rich in protein, lipid and mineral substance, also containing various active material, as proteinase, astaxanthin and chitin etc.At present, study separation and the property analysis of the composition such as enzyme, lipid acid in more concern krill shrimp both at home and abroad, less to the research of krill shell resource.Shrimp shell abandons as waste mostly, causes the waste to astaxanthin resource.
Astaxanthin very easily with free radical reaction, can active oxygen effectively in purged body, there is extremely strong anti-oxidant activity.Astaxanthin energy force rate β-carotene in the activity, scavenging free radicals of quenching activity oxygen is high more than 10 times, is 80-550 times of VE.Astaxanthin has anti-lipid peroxidation effect simultaneously, and the energy force rate VE of anti-lipid peroxidation is high more than 100 times, and phosphatidylcholine lipid can also be protected from oxidation.
A large amount of experimentation on animals shows, astaxanthin has effect that is anticancer, hypotensive, the breeding of strengthening immunity, promotion aquatic animal to grow, and coloring effect is remarkable.The activity of astaxanthin variable partition gene, the generation of enhancing antibody, suppresses the transfer of malignant tumour, thus effectively prevents or slow down the effect of cancer.Experimentation on animals shows, astaxanthin all has obvious restraining effect to liver cancer, oral carcinoma and bladder cancer.The effect that astaxanthin has growth promoting effects to breed, can promote the Reproduction of fish, and promote fish-egg fertilization, reduce the mortality ratio of fetal development, very fast individual growth, increases ripe speed and fecundity, can also improve the laying rate of poultry.Astaxanthin is used for makeup can anti-ageing, the skin whitening of smoothing wrinkle, also can remove the chloasma produced because of age growth.Astaxanthin also can be used as excellent fodder additives, can not only be that animal body is painted, can also promote body growth, the immunizing power improving animal and fecundity.Astaxanthin is bright-colored, belongs to fat-soluble pigment, and therefore for the food that food especially lipid content is higher, not only coloring effect is good, can also prevent variable color, spoiledly and rotten play freshening effect.Astaxanthin goods be applied to vegetables, marine alga and fruit the aspect such as to pickle.
Astaxanthin is widely used in food, medicine, makeup and feedstuff industry.Natural astaxanthin is high because of its security, is more received by the market.Current natural astaxanthin is mainly derived from algae, phaffia rhodozyma and Crustacean.The large-scale production of algae, yeast needs superior strain, strict envrionment conditions and broken wall measure efficiently, and production cost is higher; And Crustacean due to the content of astaxanthin lower, exploitation are in this respect less.And mainly with whole euphausia superba as raw material, in extraction purification process, easily cause the waste to protein; Simultaneously because purification process mostly is high performance liquid phase, suitability for industrialized production is restricted.Therefore, need a kind of method exploring separation and purification astaxanthin from krill shell badly, to meet comprehensive utilization and the suitability for industrialized production of prawn shell resource.
Summary of the invention
The object of this part is some aspects of general introduction embodiments of the invention and briefly introduces some preferred embodiments.May do in the specification digest and denomination of invention of this part and the application a little simplify or omit with avoid making this part, specification digest and denomination of invention object fuzzy, and this simplification or omit and can not be used for limiting the scope of the invention.
In view of Problems existing in the method for above-mentioned and/or existing separation and purification astaxanthin from krill shell, propose the present invention.
Therefore, the present invention's object be for existing from krill shell the method degree of purification of separation and purification astaxanthin low and be unfavorable for the shortcoming of suitability for industrialized production, there is provided one with krill shell for raw material, utilize Alcalase enzymic hydrolysis krill shell, obtain protein enzymatic hydrolyzate, shrimp shell residue and pigementation respectively, pigment is extracted again from shrimp shell residue and pigementation, by saponification reaction, astaxanthin ester is converted into free astaxanthin, and the high purification degree of application punching polymeric adsorbent and silica gel column chromatography technology separation purifying astaxanthin and industrial method.
