CN108753623A - A kind of chlorella store method - Google Patents
A kind of chlorella store method Download PDFInfo
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- CN108753623A CN108753623A CN201810599963.6A CN201810599963A CN108753623A CN 108753623 A CN108753623 A CN 108753623A CN 201810599963 A CN201810599963 A CN 201810599963A CN 108753623 A CN108753623 A CN 108753623A
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- chlorella
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
Abstract
The invention discloses a kind of chlorella store methods, including：It is for use to exponential phase to be inoculated in closing culture in culture solution by 1 culture for chlorella；2 concentrations, centrifugation chlorella algae solution obtain concentrate, and algae powder must be dried by being subsequently placed in dry to constant weight in micro-wave oven；3 preserve, and dry algae powder is placed in distilled water in Tissue Culture Flask, glycerine, tea extract, aloe extract are then added, and are preserved with the fully wrapped around firmly Tissue Culture Flask of masking foil.It has the beneficial effect that：This method is easy to operate, it effectively preserves medium golden camellia tea extract and the purity and content of aloe extract is higher, with higher biological activity, chlorella is enable to keep complete eucaryotic cell structure, high-survival rate, the important indicators such as its biomass, chlorophyll and active polysaccharide are without being remarkably decreased after resurrection, are that a kind of long timeliness, activity be high, sustainable chlorella store method.
Description
Technical field
The present invention relates to algae preservation fields, more particularly, to a kind of chlorella store method.
Technical background
Chlorella（Chlorella vulgaris）Chlorophyceae, chlorella section.Single-cell algae, Chang Dansheng also have many cells
Aggregation.Cell is spherical, oval, and interior there are one Zhousheng, the chromatoplasts of cup-shaped or sheet.Vegetative propagation, each cell can produce
Raw 2,4,8 or 16 autospores, mother cell ruptures when ripe, spore effusion, is new individual after growing up.Have all over the world
Distribution, moves in smaller shallow water more, also there is marine breeds.Individual is less under natural endowment, manually cultivates mass propagation.Into the cell
Protein, fat and carbohydrate content it is all very high, and there are many vitamin, edible and as bait.
In industrial aspect, Japan has begun to the research and development to chlorella very early, and it is food successfully to make chlorella extract CGF
Product additive is quickly applied in numerous food product because it has the advantages that specific to food additives.And in fermentation industry
Advantageous microorganism is set quickly to breed using the fermentation of chlorella.Also, in terms of aquaculture, chlorella is alternatively arranged as
The feed of fish, shrimp, crab and shellfish etc. feeds effect with good.With the fast development of cryobiology, Cord blood
Technology medicine, food industry and agriculturally especially microorganism preservation, sperm freezing etc. it is many-sided have be widely applied.
Marine microalgae is full of nutrition, wide containing nutriments such as a large amount of protein, polysaccharide, unsaturated fatty acid, multivitamins
General is applied to aquaculture, but the growth of microalgae itself is easily influenced by factors such as environment, weathers, often gives aquaculture
Industry brings big inconvenience, so application of the research and development cryobiology in microalgae is also increasingly taken seriously.Currently, lacking
A kind of good chlorella store method of weary preservation effect.
Invention content
The purpose of the present invention is to provide a kind of chlorella store methods, and this method is easy to operate, effectively preserve medium gold
The purity of jasmine tea leaf extract and aloe extract is higher with content, has higher biological activity so that chlorella can
Keep complete eucaryotic cell structure, high-survival rate, after resurrection the important indicators such as its biomass, chlorophyll and active polysaccharide without significantly under
Drop is that a kind of long timeliness, activity be high, sustainable chlorella store method.
