CN113730950A - Multistage circulating extraction equipment and preparation method for chlorella vulgaris and chlorella vulgaris deep purification factor - Google Patents
Multistage circulating extraction equipment and preparation method for chlorella vulgaris and chlorella vulgaris deep purification factor Download PDFInfo
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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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9706—Algae
- A61K8/9722—Chlorophycota or Chlorophyta [green algae], e.g. Chlorella
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/005—Preparations for sensitive skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
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Abstract
The invention discloses a chlorella tianshan, which is named as: the chlorella pyrenoidosa, JY-QWZ-1900386, Sinkiang, is preserved in the China general microbiological culture Collection center of the culture Collection management Committee of microorganisms in 6 months in 2021, and the preservation number is CGMCC NO: 17926. The newly discovered chlorella variabilis is subjected to enlarged culture by a bioreactor, and the chlorella variabilis is extracted to prepare the chlorella deep purification factor. The newly developed multi-stage external circulation extraction equipment comprises an extraction tank, wherein a rotating shaft is rotatably mounted in the extraction tank, and a servo motor for rotating the rotating shaft is arranged at the top of the extraction tank; the method can improve the extraction efficiency of the compound, reduce the use of solvents and the dissolution of impurities, control the extraction end point by monitoring the effective components and the dissolution of the impurities in real time, more accurately control the component stability of the product, further control the effect stability of the product, and ensure that the prepared chlorella deep purification factor is applied to the field of daily chemicals and has the effects of moisturizing, repairing and improving the immunity.
Description
Technical Field
The invention relates to chlorella vulgaris and the technical field of extraction thereof, in particular to a preparation method, application and multistage circulating extraction equipment of a chlorella vulgaris deep purification factor, and belongs to the technical field of daily cosmetics.
Background
The chlorella deep purification factor is a mixture containing various substances such as protein, polysaccharide, nucleic acid, polypeptide, vitamin, trace elements and the like, and the diversified substances make the chlorella deep purification factor have various skin care effects: the composition can be used for removing free radicals, chelating redox heavy metal ions, strengthening the barrier of the stratum corneum, regulating the in vitro immune function of macrophages, repairing the damage of cells and DNA, resisting pollution and radiation and being extremely safe and non-irritant.
Nowadays, the skin of children is increasingly damaged due to the problems of environmental influence, living habits, bad cosmetics and the like, and the chlorella deep purification factor has a good protection effect on human skin, particularly the skin of children. The chlorella pyrenoidosa variety discovered from Tianshan mountain of Xinjiang contains more stress-resistant active ingredients, mainly C.G.F-chlorella growth factors, and has the effect of repairing damaged skin of children due to the fact that the chlorella pyrenoidosa variety grows in extreme environments. The preparation method of the cosmetics determines that liquid raw materials are preferred, and the practical guiding significance is provided for how to prepare the chlorella deep purification factor which is stable and has good anti-adversity restoration active substance retention.
The existing extraction equipment for plant extraction has the following problems:
(1) the existing extraction equipment has a relatively common mixing effect on materials and extraction liquid, and generally only uses a single shaft-shaped component for mixing operation, so that the efficiency is relatively low;
(2) at present, the extraction equipment lacks the compounding effect in the vertical direction in the mixing process, leads to the material to be insufficient with the contact of extraction liquid, leads to the extraction time longer, further influences its extraction efficiency.
Disclosure of Invention
Objects of the invention
In order to solve the technical problems in the background technology, the invention provides a preparation method of a deep purification factor of chlorella vulgaris and chlorella vulgaris, application and multi-stage circulating extraction equipment.
(II) technical scheme
In order to solve the above problems, the present invention provides a chlorella tianshan named as: the Xinjiang Tianshan Chlorella JY-QWZ-1900386 is preserved in China general microbiological culture Collection center (CGMCC) 6, 7 days 2021, the preservation number is CGMCC No.22396, the preservation address is No. 3 of West Lu No. 1 of the morning area of Beijing city, the sample is a survival sample, and the classification name is Chlorella (Chlorella sp.).
