CN117017895A - Preparation method of blackcurrant stem cell extract - Google Patents
Preparation method of blackcurrant stem cell extract Download PDFInfo
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- CN117017895A CN117017895A CN202311012701.2A CN202311012701A CN117017895A CN 117017895 A CN117017895 A CN 117017895A CN 202311012701 A CN202311012701 A CN 202311012701A CN 117017895 A CN117017895 A CN 117017895A
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- 240000001890 Ribes hudsonianum Species 0.000 title claims abstract description 113
- 235000016954 Ribes hudsonianum Nutrition 0.000 title claims abstract description 103
- 235000001466 Ribes nigrum Nutrition 0.000 title claims abstract description 103
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 95
- 238000002360 preparation method Methods 0.000 title abstract description 14
- 238000012258 culturing Methods 0.000 claims abstract description 40
- 210000004027 cell Anatomy 0.000 claims abstract description 33
- 239000006285 cell suspension Substances 0.000 claims abstract description 24
- 239000012879 subculture medium Substances 0.000 claims abstract description 23
- 238000004108 freeze drying Methods 0.000 claims abstract description 6
- 239000001963 growth medium Substances 0.000 claims description 34
- 239000000706 filtrate Substances 0.000 claims description 20
- ZNJFBWYDHIGLCU-HWKXXFMVSA-N jasmonic acid Chemical compound CC\C=C/C[C@@H]1[C@@H](CC(O)=O)CCC1=O ZNJFBWYDHIGLCU-HWKXXFMVSA-N 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 20
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 claims description 20
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 claims description 16
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 12
- 229930006000 Sucrose Natural products 0.000 claims description 12
- 239000005720 sucrose Substances 0.000 claims description 12
- 229940088594 vitamin Drugs 0.000 claims description 12
- 229930003231 vitamin Natural products 0.000 claims description 12
- 235000013343 vitamin Nutrition 0.000 claims description 12
- 239000011782 vitamin Substances 0.000 claims description 12
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 claims description 11
- 235000019393 L-cystine Nutrition 0.000 claims description 11
- 239000004158 L-cystine Substances 0.000 claims description 11
- 229960003067 cystine Drugs 0.000 claims description 11
- ZNJFBWYDHIGLCU-UHFFFAOYSA-N jasmonic acid Natural products CCC=CCC1C(CC(O)=O)CCC1=O ZNJFBWYDHIGLCU-UHFFFAOYSA-N 0.000 claims description 10
- 239000002048 multi walled nanotube Substances 0.000 claims description 10
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 claims description 10
- 229960004889 salicylic acid Drugs 0.000 claims description 10
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 claims description 8
- HXKWSTRRCHTUEC-UHFFFAOYSA-N 2,4-Dichlorophenoxyaceticacid Chemical compound OC(=O)C(Cl)OC1=CC=C(Cl)C=C1 HXKWSTRRCHTUEC-UHFFFAOYSA-N 0.000 claims description 8
- 229920001817 Agar Polymers 0.000 claims description 8
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims description 8
- 239000008272 agar Substances 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 8
- 150000003839 salts Chemical class 0.000 claims description 8
- 238000007710 freezing Methods 0.000 claims description 7
- 230000008014 freezing Effects 0.000 claims description 7
- 238000000108 ultra-filtration Methods 0.000 claims description 7
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 6
- 150000001720 carbohydrates Chemical class 0.000 claims description 6
- KRVSOGSZCMJSLX-UHFFFAOYSA-L chromic acid Substances O[Cr](O)(=O)=O KRVSOGSZCMJSLX-UHFFFAOYSA-L 0.000 claims description 6
- AWJWCTOOIBYHON-UHFFFAOYSA-N furo[3,4-b]pyrazine-5,7-dione Chemical compound C1=CN=C2C(=O)OC(=O)C2=N1 AWJWCTOOIBYHON-UHFFFAOYSA-N 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- 238000010008 shearing Methods 0.000 claims description 6
- 238000002791 soaking Methods 0.000 claims description 6
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 6
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- 230000001954 sterilising effect Effects 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000005286 illumination Methods 0.000 claims description 5
- 229930091371 Fructose Natural products 0.000 claims description 4
- 239000005715 Fructose Substances 0.000 claims description 4
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 150000001413 amino acids Chemical class 0.000 claims description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims description 2
- 238000003287 bathing Methods 0.000 claims 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 abstract description 11
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- 239000004480 active ingredient Substances 0.000 abstract description 2
- 229910017053 inorganic salt Inorganic materials 0.000 description 15
- 239000002537 cosmetic Substances 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 239000006870 ms-medium Substances 0.000 description 4