For solving the problems of the technologies described above, the invention provides following technical scheme: a kind of method of separation and purification astaxanthin from krill shell, krill shell after enzymolysis more after filtration, centrifugal, extraction, saponification, resin absorption, silica gel column chromatography, lyophilize step, obtain highly purified astaxanthin powder; Wherein, described enzymolysis is under pH is the condition of 8.0 ~ 8.5, adopt Alcalase enzyme to carry out enzymolysis; Described filtration is filtered by enzymolysis solution to obtain shrimp shell residue and filtrate; Described centrifugal be that described filtrate is centrifugal under the rotating speed of 6000rap/min ~ 8000rap/min, obtain enzymolysis supernatant liquor and pigementation; Described extraction described shrimp shell residue by acetone suction filtration is obtained extracting solution and described pigementation is obtained extracting solution through ethanol suction filtration to merge, concentrated except desolventizing under 40 ° of C ~ 45 ° C conditions, obtains thick pigment oil; Described saponification be first with dehydrated alcohol by the dilution of described thick pigment oil, then add NaOH-ethanolic soln and carry out saponification and obtain saponification liquor; Described resin absorption is diluted by described saponification liquor, then by macroporous adsorptive resins, during adsorption equilibrium, use ethyl acetate desorb, obtain stripping workshop, concentrated except desolventizing; Described silica gel column chromatography gets described stripping workshop concentrated solution loading, carries out gradient elution according to the normal hexane → normal hexane-acetone → normal hexane-acetone of 100%, collects red component, concentrated except desolventizing, obtains free astaxanthin concentrated solution; Described lyophilize be under-20 DEG C ~-18 DEG C conditions by the lyophilize of described free astaxanthin concentrated solution, obtain astaxanthin powder.
As a kind of preferred version of the method for separation and purification astaxanthin from krill shell of the present invention, wherein: described enzymolysis krill shell is used in Alcalase2.4L enzyme carry out enzymolysis, regulate pH8.2 ~ 8.5, to add Alcalase enzyme than 3000U/g at the bottom of enzyme, lucifuge water-bath vibration 2.5h ~ 3.5h at 55 DEG C.
As a kind of preferred version of the method for separation and purification astaxanthin from krill shell of the present invention, wherein: described filtration is filtered with 50 ~ 100 object filter materials by enzymolysis solution to obtain shrimp shell residue and filtrate.
As a kind of preferred version of the method for separation and purification astaxanthin from krill shell of the present invention, wherein: described centrifugal be by described filtrate at 4 DEG C, centrifugal 20min under 7000rap/min condition, obtains enzymolysis supernatant liquor and pigementation.
As a kind of preferred version of the method for separation and purification astaxanthin from krill shell of the present invention, wherein: described shrimp shell residue is got in described extraction, add acetone with solid-liquid ratio (w:v) 1:4 ~ 1:6,40 DEG C of lucifuge water-bath vibration 3.0h ~ 4.0h, suction filtration obtains extracting solution; Get described pigementation, add ethanol with solid-liquid ratio (w:v) 1:3 ~ 1:5,40 DEG C of lucifuge water-bath vibration 1.5h ~ 2.5h, suction filtration obtains extracting solution; Merge the extracting solution of described shrimp shell residue and described pigementation, concentrated except desolventizing at 40 DEG C, obtain thick pigment oil.
As a kind of preferred version of the method for separation and purification astaxanthin from krill shell of the present invention, wherein: described saponification is that with dehydrated alcohol, described thick pigment oil to be diluted to concentration be 0.1g/mL, add the NaOH-ethanolic soln of 1/5 volume 0.020mol/L, lucifuge saponification 11h ~ 13h at 5 DEG C.
As a kind of preferred version of the method for separation and purification astaxanthin from krill shell of the present invention, wherein: described resin absorption is dilution saponification liquor to astaxanthin concentration is 2 μ g/mL, with the AB-8 macroporous adsorptive resins that the flow velocity of 4BV/h is 10mm × 200mm by specification, during adsorption equilibrium, use ethyl acetate desorb, obtain stripping workshop, and concentrated except desolventizing at 40 DEG C.
As a kind of preferred version of the method for separation and purification astaxanthin from krill shell of the present invention, wherein: described silica gel column chromatography selects 100 ~ 200 object silica gel dress posts, adds normal hexane, wet method dress post; Get stripping workshop concentrated solution loading, the normal hexane-acetone of the normal hexane-acetone → volume ratio 4:1 of the normal hexane → volume ratio 23:2 according to 100% carries out gradient elution, collects red component, and concentrated except desolventizing at 40 DEG C, obtains free astaxanthin concentrated solution.
As a kind of preferred version of the method for separation and purification astaxanthin from krill shell of the present invention, wherein: described lyophilize be under-18 DEG C of conditions by the lyophilize of free astaxanthin concentrated solution, obtain astaxanthin powder.
First the present invention carries out enzymolysis processing to krill shell, and the shrimp shell residue obtained and pigementation are for extracting astaxanthin, and protein enzymatic hydrolyzate can be used for preparing protein powder, thus realize the comprehensive utilization of shrimp shell resource; By saponification reaction, astaxanthin ester is converted into free astaxanthin, is conducive to the quantitative analysis of astaxanthin; Absorption with macroporous adsorbent resin is used to be separated astaxanthin, for suitability for industrialized production provides Technical Reference; By the further separation and purification astaxanthin of silica gel column chromatography, obtain the astaxanthin of purity 97.1%, meet the application of astaxanthin at food, healthcare products and cosmetic industry.