Still provide a multistage circulation extraction equipment of deep purification factor of Tianshan chlorella and chlorella, including the extraction jar, the pivot is installed to the extraction jar internal rotation, the top of extraction jar is equipped with and is used for pivot pivoted servo motor, the both sides of pivot are equipped with the material mixing pole, the bottom inner wall of extraction jar is equipped with the transmission case, and the bottom and the transmission case of pivot rotate to be connected, the bottom of extraction jar is connected with the driving tube, and the driving tube internal rotation is installed and is carried screw rod, carrying screw rod is connected with the pivot transmission, the one end of driving tube is connected with the circulating pipe, swing joint has the reposition of redundant personnel bucket that the top set up for uncovered in the pivot, the one end of circulating pipe extends to in the extraction jar and is located the top of reposition of redundant personnel bucket, and the bottom of redundant personnel bucket is provided with a plurality of shunt tubes that are annular evenly distributed, and the shunt tubes is located the top of material mixing pole, the one end fixed mounting that the pivot is close to reposition of redundant personnel bucket has the end piece, and the top outer wall fixed mounting of end piece has vibrations spring, the top of vibrations spring offsets with the bottom of reposition of redundant personnel bucket, the interior bottom of reposition of redundant personnel bucket is the inclined plane form, and the interior bottom fixed mounting of reposition of redundant personnel bucket has the arc muscle piece that the equidistance distributes.
Preferably, the top of extraction jar is equipped with the inlet pipe, the bottom of extraction jar is equipped with the discharging pipe, and the top of discharging pipe is equipped with the current-limiting stopper, the bottom of extraction jar is equipped with the push rod motor, and the push rod transmission of push rod motor is connected with the U-shaped pole, the one end of U-shaped pole runs through the discharging pipe and is connected with the current-limiting stopper.
Preferably, the bottom of the rotating shaft extends into the transmission box and is connected with a first bevel gear in a transmission mode, one side of the transmission box is rotated to be provided with the linkage rod, one end of the linkage rod is in transmission connection with the conveying screw rod, the other end of the linkage rod extends into the transmission box and is connected with a second bevel gear, the first bevel gear is meshed with the second bevel gear, one side of the mixing rod is provided with a water inlet hole and a water outlet hole respectively, a communication hole is formed in the mixing rod, two ends of the communication hole are communicated with the water inlet hole and the water outlet hole respectively, the communication hole is of a wave-shaped structure, the outer wall of the mixing rod is connected with a sleeve in a rotating mode through a bearing, and wave-shaped sheets distributed equidistantly are fixedly arranged on the outer wall of the circumference of the sleeve.
The invention also discloses a preparation method of the chlorella deep purification factor, which comprises the following steps:
s1, preparing raw materials: performing wall breaking treatment on the chlorella to obtain 50-60% of chlorella wall-broken chlorella powder;
s2, adding water for external circulation extraction, adding 5-6 times of water into chlorella wall-broken algae powder for external circulation extraction, wherein the extraction temperature is 70-90 ℃, the extraction time is 0.5-1h, judging an end point according to absorbance by monitoring an online spectrophotometer, and discharging an extract to obtain A1, wherein the specific range is that the absorbance value of 240nm wavelength is ten times diluted to be more than or equal to 0.9, and the absorbance of 550nm is less than or equal to 1.6;
s3, adding 2% glycerol solution for external circulation extraction, adding 3-4 times of water for external circulation extraction, wherein the extraction temperature is 70-90 ℃, the extraction time is 30-1.5h, specifically, monitoring by using an online spectrophotometer, judging an end point according to absorbance, wherein the specific range is that the absorbance value of 280nm wavelength is ten times diluted to be more than or equal to 1.5, the absorbance of 550nm is less than or equal to 10, and discharging extract liquor to obtain A2;
s4, adding 3% glycerol solution for external circulation extraction, discharging the extract, adding 2-3 times of water for external circulation extraction at 90-100 ℃ for 0.5-1.0h, specifically monitoring by using an online spectrophotometer, judging an end point according to absorbance, wherein the specific range is that the absorbance value of 280nm wavelength is ten times diluted to be more than or equal to 1.0, the absorbance of 550nm is less than or equal to 1.2, and discharging the extract to obtain A3;
s5, mixing the extract, and mixing A1, A2 and A3 by an extraction tank to prepare mixed extract;
s6, centrifuging at 10000rpm for 30min, and taking supernatant;
s7, performing ultrafiltration on the supernatant, namely passing the mixed extract through a 5000-10000 Dalton ultrafiltration membrane, circulating until 25-30% of the concentrated solution is left, supplementing 5% of glycerol solution with the concentration of 50-60% into the concentrated solution, and continuously performing ultrafiltration until the crude drug content is 15%;
s8, concentrating the crude drug, and concentrating with a nanofiltration membrane of 100-.
