CN106221887B - A kind of method of the total rouge of high efficiency extraction ocean ballstone frustule - Google Patents
A kind of method of the total rouge of high efficiency extraction ocean ballstone frustule Download PDFInfo
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- CN106221887B CN106221887B CN201610600342.6A CN201610600342A CN106221887B CN 106221887 B CN106221887 B CN 106221887B CN 201610600342 A CN201610600342 A CN 201610600342A CN 106221887 B CN106221887 B CN 106221887B
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- 238000011084 recovery Methods 0.000 abstract description 23
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- 238000002474 experimental method Methods 0.000 description 10
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- 229930001119 polyketide Natural products 0.000 description 1
- 125000000830 polyketide group Chemical group 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 1
- 235000019353 potassium silicate Nutrition 0.000 description 1
- 244000038651 primary producers Species 0.000 description 1
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- NTHWMYGWWRZVTN-UHFFFAOYSA-N sodium silicate Chemical compound [Na+].[Na+].[O-][Si]([O-])=O NTHWMYGWWRZVTN-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
- C11B1/02—Pretreatment
- C11B1/025—Pretreatment by enzymes or microorganisms, living or dead
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Medicines Containing Plant Substances (AREA)
- Cosmetics (AREA)
- Fats And Perfumes (AREA)
Abstract
The invention discloses a kind of methods of the total rouge of high efficiency extraction ocean ballstone frustule, the following steps are included: Step 1: extraction: using concentration for the ethanol solution of 65-85%, extract ocean ballstone algae powder, liquid-to-solid ratio 5:1 to 10:1(mL/g), 25-45 DEG C of extraction temperature, extraction time 60-120min；Step 2: centrifugation: centrifuging and taking supernatant, centrifugal condition: 4 DEG C, 6000rpm is centrifuged 10 minutes；Step 3: drying: the total rouge of ocean ballstone frustule is made with being dried with nitrogen to constant weight in supernatant.The present invention determines the optimum process condition of total rouge extraction in the ballstone algae of ocean by single factor test and orthogonal test simultaneously are as follows: concentration of alcohol 75%, liquid-to-solid ratio 10:1(mL/g), 35 DEG C of extraction temperature, 120 min of extraction time, the total rouge recovery rate of ocean ballstone frustule is the 26.38% of dry cell weight under this condition, and experimental repeatability is good.
Description
Technical field
The present invention relates to the technical field of bio-pharmaceuticals more particularly to a kind of total rouge of high efficiency extraction ocean ballstone frustule
Method.
Background technique
As phytoplankton most important in living marine resources and primary producer, marine microalgae is not only in type sum number
Measure it is various, be easy for workers to very fast etc. the advantage for having itself of culture, breeding, and in the matter energy of marine ecosystems
And play the role of in global global chemical recycle very important.Possess this unique ecosystem of ocean again simultaneously as breeding ground,
Therefore the chemical composition of its metabolite and terrestrial organism differ widely, they can generate some new knots by metabolic pathway
Structure.
Marine microalgae has the bioactive substance of its grain husk as high photosynthetic organism, such as pigment, sterol, protein, alkaloid
Etc. bioactivity such as antitumor, anti-oxidant, antibacterial with higher and immunological regulations.β-the carrot separated from marine microalgae
Element, astaxanthin, algocyan, lutein and polyphenols have the effects that good anti-oxidant, anti-aging, pre- anti-cancer,
It can be used as the special cost of material advantage of novel healthy food and new drug source, and lipid content can in marine microalgae cell
Up to the 80% of dry cell weight.EPA, DHA and AA isoreactivity substance rich in the marine microalgae of part, can be used for health care,
Food, cosmetics and medicine and other fields, to maintain human body polyunsaturated fatty acid balance, prevent certain diseases, maintenance and tune
Managing physiological health etc. has remarkable efficacy.