Accompanying drawing explanation
The process flow diagram of Fig. 1 separation and purification astaxanthin from krill shell.
Embodiment
Below in conjunction with specific embodiment, frock of the present invention is described in detail.
Be described below in detail embodiments of the invention, the example of described embodiment is shown in the drawings, and wherein same or similar label represents same or similar element or has element that is identical or similar functions from start to finish.Being exemplary below by the embodiment be described with reference to the drawings, only for explaining the present invention, and can not limitation of the present invention being interpreted as.
Embodiment 1
Get 100g krill shell, moisture content about 78.2%.PH is regulated to be adjusted to 8.2, to add Alcalase enzyme than 3000U/g at the bottom of enzyme, lucifuge water-bath vibration 2.5h at 55 DEG C; Enzymolysis solution obtains shrimp shell residue 74.2g and filtrate through filtered through gauze, and filtrate centrifugal (4 DEG C, 6000rap/min, 20min), obtains enzymolysis supernatant liquor and pigementation 16.5g.In shrimp shell residue, add acetone with solid-liquid ratio (w:v) 1:5,40 DEG C of lucifuge water-bath vibration 3.5h, suction filtration obtains extracting solution; Add ethanol with solid-liquid ratio (w:v) to pigementation 1:4,40 DEG C of lucifuge water-bath vibration 2.0h, suction filtration obtains extracting solution.Merge the extracting solution of shrimp shell residue and pigementation, concentrated except desolventizing at 40 DEG C, obtain thick pigment oil 0.70g.Then with dehydrated alcohol, thick pigment oil being diluted to concentration is 0.1g/mL, add the 0.020mol/L NaOH-ethanol of 1/5 volume, at 5 DEG C after lucifuge saponification 12h, dilution saponification liquor to astaxanthin concentration is 2 μ g/mL, with the flow velocity of 4BV/h by AB-8 macroporous adsorbent resin, during adsorption equilibrium, use ethyl acetate desorb, and concentrated except desolventizing at 40 DEG C, in stripping workshop concentrated solution, free content astaxanthin is 284.4 μ g.
Take the 100-200 object column chromatography silica gel of 30-50g, add normal hexane, wet method dress post; Get stripping workshop concentrated solution loading, normal hexane according to 100% → normal hexane-acetone (92:8) → normal hexane-acetone (80:20) carries out gradient elution, first be that two lurid colour bands are eluted, when with normal hexane-acetone (80:20) wash-out, a red zone is got off by washed, collect this component, concentrated except desolventizing at 40 DEG C, recording free astaxanthin content is 283.0 μ g.By the lyophilize of free astaxanthin concentrated solution under-18 DEG C of conditions, obtain the astaxanthin powder of purity 99.2%.AB-8 macroporous resin can Reusability.
Embodiment 2
Get 100g krill shell, moisture content 75.3%.Regulate pH to 8.5, to add Alcalase enzyme than 3000U/g at the bottom of enzyme, lucifuge water-bath vibration 3.0h at 55 DEG C; Enzymolysis solution obtains shrimp shell residue 72.6g and filtrate through filtered through gauze, and filtrate centrifugal (4 DEG C, 7000rap/min, 20min), obtains enzymolysis supernatant liquor and pigementation 20.8g.In shrimp shell residue, add acetone with solid-liquid ratio (w:v) 1:4,40 DEG C of lucifuge water-bath vibration 4.0h, suction filtration obtains extracting solution; Add ethanol with solid-liquid ratio (w:v) to pigementation 1:5,40 DEG C of lucifuge water-bath vibration 1.5h, suction filtration obtains extracting solution.Merge the extracting solution of shrimp shell residue and pigementation, concentrated except desolventizing at 40 DEG C, obtain thick pigment oil 0.75g.Then with dehydrated alcohol, thick pigment oil being diluted to concentration is 0.1g/mL, add the 0.020mol/L NaOH-ethanol of 1/5 volume, at 5 DEG C after lucifuge saponification 13h, dilution saponification liquor to astaxanthin concentration is 2 μ g/mL, with the flow velocity of 4BV/h by AB-8 macroporous adsorbent resin, during adsorption equilibrium, use ethyl acetate desorb, and concentrated except desolventizing at 45 DEG C, in stripping workshop concentrated solution, free content astaxanthin is 306.6 μ g.