The technical scheme of the invention has the following beneficial technical effects:
1. the extraction process of the multistage external circulation extraction equipment for extracting the chlorella is initiated by adopting a gradient extraction method, the polar chlorella is extracted by a single medium proportion solvent due to the polarity difference of components to influence the extraction rate of the whole components, and the method can improve the extraction efficiency of the polar chlorella and reduce the use of the solvent and the dissolution of impurities;
2. the extraction process of the multistage external circulation extraction equipment for extracting the chlorella adopts a means of monitoring the dissolution of effective components and impurities in real time to control the extraction end point, can more accurately control the component stability of the product, and further control the efficacy stability of the product;
3. according to the multistage circulating extraction equipment for the chlorella deep purification factor, the extracted chlorella stress-resistant factor is applied to the fields of moisturizing, skin stratum corneum barrier enhancing and repairing of children cosmetics, and obvious synergistic effect can be generated under the matching condition;
4. this multistage circulation extraction equipment of deep purification factor of chlorella can utilize the pivot to rotate and drive bevel gear one and rotate and make bevel gear two rotate for the gangbar rotates, drive conveying screw rotates, guide extraction liquid enters into the reposition of redundant personnel bucket through drive tube and circulating pipe, carry for the repeated stirring of mixing rod through a plurality of reposition of redundant personnel pipes, realize that the extraction liquid from bottom to top in the extraction jar is being stirred, the horizontal stirring of cooperation mixing rod, realize the extraction liquid homogeneous mixing, improve the mixture degree of consistency of extraction liquid
5. The multistage circulating extraction equipment for the chlorella deep purification factors utilizes the arc ribs to drive the flow distribution barrel to rotate under the liquid feeding impact of the circulating pipe, so that the flow distribution operation is uniform; on the other hand, the arranged vibration spring can drive the shunting barrel to vibrate up and down in a small amplitude in practical use, so that the randomness and the uniformity of shunting are further improved, and the extraction effect of the device is favorably ensured;
6. according to the multistage circulating extraction equipment for the chlorella deep purification factors, the linear speeds of the water inlet hole and the water outlet hole are different when the mixing rod rotates in the rotating process of the mixing rod, so that the extraction liquid enters through the water inlet hole during stirring and flows out of the water outlet hole through the communication hole, the convection of the extraction liquid is realized, and the full mixing of the extraction liquid is accelerated; the sleeve pipe and the wavy sheet that set up can be at the rotatory in-process of compounding pole, under the effect of liquid, and self takes place to rotate, improve the mixed effect of material and extraction liquid, guarantee extraction efficiency.
Drawings
FIG. 1 is a flow chart of the extraction process of the multi-stage external circulation extraction equipment for plant extraction of the present invention.
Fig. 2 is a schematic diagram of the structure of the extraction tank of the present invention.
Fig. 3 is a schematic view of the shunt bucket structure of the present invention.
FIG. 4 is a schematic view of a semi-sectional structure of the split-flow barrel of the present invention
FIG. 5 is a schematic sectional view of a top view of the mixing rod of the present invention.
Fig. 6 is a schematic partial three-dimensional structure of the mixing rod of the present invention.
FIG. 7 is a diagram of single cell gel electrophoresis test (comet electrophoresis) -DNA damage repair according to the present invention.
Description of reference numerals:
1. an extraction tank; 2. a rotating shaft; 201. vibrating the spring; 202. an end piece; 3. a mixing rod; 301. a water inlet hole; 302. a water outlet hole; 303. a communicating hole; 4. a servo motor; 5. a transmission case; 6. a drive tube; 7. a circulation pipe; 8. a conveying screw; 9. a shunt barrel; 901. arc rib pieces; 10. a shunt tube; 11. a linkage rod; 12. a discharge pipe; 13. a flow-limiting plug; 14. a push rod motor; 15. A U-shaped rod; 16. a feed pipe; 17. a sleeve; 18. a corrugated sheet.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings in conjunction with the following detailed description. It should be understood that the description is intended to be exemplary only, and is not intended to limit the scope of the present invention. Moreover, in the following description, descriptions of well-known structures and techniques are omitted so as to not unnecessarily obscure the concepts of the present invention.