The present invention is directed to the problem of being mentioned in background technology, and the technical solution taken is：
A kind of chlorella store method, including：Culture, preserves concentration, specifically includes following steps：
Culture：Chlorella is inoculated in culture solution and closes culture, culture periodicity of illumination is 10~12L:12~14D, temperature are
20~22 DEG C, after inoculation and add that apply the mass ratio of inorganic fertilizer ammonium hydrogen carbonate and calcium superphosphate after water expanding species be 3~4:1, according to
Every square metre of water surface full ponds 25~35g of splashing uniformly are splashed, daily fertilising 1 time, the algae cell density in daily monitoring test pond and
Chemical measurements of water, by microdisk electrode to exponential phase, for use；Microalgae strain is cultivated under suitable culture conditions, can be promoted
The growth and breeding of microalgae strain health carry out concentration and preserve conducive to keeping chlorella more prosperous after culture to exponential phase
The vitality of Sheng is easy to improve its activity and survival rate；
Concentration：Logarithmic phase chlorella algae solution is taken, centrifuges 15~35min in 500~800r/min rotating speeds, removal supernatant obtains bead
Algae concentrate, is subsequently placed in micro-wave oven, microwave power is adjusted to the 20~30% of maximum power, drying is obtained to constant weight
Microwave drying algae powder；Using micro-wave oven dried pellet algae algae solution, it can be dehydrated with the realization of greater efficiency, chlorella is made to keep
In the vigorous life state of exponential phase, while can also reduce processing time, it is energy saving；
It preserves：According to solid-to-liquid ratio 1:Dry algae powder is placed in distilled water in Tissue Culture Flask by 2.5~5 ratio, is then added
Glycerine, tea extract, aloe extract, the mass fraction of glycerine are that the mass fraction of 0.5~0.8%, golden flower tea extractions is
1.2~1.5%, the mass fraction of aloe extract is 5.5~6.0%, with masking foil it is fully wrapped around live Tissue Culture Flask, by cell
Culture bottle is placed in 1~2 DEG C, sterile, oxygenation, preserves in dark surrounds；By optimization, the holding time of chlorella has obtained significantly
Extension, the Free water in the frustule in temperature above freezing will not form ice crystal, and most cells can keep completely tying
The ability of structure, survival rate and restoration ecosystem；The important indicators such as bead algae biomass, chlorophyll and active polysaccharide are without notable after resurrection
Decline, and can be stored and be transported at normal temperatures, avoids low temperature energy consumption and transport inconvenient, and chlorella vigor is preferable, loses
Perfect in shape and function is passed, therefore is that a kind of long timeliness, activity be high, sustainable chlorella store method.
Preferably, the culture solution in algae solution incubation step is NaNO378~80mg/L, NaH2PO4·H2O3~4mg/L,
Na2SiO3·9H2O22~25mg/L, Na2EDTA4.36~4.4mg/L, FeCl3·6H2O3.16~3.2mg/L, CuSO4·
5H2O0.01~0.015mg/L, ZnSO4·7H2O0.023~0.025mg/L, CoCL2·6H2O0.012~0.015mg/L,
MnCL·4H2O0.18~0.2mg/L, Na2MoO4·2H2O0.07~0.08mg/L, 0.1~0.12 μ g/L of vitamin B1, dimension
0.5~0.6 μ g/L of raw element B12,0.8~1 μ g/L of biotin, surplus is desinfection chamber, and sterilize 20min at 121 DEG C.
Preferably, main chemical measurements of water is during culture：Water temperature is 10.0~12.5 DEG C, and salinity is 19~21, pH
Be 8.55~9.33, dissolved oxygen be 15.2~20.0mg/L, ammoniacal nitrogen be 24.29~70.95mg/L, active phosphorus be 0.71~
2.37mg/L, transparency are 11~18cm.
Preferably, the preparation method of golden flower tea extractions is：According to 1:2.1~2.5 weight ratio is by golden camellia tea powder
Medicinal extract is extracted to obtain with the diafiltration of 75~90% ethanol solutions, medicinal extract is placed in the mixed of 3~4 times of 2,2- diphenyl cyclopentanol and n-butanol
It is extracted in bonding solvent, the content of 2,2- diphenyl cyclopentanol is 0.85~0.88 ‰, and extract is that golden camellia tea n-butanol carries
Take object, vacuum dried that medicinal extract is made is for use；The extract of golden camellia tea because containing a large amount of flavones, tea polyphenols, tea polysaccharide and
Saponin component has extensive pharmacological action, chlorella during can effectively inhibiting to preserve with barbaloin synergistic effect
Cell activity reduces metabolism, reduce during Cord blood the decomposition of substances such as bead algae biomass, polysaccharide and chlorophyll with
Metabolism, maintains its content content, and make the rapid activity recovery of chlorella and life after resurrection；Golden camellia tea medicinal extract is placed in
The extraction of the in the mixed solvent of 2,2- diphenyl cyclopentanol and n-butanol can greatly promote extraction efficiency, 2,2- diphenyl cyclopentanol
The fibre fractionation and protein ingredient that golden camellia tea is acted synergistically on n-butanol make its fiber degradation be denaturalized, release flavones, tea
The pharmaceutical components such as polyphenol, tea polysaccharide, saponin(e and terpene, while decomposition of the enzyme to pharmacology component is reduced, improve the positive fourth of golden camellia tea
The content and purity of effective medicinal ingredient in alcohol extracting thing, and further increase golden camellia tea extract and the preservation of chlorella is imitated
Fruit.