Ocean ballstone algae (Coccolithophores) is the miniature phytoplankton of a kind of single-cell sea (3~10 μ of diameter
M), it is distributed widely in worldwide coastal waters and ocean waters.Rich in nutrition content in the ballstone frustule of ocean contains 20
Two carbon acids (DHA), polyketides isoreactivity substance have the biological activities such as good antibacterial, pest-resistant, antitumor,
Have broad application prospects in technical field of research such as biology, food, medicine.Research finds that ocean ballstone algae total lipid content is thin
The 31.83% of born of the same parents' dry weight.Therefore, application potential of the ballstone algae in ocean in fields such as medicine, food, agricultural and environmental protection is urgently opened
Hair.
The present invention using ocean ballstone algae as material, by comparing and optimize the extraction conditions of its total rouge, establish it is a kind of efficiently
The method for extracting the total rouge of ocean ballstone frustule.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of methods of the total rouge of high efficiency extraction ocean ballstone frustule.
To achieve the goals above, the technical scheme adopted by the invention to solve the technical problem is that:
A kind of method of the total rouge of high efficiency extraction ocean ballstone frustule, comprising the following steps:
Step 1: extraction: it uses concentration for the ethanol solution of 65-85%, extracts ocean ballstone algae powder, liquid-to-solid ratio 5:
1 to 10:1 (mL/g), 25-45 DEG C of extraction temperature, extraction time 60-120min；
Step 2: centrifugation: centrifuging and taking supernatant, centrifugal condition: 4 DEG C, 6000rpm is centrifuged 10 minutes；
Step 3: drying: the total rouge of ocean ballstone frustule is made with being dried with nitrogen to constant weight in supernatant.
As the preferred embodiment of embodiment, uses concentration for 75% ethanol solution, extract ocean ballstone algae powder, liquid-to-solid ratio
10:1 (mL/g), 35 DEG C of extraction temperature, extraction time 120min.
As the preferred embodiment of embodiment, the ocean ballstone algae powder stablizes growth period the preparation method comprises the following steps: harvesting and being in
Ocean ballstone frustule suspension frustule is gently scraped from filter membrane using disk filter through 2.0 μm of membrane filtrations；
For algal gel after ultrapure water desalination, freeze-drying obtains algae powder, is stored in spare in drier.
As the preferred embodiment of embodiment, the cultural method of the ocean ballstone frustule are as follows: after 0.45 μm of filter membrane
Seawater be basic medium, using f/2 culture medium culture, wherein the sea for being 70% containing volume fraction in the culture medium culture
Water, 121 DEG C of sterilizings；It is 40-50E/ (m that algae strain, which is grown on intensity of illumination,2S) in environment, brightness time ratio is 14h:10h, temperature
18 DEG C of degree, daily manual shaking flask 3~5 times.
The present invention using ocean ballstone frustule as material, by comparing and optimize its total rouge extraction conditions, establish one
The technique of the kind total rouge of high efficiency extraction ballstone frustule, using concentration of alcohol 75%, liquid-to-solid ratio 10:1 (mL/g), extraction temperature 35
DEG C, extraction time 120min, the total rouge recovery rate of ocean ballstone frustule is the 26.38% of dry cell weight under this condition, and real
It tests reproducible.
Detailed description of the invention
Fig. 1 is total rouge recovery rate of different solvents of the present invention；
Fig. 2 is influence of the Different front processing method of the present invention to the total rouge recovery rate of ballstone algae；
Fig. 3 is influence of the different ethanol concentration of the present invention to the total rouge crude extract of ocean ballstone algae；
Fig. 4 is influence of the different liquid-to-solid ratios of the present invention to rouge crude extract total in the ballstone algae of ocean；
Fig. 5 is influence of the different Extracting temperatures of the present invention to rouge crude extract total in the ballstone algae of ocean；
Fig. 6 is influence of the present invention different extraction times to rouge crude extract total in the ballstone algae of ocean.
Specific embodiment
Below in conjunction with the drawings and specific embodiments with the invention will be further described.
The extraction of the 1 total rouge of ocean ballstone frustule of embodiment
The culture of 1.1 ballstone algaes
It is 40-50E/ (m that algae strain, which is grown on intensity of illumination,2S) in environment, the photoperiod is 14h:10h (light: dark), temperature
18 DEG C, daily manual shaking flask 3~5 times.Culture is derived from Xiamen fair U.S. dragon boat pond sand filtration seawater with seawater, after 0.45 μm of filter membrane
Seawater is basic medium.It is cultivated using f/2 culture medium (70% seawater), 121 DEG C of sterilizings.