Referring to fig. 2 to 7, the present invention provides chlorella tianshan named as: the chlorella pyrenoidosa JY-QWZ-1900386, which is preserved in China general microbiological culture Collection center (CGMCC) in 2021 at 6 months, has the preservation number of CGMCC NO: 17926.
The invention also provides multistage circulating extraction equipment for the deep purification factors of the chlorella vulgaris and the chlorella vulgaris, which comprises an extraction tank 1, wherein a rotating shaft 2 is rotatably arranged in the extraction tank 1, a servo motor 4 for rotating the rotating shaft 2 is arranged at the top of the extraction tank 1, mixing rods 3 are arranged at two sides of the rotating shaft 2, a transmission case 5 is arranged on the inner wall of the bottom of the extraction tank 1, the bottom of the rotating shaft 2 is rotatably connected with the transmission case 5, a driving pipe 6 is connected with the bottom of the extraction tank 1, a conveying screw 8 is rotatably arranged in the driving pipe 6, the conveying screw 8 is in transmission connection with the rotating shaft 2, one end of the driving pipe 6 is connected with a circulating pipe 7, a shunting barrel 9 with an open top is movably connected on the rotating shaft 2, one end of the circulating pipe 7 extends into the extraction tank 1 and is positioned above the shunting barrel 9, a plurality of shunting pipes 10 which are uniformly distributed in an annular shape are arranged at the bottom of the shunting barrel 9, and the shunting pipes 10 are positioned above the mixing rods 3, an end plate 202 is fixedly installed at one end, close to the shunting barrel 9, of the rotating shaft 2, a vibration spring 201 is fixedly installed on the outer wall of the top of the end plate 202, the top end of the vibration spring 201 abuts against the bottom end of the shunting barrel 9, the inner bottom of the shunting barrel 9 is in an inclined plane shape, and arc rib plates 901 which are distributed equidistantly are fixedly installed at the inner bottom of the shunting barrel 9; the inner bottom of the shunting barrel 9 is inclined, so that shunting operation is convenient to carry out; the arranged arc rib pieces 901 can drive the shunting barrel 9 to rotate under the liquid feeding impact of the circulating pipe 7, so as to uniformly shunt; on the other hand, the vibrations spring 201 that sets up can drive the vibrations from top to bottom of reposition of redundant personnel bucket 9 small-amplitude when in actual use to further improve the randomness, the homogeneity of reposition of redundant personnel.
As shown in fig. 2, a feeding pipe 16 is arranged at the top of the extraction tank 1, a discharging pipe 12 is arranged at the bottom of the extraction tank 1, a flow-limiting plug 13 is arranged at the top of the discharging pipe 12, a push rod motor 14 is arranged at the bottom of the extraction tank 1, a push rod of the push rod motor 14 is in transmission connection with a U-shaped rod 15, and one end of the U-shaped rod 15 penetrates through the discharging pipe 12 and is connected with the flow-limiting plug 13; the U-shaped rod 15 is driven by the push rod motor 14 to be lifted and adjusted, so that the flow limiting plug 13 is lifted, the flow limiting plug 13 is separated from and contacted with the discharge pipe 12, the material is discharged, and the discharge is convenient.
As shown in fig. 2, the bottom of the rotating shaft 2 extends into the transmission case 5 and is connected with a first bevel gear in a transmission manner, one side of the transmission case 5 is rotatably provided with a linkage rod 11, one end of the linkage rod 11 is connected with the conveying screw 8 in a transmission manner, the other end of the linkage rod 11 extends into the transmission case 5 and is connected with a second bevel gear, and the first bevel gear is meshed with the second bevel gear; the rotating shaft 2 rotates to drive the bevel gear I to rotate so that the bevel gear II rotates, the linkage rod 11 rotates to drive the conveying screw rod 8 to rotate, one side of the mixing rod 3 is provided with a water inlet hole 301 and a water outlet hole 302 respectively, the mixing rod 3 is provided with a communication hole 303, two ends of the communication hole 303 are communicated with the water inlet hole 301 and the water outlet hole 302 respectively, the communication hole 303 is of a wave-shaped structure, the outer wall of the mixing rod 3 is rotatably connected with a sleeve 17 through a bearing, and the outer wall of the circumference of the sleeve 17 is fixedly provided with wave-shaped sheets 18 which are distributed at equal intervals; in the rotating process of the mixing rod 3, linear velocities of the water inlet hole 301 and the water outlet hole 302 are different when the mixing rod 3 rotates, so that extraction liquid enters through the water inlet hole 301 during stirring and flows out of the water outlet hole 302 through the communication hole 303, convection of the extraction liquid is realized, and the extraction liquid is accelerated to be fully mixed; the sleeve 17 and the wavy sheet 18 that set up can be at the rotatory in-process of compounding pole 3, under the effect of liquid, and self takes place to rotate, improves the mixed effect of material and extraction liquid, guarantees extraction efficiency.
