CN111318050B - Method for comprehensively extracting polyunsaturated fatty acid and polysaccharide from seaweed - Google Patents

Method for comprehensively extracting polyunsaturated fatty acid and polysaccharide from seaweed Download PDF

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CN111318050B
CN111318050B CN202010147243.3A CN202010147243A CN111318050B CN 111318050 B CN111318050 B CN 111318050B CN 202010147243 A CN202010147243 A CN 202010147243A CN 111318050 B CN111318050 B CN 111318050B
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extraction
polysaccharide
seaweed
polyunsaturated fatty
sargassum
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CN111318050A (en
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柯珂
王一兵
王俊
庄军莲
张荣灿
黄晓煦
雷富
赖俊翔
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Guangxi University for Nationalities
Guangxi Academy of Sciences
Guangxi Research Institute of Chemical Industry
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Guangxi Academy of Sciences
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • B01D11/028—Flow sheets
    • B01D11/0284—Multistage extraction
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
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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
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/10—Production of fats or fatty oils from raw materials by extracting
    • C11B1/108—Production of fats or fatty oils from raw materials by extracting after-treatment, e.g. of miscellae

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Abstract

The invention discloses a method for comprehensively extracting polyunsaturated fatty acid and polysaccharide from seaweed. The invention can improve the extraction rate, reduce the consumption of seaweed and extraction solvent and the generation of waste, improve the utilization efficiency of resources, reduce the cost and improve the economic value of the seaweed.

Description

Method for comprehensively extracting polyunsaturated fatty acid and polysaccharide from seaweed
Technical Field
The invention relates to the technical field of marine organism processing, in particular to a method for comprehensively extracting polyunsaturated fatty acid and polysaccharide from seaweed.
Background
Seaweeds (Algae or Seaweeds) are important components of marine biological resources, are original producers of organic matters in the sea and natural enrichers of inorganic matters, and contain abundant amino acids, cellulose, vitamins, minerals, trace elements and other nutrient components, and a plurality of active substances with novel structures and unique effects, such as proteins, polysaccharides, lipids, terpenoids, sterols, alkaloids, saponins, enzymes and the like.
The polyunsaturated fatty acids (PUFAs) in algae have strong bioactivity, and can promote development of nervous system and visual system, prevent and treat cardiovascular diseases, and resist cancer, inflammation, and oxidation. The PUFAs content in the seaweed is high, compared with PUFAs extracted from marine fishes, the PUFAs has the advantages of good oxidation stability, no fishy smell, simple composition of seaweed fatty acid and easy separation and purification; meanwhile, the seaweed resources are rich,
can be used for large-scale cultivation, and has low cost. Thus, seaweed is receiving increasing attention as a new source of polyunsaturated fatty acids from scholars both at home and abroad.
Algal polysaccharide is a general term of all high molecular carbohydrates in algae, generally has the characteristics of water solubility, high viscosity and the like, and has strong physiological activity, such as immunity promotion and anti-tumor effect; can resist virus and has certain effect on treating AIDS; has effects in lowering blood sugar, reducing blood lipid, resisting blood coagulation, resisting oxidation, and resisting aging; in addition, the seaweed polysaccharide also has good moisture absorption and retention and whitening effects. Therefore, the algal polysaccharide is a marine medicine with great development potential, and a health-care product and a beauty product resource.
At present, the PUFAs and the polysaccharide of the seaweed are extracted separately, the seaweed after the PUFAs is extracted can be directly discarded, in order to improve the extraction rate of the polysaccharide, the seaweed needs to be degreased by using an organic solvent before the polysaccharide is extracted, and the fatty acid component and the solvent which can not be completely recovered in the degreasing solvent are discarded. Thus not only wasting seaweed and organic solvent, but also polluting environment.
Disclosure of Invention
Aiming at the problems, the method for comprehensively extracting the polyunsaturated fatty acid and the polysaccharide of the seaweed is provided, the seaweed is taken as a raw material, the polyunsaturated fatty acid is extracted firstly, the seaweed from which the polyunsaturated fatty acid is extracted is separated, and then the polysaccharide is extracted, so that the extraction rate can be improved, the use amount of the seaweed and an extraction solvent and the generation of wastes are reduced, the resource utilization efficiency is improved, the cost is reduced, and the economic value of the seaweed is improved.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the invention relates to a method for comprehensively extracting polyunsaturated fatty acid and polysaccharide from seaweed.
Further, the method for comprehensively extracting the polyunsaturated fatty acids and the polysaccharides from the seaweeds comprises the steps of taking the seaweeds as raw materials, firstly extracting the polyunsaturated fatty acids by using petroleum ether, then separating the seaweeds with the extracted polyunsaturated fatty acids, washing the seaweeds by using absolute ethyl alcohol, drying the seaweed, and then extracting the polysaccharides by using water.
Further, the extraction of the polyunsaturated fatty acids comprises the following steps:
A. pulverizing pretreated Sargassum into 10-80 mesh Sargassum powder, dispersing Sargassum powder in petroleum ether, and ultrasonic pulverizing at temperature below 80 deg.C;
B. performing solid-liquid separation on the suspension after ultrasonic crushing, supplementing petroleum ether into the separated liquid as an extraction solvent to ensure that the weight volume ratio of the seaweed meal to the extraction solvent is 1:5-20, respectively placing the extraction solvent and the solid residues into corresponding vessels of a Soxhlet extraction device, performing reflux extraction at 80-90 ℃ for more than 8 hours, collecting the extraction solution, and separating extraction residues for later use;
C. evaporating the extracting solution to dryness after extraction is finished, wherein the residue after evaporation to dryness is the total lipid of the seaweed;
D. dissolving total lipids of Sargassum with petroleum ether 1-4 times of its weight, transferring to drying container, blow-drying with nitrogen gas to obtain dried substance, adding KOH-CH of 0.4-0.8mol/L to the same volume of the dried substance3Heating OH solution at 50-70 deg.C for more than 50min under nitrogen charging, adjusting pH to 1-2 with 10% (V/V) HCl dilute solution, adding water, and making into beverageMixing to separate the solution, and centrifuging to obtain upper oil layer as total fatty acid.
Further, the pretreatment method in the step A comprises the following steps: soaking in tap water 20-50 times of the weight of Sargassum for 5-30min, washing with tap water and deionized water for more than 1 times, and baking at 80 deg.C for 10-30 hr.
Further, the ratio of the seaweed meal to the petroleum ether in the step A is 1: 5-20.
Further, the ultrasonic power of the ultrasonic crushing in the step A is 60-90W, and the crushing time is 5-40 min.
Further, in the step C, the temperature for evaporating the extracting solution is 30-40 ℃, and the extracting solution is evaporated by rotary evaporation.