Take the 100-200 object column chromatography silica gel of 30-50g, add normal hexane, wet method dress post; Get stripping workshop concentrated solution loading, normal hexane according to 100% → normal hexane-acetone (92:8) → normal hexane-acetone (80:20) carries out gradient elution, first be that two lurid colour bands are eluted, when with normal hexane-acetone (80:20) wash-out, a red zone is got off by washed, collect this component, and concentrated except desolventizing at 40 DEG C, and recording free astaxanthin content is 305.0 μ g.By the lyophilize of free astaxanthin concentrated solution under-18 DEG C of conditions, obtain the astaxanthin powder of purity 98.4%.AB-8 macroporous resin can Reusability.
Embodiment 3
Get 100g krill shell, moisture content 79%.Regulate pH8.0-8.5, to add Alcalase enzyme than 3000U/g at the bottom of enzyme, lucifuge water-bath vibration 3.5h at 55 DEG C; Enzymolysis solution obtains shrimp shell residue 70.5g and filtrate through filtered through gauze, and filtrate centrifugal (4 DEG C, 8000rap/min, 20min), obtains enzymolysis supernatant liquor and pigementation 18.2g.In shrimp shell residue, add acetone with solid-liquid ratio (w:v) 1:6,40 DEG C of lucifuge water-bath vibration 3.0h, suction filtration obtains extracting solution; Add ethanol with solid-liquid ratio (w:v) to pigementation 1:3,40 DEG C of lucifuge water-bath vibration 2.5h, suction filtration obtains extracting solution.Merge the extracting solution of shrimp shell residue and pigementation, concentrated except desolventizing at 40 DEG C, obtain thick pigment oil 0.72g.Then with dehydrated alcohol, thick pigment oil being diluted to concentration is 0.1g/mL, add the 0.020mol/L NaOH-ethanol of 1/5 volume, at 5 DEG C after lucifuge saponification 11h, dilution saponification liquor to astaxanthin concentration is 2 μ g/mL, with the flow velocity of 4BV/h by AB-8 macroporous adsorbent resin, during adsorption equilibrium, use ethyl acetate desorb, and concentrated except desolventizing at 40 DEG C, in stripping workshop concentrated solution, free content astaxanthin is 294.5 μ g.
Take the 100-200 object column chromatography silica gel of 30-50g, add normal hexane, wet method dress post; Get stripping workshop concentrated solution loading, normal hexane according to 100% → normal hexane-acetone (92:8) → normal hexane-acetone (80:20) carries out gradient elution, first be that two lurid colour bands are eluted, when with normal hexane-acetone (80:20) wash-out, a red zone is got off by washed, collect this component, and concentrated except desolventizing at 40 DEG C, and recording free astaxanthin content is 294.0 μ g.By the lyophilize of free astaxanthin concentrated solution under-18 DEG C of conditions, obtain the astaxanthin powder of purity 97.1%.AB-8 macroporous resin can Reusability.
In sum, the present invention with krill shell for raw material, through the enzymolysis processing of Alcalase, obtain protein enzymatic hydrolyzate, shrimp shell residue and pigementation, from shrimp shell residue and pigementation, extract pigment, united extraction liquid with acetone and ethanol respectively, saponification through NaOH-ethanol obtains saponification liquor, utilize the free astaxanthin in AB-8 macroporous resin adsorption saponification liquor, then through the further separation and purification of silicagel column, obtained the astaxanthin powder of purity 97.1% ~ 99.2% by freeze-drying.The present invention utilizes Alcalase ferment treatment shrimp shell, is conducive to the release of astaxanthin, protects protein simultaneously; The method of application macroporous adsorbent resin binding silica gel column chromatography, is convenient to realize producing in enormous quantities.
It should be noted that, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.