The working principle is as follows: extract liquor is added into an extraction tank 1 from a feed pipe 16, a rotating shaft 2 is driven to rotate through a servo motor 4, a mixing rod 3 is driven to rotate, materials are transversely mixed, the rotating shaft 2 drives a bevel gear I to rotate, a bevel gear II rotates, a linkage rod 11 rotates, a conveying screw rod 8 is driven to rotate, the extract liquor is guided to enter a shunting barrel 9 through a driving pipe 6 and a circulating pipe 7 and is conveyed to the mixing rod 3 through a plurality of shunting pipes 10 to be repeatedly mixed, the extract liquor in the extraction tank 1 is conveyed from bottom to top to be mixed, the extract liquor is uniformly mixed by matching with transverse mixing of the mixing rod 3, and the mixing uniformity of the extract liquor is improved; after the materials and the extraction liquid are fully mixed, the mixture is kept stand for a period of time, after the liquid in the extraction tank 1 is layered, an operator drives the U-shaped rod 15 to rise through the push rod motor 14, so that the flow limiting plug 13 rises, the flow limiting plug 13 is separated from the discharge pipe 12, and the layered liquid at the bottom end is discharged.
The invention also discloses a preparation method of the chlorella deep purification factor, which comprises the following steps:
example one
As shown in fig. 1, performing wall breaking treatment on the chlorella to obtain 50-60% of chlorella wall-broken chlorella powder, specifically, performing wall breaking treatment, performing cell disruption by using a grinder, adding 6 times of water for external circulation extraction, performing external circulation extraction at the extraction temperature of 70 ℃, monitoring and judging an end point according to an online spectrophotometer, diluting the specific range of absorbance value of 240nm in ten times of the absorbance value of not less than 0.9,550nm to obtain the absorbance of not more than 1.3, and discharging an extract to obtain A1; adding 3 times of 2% glycerol solution into algae residue, performing external circulation extraction at 80 deg.C, diluting with absorbance value of 280nm at ten times of that of 1.2,550nm at the extraction end point to obtain absorbance value of 1.4, and discharging the extractive solution to obtain A2; adding 2 times of 5% glycerol solution into algae residue, performing external circulation extraction at 80 deg.C, diluting with absorbance value of 280nm at ten times of that of 1.0,550nm at the extraction end point to obtain absorbance value of 1.2, and discharging the extractive solution to obtain A3. Mixing A1, A2 and A3, filtering with 10000 Dalton ultrafiltration membrane, circulating to concentrate with 25-30% of the concentrate, adding 3% glycerol solution with concentration of 50%, continuously ultrafiltering to crude drug content of 15%, and concentrating with 100 Dalton nanofiltration membrane to 100% crude drug to obtain Chlorella deep purification factor.