Preferably, the preparation method of aloe extract is：Aloe medicinal material is ground into the molecule of 300~360 mesh,
It is placed in container, according to solid-liquid ratio 1:15~18 are added deionized water, and it is 7.4~7.6 to adjust pH, and aloe medicinal material weight 3 is added
~12% complex enzyme, complex enzyme are that weight ratio is 1:3~5 trypsase and papain, adds compound protease weight
Measure 15~18 ‰ pentanediol and 3~3.6 ‰ t- leucine tert-butyl esters, enzymolysis and extraction at a temperature of 42~45 DEG C, enzymolysis 25
Enzyme deactivation after~45min, enzymolysis liquid cross 0.35~0.45 μm of membrane filtration, and it is 1.05~1.08 to take filtrate to dry to relative density
Up to extract solution from aloe；Aloe proteins can be digested with compound protease, enzymolysis is more thorough, is added in enzymatic hydrolysis system special
The pentanediol very matched can be acted on t- leucine tert-butyl esters and the sulphydryl activity center of veraldon, can drop significantly
Energy barrier between low enzyme-to-substrate increases the reactivity between enzyme-to-substrate, improves reaction efficiency, accelerates enzyme digestion reaction
Progress, reduce enzymolysis cost and energy expenditure, while keeping enzymolysis more thorough, weaken packet of the aloe proteins to barbaloin significantly
It covers, accelerates the release of barbaloin, the content and purity of barbaloin in extract solution from aloe is greatly improved, improves its biological effectiveness.
Compared with the prior art, the advantages of the present invention are as follows：
1）Using micro-wave oven dried pellet algae algae solution, it can be dehydrated with the realization of greater efficiency, chlorella is made to be maintained at logarithm
The vigorous life state in growth period, while can also reduce processing time, it is energy saving；
2）2,2- diphenyl cyclopentanol act synergistically on the fibre fractionation and protein ingredient of golden camellia tea with n-butanol, make its fiber
Degradation denaturation, releases the pharmaceutical components such as flavones, tea polyphenols, tea polysaccharide, saponin(e and terpene, while reducing enzyme to pharmacology component
It decomposes, improves the content and purity of effective medicinal ingredient in golden camellia tea n-butanol extract, and further increase golden camellia tea
Preservation effect of the extract to chlorella；
3）Be added the pentanediol of special proportioning in enzymolysis aloe system and t- leucine tert-butyl esters can substantially reduce enzyme-to-substrate it
Between energy barrier, increase the reactivity between enzyme-to-substrate, accelerate the progress of enzyme digestion reaction, reduce enzymolysis cost and energy
Consumption weakens aloe proteins to the cladding of barbaloin, accelerates the release of barbaloin, aloe in extract solution from aloe is greatly improved significantly
The content and purity of glycosides, improve its biological effectiveness；
4）Storage temperature is in above freezing, and the Free water in chlorella cells will not form ice crystal, and most cells can be kept
The ability of complete structure, survival rate and restoration ecosystem；The important indicators such as bead algae biomass, chlorophyll and active polysaccharide after resurrection
Nothing is remarkably decreased；
5）It can be stored and be transported at normal temperatures, avoid low temperature energy consumption and transport inconvenient, and chlorella vigor is preferable, heredity
Perfect in shape and function, therefore be that a kind of long timeliness, activity be high, sustainable chlorella store method.
Specific implementation mode
The present invention program is described further below by embodiment：
Embodiment 1：
A kind of chlorella store method, specifically includes following steps：
S1：Chlorella is inoculated in culture solution and closes culture, culture periodicity of illumination is 10L:14D, temperature are 20 DEG C, after inoculation
And add that apply the mass ratio of inorganic fertilizer ammonium hydrogen carbonate and calcium superphosphate after water expanding species be 3:1, it splashes 25g according to every square metre of water surface
Full pond is uniformly splashed, daily fertilising 1 time, the algae cell density and chemical measurements of water in daily monitoring test pond, extremely by microdisk electrode
Exponential phase, for use；
S2：Logarithmic phase chlorella algae solution is taken, centrifuges 15min in 500r/min rotating speeds, removal supernatant obtains chlorella concentrate, so
It is placed in micro-wave oven, microwave power is adjusted to the 20% of maximum power, drying obtains microwave drying algae powder to constant weight；
S3：According to solid-to-liquid ratio 1:Dry algae powder is placed in distilled water in Tissue Culture Flask by 2.5 ratio, then add glycerine,
Tea extract, aloe extract, the mass fraction of glycerine are that the mass fraction of 0.5%, golden flower tea extractions is 1.2%, aloe
The mass fraction of extract be 5.5%, with masking foil it is fully wrapped around live Tissue Culture Flask, by Tissue Culture Flask be placed in 1 DEG C, it is sterile,
It is preserved in oxygenation, dark surrounds.