The acquisition of 1.2 ballstone algae powders
Harvesting is in stablizing the ballstone frustule suspension in growth period, using disk filter through 2.0 μm of membrane filtrations, from
Frustule is gently scraped on filter membrane.For algal gel after ultrapure water desalination, freeze-drying obtains algae powder, is stored in spare in drier.
The extracting method of 1.3 total rouge
A kind of method of the total rouge of high efficiency extraction ocean ballstone frustule, comprising the following steps:
Step 1: extraction；Step 2: centrifugation: centrifuging and taking supernatant, centrifugal condition: 4 DEG C, 6000rpm is centrifuged 10 points
Clock；Step 3: drying: the total rouge of ocean ballstone frustule is made with being dried with nitrogen to constant weight in supernatant.
The recovery rate of 1.4 total rouge crude extracts
The selection of 1.5 Extraction solvents
200mg algae powder is accurately weighed, petroleum ether, ethyl acetate, acetone, ethyl alcohol, chloroform: methanol (2:1), stone are separately added into
Oily ether: ether (2:1) 35 DEG C of temperature, is extracted under conditions of extraction time 30min at liquid-to-solid ratio 5:1 (mL/g), in order to
Guarantee that the accuracy of experimental data, three parallel groups of every group of experimental setup calculate total rouge crude extract yield, so that it is determined that extracting molten
Agent.
The selection of 1.6 Different front processing methods
200mg algae powder is accurately weighed, using dehydrated alcohol as extractant, at liquid-to-solid ratio 5:1 (mL/g), 35 DEG C of temperature, is extracted
Under conditions of time is 30min, respectively with ultrasonic method, crush method, acid heat method and usual vehicle extraction to total rouge in algae powder
Substance is stripped, more total rouge crude extract yield, so that it is determined that pre-treating method.
1.7 single factor experiment
By after testing pre-treating method and suitable Extraction solvent has been determined above, select concentration of alcohol, extraction time,
Extracting temperature and liquid-to-solid ratio etc. 4 have the factor of larger impact to carry out experiment of single factor total rouge crude extract yield.It accurately weighs
The ethanol solution of various concentration is added by certain liquid-to-solid ratio, extracts certain time at a certain temperature for the algae powder of 200mg,
Supernatant is collected by centrifugation, is dried up under nitrogen protection to constant weight.When measuring another factor, fixed other factors.Every group
Experiment does three in parallel, calculates total rouge crude extract yield.
1.8 orthogonal test
On the basis of experiment of single factor, selecting the time being affected, temperature, concentration of alcohol, liquid-to-solid ratio is principal element,
Using total rouge crude extract yield as index, L is designed9(34) orthogonal test optimizes extraction process, it is true as a result to carry out range analysis
Surely the condition optimized.
1 L of table9(34) quadrature factor water-glass
Reproducibility is investigated under 1.9 optimum extraction conditions
Under optimum extraction condition, 5 parallel tests are set, the relative standard deviation by calculating 5 experiments investigates experiment
Reproducibility.
2 experimental result of embodiment
The selection of 2.1 Extraction solvents
This experiment has selected 6 kinds of organic solvents to extract total lipid in ballstone frustule according to the difference of solvent polarity,
In include 4 kinds of single solvents and 2 kinds of mixed solvents.At liquid-to-solid ratio 5:1 (mL/g), 35 DEG C of temperature, the condition of extraction time 30min
Under, respectively with 4 kinds of single solvents and chloroforms such as petroleum ether, ethyl acetate, acetone and ethyl alcohol: methanol (2:1), petroleum ether: ether
(2:1) two kinds of mixed solvents extract, total rouge recovery rate calculated result such as Fig. 1.
The result shows that petroleum ether is minimum to the recovery rate of rouge total in the ballstone algae of ocean, and it is 11.3%, ethyl acetate and acetone
Recovery rate difference it is little, chloroform methanol system is maximum to the recovery rate of rouge total in ballstone algae, can reach 24.67%, but by
In this system severe toxicity, it is unfavorable for industrialized production.And ethyl alcohol as solvent recovery rate up to 20.33%, and this solvent pacify
Entirely, difference is not significant (P > 0.05) and compared with chloroform methanol system, therefore subsequent experimental selects ethyl alcohol molten as extracting
Agent.