The total sugar content of the obtained chlorella deep purification factor is 1.05%, the stability of an accelerated test is 3 months, the repair capacity of 1% of the chlorella deep purification factor on DNA damage of fibroblasts induced by UVB reaches 80%, the phagocytosis of macrophages is increased to 188.2% by 0.01% of the chlorella deep purification factor, and a 3D skin efficacy evaluation experiment is shown in Table 1:
TABLE 1
Example two
As shown in figure 1, performing wall breaking treatment on the chlorella to obtain 50-60% of chlorella wall-broken chlorella powder, specifically, performing cell disruption by using a grinder, adding 5 times of water for external circulation extraction, performing external circulation extraction at the extraction temperature of 80 ℃, monitoring and judging an end point according to an online spectrophotometer, wherein the specific range is that the absorbance value of 240nm wavelength is diluted ten times and is not less than 1.3,550nm absorbance and is not more than 1.6, and discharging extract liquid to obtain A1; adding 3 times of 3% glycerol solution into the residue, performing external circulation extraction at 70 deg.C, diluting with absorbance value of 280nm at ten times of that of 1.2,550nm at the end point of extraction to obtain absorbance value of less than or equal to 1.3, and discharging the extractive solution to obtain A2; adding 2 times of 5% glycerol solution into the residue, performing external circulation extraction at 850 deg.C, diluting with absorbance value of 280nm at ten times of that of 1.0,550nm at the end point of extraction to obtain absorbance value of less than 1.2, and discharging the extractive solution to obtain A3. And (3) combining the A1, the A2 and the A3, mixing, passing through a 5000 dalton ultrafiltration membrane, circulating until 25-30% of the concentrated solution is remained, supplementing 2% of glycerol solution into the concentrated solution, keeping the concentration at 60%, continuously performing ultrafiltration until the content of the crude drug is 15%, and concentrating by using a 1000 dalton nanofiltration membrane to 100% of the crude drug to obtain the chlorella deep purification factor.
The total sugar content of the obtained chlorella deep purification factor is 1.1%, the stability of an accelerated test is 3 months, the repair capacity of 1% of the chlorella deep purification factor on DNA damage of fibroblasts induced by UVB reaches 83%, the phagocytosis of macrophages is increased to 206.9% by 0.01% of the chlorella deep purification factor, and a 3D skin efficacy evaluation experiment is shown in Table 2:
TABLE 2
EXAMPLE III
As shown in figure 1, performing wall breaking treatment on the chlorella to obtain 50-60% of chlorella wall-broken chlorella powder, performing cell disruption by using a grinder, adding 5 times of water for external circulation extraction, performing external circulation extraction at 90 ℃, judging an end point according to monitoring of an online spectrophotometer, and discharging an extract to obtain A1, wherein the specific range is that the absorbance value of 240nm wavelength is ten times diluted to be not less than 1.3,550nm and the absorbance is not more than 1.5; adding 4 times of 4% glycerol into the algae residue for external circulation extraction, diluting with absorbance value of 280nm at 80 deg.C to obtain absorbance value of 1.5,550nm or more and less than 1.4, and discharging the extractive solution to obtain A2; adding 3 times of 5% glycerol solution into algae residue, performing external circulation extraction at 95 deg.C, diluting with absorbance value of 280nm at ten times of that of 1.0,550nm at the end point of extraction to obtain absorbance value of 1.2, and discharging the extractive solution to obtain A3. And (3) mixing the A1, the A2 and the A3, filtering the mixture through an ultrafiltration membrane of 8000 dalton after mixing, circulating until 25-30% of the concentrated solution is remained, supplementing a glycerol solution of 3% into the concentrated solution, keeping the concentration at 550%, continuously performing ultrafiltration until the content of the crude drug is 15%, and concentrating the crude drug to 100% by using a nanofiltration membrane of 500 dalton to obtain the chlorella deep purification factor.
The total sugar content of the obtained chlorella deep purification factor is 1.09%, the stability of an accelerated test is 3 months, the repair capacity of 1% of the chlorella deep purification factor on DNA damage of fibroblasts induced by UVB reaches 79%, the phagocytosis of macrophages is increased to 178.4% by 0.01% of the chlorella deep purification factor, and a 3D skin efficacy evaluation experiment is shown in Table 3:
TABLE 3
The detection method of the active ingredients in each example is as follows:
total sugar: phenol-sulfuric acid process;
single cell gel electrophoresis assay (comet electrophoresis) -DNA Damage repair, see FIG. 7
The test method comprises the steps of embedding processed cells in agarose, paving the agarose on a glass slide, immersing the agarose in cold cracking liquid after solidification to crack the cells, horizontally performing electrophoresis after unwinding in an electrophoresis liquid, neutralizing a buffer solution, drying the neutralized buffer solution, dyeing the cells by using EB (electron beam), observing a fluorescence image of the cells on the glass slide under a fluorescence microscope, and evaluating the damage degree of DNA.