Further, the polysaccharide extraction comprises the following steps:
E. washing the residue obtained after Soxhlet extraction in the step B with absolute ethyl alcohol for more than 2 times, then drying at 40-70 ℃, adding pure water which is 20-50 times of the weight of the residue, stirring and extracting at 60-90 ℃ for more than 8h, filtering, collecting filtrate, and then centrifuging the filtrate to obtain centrifugate;
F. evaporating and concentrating the centrifugate at 40-70 deg.C to 1/4 of original volume, adding 80-100% ethanol 1-4 times of the volume of the concentrate, mixing, refrigerating at 0-6 deg.C for more than 10 hr, and centrifuging to obtain precipitate; freeze-drying the precipitate to obtain crude algal polysaccharide;
G. dissolving the crude seaweed polysaccharide product into pure water with the weight 1-4 times of that of the crude seaweed polysaccharide product, adding Sevag reagent, uniformly mixing, performing centrifugal separation to obtain a water layer and a Sevag reagent layer, discarding the Sevag reagent layer, then adding the Sevag reagent into the water layer again, performing centrifugal separation, discarding the Sevag reagent layer, and repeating the steps of adding the Sevag reagent into the water layer and performing centrifugal separation until no organic matter or protein appears in the Sevag reagent layer;
H. adding 80-100% ethanol with volume percentage of 1-4 times of the volume of the water layer after the centrifugal treatment, refrigerating at 0-6 ℃ for more than 10h, and then performing centrifugal separation to obtain a precipitate; freeze drying the precipitate to obtain algal polysaccharide.
Further, the temperature of freeze drying in the step F and the step H is-35 ℃ to-25 ℃.
Further, the seaweed is gulfweed.
The Sevage reagent is a mixed solution of chloroform and n-butanol, wherein the weight ratio of chloroform: n-butanol (V/V) ═ 5: 1.
In view of the above, the present invention has the following remarkable effects:
according to the method for comprehensively extracting the polyunsaturated fatty acid and the polysaccharide of the seaweed, the seaweed is taken as a raw material, the polyunsaturated fatty acid is extracted firstly, the seaweed from which the polyunsaturated fatty acid is extracted is separated, then the polysaccharide is extracted, compared with the method for directly extracting the fatty acid and the polysaccharide respectively, the consumption of the seaweed raw material is reduced by half, the consumption of an extraction solvent and the generation of wastes can be effectively reduced, the resource utilization efficiency is greatly improved, the cost is reduced, and the economic value of the seaweed is improved. More importantly, the extraction of the polysaccharide is carried out after the fatty acid is extracted, so that the extraction rate of the polysaccharide can be improved while the extraction rate of the fatty acid is ensured.
The inventor finds that the content of the extracted crude polysaccharide at the rear end of fatty acid is reduced compared with the content of the extracted crude polysaccharide directly through experimental research of the embodiment 11 of the invention, but the extraction frequency of the extracted crude polysaccharide is less, and the extraction rate of the extracted polysaccharide is greatly improved. Therefore, it can be considered that the presence of fatty acid in the process of the secondary extraction interferes with the extraction of polysaccharide, which affects the extraction rate of polysaccharide, and because the obtained crude polysaccharide needs to be extracted for many times to obtain the polysaccharide with higher purity, the loss of polysaccharide is also caused in the process of the multiple extraction.
After fatty acid is extracted, the polysaccharide can be obtained by only two times of extraction, and the obtained polysaccharide amount is far larger than that of polysaccharide obtained by direct extraction. The comprehensive extraction method has the advantages of low cost and high extraction efficiency, effectively avoids resource waste, reduces the discharge of waste solvent, and has wide application prospect.
Before the polyunsaturated fatty acid is extracted, petroleum ether is used as a dispersing carrier for ultrasonic crushing, so that the polyunsaturated fatty acid ultrasonic-crushing agent has the effects of fully mixing and increasing the contact surface.
In the process of extracting the polyunsaturated fatty acid, the Soxhlet extraction method is preferably adopted, so that the extraction efficiency is high, the energy consumption is low, and the operation is simple.
According to the invention, after the polyunsaturated fatty acid is extracted, the seaweed residue is washed by absolute ethyl alcohol and then dried, so that the interference components such as monosaccharide, oligosaccharide and glycoside can be effectively degreased and removed, a good foundation is laid for extracting polysaccharide by subsequent water, fewer impurities are ensured to be extracted by subsequent water, and the extraction rate is higher.
In the process of extracting the polysaccharide, the invention carries out refrigeration and freeze drying twice, and has precipitation and purification effects.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more concise and clear, the present invention is described with reference to the following specific examples, but the present invention is by no means limited to these examples. The following are only preferred embodiments of the present invention, which are intended to illustrate the present invention and should not be construed as limiting the scope of the present invention. It should be understood that any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Example 1
The method for comprehensively extracting the polyunsaturated fatty acid and the polysaccharide from the seaweed comprises the following steps:
extracting polyunsaturated fatty acid:
A. soaking Sargassum in tap water with weight 20 times of Sargassum for 5min, washing with tap water and deionized water for 1 time, and baking at 80 deg.C for 10 hr; drying, pulverizing into 10-mesh sargassum powder, dispersing the sargassum powder in petroleum ether according to the material-liquid ratio of 1:5, and performing ultrasonic crushing at the ultrasonic power of 60W for 5min at the temperature of 80 ℃;
B. performing solid-liquid separation on the suspension after ultrasonic crushing, supplementing petroleum ether into the separated liquid as an extraction solvent to ensure that the material-liquid ratio of the seaweed meal to the extraction solvent is 1:5, respectively placing the extraction solvent and the solid residues into corresponding vessels of a Soxhlet extraction device, performing reflux extraction at 80 ℃ for 8 hours, collecting the extraction solution, and separating the extraction residues for later use;
C. evaporating the extractive solution by rotary evaporation at 30 deg.C to dryness to obtain Sargassum total lipid as residue;
D. dissolving total lipids of Sargassum with 1 times of petroleum ether, transferring into drying container, blow-drying with nitrogen gas to obtain dried substance, and adding KOH-CH with volume equal to 0.4mol/L3Heating OH solution at 50 deg.C for 50min under nitrogen charging, adjusting pH to 1 with 10% (V/V) HC1 dilute solution, adding water, mixing to separate the solution, and centrifuging to obtain upper oil layer as total fatty acid.