Claims (6)
1. the method for separation and purification astaxanthin from krill shell, is characterized in that:
Krill shell after enzymolysis more after filtration, centrifugal, extraction, saponification, resin absorption, silica gel column chromatography, lyophilize step, obtain highly purified astaxanthin powder; Wherein,
Described enzymolysis krill shell is used in Alcalase 2.4L enzyme to carry out enzymolysis, regulates pH8.2 ~ 8.5, to add Alcalase enzyme than 3000U/g at the bottom of enzyme, and lucifuge water-bath vibration 2.5h ~ 3.5h at 55 DEG C;
Described filtration is filtered by enzymolysis solution to obtain shrimp shell residue and filtrate;
Described centrifugal be that described filtrate is centrifugal under the rotating speed of 6000rap/min ~ 8000rap/min, obtain enzymolysis supernatant liquor and pigementation;
Described shrimp shell residue is got in described extraction, adds acetone with solid-liquid ratio w:v1:4 ~ 1:6,40 DEG C of lucifuge water-bath vibration 3.0h ~ 4.0h, and suction filtration obtains extracting solution; Get described pigementation, add ethanol with solid-liquid ratio w:v1:3 ~ 1:5,40 DEG C of lucifuge water-bath vibration 1.5h ~ 2.5h, suction filtration obtains extracting solution; Merge the extracting solution of described shrimp shell residue and described pigementation, concentrated except desolventizing at 40 DEG C, obtain thick pigment oil;
Described saponification be first with dehydrated alcohol by the dilution of described thick pigment oil, then add NaOH-ethanolic soln and carry out saponification and obtain saponification liquor;
Described resin absorption is diluted by described saponification liquor, then by macroporous adsorptive resins, during adsorption equilibrium, use ethyl acetate desorb, obtain stripping workshop, concentrated except desolventizing;
Described silica gel column chromatography selects 100 ~ 200 object silica gel dress posts, adds normal hexane, wet method dress post; Get stripping workshop concentrated solution loading, the normal hexane-acetone of the normal hexane-acetone → volume ratio 4:1 of the normal hexane → volume ratio 23:2 according to 100% carries out gradient elution, collects red component, and concentrated except desolventizing at 40 DEG C, obtains free astaxanthin concentrated solution;
Described lyophilize be under-20 DEG C ~-18 DEG C conditions by the lyophilize of described free astaxanthin concentrated solution, obtain astaxanthin powder.
2. the method for separation and purification astaxanthin from krill shell according to claim 1, is characterized in that: described filtration is filtered with 50 ~ 100 object filter materials by enzymolysis solution to obtain shrimp shell residue and filtrate.
3. the method for separation and purification astaxanthin from krill shell according to claim 1, is characterized in that: described centrifugal be by described filtrate at 4 DEG C, centrifugal 20min under 6000-8000rap/min condition, obtains enzymolysis supernatant liquor and pigementation.
4. the method for separation and purification astaxanthin from krill shell according to claim 1, it is characterized in that: described saponification is that with dehydrated alcohol, described thick pigment oil to be diluted to concentration be 0.1g/mL, add the NaOH-ethanolic soln of 1/5 volume 0.020mol/L, lucifuge saponification 11h ~ 13h at 5 DEG C.
5. the method for separation and purification astaxanthin from krill shell according to claim 1, it is characterized in that: described resin absorption is dilution saponification liquor to astaxanthin concentration is 2 μ g/mL, with the AB-8 macroporous adsorptive resins that the flow velocity of 4BV/h is 10mm × 200mm by specification, during adsorption equilibrium, use ethyl acetate desorb, obtain stripping workshop, and concentrated except desolventizing at 40 DEG C.
6. the method for separation and purification astaxanthin from krill shell according to claim 1, is characterized in that: described lyophilize be under-18 DEG C of conditions by the lyophilize of free astaxanthin concentrated solution, obtain astaxanthin powder.
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| CN104529851A (en) * | 2014-12-06 | 2015-04-22 | 黑龙江众生生物工程有限公司 | New technology for extracting astaxanthin from Haematococcus pluvialis through enzyme method |
| CN106591410A (en) * | 2016-12-30 | 2017-04-26 | 深圳市江牧实业有限公司 | Method for preparing polypeptide and astaxanthin from shrimp and crab processing waste |
| CN107033049A (en) * | 2017-06-09 | 2017-08-11 | 中国水产科学研究院南海水产研究所 | A kind of method that combined-enzyme method extracts krill astaxanthin |
| CN108863881A (en) * | 2018-08-23 | 2018-11-23 | 济宁医学院 | A method of extracting astaxanthin from shrimp shell and crab shell |
| CN108752252B (en) * | 2018-08-23 | 2021-12-14 | 济宁医学院 | Method for extracting and purifying astaxanthin in shrimp shells and crab shells by using neutral protease |
| CN111978231A (en) * | 2019-05-21 | 2020-11-24 | 清馨(北京)科技有限公司 | Purification method of astaxanthin oil |
| CN110720623A (en) * | 2019-10-14 | 2020-01-24 | 浙江海洋大学 | Method for preparing seasoning by using euphausia superba processing by-product |
| CN110724082A (en) * | 2019-10-15 | 2020-01-24 | 浙江海洋大学 | Method for extracting astaxanthin from antarctic krill |
| CN117604060A (en) * | 2024-01-24 | 2024-02-27 | 逢时(青岛)海洋科技有限公司 | Free astaxanthin based on immobilized cholesterol esterase and preparation method thereof |
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