Culture solution in algae solution incubation step is NaNO378mg/L、NaH2PO4·H2O3mg/L、Na2SiO3·
9H2O22mg/L、Na2EDTA4.36mg/L、FeCl3·6H2O3.16mg/L、CuSO4·5H2O0.01mg/L、ZnSO4·
7H2O0.023mg/L、CoCL2·6H2O0.012mg/L、MnCL·4H2O0.18mg/L、Na2MoO4·2H2O0.07mg/L, dimension
0.1 μ g/L of raw element B1,0.5 μ g/L of vitamin B12,0.8 μ g/L of biotin, surplus is desinfection chamber, is sterilized at 121 DEG C
20min。
Main chemical measurements of water is during culture：Water temperature is 10 DEG C, salinity 19, pH 8.55, dissolved oxygen 15.2mg/
L, ammoniacal nitrogen 24.29mg/L, active phosphorus 0.71mg/L, transparency 11cm.
The preparation method of golden flower tea extractions is：According to 1:2.1 weight ratio oozes golden camellia tea powder with 75% ethanol solution
Medicinal extract is extracted to obtain in filter, and the in the mixed solvent that medicinal extract is placed in 3 times of n-butanols extracts, and extract is the extraction of golden camellia tea n-butanol
Object, vacuum dried that medicinal extract is made is for use；The extract of golden camellia tea is because containing a large amount of flavones, tea polyphenols, tea polysaccharide and soap
Methods of glycosides has extensive pharmacological action, and chlorella is thin during can effectively inhibiting to preserve with barbaloin synergistic effect
Cytoactive reduces metabolic, the decomposition of the substances such as bead algae biomass, polysaccharide and chlorophyll and generation during reduction Cord blood
It thanks, maintains its content content, and make the rapid activity recovery of chlorella and life after resurrection.
The preparation method of aloe extract is：Aloe medicinal material is ground into the molecule of 300 mesh, is placed in container, is pressed
Take care of liquor ratio 1:15 are added deionized water, and it is 7.4 to adjust pH, the complex enzyme of aloe medicinal material weight 3% is added, complex enzyme is weight
Than being 1:3 trypsase and papain adds the pentanediol of compound protease weight 15 ‰ and 3 ‰ t- leucines
The tert-butyl ester, enzymolysis and extraction at a temperature of 42 DEG C digest enzyme deactivation after 25min, and enzymolysis liquid crosses 0.35 μm of membrane filtration, take filtrate dry
It is dry to relative density be 1.05 up to extract solution from aloe；Aloe proteins can be digested with compound protease, enzymolysis is more thorough
Bottom, the pentanediol of special proportioning is added in enzymatic hydrolysis system can act on and the mercapto of veraldon with t- leucine tert-butyl esters
Base activated centre can substantially reduce the energy barrier between enzyme-to-substrate, increase the reactivity between enzyme-to-substrate, improve anti-
Efficiency is answered, the progress of enzyme digestion reaction is accelerated, enzymolysis cost and energy expenditure is reduced, while keeping enzymolysis more thorough, weakens significantly
Aloe proteins accelerate the release of barbaloin to the cladding of barbaloin, be greatly improved in extract solution from aloe the content of barbaloin with it is pure
Degree, improves its biological effectiveness.
By optimization, the holding time of chlorella has obtained greatly extending, and is in oneself in the frustule of temperature above freezing
Ice crystal will not be formed by water, most cells can keep the ability of complete structure, survival rate and restoration ecosystem；It is small after resurrection
The important indicators such as ball algae biomass, chlorophyll and active polysaccharide can be stored and be transported at normal temperatures without being remarkably decreased, and be kept away
Exempt from low temperature energy consumption and transport be inconvenient, and chlorella vigor is preferable, genetic function is perfect, therefore be a kind of long timeliness, activity it is high,
Sustainable chlorella store method.