The polysaccharide extraction comprises the following steps:
E. washing the residue obtained after Soxhlet extraction in the step B with absolute ethyl alcohol for 2 times, then drying at 40 ℃, adding pure water which is 20 times of the weight of the residue, stirring and extracting for 8 hours at 60 ℃, filtering, collecting filtrate, and then centrifuging the filtrate to obtain centrifugate;
F. evaporating and concentrating the centrifugate at 40 deg.C to 1/4 of original volume, adding 80% ethanol with volume percentage of 1 time of the volume of the concentrated solution, mixing well, refrigerating at 0 deg.C for 10h, and centrifuging to obtain precipitate; freeze drying the precipitate at-35 deg.C to obtain crude Sargassum polysaccharide product;
G. dissolving a sargassum polysaccharide crude product into pure water with the weight 1 time of that of the sargassum polysaccharide crude product, adding Sevag reagent, uniformly mixing, performing centrifugal separation to obtain a water layer and a Sevag reagent layer, discarding the Sevag reagent layer, then adding the Sevag reagent into the water layer again, performing centrifugal separation, discarding the Sevag reagent layer, and repeating the steps of adding the Sevag reagent into the water layer and performing centrifugal separation until no organic matter and protein appear in the Sevag reagent layer;
H. adding 80% ethanol with volume percentage 1 time of the volume of the water layer after the centrifugal treatment, refrigerating the water layer at 0 ℃ for 10 hours, and then performing centrifugal separation to obtain a precipitate; freeze drying the precipitate at-35 deg.C to obtain Sargassum polysaccharide.
Example 2
The method for comprehensively extracting the polyunsaturated fatty acid and the polysaccharide from the seaweed comprises the following steps:
extracting polyunsaturated fatty acid:
A. soaking Sargassum in tap water 30 times its weight for 10min, washing with tap water and deionized water for 2 times, and baking at 78 deg.C for 12 hr; drying, pulverizing into sargassum powder of 20 meshes, dispersing the sargassum powder in petroleum ether according to the material-liquid ratio of 1:8, and performing ultrasonic crushing, wherein the ultrasonic power is 65W, the crushing time is 10min, and the temperature is kept at 79 ℃ during crushing;
B. performing solid-liquid separation on the suspension after ultrasonic crushing, supplementing petroleum ether into the separated liquid as an extraction solvent to ensure that the material-liquid ratio of the seaweed meal to the extraction solvent is 1:8, respectively placing the extraction solvent and the solid residues into corresponding vessels of a Soxhlet extraction device, performing reflux extraction at 82 ℃ for 9h, collecting the extraction solution, and separating the extraction residues for later use
C. After extraction, evaporating the extracting solution to dryness in a rotary evaporation mode at 32 ℃, wherein the residue after evaporation to dryness is the total lipids of the gulfweed;
D. dissolving total lipids with petroleum ether 2 times of the weight of seaweed meal, transferring to a drying container, blow-drying with nitrogen gas to obtain dried product, and adding KOH-CH with the same volume of 0.5mol/L of the dried product3Heating OH solution at 52 deg.C for 52min under nitrogen charging, adjusting pH to 1.2 with 10% (V/V) HC1 dilute solution, adding water, mixing, layering, and centrifuging to obtain upper oil layer as total fatty acid.
The polysaccharide extraction comprises the following steps:
E. washing the residue obtained after Soxhlet extraction in the step B with absolute ethyl alcohol for 3 times, then drying at 45 ℃, adding pure water with the weight being 25 times of that of the residue, stirring and extracting for 9 hours at 65 ℃, filtering, collecting filtrate, and then centrifuging the filtrate to obtain centrifugate;
F. the centrate was concentrated by evaporation at 45 ℃ to 1/4; adding 85% ethanol 2 times the volume of the concentrated solution, mixing, refrigerating at 1 deg.C for 11 hr, and centrifuging to obtain precipitate; freeze drying the precipitate at-32 deg.C to obtain crude Sargassum polysaccharide product;
G. dissolving the crude sargassum polysaccharide product into pure water which is 2 times of the weight of the crude sargassum polysaccharide product, adding Sevag reagent, uniformly mixing, performing centrifugal separation to obtain a water layer and a Sevag reagent layer, removing the Sevag reagent layer, then adding the Sevag reagent into the water layer again, performing centrifugal separation, removing the Sevag reagent layer, and repeating the steps of adding the Sevag reagent into the water layer and performing centrifugal separation until no organic matter and protein appear in the Sevag reagent layer;
H. adding 85% ethanol 2 times the volume of the water layer, refrigerating at 1 deg.C for 11 hr, and centrifuging to obtain precipitate; freeze drying the precipitate at-32 deg.C to obtain Sargassum polysaccharide.
Example 3
The method for comprehensively extracting the polyunsaturated fatty acid and the polysaccharide from the seaweed comprises the following steps:
extracting polyunsaturated fatty acid:
A. soaking gulfweed in tap water with the weight 40 times of that of gulfweed for 15min, washing with tap water and deionized water for 3 times respectively, and baking at 76 ℃ for 14 h; drying, pulverizing into 30-mesh sargassum powder, dispersing the sargassum powder in petroleum ether according to the material-liquid ratio of 1:10, and performing ultrasonic crushing at the ultrasonic power of 70W for 15min at the temperature of 78 ℃;
B. performing solid-liquid separation on the suspension after ultrasonic crushing, supplementing petroleum ether into the separated liquid as an extraction solvent to ensure that the material-liquid ratio of the seaweed meal to the extraction solvent is 1:10, respectively placing the extraction solvent and the solid residues into corresponding vessels of a Soxhlet extraction device, performing reflux extraction at 84 ℃ for 10 hours, collecting the extraction solution, and separating the extraction residues for later use;
C. evaporating the extractive solution by rotary evaporation at 34 deg.C to obtain residue of Sargassum total lipids;
D. dissolving total lipids with petroleum ether 3 times of the weight of seaweed meal, transferring to a drying container, blow-drying with nitrogen gas to obtain dried product, and adding KOH-CH with the same volume of 0.6mol/L of the dried product3Heating OH solution at 55 deg.C for 55min under nitrogen charging, adjusting pH to 1.5 with 10% (V/V) HC1 dilute solution, adding water, mixing to separate the solution, and centrifuging to obtain upper oil layer as total fatty acid.
The polysaccharide extraction comprises the following steps:
E. washing the residue obtained after Soxhlet extraction in the step B with absolute ethyl alcohol for 4 times, then drying at 50 ℃, adding pure water with the weight 30 times that of the residue, stirring and extracting at 60 ℃ for 10 hours, filtering, collecting filtrate, and then centrifuging the filtrate to obtain centrifugate;
F. the centrate was concentrated by evaporation at 50 ℃ to 1/4; then adding 90% ethanol with volume percentage which is 3 times of the volume of the concentrated solution, uniformly mixing, refrigerating at 2 ℃ for 12h, and then carrying out centrifugal separation to obtain a precipitate; freeze drying the precipitate at-30 deg.C to obtain crude Sargassum polysaccharide product;
G. dissolving the crude sargassum polysaccharide product into pure water with the weight 3 times of that of the sargassum polysaccharide product, adding Sevag reagent, uniformly mixing, performing centrifugal separation to obtain a water layer and a Sevag reagent layer, discarding the Sevag reagent layer, then adding the Sevag reagent into the water layer again, performing centrifugal separation, discarding the Sevag reagent layer, and repeating the steps of adding the Sevag reagent into the water layer and performing centrifugal separation until no organic matter and protein appear in the Sevag reagent layer;
H. adding 90% ethanol with volume percentage 3 times of the volume of the water layer after the centrifugal treatment, refrigerating the water layer at 4 ℃ for 12 hours, and then performing centrifugal separation to obtain a precipitate; freeze drying the precipitate at-30 deg.C to obtain Sargassum polysaccharide.