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- 206010020649 Hyperkeratosis Diseases 0.000 description 1
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- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000019990 fruit wine Nutrition 0.000 description 1
- 235000015094 jam Nutrition 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
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- 239000007787 solid Substances 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
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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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
-
- 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
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
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- 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
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- 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 belongs to the field of biotechnology. The invention relates to a preparation method of a blackcurrant stem cell extract, which comprises the following steps: i: a step of harvesting cambium-containing tissue from a blackcurrant plant; ii: a step of culturing the harvested cambium-containing tissue in a medium; iii: a step of isolating cells from the cambium, thereby harvesting cambium-derived blackcurrant stem cells; iv: placing the blackcurrant stem cells in a cell subculture medium, and culturing to obtain a blackcurrant stem cell suspension; v: performing an amplification culture step on the blackcurrant stem cell suspension; vi: and crushing, concentrating and freeze-drying the obtained amplified blackcurrant stem cells. The blackcurrant stem cell extract obtained by the preparation method has high active ingredients, particularly improves the content of phenolic substances in the blackcurrants obviously, and has obvious economic value and use value.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method of a blackcurrant stem cell extract.
Background
At present, the research on the blackcurrant deep-processed products is not much, most of the products are primary processing, the productivity is very small, and the research is relatively weak. Mainly focuses on blackcurrant fruit wine, fruit vinegar, jam, jelly, beverage and the like.
Disclosure of Invention
In view of the above, the invention provides a method for extracting and separating blackcurrant stem cells with higher physiological activity from stem tip tissues of blackcurrant plants, and adding the blackcurrant stem cells into a repair essence product for application after amplification culture, crushing, concentration and freeze-drying.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a method for preparing a blackcurrant stem cell extract, comprising the steps of:
i: a step of harvesting cambium-containing tissue from a blackcurrant plant;
ii: a step of culturing the harvested cambium-containing tissue in a medium;
iii: a step of isolating cells from the cambium, thereby harvesting cambium-derived blackcurrant stem cells;
iv: placing the blackcurrant stem cells in a cell subculture medium, and culturing to obtain a blackcurrant stem cell suspension;
v: performing an amplification culture step on the blackcurrant stem cell suspension;
vi: and crushing, concentrating and freeze-drying the obtained amplified blackcurrant stem cells.
Further, the step of harvesting cambium-containing tissue from a blackcurrant plant as described above is specifically performed as:
the method comprises the steps of cutting off delignified tender stems from tender branches of annual blackcurrant plants to serve as explants, and cutting off all leaves on the tender stems;
soaking the explant in the step of preparing the implant with 75% alcohol for 5min, sterilizing with 30% sodium hypochlorite for 5min, washing with sterile water for 3 times, shearing into stem tips with the length of 0.2-0.5cm with sterile scissors, and stripping from the stem tips to obtain cambium tissues.
Further, the step of culturing the harvested cambium-containing tissue in a culture medium comprises the following specific operation method:
third, the cambium tissue obtained in the step II is inoculated on a culture medium for culture.
Further, the culture medium comprises inorganic salts, vitamins, agar and sucrose; wherein the inorganic salt comprises: caCl (CaCl) 2 、KH 2 PO 4 、KNO 3 、MgSO 4 、NH 4 NO 3 、CuSO 4 ·5H 2 O, etc., preferably, the medium is MS medium; and the concentration of agar is 5g/L, and the concentration of sucrose is 30g/L; more preferably, the culture medium further comprises biochar, 6-benzylaminopurine, 3-indoleacetic acid and L-cystine; wherein, the concentration of 6-benzyl amino purine is 0.8mg/L, the concentration of 3-indoleacetic acid is 0.2mg/L, the concentration of biochar is 1g/L, and the concentration of L-cystine is 5mg/L.