Embodiment 2：
A kind of chlorella store method, specifically includes following steps：
Culture：Chlorella is inoculated in culture solution and closes culture, culture periodicity of illumination is 12L:12D, temperature are 22 DEG C, inoculation
Afterwards and add that apply the mass ratio of inorganic fertilizer ammonium hydrogen carbonate and calcium superphosphate after water expanding species be 4:1, it splashes according to every square metre of water surface
35g is complete, and pond is uniformly splashed, daily fertilising 1 time, the algae cell density and chemical measurements of water in daily monitoring test pond, by microdisk electrode
To exponential phase, for use；Cultivate microalgae strain under suitable culture conditions, can promote the growth of microalgae strain health with it is numerous
It grows, concentration is carried out after cultivating to exponential phase and is preserved conducive to the vitality for keeping chlorella more vigorous, is easy to improve it
Activity and survival rate；
Concentration：Logarithmic phase chlorella algae solution is taken, 35min is centrifuged in 800r/min rotating speeds, removal supernatant obtains chlorella concentrate,
It is subsequently placed in micro-wave oven, microwave power is adjusted to the 30% of maximum power, drying obtains microwave drying algae powder to constant weight；
Using micro-wave oven dried pellet algae algae solution, it can be dehydrated with the realization of greater efficiency, chlorella is made to be maintained at exponential phase
Vigorous life state, while can also reduce processing time, it is energy saving；
It preserves：According to solid-to-liquid ratio 1:Dry algae powder is placed in distilled water in Tissue Culture Flask by 5 ratio, then add glycerine,
Tea extract, aloe extract, the mass fraction of glycerine are that the mass fraction of 0.8%, golden flower tea extractions is 1.5%, aloe
The mass fraction of extract be 6.0%, with masking foil it is fully wrapped around live Tissue Culture Flask, by Tissue Culture Flask be placed in 2 DEG C, it is sterile,
It is preserved in oxygenation, dark surrounds；By optimization, the holding time of chlorella has obtained greatly extending, in temperature above freezing
Free water in frustule will not form ice crystal, and most cells can keep complete structure, survival rate and restoration ecosystem
Ability；The important indicators such as bead algae biomass, chlorophyll and active polysaccharide are without being remarkably decreased after resurrection, and can carry out at normal temperatures
Storage and transport avoid low temperature energy consumption and transport inconvenient, and chlorella vigor is preferable, and genetic function is perfect, therefore is a kind of
Long timeliness, activity height, sustainable chlorella store method.
Culture solution in algae solution incubation step is NaNO380mg/L、NaH2PO4·H2O4mg/L、Na2SiO3·
9H2O25mg/L、Na2EDTA4.4mg/L、FeCl3·6H2O3.2mg/L、CuSO4·5H2O0.015mg/L、ZnSO4·
7H2O0.025mg/L、CoCL2·6H2O0.015mg/L、MnCL·4H2O0.2mg/L、Na2MoO4·2H2O0.08mg/L, dimension life
0.12 μ g/L of plain B1,0.6 μ g/L of vitamin B12,1 μ g/L of biotin, surplus is desinfection chamber, and sterilize 20min at 121 DEG C.
Main chemical measurements of water is during culture：Water temperature is 12.5 DEG C, salinity 21, pH 9.33, and dissolved oxygen is
20.0mg/L, ammoniacal nitrogen 70.95mg/L, active phosphorus 2.37mg/L, transparency 18cm.
The preparation method of golden flower tea extractions is：According to 1:2.5 weight ratio oozes golden camellia tea powder with 90% ethanol solution
Medicinal extract is extracted to obtain in filter, and the in the mixed solvent that medicinal extract is placed in 4 times of 2,2- diphenyl cyclopentanol and n-butanol extracts, 2,2- diphenyl
The content of cyclopentanol is 0.88 ‰, and extract is golden camellia tea n-butanol extract, and vacuum dried that medicinal extract is made is for use；Gold
The extract of jasmine tea leaf because contain a large amount of flavones, tea polyphenols, tea polysaccharide and saponin component, have extensive pharmacological action,
The cell activity of chlorella, reduces metabolism during it can effectively inhibit to preserve with barbaloin synergistic effect, reduces low temperature
The decomposition and metabolism of the substances such as chlorella biomass, polysaccharide and chlorophyll during preservation, maintain its content content, and bringing back to life
After make the rapid activity recovery of chlorella and life；Golden camellia tea medicinal extract is placed in the mixing of 2,2- diphenyl cyclopentanol and n-butanol
Extraction can greatly promote extraction efficiency in solvent, and 2,2- diphenyl cyclopentanol act synergistically on the fibre of golden camellia tea with n-butanol
Tie up component and protein ingredient, its fiber degradation made to be denaturalized, release the pharmacology such as flavones, tea polyphenols, tea polysaccharide, saponin(e and terpene at
Point, while reducing decomposition of the enzyme to pharmacology component, improve in golden camellia tea n-butanol extract effectively the content of medicinal ingredient with
Purity, and further increase preservation effect of the golden camellia tea extract to chlorella.
The preparation method of aloe extract is：Aloe medicinal material is ground into the molecule of 360 mesh, is placed in container, is pressed
Take care of liquor ratio 1:18 are added deionized water, and it is 7.6 to adjust pH, the complex enzyme of aloe medicinal material weight 12% is added, complex enzyme is weight
Than being 1:5 trypsase and papain, enzymolysis and extraction at a temperature of 45 DEG C digest enzyme deactivation after 45min, enzymolysis liquid mistake
0.45 μm of membrane filtration takes filtrate to dry to relative density as 1.08 up to extract solution from aloe；It can be incited somebody to action with compound protease
Aloe proteins are digested, and enzymolysis is more thorough.