Example 4
The method for comprehensively extracting the polyunsaturated fatty acid and the polysaccharide from the seaweed comprises the following steps:
extracting polyunsaturated fatty acid:
A. soaking Sargassum in tap water 50 times its weight for 20min, washing with tap water and deionized water for 4 times, and baking at 74 deg.C for 16 hr; drying, pulverizing into sargassum powder of 40 meshes, dispersing the sargassum powder in petroleum ether according to the material-liquid ratio of 1:12, and performing ultrasonic crushing, wherein the ultrasonic power is 75W, the crushing time is 20min, and the temperature is kept at 77 ℃ during crushing;
B. performing solid-liquid separation on the suspension after ultrasonic crushing, supplementing petroleum ether into the separated liquid as an extraction solvent to ensure that the material-liquid ratio of the seaweed meal to the extraction solvent is 1:12, respectively placing the extraction solvent and the solid residues into corresponding vessels of a Soxhlet extraction device, performing reflux extraction at 85 ℃ for 11 hours, collecting the extraction solution, and separating the extraction residues for later use;
C. evaporating the extractive solution by rotary evaporation at 35 deg.C to dryness to obtain Sargassum total lipid as residue;
D. dissolving total lipids with petroleum ether 4 times of the weight of seaweed meal, transferring to a drying container, blow-drying with nitrogen gas to obtain dried product, and adding KOH-CH with the same volume of 0.7mol/L of the dried product3Heating OH solution at 56 deg.C for 56min under nitrogen charging, adjusting pH to 1.6 with 10% (V/V) HC1 dilute solution, adding water, mixing to separate the solution, and centrifuging to obtain upper oil layer as total fatty acid.
The polysaccharide extraction comprises the following steps:
E. washing the residue after Soxhlet extraction in the step B with absolute ethyl alcohol for 5 times, then drying at 55 ℃, adding pure water which is 35 times of the weight of the residue, stirring and extracting at 75 ℃ for 11 hours, filtering, collecting filtrate, and then centrifuging the filtrate to obtain centrifugate;
F. the centrate was concentrated by evaporation at 55 ℃ to 1/4; then adding 95% ethanol with volume percentage which is 4 times of the volume of the concentrated solution, uniformly mixing, refrigerating at 3 ℃ for 13h, and then carrying out centrifugal separation to obtain a precipitate; freeze drying the precipitate at-29 deg.C to obtain crude Sargassum polysaccharide product;
G. dissolving the crude sargassum polysaccharide product into pure water with the weight 4 times of that of the sargassum polysaccharide product, adding Sevag reagent, uniformly mixing, performing centrifugal separation to obtain a water layer and a Sevag reagent layer, discarding the Sevag reagent layer, then adding the Sevag reagent into the water layer again, performing centrifugal separation, discarding the Sevag reagent layer, and repeating the steps of adding the Sevag reagent into the water layer and performing centrifugal separation until no organic matter and protein appear in the Sevag reagent layer;
H. adding 95% ethanol with volume percentage 4 times of the volume of the water layer after the centrifugal treatment, refrigerating the water layer at 3 ℃ for 13h, and then performing centrifugal separation to obtain a precipitate; freeze drying the precipitate at-29 deg.C to obtain Sargassum polysaccharide.
Example 5
The method for comprehensively extracting the polyunsaturated fatty acid and the polysaccharide from the seaweed comprises the following steps:
extracting polyunsaturated fatty acid:
A. soaking gulfweed in tap water with the weight 20 times of that of the gulfweed for 25min, washing the gulfweed with tap water and deionized water for 5 times respectively, and then baking the gulfweed at 72 ℃ for 18 h; drying, pulverizing into 50 mesh sargassum powder, dispersing sargassum powder in petroleum ether at a material-to-liquid ratio of 1:14, and performing ultrasonic crushing at an ultrasonic power of 80W for 25min at a temperature of 72 deg.C;
B. performing solid-liquid separation on the suspension after ultrasonic crushing, supplementing petroleum ether into the separated liquid as an extraction solvent to ensure that the material-liquid ratio of seaweed powder to the extraction solvent is 1:14, respectively placing the extraction solvent and solid residues into corresponding vessels of a Soxhlet extraction device, performing reflux extraction for 12 hours at 86 ℃, collecting the extraction solution, and separating the extraction residues for later use;
C. evaporating the extractive solution by rotary evaporation at 36 deg.C to dryness to obtain residue of Sargassum total lipids;
D. dissolving total lipid with petroleum ether 1 times of the weight of Sargassum powder, transferring into drying container, blow-drying with nitrogen gas to obtain dried product, and adding into the dried productThe same volume of the product is 0.8mol/L KOH-CH3Heating OH solution at 58 deg.C for 58min under nitrogen charging, adjusting pH to 1.8 with 10% (V/V) HC1 dilute solution, adding water, mixing to separate the solution, and centrifuging to obtain upper oil layer as total fatty acid.
The polysaccharide extraction comprises the following steps:
E. washing the residue obtained after Soxhlet extraction in the step B with absolute ethyl alcohol for 2 times, then drying at 60 ℃, adding pure water which is 40 times of the weight of the residue, stirring and extracting for 12 hours at 80 ℃, filtering, collecting filtrate, and then centrifuging the filtrate to obtain centrifugate;
F. the centrate was concentrated by evaporation at 60 ℃ to 1/4; then adding 96% ethanol with volume percentage which is 1 time of the volume of the concentrated solution, uniformly mixing, refrigerating at 4 ℃ for 14h, and then carrying out centrifugal separation to obtain a precipitate; freeze drying the precipitate at-28 deg.C to obtain crude Sargassum polysaccharide product;
G. dissolving a sargassum polysaccharide crude product into pure water which is 1 time of the sargassum polysaccharide crude product in weight, adding Sevag reagent, uniformly mixing, carrying out centrifugal separation to obtain a water layer and a Sevag reagent layer, removing the Sevag reagent layer, then adding the Sevag reagent into the water layer again, carrying out centrifugal separation, removing the Sevag reagent layer, and repeating the steps of adding the Sevag reagent into the water layer and carrying out centrifugal separation until no organic matter and protein appear in the Sevag reagent layer;
H. adding 96% ethanol with volume percentage 1 time of the volume of the water layer after the centrifugal treatment, refrigerating the water layer at 4 ℃ for 14h, and then performing centrifugal separation to obtain a precipitate; freeze drying the precipitate at-28 deg.C to obtain Sargassum polysaccharide.