The selection of 2.2 Different front processing methods
Ultrasonic method, crush method, total rouge in acid heat method and usual vehicle restricted-access media ballstone algae is respectively adopted, as a result as schemed
Shown in 2.
Figure it is seen that crush method is 22.42% to total rouge recovery rate highest in ballstone algae in 4 kinds of methods, acid
The recovery rate of thermal method is minimum, is 16.3%, and the recovery rate of usual vehicle extraction is 20.46%, and the difference compared with crush method
Not significant (P > 0.05), it is contemplated that crush method needs more energy in industrialized production, therefore this experimental selection is with commonly
Solvent extraction method extract, it is possible to reduce the cost of industrialized production.
2.3 experiment of single factor
2.3.1 influence of the different ethanol concentration to rouge crude extract total in ballstone algae
A certain amount of algae powder of precise, at liquid-to-solid ratio 5:1 (mL/g), 35 DEG C of temperature, the condition of extraction time 30min
Under, 55%, 65%, 75%, 85% and 95% concentration of alcohol is chosen respectively as extractant, is carried out with usual vehicle extraction
It extracts.As a result as shown in figure 3, when concentration of alcohol is 75% total rouge in ballstone algae can be extracted to the greatest extent, recovery rate is most
Height can reach 26.33%.Therefore 75% concentration of alcohol is selected to extract.
2.3.2 influence of the different liquid-to-solid ratios to rouge crude extract total in ballstone algae
A certain amount of algae powder of precise is 75% in concentration of alcohol, 35 DEG C of temperature, under conditions of extraction time 30min,
Choosing liquid-to-solid ratio respectively is 5:1,10:1,15:1, and 20:1,25:1 (mL/g) are extracted with usual vehicle extraction.As a result such as
Shown in Fig. 4, growth trend is presented with the increase of liquid-to-solid ratio in total rouge recovery rate, and under the conditions of 15:1 (mL/g), total lipid content reaches
To 24%, and later as the increase total lipid content variation of solvent volume is not significant.Therefore it can guarantee extraction again in saving solvent
Select 15:1 as optimal process conditions in the case that rate is high.
2.3.3 influence of the different Extracting temperatures to rouge crude extract total in ballstone algae
A certain amount of algae powder of precise is 75% in concentration of alcohol, and liquid-to-solid ratio is 15:1 (mL/g), respectively at 4,25,
30min is extracted under 35,45,55,65,75 DEG C of water bath conditions, is extracted with usual vehicle extraction.As a result as shown in figure 5,
Bath temperature is 35 DEG C of recovery rate highests, and later as the temperature rises, total rouge recovery rate reduces instead, and analysis reason may be
Since temperature is excessively high, the ingredient in frustule is destroyed, is changed so as to cause the space structure of Cucumber.Therefore it selects
Extracting temperature is 35 DEG C.
2.3.4 influence of the different extraction times to rouge crude extract total in ballstone algae
A certain amount of algae powder of precise is 75% in concentration of alcohol, and liquid-to-solid ratio is 15:1 (mL/g), 35 DEG C of temperature, is mentioned
Taking the time is respectively 30,60,90,120,150,180min, is extracted with usual vehicle extraction.As a result as shown in fig. 6, reason
By upper with the extension of time, total lipid content should increase, but experimental result is shown, in 60min, total rouge recovery rate is
21.66%, then total rouge recovery rate is not significantly improved.In view of industrialized energy consumption choose 60min be optimum extraction when
Between.
2.4 orthogonal test
Experimental result based on single factor test devises temperature, and the time, 4 factor such as liquid-to-solid ratio and concentration of alcohol 3 is horizontal just
Hand over test, experimental result such as table 2.