Embodiment 3：
A kind of chlorella store method, including：Culture, preserves concentration, specifically includes following steps：
Culture：Chlorella is inoculated in culture solution and closes culture, culture periodicity of illumination is 12L:12D, temperature are 20 DEG C, inoculation
Afterwards and add that apply the mass ratio of inorganic fertilizer ammonium hydrogen carbonate and calcium superphosphate after water expanding species be 3.5:1, it is sprinkled according to every square metre of water surface
It spills the full ponds 32g uniformly to splash, daily fertilising 1 time, the algae cell density and chemical measurements of water in daily monitoring test pond train microalgae
It supports to exponential phase, for use；Under suitable culture conditions cultivate microalgae strain, can promote microalgae strain health growth and
Breeding carries out concentration and preserves conducive to the vitality for keeping chlorella more vigorous, is easy to improve after cultivating to exponential phase
Its activity and survival rate；
Concentration：Logarithmic phase chlorella algae solution is taken, 20min is centrifuged in 600r/min rotating speeds, removal supernatant obtains chlorella concentrate,
It is subsequently placed in micro-wave oven, microwave power is adjusted to the 25% of maximum power, drying obtains microwave drying algae powder to constant weight；
Using micro-wave oven dried pellet algae algae solution, it can be dehydrated with the realization of greater efficiency, chlorella is made to be maintained at exponential phase
Vigorous life state, while can also reduce processing time, it is energy saving；
It preserves：According to solid-to-liquid ratio 1:Dry algae powder is placed in distilled water in Tissue Culture Flask by 4 ratio, then add glycerine,
Tea extract, aloe extract, the mass fraction of glycerine are that the mass fraction of 0.6%, golden flower tea extractions is 1.4%, aloe
The mass fraction of extract be 5.5%, with masking foil it is fully wrapped around live Tissue Culture Flask, by Tissue Culture Flask be placed in 1 DEG C, it is sterile,
It is preserved in oxygenation, dark surrounds；By optimization, the holding time of chlorella has obtained greatly extending, in temperature above freezing
Free water in frustule will not form ice crystal, and most cells can keep complete structure, survival rate and restoration ecosystem
Ability；The important indicators such as bead algae biomass, chlorophyll and active polysaccharide are without being remarkably decreased after resurrection, and can carry out at normal temperatures
Storage and transport avoid low temperature energy consumption and transport inconvenient, and chlorella vigor is preferable, and genetic function is perfect, therefore is a kind of
Long timeliness, activity height, sustainable chlorella store method.
Culture solution in algae solution incubation step is NaNO378mg/L、NaH2PO4·H2O3.5mg/L、Na2SiO3·
9H2O24mg/L、Na2EDTA4.4mg/L、FeCl3·6H2O3.2mg/L、CuSO4·5H2O0.012mg/L、ZnSO4·
7H2O0.024mg/L、CoCL2·6H2O0.014mg/L、MnCL·4H2O0.18mg/L、Na2MoO4·2H2O0.07mg/L, dimension
0.11 μ g/L of raw element B1,0.5 μ g/L of vitamin B12,0.8 μ g/L of biotin, surplus is desinfection chamber, is sterilized at 121 DEG C
20min。
Main chemical measurements of water is during culture：Water temperature is 12 DEG C, salinity 20.5, pH 8.85, and dissolved oxygen is
18.2mg/L, ammoniacal nitrogen 45.0mg/L, active phosphorus 1.2mg/L, transparency 15cm.
The preparation method of golden flower tea extractions is：According to 1:2.2 weight ratio oozes golden camellia tea powder with 85% ethanol solution
Medicinal extract is extracted to obtain in filter, and the in the mixed solvent that medicinal extract is placed in 3 times of 2,2- diphenyl cyclopentanol and n-butanol extracts, 2,2- diphenyl
The content of cyclopentanol is 0.88 ‰, and extract is golden camellia tea n-butanol extract, and vacuum dried that medicinal extract is made is for use；Gold
The extract of jasmine tea leaf because contain a large amount of flavones, tea polyphenols, tea polysaccharide and saponin component, have extensive pharmacological action,
The cell activity of chlorella, reduces metabolism during it can effectively inhibit to preserve with barbaloin synergistic effect, reduces low temperature
The decomposition and metabolism of the substances such as chlorella biomass, polysaccharide and chlorophyll during preservation, maintain its content content, and bringing back to life
After make the rapid activity recovery of chlorella and life；Golden camellia tea medicinal extract is placed in the mixing of 2,2- diphenyl cyclopentanol and n-butanol
Extraction can greatly promote extraction efficiency in solvent, and 2,2- diphenyl cyclopentanol act synergistically on the fibre of golden camellia tea with n-butanol
Tie up component and protein ingredient, its fiber degradation made to be denaturalized, release the pharmacology such as flavones, tea polyphenols, tea polysaccharide, saponin(e and terpene at
Point, while reducing decomposition of the enzyme to pharmacology component, improve in golden camellia tea n-butanol extract effectively the content of medicinal ingredient with
Purity, and further increase preservation effect of the golden camellia tea extract to chlorella.