Example 6
The method for comprehensively extracting the polyunsaturated fatty acid and the polysaccharide from the seaweed comprises the following steps:
extracting polyunsaturated fatty acid:
A. soaking gulfweed in tap water with the weight 30 times of that of gulfweed for 30min, washing the gulfweed with tap water and deionized water for 1 time respectively, and then baking the gulfweed at 70 ℃ for 20 h; drying, pulverizing into sargassum powder of 60 meshes, dispersing the sargassum powder in petroleum ether according to the material-liquid ratio of 1:16, and carrying out ultrasonic crushing, wherein the ultrasonic power is 85W, the crushing time is 30min, and the temperature is kept at 70 ℃ during crushing;
B. performing solid-liquid separation on the suspension after ultrasonic crushing, supplementing petroleum ether into the separated liquid as an extraction solvent to ensure that the material-liquid ratio of the seaweed meal to the extraction solvent is 1:16, respectively placing the extraction solvent and the solid residues into corresponding vessels of a Soxhlet extraction device, performing reflux extraction at 88 ℃ for 13h, collecting the extraction solution, and separating the extraction residues for later use;
C. evaporating the extractive solution by rotary evaporation at 38 deg.C to dryness to obtain Sargassum total lipid as residue;
D. dissolving total lipids of Sargassum with petroleum ether 2 times of its weight, transferring to drying container, blow-drying with nitrogen gas to obtain dried substance, and adding KOH-CH with volume equal to 0.4mol/L3Heating OH solution at 60 deg.C for 60min under nitrogen gas filling, adjusting pH to 2 with 10% (V/V) dilute HC1 solution, adding water, mixing to allow solution to separate, and centrifuging to obtain upper oil layer as total fatty acid.
The polysaccharide extraction comprises the following steps:
E. washing the residue obtained after Soxhlet extraction in the step B with absolute ethyl alcohol for 3 times, then drying at 65 ℃, adding pure water which is 35 times of the weight of the residue, stirring and extracting for 13 hours at 85 ℃, filtering, collecting filtrate, and then centrifuging the filtrate to obtain centrifugate;
F. the centrate was concentrated to 1/4 of the original volume by evaporation at 65 ℃; then adding 100% ethanol with volume percentage which is 2 times of the volume of the concentrated solution, uniformly mixing, refrigerating for 15h at 6 ℃, and then carrying out centrifugal separation to obtain a precipitate; freeze drying the precipitate at-27 deg.C to obtain Sargassum polysaccharide crude product;
G. dissolving a sargassum polysaccharide crude product into pure water with the weight 2 times that of the sargassum polysaccharide crude product, adding Sevag reagent, uniformly mixing, performing centrifugal separation to obtain a water layer and a Sevag reagent layer, discarding the Sevag reagent layer, then adding the Sevag reagent into the water layer again, performing centrifugal separation, discarding the Sevag reagent layer, and repeating the steps of adding the Sevag reagent into the water layer and performing centrifugal separation until no organic matter and protein appear in the Sevag reagent layer;
H. adding 90% ethanol with volume percentage 2 times of the volume of the water layer after the centrifugal treatment, refrigerating the water layer at 6 ℃ for 15h, and then performing centrifugal separation to obtain a precipitate; freeze drying the precipitate at-27 deg.C to obtain Sargassum polysaccharide.
Example 7
The method for comprehensively extracting the polyunsaturated fatty acid and the polysaccharide from the seaweed comprises the following steps:
extracting polyunsaturated fatty acid:
A. soaking Sargassum in tap water 40 times its weight for 5min, washing with tap water and deionized water for 2 times, and baking at 68 deg.C for 22 hr; drying, pulverizing into 70 mesh sargassum powder, dispersing sargassum powder in petroleum ether according to a material-liquid ratio of 1:18, and performing ultrasonic crushing at an ultrasonic power of 90W for 35min at a temperature of 68 ℃;
B. performing solid-liquid separation on the suspension after ultrasonic crushing, supplementing petroleum ether into the separated liquid as an extraction solvent to ensure that the material-liquid ratio of the seaweed meal to the extraction solvent is 1:18, respectively placing the extraction solvent and the solid residues into corresponding vessels of a Soxhlet extraction device, performing reflux extraction at 90 ℃ for 14 hours, collecting the extraction solution, and separating the extraction residues for later use;
C. evaporating the extractive solution by rotary evaporation at 40 deg.C to dryness to obtain Sargassum total lipid as residue;
D. dissolving the total lipids with 3 times of petroleum ether, transferring to a drying container, blow-drying with nitrogen gas to obtain dried product, and adding KOH-CH with the same volume of 0.5mol/L3Heating OH solution at 62 deg.C for 62min under nitrogen charging, adjusting pH to 1 with 10% (V/V) HC1 dilute solution, adding water, mixing to separate the solution, and centrifuging to obtain upper oil layer as total fatty acid.
The polysaccharide extraction comprises the following steps:
E. washing the residue obtained after Soxhlet extraction in the step B with absolute ethyl alcohol for 2 times, then drying at 70 ℃, adding pure water which is 40 times of the weight of the residue, stirring and extracting for 14h at 88 ℃, filtering, collecting filtrate, and then centrifuging the filtrate to obtain centrifugate;
F. the centrate was concentrated by evaporation at 70 ℃ to 1/4; then adding 80 percent ethanol with volume percentage being equal to 3 times of the volume of the concentrated solution, uniformly mixing, refrigerating for 16h at 0 ℃, and then carrying out centrifugal separation to obtain a precipitate; freeze drying the precipitate at-26 deg.C to obtain crude Sargassum polysaccharide product;
G. dissolving the crude sargassum polysaccharide product into pure water with the weight 3 times of that of the sargassum polysaccharide product, adding Sevag reagent, uniformly mixing, performing centrifugal separation to obtain a water layer and a Sevag reagent layer, discarding the Sevag reagent layer, then adding the Sevag reagent into the water layer again, performing centrifugal separation, discarding the Sevag reagent layer, and repeating the steps of adding the Sevag reagent into the water layer and performing centrifugal separation until no organic matter and protein appear in the Sevag reagent layer;
H. adding 80% ethanol with volume percentage of 3 times of the water layer after the centrifugal treatment, refrigerating at 0 ℃ for 16h, and then performing centrifugal separation to obtain a precipitate; freeze drying the precipitate at-26 deg.C to obtain Sargassum polysaccharide.