2 orthogonal experiments analytical table of table
3 orthogonal test analysis of variance table of table
Note: * is significant difference (P < 0.05)
Such as table 3, very poor and variance analysis shows in the total rouge extraction process of ballstone algae that concentration of alcohol is for ocean ballstone algae
The recovery rate of total rouge has a significant impact, when the primary and secondary sequence that each factor influences is followed successively by concentration of alcohol > temperature > liquid-to-solid ratio >
Between, optimised process group is combined into A2B2C2D3That is concentration of alcohol 75%, liquid-to-solid ratio 10:1,35 DEG C of temperature, the condition of time 120min
Under, extraction process is best, can reach 26.38%.
According to the experiment condition of above-mentioned optimization, 5 parallel laboratory tests are carried out, the results are shown in Table 4, ballstone algae total lipid content average value
For 26.32%, RSD 1.9%, reproducibility is preferable.
4 reproducibility experimental result of table
This result of study shows chloroform methanol system to the extraction efficiency highest of the total rouge of ocean ballstone algae, and ink is presented in grease
Green.Although the recovery rate of ethyl alcohol is not so good as chloroform methanol system, it is contemplated that safety and its commercial cost, therefore this research
Select ethyl alcohol as Extraction solvent.Ocean ballstone frustule periphery is wrapped in a thick layer coccolith, after clasmatosis its
The recovery rate highest of total rouge, but it is not significant with the recovery rate difference of usual vehicle extraction, and microalgae cell shattering process needs
Energy is consumed, it is very high for the cost requirement of industrialization production, so follow-up test finally uses usual vehicle extraction to carry out
It extracts.The optimum process condition of optimization of orthogonal test is: 75% concentration of alcohol, the liquid-to-solid ratio of 10:1 (mL/g), Extracting temperature 35
DEG C, extraction time 120min, total lipid content can reach 26.38% in ocean ballstone frustule with this condition, and tests and repeat
Property is good.Total rouge that the method for this Experimental Research can be used for high density ocean ballstone algae extracts, and is that the total rouge of ocean ballstone algae extracts
Industrialized production provides certain reference frame.
All deformations that those skilled in the art directly can export or associate from the disclosure of invention, should all
It is considered protection scope of the present invention.
Claims (4)
1. a kind of method of the total rouge of high efficiency extraction ocean ballstone frustule, it is characterised in that: the following steps are included:
Step 1: extraction: using concentration for the ethanol solution of 65-85%, extract ocean ballstone algae powder, liquid-to-solid ratio 5:1 is extremely
10:1(mL/g), 25-45 DEG C of extraction temperature, extraction time 60-120min；
Step 2: centrifugation: centrifuging and taking supernatant, centrifugal condition: 4 DEG C, 6000rpm is centrifuged 10 minutes；
Step 3: drying: the total rouge of ocean ballstone frustule is made with being dried with nitrogen to constant weight in supernatant.
2. a kind of method of the total rouge of high efficiency extraction ocean ballstone frustule as described in claim 1, it is characterised in that: using dense
The ethanol solution that degree is 75% extracts ocean ballstone algae powder, liquid-to-solid ratio 10:1(mL/g), 35 DEG C of extraction temperature, extraction time 120
min。
3. a kind of method of the total rouge of high efficiency extraction ocean ballstone frustule as described in claim 1, it is characterised in that: the sea
Foreign ballstone algae powder the preparation method comprises the following steps: harvesting uses disk filtration in the ocean ballstone frustule suspension in growth period is stablized
Device gently scrapes frustule through 2.0 μm of membrane filtrations from filter membrane；For algal gel after ultrapure water desalination, freeze-drying obtains algae
Powder is stored in spare in drier.
4. a kind of method of the total rouge of high efficiency extraction ocean ballstone frustule as claimed in claim 3, it is characterised in that: the sea
The cultural method of foreign ballstone frustule are as follows: seawater after using 0.45 μm of filter membrane is basic medium, using f/2 culture medium culture,
The seawater for being wherein 70% containing volume fraction in the culture medium culture, 121 DEG C of sterilizings；It is 40- that algae strain, which is grown on intensity of illumination,
50 E/(m2S) in environment, brightness time ratio is 14 h:10 h, 18 DEG C of temperature, daily manual shaking flask 3 ~ 5 times.
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