The preparation method of aloe extract is：Aloe medicinal material is ground into the molecule of 300 mesh, is placed in container, is pressed
Take care of liquor ratio 1:16 are added deionized water, and it is 7.5 to adjust pH, the complex enzyme of aloe medicinal material weight 10% is added, complex enzyme is weight
Than being 1:4 trypsase and papain adds the pentanediol of compound protease weight 16 ‰ and the 3.5 ‰ bright ammonia of t-
Tert-butyl acrylate, enzymolysis and extraction at a temperature of 44 DEG C digest enzyme deactivation after 30min, and enzymolysis liquid crosses 0.4 μm of membrane filtration, takes filtrate
Drying to relative density is 1.06 up to extract solution from aloe；Aloe proteins can be digested with compound protease, enzymolysis is more
Thoroughly, the pentanediol of special proportioning and t- leucine tert-butyl esters are added in enzymatic hydrolysis system can act on and veraldon
Sulphydryl activity center can substantially reduce the energy barrier between enzyme-to-substrate, increase the reactivity between enzyme-to-substrate, improve
Reaction efficiency accelerates the progress of enzyme digestion reaction, reduces enzymolysis cost and energy expenditure, while keeping enzymolysis more thorough, cuts significantly
Weak aloe proteins accelerate the release of barbaloin to the cladding of barbaloin, be greatly improved in extract solution from aloe the content of barbaloin with
Purity improves its biological effectiveness.
Comparative example 1：
The chlorella algae solution normally cultivated is taken, 20min is centrifuged through 120r/min, discards supernatant to obtain chlorella concentrate, control is small
A concentration of 0.2g/100mL of chlorella in ball algae concentrate；Chlorella concentrate is taken, the sterile water of 3 times of weight is added, then add
Enter 0.5% glycerine, 0.03% sodium sorbate, mixture is then placed in 15 DEG C of preservations.
Experimental example：
Biomass, polyoses content and chlorophyll content that following measurement method measures chlorella is respectively adopted：
A biomass estimation methods：5% sodium citrate of 5mL is added in the glueballs for taking 5mL amounts, and the 2~3min that is vortexed is whole to algae pearl
Dissolving, centrifugation (8000r/min, 5min) discard supernatant liquid, and appropriate pure water is added and is resuspended, cleaning is several times.By algal gel again constant volume
To 5mL, its A667 value is surveyed, its biomass is calculated；
B polyoses contents：1ml algae solutions are taken, are first diluted with water 10 times, Phenol sulfuric acid procedure is reused and surveys polysaccharide, according to standard curve meter
Calculate polyoses content；
C chlorophyll contents：5ml algae solutions, 0.45 μm of filter membrane is taken to filter, suck dry moisture is washed down with a small amount of absolute ethyl alcohol, is ground to nothing
Water-ethanol volatilizees, and continues to use liquid nitrogen grinding 10min, is settled to 5mL with absolute ethyl alcohol, is protected from light and is statically placed in 4 DEG C of refrigerators, afterwards will for 24 hours
Extracting solution is centrifuged with 4000r/min, 10min, and supernatant is taken to measure A645、A663, calculate chlorophyll content, according to formula calculate C=
20.3A645+8.04A663, A in formula645、A663Respectively represent the OD values in the case where wavelength is 645nm, 663nm.
The measurement result of chlorella in comparative example 1 and embodiment 1-3 is organized into shown in table 1.As shown in Table 1, this hair
No matter the chlorella in embodiment 1-3 in bright method is in the side such as survival rate or biomass, polyoses content and chlorophyll content
Face is significantly better than comparative example 1, illustrates that the store method of this chlorella is to be applicable in and efficiently, should also be seen that simultaneously, embodiment 3
Every measurement index of middle chlorella is significantly better than embodiment 2, embodiment 3.
The measurement data of 1 chlorella of table
Routine operation in operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme of the present invention is described in detail in embodiment described above, it should be understood that the above is only
For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in the spirit of the present invention,
Supplement or similar fashion replacement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of chlorella store method, it is characterised in that：Including：
Culture：Chlorella is inoculated in culture solution and closes culture, inorganic fertilizer ammonium hydrogen carbonate is applied after inoculation and after adding water expanding species
With calcium superphosphate, the algae cell density and chemical measurements of water in daily monitoring test pond wait for microdisk electrode to exponential phase
With；
Concentration：Logarithmic phase chlorella algae solution, centrifugation removal supernatant is taken to obtain chlorella concentrate, be subsequently placed in micro-wave oven dry
To constant weight, microwave drying algae powder is obtained；
It preserves：Dry algae powder is placed in distilled water in Tissue Culture Flask, glycerine, tea extract, aloe extraction are then added
Object is preserved with the fully wrapped around firmly Tissue Culture Flask of masking foil.