Example 8
The method for comprehensively extracting the polyunsaturated fatty acid and the polysaccharide from the seaweed comprises the following steps:
extracting polyunsaturated fatty acid:
A. soaking Sargassum in tap water 50 times its weight for 15min, washing with tap water and deionized water for 3 times, and baking at 66 deg.C for 24 hr; drying, pulverizing into sargassum powder of 80 meshes, dispersing the sargassum powder in petroleum ether according to the material-liquid ratio of 1:20, and performing ultrasonic crushing, wherein the ultrasonic power is 60W, the crushing time is 40min, and the temperature is kept at 66 ℃ during crushing;
B. performing solid-liquid separation on the suspension after ultrasonic crushing, supplementing petroleum ether into the separated liquid as an extraction solvent to ensure that the material-liquid ratio of the seaweed meal to the extraction solvent is 1:20, respectively placing the extraction solvent and the solid residues into corresponding vessels of a Soxhlet extraction device, performing reflux extraction at 80 ℃ for 15h, collecting the extraction solution, and separating the extraction residues for later use;
C. evaporating the extractive solution by rotary evaporation at 30 deg.C to dryness to obtain Sargassum total lipid as residue;
D. dissolving the total lipids with petroleum ether 4 times its weight, transferring to a drying container, blow-drying with nitrogen gas to obtain dried product, and adding KOH-CH with the same volume of 0.6mol/L3Heating OH solution at 64 deg.C for 64min under nitrogen charging, adjusting pH to 1.2 with 10% (V/V) HC1 dilute solution, adding water, mixing to separate the solution, and centrifuging to obtain upper oil layer as total fatty acid.
The polysaccharide extraction comprises the following steps:
E. washing the residue obtained after Soxhlet extraction in the step B with absolute ethyl alcohol for 3 times, then drying at 40 ℃, adding pure water with the weight 45 times that of the residue, stirring and extracting for 15h at 90 ℃, filtering, collecting filtrate, and then centrifuging the filtrate to obtain centrifugate;
F. concentrating the centrifugate at 40 deg.C by evaporation to 1/4; then adding 88% ethanol with volume percentage which is 4 times of the volume of the concentrated solution, uniformly mixing, refrigerating for 17h at 1 ℃, and then carrying out centrifugal separation to obtain a precipitate; freeze drying the precipitate at-25 deg.C to obtain Sargassum polysaccharide crude product;
G. dissolving the crude sargassum polysaccharide product into pure water with the weight 4 times of that of the sargassum polysaccharide product, adding Sevag reagent, uniformly mixing, performing centrifugal separation to obtain a water layer and a Sevag reagent layer, discarding the Sevag reagent layer, then adding the Sevag reagent into the water layer again, performing centrifugal separation, discarding the Sevag reagent layer, and repeating the steps of adding the Sevag reagent into the water layer and performing centrifugal separation until no organic matter and protein appear in the Sevag reagent layer;
H. adding 88% ethanol with volume percentage 4 times of the volume of the water layer after the centrifugal treatment, refrigerating the water layer at 1 ℃ for 17 hours, and then performing centrifugal separation to obtain a precipitate; freeze drying the precipitate at-25 deg.C to obtain Sargassum polysaccharide.
Example 9
The method for comprehensively extracting the polyunsaturated fatty acid and the polysaccharide from the seaweed comprises the following steps:
extracting polyunsaturated fatty acid:
A. soaking gulfweed in tap water with the weight 20 times of that of gulfweed for 25min, washing with tap water and deionized water for 3 times respectively, and baking at 65 ℃ for 26 h; drying, pulverizing into 10-mesh sargassum powder, dispersing the sargassum powder in petroleum ether according to the material-liquid ratio of 1:5, and performing ultrasonic crushing at the ultrasonic power of 90W for 10min at the temperature of 65 ℃;
B. performing solid-liquid separation on the suspension after ultrasonic crushing, supplementing petroleum ether into the separated liquid as an extraction solvent to ensure that the material-liquid ratio of the seaweed meal to the extraction solvent is 1:5, respectively placing the extraction solvent and the solid residues into corresponding vessels of a Soxhlet extraction device, performing reflux extraction at 83 ℃ for 16h, collecting the extraction solution, and separating the extraction residues for later use;
C. evaporating the extractive solution by rotary evaporation at 33 deg.C to dryness to obtain Sargassum total lipid as residue;
D. dissolving the total lipids with 1 time of petroleum ether, transferring to a drying container, blow-drying with nitrogen gas to obtain dried product, and adding KOH-CH with the same volume of 0.7mol/L3Heating OH solution at 66 deg.C for 65min under nitrogen charging, adjusting pH to 2 with 10% (V/V) HC1 dilute solution, adding water, mixing to separate the solution, and centrifuging to obtain upper oil layer as total fatty acid.
The polysaccharide extraction comprises the following steps:
E. washing the residue obtained after Soxhlet extraction in the step B with absolute ethyl alcohol for 4 times, then drying at 50 ℃, adding pure water which is 50 times of the weight of the residue, stirring and extracting at 60 ℃ for 6 hours, filtering, collecting filtrate, and then centrifuging the filtrate to obtain centrifugate;
F. the centrate was concentrated by evaporation at 50 ℃ to 1/4; then adding 92% ethanol with volume percentage which is 1 time of the volume of the concentrated solution, uniformly mixing, refrigerating at 2 ℃ for 18h, and then carrying out centrifugal separation to obtain a precipitate; freeze drying the precipitate at-35 deg.C to obtain crude Sargassum polysaccharide product;
G. dissolving a sargassum polysaccharide crude product into pure water with the weight 1 time of that of the sargassum polysaccharide crude product, adding Sevag reagent, uniformly mixing, performing centrifugal separation to obtain a water layer and a Sevag reagent layer, discarding the Sevag reagent layer, then adding the Sevag reagent into the water layer again, performing centrifugal separation, discarding the Sevag reagent layer, and repeating the steps of adding the Sevag reagent into the water layer and performing centrifugal separation until no organic matter and protein appear in the Sevag reagent layer;
H. adding 92% ethanol with volume percentage of 1 time of the volume of the water layer after the centrifugal treatment, refrigerating the water layer at 2 ℃ for 18 hours, and then performing centrifugal separation to obtain a precipitate; freeze drying the precipitate at-35 deg.C to obtain Sargassum polysaccharide.
Example 10
The method for comprehensively extracting the polyunsaturated fatty acid and the polysaccharide from the seaweed comprises the following steps:
extracting polyunsaturated fatty acid:
A. soaking gulfweed in tap water with the weight 30 times of that of gulfweed for 30min, washing with tap water and deionized water for 5 times respectively, and baking at 80 ℃ for 30 h; drying, pulverizing into sargassum powder of 80 meshes, dispersing the sargassum powder in petroleum ether according to the material-liquid ratio of 1:20, and carrying out ultrasonic crushing, wherein the ultrasonic power is 90W, the crushing time is 40min, and the temperature is kept at 80 ℃ during crushing;
B. performing solid-liquid separation on the suspension after ultrasonic crushing, supplementing petroleum ether into the separated liquid as an extraction solvent to ensure that the material-liquid ratio of the seaweed meal to the extraction solvent is 1:20, respectively placing the extraction solvent and the solid residues into corresponding vessels of a Soxhlet extraction device, performing reflux extraction at 90 ℃ for 8 hours, collecting the extraction solution, and separating the extraction residues for later use;
C. evaporating the extractive solution by rotary evaporation at 40 deg.C to dryness to obtain Sargassum total lipid as residue;
D. dissolving the total lipids with petroleum ether 2 times its weight, transferring to a drying container, blow-drying with nitrogen gas to obtain dried product, and adding KOH-CH with the same volume of 0.8mol/L3Heating OH solution at 70 deg.C for 50min under nitrogen charging, adjusting pH to 1 with 10% (V/V) HC1 dilute solution, adding water, mixing to separate the solution, and centrifuging to obtain upper oil layer as total fatty acid.