Effect on phagocytic Activity of macrophage Ana-1
And taking cells in logarithmic phase, blowing and beating the cells to prepare 2 x 105/mL single cell suspension, inoculating 100 mu L of the single cell suspension into a 96-well cell plate, removing culture supernatant after the cells are attached to the wall, setting a control group and an addition medicine group, adding 20 mu L of freshly prepared 0.1% NR and PM2.5 suspension into each well after culturing for 24, 48 and 72 hours respectively, and continuing culturing for 3 hours after gently mixing the suspension uniformly. The NR-containing medium was then carefully aspirated, and the non-phagocytosed NR was gently washed off with physiological PBS. mu.L of cell lysate (absolute ethanol: acetic acid: 1(v/v)) was added to each well, and the mixture was allowed to stand for 2 hours to lyse the cells, shaken, and mixed well, and then OD at 540nm was measured [90 ]. The phagocytosis of NR by cells is as follows:
phagocytosis rate was 100% for dosing/control.
3D skin efficacy evaluation experiment: 3 pieces of 3D skin were immuno-labeled.
A. Culturing human 3D skin directly for 5 days; B. treating with heavy metal complex solution (water solution with lead-mercury ratio of 1:2 and 0.1%), and culturing human 3D skin for 5 days; C. uniformly smearing chlorella deep purification factor on 3D skin, treating with heavy metal complex solution (water solution with lead-mercury ratio of 1:2, 0.1%), and culturing 3D skin for 5 days
B. Human 3D skin thickness was measured after 5 days using Leica's Q500 imaging analysis software for three cases A/B/C;
C. detecting the expression level of the silk fibroin serving as a marker.
1. The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and the preferred embodiments of the present invention are described in the above embodiments and the description, and are not intended to limit the present invention.
Claims (10)
1. A chlorella tianshanensis named as: the chlorella pyrenoidosa JY-QWZ-1900386, which is preserved in China general microbiological culture Collection center (CGMCC) 6 months in 2021, has the preservation number of CGMCC No. 22396.
2. The multistage circulation extraction equipment of the chlorella deep purification factor, according to claim 1, comprising an extraction tank (1), wherein a rotating shaft (2) is rotatably installed in the extraction tank (1), a servo motor (4) for rotating the rotating shaft (2) is arranged at the top of the extraction tank (1), mixing rods (3) are arranged at two sides of the rotating shaft (2), a transmission box (5) is arranged on the inner wall of the bottom of the extraction tank (1), the bottom of the rotating shaft (2) is rotatably connected with the transmission box (5), a driving pipe (6) is connected at the bottom of the extraction tank (1), a conveying screw (8) is rotatably installed in the driving pipe (6), the conveying screw (8) is in transmission connection with the rotating shaft (2), a circulating pipe (7) is connected at one end of the driving pipe (6), a shunt barrel (9) with an open top is movably connected on the rotating shaft (2), the one end of circulating pipe (7) extends to in extraction jar (1) and is located the top of reposition of redundant personnel bucket (9), and the bottom of reposition of redundant personnel bucket (9) is provided with a plurality of shunt tubes (10) that are annular evenly distributed, and shunt tubes (10) are located the top of mixing rod (3), pivot (2) are close to the one end fixed mounting of reposition of redundant personnel bucket (9) and have end piece (202), and the top outer wall fixed mounting of end piece (202) has vibrations spring (201), the top of vibrations spring (201) offsets with the bottom of reposition of redundant personnel bucket (9), the interior bottom of reposition of redundant personnel bucket (9) is the inclined plane form, and the interior bottom fixed mounting of reposition of redundant personnel bucket (9) has arc muscle piece (901) that the equidistance distributes.
3. The multistage circulation extraction equipment for the chlorella deep purification factor according to claim 2, wherein a feeding pipe (16) is arranged at the top of the extraction tank (1), a discharging pipe (12) is arranged at the bottom of the extraction tank (1), a flow limiting plug (13) is arranged at the top of the discharging pipe (12), a push rod motor (14) is arranged at the bottom of the extraction tank (1), a U-shaped rod (15) is connected to a push rod of the push rod motor (14) in a transmission manner, and one end of the U-shaped rod (15) penetrates through the discharging pipe (12) and is connected with the flow limiting plug (13).