2. a kind of chlorella store method according to claim 1, it is characterised in that：In the incubation step, culture solution
For NaNO378~80mg/L, NaH2PO4·H2O3~4mg/L, Na2SiO3·9H2O22~25mg/L, Na2EDTA4.36~
4.4mg/L、FeCl3·6H2O3.16~3.2mg/L, CuSO4·5H2O0.01~0.015mg/L, ZnSO4·7H2O0.023~
0.025mg/L、CoCL2·6H2O0.012~0.015mg/L, MnCL4H2O0.18~0.2mg/L, Na2MoO4·2H2O0.07
~0.08mg/L, 0.1~0.12 μ g/L of vitamin B1,0.5~0.6 μ g/L of vitamin B12, biotin 0.8~1 μ g/L, it is remaining
Amount is desinfection chamber, and sterilize 20min at 121 DEG C.
3. a kind of chlorella store method according to claim 1, it is characterised in that：In the incubation step, light is cultivated
It is 10~12L according to the period:12~14D, temperature are 20~22 DEG C.
4. a kind of chlorella store method according to claim 1, it is characterised in that：In the incubation step, culture period
Between main chemical measurements of water be：Water temperature is 10.0~12.5 DEG C, and salinity is that 19~21, pH is 8.55~9.33, and dissolved oxygen is
15.2~20.0mg/L, ammoniacal nitrogen be 24.29~70.95mg/L, active phosphorus be 0.71~2.37mg/L, transparency be 11~
18cm。
5. a kind of chlorella store method according to claim 1, it is characterised in that：In the preservation step, glycerine
Mass fraction is that the mass fraction of 0.5~0.8%, golden flower tea extractions is 1.2~1.5%, and the mass fraction of aloe extract is
5.5~6.0%.
6. a kind of chlorella store method according to claim 1 or 5, it is characterised in that：In the preservation step, golden flower
The preparation method of tea extraction is：Golden camellia tea powder is extracted into obtain medicinal extract with ethanol solution diafiltration, medicinal extract is placed in containing 0.85
It is extracted in the n-butanol solvent of~0.88 ‰ 2,2- diphenyl cyclopentanol, extract is golden camellia tea n-butanol extract, warp
It is for use that medicinal extract is made in vacuum drying.
7. a kind of chlorella store method according to claim 1 or 5, it is characterised in that：In the preservation step, aloe
The preparation method of extract is：Aloe medicinal material is ground into molecule, is placed in container, deionized water, complex enzyme, penta is added
Glycol is digested with t- leucine tert-butyl esters, and enzymolysis liquid crosses 0.35~0.45 μm of membrane filtration, filtrate is taken to dry to relative density
For 1.05~1.08 up to extract solution from aloe.
8. a kind of chlorella store method according to claim 7, it is characterised in that：The preparation side of the aloe extract
In method, enzymolysis pH is 7.4~7.6, and hydrolysis temperature is 42~45 DEG C, and enzymolysis time is 25~45min.
9. a kind of chlorella store method according to claim 7, it is characterised in that：The preparation side of the aloe extract
In method, the additive amount of complex enzyme is the 3~12% of aloe medicinal material weight, the additive amount of pentanediol be complex enzyme weight 15~
The additive amount of 18 ‰, t- leucine tert-butyl ester is 3~the 3.6 ‰ of complex enzyme weight, and complex enzyme is that weight ratio is 1:3~5 pancreas
Protease and papain.
10. a kind of chlorella store method according to claim 1, it is characterised in that：In the preservation step, cell training
Foster bottle is placed in 1~2 DEG C, sterile, oxygenation, preserves in dark surrounds.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628632A (en) * | 2019-10-29 | 2019-12-31 | 岳阳渔美康生物科技有限公司 | Method for preserving chlorella at normal temperature |
| CN115678785A (en) * | 2021-11-18 | 2023-02-03 | 珠海光藻生命科学有限公司 | Food-grade chlorella culture medium and culture method |
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| CN110628632A (en) * | 2019-10-29 | 2019-12-31 | 岳阳渔美康生物科技有限公司 | Method for preserving chlorella at normal temperature |
| CN115678785A (en) * | 2021-11-18 | 2023-02-03 | 珠海光藻生命科学有限公司 | Food-grade chlorella culture medium and culture method |
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