The polysaccharide extraction comprises the following steps:
E. washing the residue obtained after Soxhlet extraction in the step B with absolute ethyl alcohol for 5 times, then drying at 70 ℃, adding pure water which is 50 times of the weight of the residue, stirring and extracting for 8 hours at 90 ℃, filtering, collecting filtrate, and then centrifuging the filtrate to obtain centrifugate;
F. the centrate was concentrated by evaporation at 70 ℃ to 1/4; then adding 100 percent ethanol which is equal to 4 times of the volume of the concentrated solution, uniformly mixing, refrigerating for 10 hours at 6 ℃, and then carrying out centrifugal separation to obtain a precipitate; freeze drying the precipitate at-30 deg.C to obtain crude Sargassum polysaccharide product;
G. dissolving a sargassum polysaccharide crude product into pure water with the weight 2 times that of the sargassum polysaccharide crude product, adding Sevag reagent, uniformly mixing, performing centrifugal separation to obtain a water layer and a Sevag reagent layer, discarding the Sevag reagent layer, then adding the Sevag reagent into the water layer again, performing centrifugal separation, discarding the Sevag reagent layer, and repeating the steps of adding the Sevag reagent into the water layer and performing centrifugal separation until no organic matter and protein appear in the Sevag reagent layer;
H. adding 100% ethanol with volume percentage 4 times of the volume of the water layer after the centrifugal treatment, refrigerating the water layer at 0 ℃ for 10 hours, and then performing centrifugal separation to obtain a precipitate; freeze drying the precipitate at-30 deg.C to obtain Sargassum polysaccharide.
Example 11 Effect of Integrated extraction and direct extraction on the amount of Sargassum polysaccharide extracted
The method comprises the following steps:
experimental groups: fatty acids and polysaccharides were prepared according to the method of example 5 of the present invention;
control group 1: the polysaccharide was directly extracted according to the method for preparing the polysaccharide in example 5 of the present invention;
control group 2: respectively extracting the total fatty acid and the polysaccharide according to a conventional ultrasonic wave auxiliary method.
Experimental groups:
A. weighing 20g of gulfweed, soaking the gulfweed in tap water with the weight 20 times of the weight of the gulfweed for 25min, washing the gulfweed with tap water and deionized water for 5 times respectively, and then baking the gulfweed at 72 ℃ for 18 h; drying, pulverizing into 50 mesh sargassum powder, dispersing sargassum powder in petroleum ether at a material-to-liquid ratio of 1:14, and performing ultrasonic crushing at an ultrasonic power of 80W for 25min at a temperature of 72 deg.C;
B. performing solid-liquid separation on the suspension after ultrasonic crushing, supplementing petroleum ether into the separated liquid as an extraction solvent to ensure that the material-liquid ratio of the seaweed meal to the extraction solvent is 1:14, respectively placing the extraction solvent and the solid residues into corresponding vessels of a Soxhlet extraction device, performing reflux extraction at 86 ℃ for 12 hours, collecting the extraction solution, and separating the extraction residues for later use;
C. evaporating the extractive solution by rotary evaporation at 36 deg.C to dryness to obtain residue of Sargassum total lipids;
D. dissolving total lipids with petroleum ether 1 times of the weight of seaweed meal, transferring to a drying container, blow-drying with nitrogen gas to obtain dried product, and adding KOH-CH with the same volume of 0.8mol/L of the dried product3Heating OH solution at 58 deg.C for 58min under nitrogen charging condition, adjusting pH to 1.8 with 10% (V/V) HC1 dilute solution, adding water, mixing to layer the solution, and centrifuging to obtain upper oil layer as total fatty acid;
E. washing the residue obtained after Soxhlet extraction in the step B with absolute ethyl alcohol for 2 times, then drying at 60 ℃, adding pure water which is 40 times of the weight of the residue, stirring and extracting for 12 hours at 80 ℃, filtering, collecting filtrate, and then centrifuging the filtrate to obtain centrifugate;
F. the centrate was concentrated by evaporation at 60 ℃ to 1/4; then adding 96% ethanol with volume percentage of 96% which is 1 time of the volume of the concentrated solution, uniformly mixing, refrigerating for 14 hours at 4 ℃, and then carrying out centrifugal separation to obtain a precipitate; freeze drying the precipitate at-28 deg.C to obtain crude Sargassum polysaccharide product.
Control group 1:
a. weighing 20g of gulfweed, soaking the gulfweed in tap water with the weight 20 times of the weight of the gulfweed for 25min, washing the gulfweed with tap water and deionized water for 5 times respectively, and then baking the gulfweed at 72 ℃ for 18 h; drying, pulverizing into 50 mesh sargassum powder, dispersing sargassum powder in petroleum ether at a material-to-liquid ratio of 1:14, and performing ultrasonic crushing at an ultrasonic power of 80W for 25min at a temperature of 72 deg.C;
b. adding pure water with the weight 40 times of that of the sargassum powder, stirring and extracting at 80 ℃ for 12 hours, filtering, collecting filtrate, and centrifuging the filtrate to obtain centrifugate;
c. the centrate was concentrated by evaporation at 60 ℃ to 1/4; then adding 96% ethanol with volume percentage which is 1 time of the volume of the concentrated solution, uniformly mixing, refrigerating at 4 ℃ for 14h, and then carrying out centrifugal separation to obtain a precipitate; freeze drying the precipitate at-28 deg.C to obtain crude Sargassum polysaccharide product.
Control group 2:
extracting fatty acid: weighing 20g of sargassum powder, extracting with 80mL of 90 vol% ethanol, extracting for 20min under the assistance of 90W ultrasonic waves, and extracting with 40mL of 0.5m L n-hexane.
And (3) extracting polysaccharide: weighing 20g of sargassum powder, extracting with 100mL of pure water, extracting for 15min under the assistance of 90W ultrasonic waves, and then incubating for 4h in 80% ethanol at 100 ℃.
The experimental group, the control group 1 and the control group 2 were each provided with 3 replicates, the amounts of total lipids of gulfweed and crude polysaccharide of gulfweed extracted after the three replicates were weighed, and the results are shown in table 1 by taking the average values.
In the experimental process, it can be observed that the color of the polysaccharide obtained by the extraction method of the experimental group is light brown, while the color of the polysaccharide obtained by the control group is darker.