4. The multistage circulation extraction equipment for the chlorella deep purification factor as claimed in claim 2, wherein the bottom of the rotating shaft (2) extends into the transmission case (5) and is in transmission connection with a first bevel gear, one side of the transmission case (5) is rotatably provided with a linkage rod (11), one end of the linkage rod (11) is in transmission connection with the conveying screw (8), the other end of the linkage rod (11) extends into the transmission case (5) and is connected with a second bevel gear, the first bevel gear is meshed with the second bevel gear, one side of the mixing rod (3) is provided with a water inlet hole (301) and a water outlet hole (302) respectively, the mixing rod (3) is provided with a communication hole (303), two ends of the communication hole (303) are respectively communicated with the water inlet hole (301) and the water outlet hole (302), the communication hole (303) is in a wave-shaped structure, the outer wall of the mixing rod (3) is rotatably connected with a sleeve (17) through a bearing, and the circumferential outer wall of the sleeve (17) is fixedly provided with wavelike sheets (18) which are distributed at equal intervals.
5. A preparation method of chlorella deep purification factor is characterized by comprising the following steps:
s1, preparing raw materials: performing wall breaking treatment on the chlorella of claim 1 to obtain 50-60% of chlorella wall-broken powder;
s2, adding water for external circulation extraction, adding 5-6 times of water into chlorella wall-broken algae powder for external circulation extraction, wherein the extraction temperature is 70-90 ℃, the extraction time is 0.5-1h, judging an end point according to absorbance by monitoring an online spectrophotometer, and discharging an extract to obtain A1, wherein the specific range is that the absorbance value of 240nm wavelength is ten times diluted to be more than or equal to 0.9, and the absorbance of 550nm is less than or equal to 1.6;
s3, adding 2% glycerol solution for external circulation extraction, adding 3-4 times of water for external circulation extraction, wherein the extraction temperature is 70-90 ℃, the extraction time is 30-1.5h, specifically, monitoring by using an online spectrophotometer, judging an end point according to absorbance, wherein the specific range is that the absorbance value of 280nm wavelength is ten times diluted to be more than or equal to 1.5, the absorbance of 550nm is less than or equal to 10, and discharging extract liquor to obtain A2;
s4, adding 3% glycerol solution for external circulation extraction, discharging the extract, adding 2-3 times of water for external circulation extraction at 90-100 ℃ for 0.5-1.0h, specifically monitoring by using an online spectrophotometer, judging an end point according to absorbance, wherein the specific range is that the absorbance value of 280nm wavelength is ten times diluted to be more than or equal to 1.0, the absorbance of 550nm is less than or equal to 1.2, and discharging the extract to obtain A3;
s5, mixing the extract, and mixing A1, A2 and A3 by an extraction tank to prepare mixed extract;
s6, centrifuging at 10000rpm for 30min, and taking supernatant;
s7, performing ultrafiltration on the supernatant, namely passing the mixed extract through a 5000-10000 Dalton ultrafiltration membrane, circulating until 25-30% of the concentrated solution is left, supplementing 5% of glycerol solution with the concentration of 50-60% into the concentrated solution, and continuously performing ultrafiltration until the crude drug content is 15%;
s8, concentrating the crude drug, and concentrating with a nanofiltration membrane of 100-.
6. The method for preparing a chlorella deep purification factor according to claim 5, wherein in step S1, raw materials are prepared: performing wall breaking treatment on the chlorella as the claim 1 to obtain 40 to 50 percent of chlorella wall-broken powder.
7. The method for preparing a chlorella deep purification factor according to claim 5, wherein in step S1, raw materials are prepared: performing wall breaking treatment on the chlorella of claim 1 to obtain 45% of chlorella wall-broken powder.
8. The method for preparing a chlorella deep purification factor according to claim 5, wherein in step S2, the chlorella is ground by a grinder, the grinder being any one of a universal grinder and a jaw grinder.
9. The method as claimed in claim 5, wherein the step S7 comprises ultrafiltering the mixed extractive solution with 6000-9000 Dalton ultrafiltering membrane, circulating to the residual 25-30% of the concentrated solution, adding 5% glycerol solution with concentration of 52-58%, and continuously ultrafiltering to reach crude drug content of 15%.
10. The method for preparing chlorella deep purification factor as claimed in claim 5, wherein step S7, ultrafiltration is carried out, the mixed extractive solution is passed through 8000 Dalton ultrafiltration membrane, and circulated to the concentration of 30%, and 5% glycerol solution is added into the concentrated solution, the concentration is 55%, and ultrafiltration is continued until crude drug content reaches 15%.
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