TABLE 1 comparison of total lipids and crude polysaccharide extracts from Sargassum
Figure BDA0002401191000000171
As can be seen from Table 1, the amount of crude polysaccharide extracted from the same weight of Sargassum as that extracted from control group 1 and control group 2 is significantly higher than that extracted by the synthetic method of the present invention; the amount of total lipid obtained by the conventional method in the control group 2 was much lower than that obtained by the extraction of the invention.
Step two:
on the basis of the substances obtained in table 1, the crude polysaccharides of the experimental group, the control group 1 and the control group 2 were respectively subjected to the following purification treatments, and the treatment steps were as follows:
1. dissolving a sargassum polysaccharide crude product into pure water with the weight 1 time of that of the sargassum polysaccharide crude product, adding Sevag reagent, uniformly mixing, performing centrifugal separation to obtain a water layer and a Sevag reagent layer, discarding the Sevag reagent layer, then adding the Sevag reagent into the water layer again, performing centrifugal separation, discarding the Sevag reagent layer, and repeating the steps of adding the Sevag reagent into the water layer and performing centrifugal separation until no organic matter and protein appear in the Sevag reagent layer;
2. adding 96% ethanol with volume percentage 1 time of the volume of the water layer after the centrifugal treatment, refrigerating the water layer at 4 ℃ for 14h, and then performing centrifugal separation to obtain a precipitate; freeze drying the precipitate at-28 deg.C to obtain Sargassum polysaccharide.
After the crude polysaccharide is purified by the steps, the quality of the sargassum polysaccharide obtained by the three extraction methods is shown in table 2, and the comparison of the yield of the crude polysaccharide and the yield of the polysaccharide obtained by the three extraction methods is shown in table 3.
TABLE 2 comparison of the quality of the purified Sargassum polysaccharide
Figure BDA0002401191000000181
TABLE 3 Sargassum polysaccharide yield obtained by two methods
Figure BDA0002401191000000182
As can be seen from tables 2 and 3, although the quality of the crude polysaccharides obtained by direct extraction of the control group 1 and the control group 2 is high, the amount of the polysaccharides obtained by purification is much lower than that of the polysaccharides extracted by the experimental group, and the crude polysaccharides obtained by direct extraction need to be extracted for many times to obtain the polysaccharides, while the crude polysaccharides obtained by comprehensive extraction can be extracted for only two times to obtain the polysaccharides, thereby greatly simplifying the operation steps.

Claims (6)

1. A method for comprehensively extracting polyunsaturated fatty acid and polysaccharide from seaweed is characterized by comprising the following steps: firstly, extracting polyunsaturated fatty acid from seaweed serving as a raw material to obtain polyunsaturated fatty acid, then separating the seaweed from which the polyunsaturated fatty acid is extracted, and extracting polysaccharide from the seaweed from which the polyunsaturated fatty acid is extracted to obtain polysaccharide;
the extraction of the polyunsaturated fatty acid comprises the following steps:
A. pulverizing pretreated Sargassum into 10-80 mesh Sargassum powder, dispersing Sargassum powder in petroleum ether, and ultrasonic pulverizing at temperature below 80 deg.C;
B. performing solid-liquid separation on the suspension after ultrasonic crushing, supplementing petroleum ether into the separated liquid as an extraction solvent to ensure that the material-liquid ratio of the seaweed meal to the extraction solvent is 1:5-20, respectively placing the extraction solvent and the solid residues into corresponding vessels of a Soxhlet extraction device, performing reflux extraction at 80-90 ℃ for more than 8 hours, collecting the extraction solution, and separating extraction residues for later use;
C. evaporating the extracting solution to dryness after extraction is finished, wherein the residue after evaporation to dryness is the total lipid of the seaweed;
D. dissolving total lipids with petroleum ether 1-4 times of the weight of Sargassum powder, transferring to drying container, blow-drying with nitrogen gas to obtain dried product, and adding 0.4 volume of the dried productKOH-CH of-0.8 mol/L3Heating OH solution at 50-70 deg.C for more than 50min under nitrogen charging, adjusting pH to 1-2 with 10% V/V dilute HCl solution, adding water, mixing to layer, and centrifuging to obtain upper oil layer as total fatty acid;
the pretreatment method in the step A comprises the following steps: soaking in tap water 20-50 times of the weight of Sargassum for 5-30min, washing with tap water and deionized water for more than 1 times, and baking at 80 deg.C for 10-30 hr;
in the step A, the ratio of the seaweed powder to the petroleum ether is 1: 5-20.
2. The method for the integrated extraction of polyunsaturated fatty acids and polysaccharides from seaweeds of claim 1, wherein: the ultrasonic power of the ultrasonic crushing in the step A is 60-90W, and the crushing time is 5-40 min.
3. The method for the integrated extraction of polyunsaturated fatty acids and polysaccharides from seaweeds of claim 1, wherein: in the step C, the temperature for evaporating the extracting solution is 30-40 ℃, and the extracting solution is evaporated by rotary evaporation.
4. The method for the integrated extraction of polyunsaturated fatty acids and polysaccharides from seaweeds of claim 1, wherein said polysaccharide extraction comprises the steps of:
E. washing the residue obtained after Soxhlet extraction in the step B with absolute ethyl alcohol for more than 2 times, then drying at 40-70 ℃, adding pure water which is 20-50 times of the weight of the residue, stirring and extracting at 60-90 ℃ for more than 8h, filtering, collecting filtrate, and then centrifuging the filtrate to obtain centrifugate;
F. evaporating and concentrating the centrifugate at 40-70 deg.C to 1/4 of original volume, adding 80-100% ethanol 1-4 times of the volume of the concentrate, mixing, refrigerating at 0-6 deg.C for more than 10 hr, and centrifuging to obtain precipitate; freeze-drying the precipitate to obtain crude algal polysaccharide;
G. dissolving the crude seaweed polysaccharide product into pure water with the weight 1-4 times of that of the crude seaweed polysaccharide product, adding Sevag reagent, uniformly mixing, performing centrifugal separation to obtain a water layer and a Sevag reagent layer, discarding the Sevag reagent layer, then adding the Sevag reagent into the water layer again, performing centrifugal separation, discarding the Sevag reagent layer, and repeating the steps of adding the Sevag reagent into the water layer and performing centrifugal separation until no organic matter or protein appears in the Sevag reagent layer in the solution;
H. adding 80-100% ethanol with volume percentage of 1-4 times of the water layer after the centrifugal treatment, refrigerating at 0-6 deg.C for more than 10 hr, and centrifuging to obtain precipitate; freeze drying the precipitate to obtain algal polysaccharide.
5. The method for the integrated extraction of polyunsaturated fatty acids and polysaccharides from seaweeds of claim 4, wherein: the temperature of the freeze drying in the step F and the step H is-35 ℃ to-25 ℃.
6. The method for the integrated extraction of polyunsaturated fatty acids and polysaccharides from seaweed as claimed in claim 1, characterized in that: the seaweed is gulfweed.
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