Further, the culture conditions of the harvested cambium-containing tissue in the culture medium are: culturing at 23-25deg.C under illumination of 1500LX-2500LX and pH of 5.2-6.2 for 14-20d.
Further, the step of isolating cells from the cambium, thereby harvesting cambium-derived blackcurrant stem cells, specifically:
transferring the upper culture medium and the cell clusters to a sterilized empty triangular flask, adding 5% -8% chromic acid solution, controlling the rotation speed to 110rpm, oscillating for 5-8h, and separating to obtain the blackcurrant stem cells.
Further, placing the blackcurrant stem cells in a cell subculture medium, and culturing to obtain a blackcurrant stem cell suspension; the specific operation method comprises the following steps:
and fifthly, placing the blackcurrant stem cells obtained in the step in a cell subculture medium, culturing in a dark constant-temperature culture table at 23-27 ℃ and at the rotating speed of 120rpm, standing the blackcurrant stem cell suspension for 24 hours every 3 days, continuously oscillating and culturing the supernatant of the subculture medium, and circulating for 4 times to obtain the blackcurrant stem cell suspension.
Further, the cell subculture medium includes: inorganic salts, vitamins, amino acids, and saccharides, wherein the inorganic salts include: caCl (CaCl) 2 、KH 2 PO 4 、KNO 3 、MgSO 4 、NH 4 NO 3 、CuSO 4 ·5H 2 O, etc., and the saccharide is any one of sucrose, glucose or fructose; preferably, the medium is preferably MS medium, more preferably, the medium further comprises: multiwall carbon nanotubes, salicylic acid, jasmonic acid, 2, 4-dichlorophenoxyacetic acid; wherein the concentration of the multi-wall carbon nano tube is 150mg/L, the concentration of salicylic acid is 75umonl/L, the concentration of jasmonic acid is 75umonl/L, and the concentration of 2, 4-dichlorophenoxyacetic acid is 2umonl/L.
Further, the step of amplifying and culturing the blackcurrant stem cell suspension is specifically as follows:
transferring the blackcurrant stem cell suspension obtained in the step (II) to a bioreactor for culturing, culturing for 4-7d in the bioreactor, adding fresh cell subculture medium, and continuously culturing for 12d to obtain the amplified blackcurrant stem cells.
Further, the steps of crushing, concentrating and freeze-drying the amplified blackcurrant stem cells are as follows:
crushing the amplified blackcurrant stem cells obtained in the step (II) by using an ultrasonic crusher, filtering by using an ultrafiltration membrane, concentrating filtrate in a rotary vacuum evaporator, pre-freezing the concentrated filtrate in a refrigerator at-80 ℃ for 6 hours, transferring the pre-frozen filtrate into a vacuum freeze-drying device at-50 ℃ under the condition of 20Pa of vacuum degree for 48 hours to obtain the blackcurrant stem cell extract.
Further, the invention protects the blackcurrant stem cell extract obtained by the preparation method of the blackcurrant stem cell extract.
Further, the invention protects the application of the blackcurrant stem cell extract in preparing cosmetics.
Furthermore, the invention protects the application of the blackcurrant stem cell extract in preparing repairing essence in the field of cosmetics.
According to the technical scheme, the invention discloses a preparation method of the blackcurrant stem cell extract, and compared with the prior art, the blackcurrant stem cell extract obtained by the preparation method has high active ingredients, particularly the content of phenolic substances in blackcurrants is obviously improved, and the preparation method has obvious economic value and use value.
The cell subculture medium adopted by the invention is an MS solid medium containing salicylic acid, jasmonic acid, multi-wall carbon nanotubes and the like. MS culture medium is a common plant culture medium which can be used for inducing callus and can also be used for culturing embryo, stem segment, stem tip and anther. The salicylic acid, the jasmonic acid and the multiwall carbon nanotubes are added as the inducing molecules to have obvious promotion effect on the increase of the content of phenolic substances in blackcurrants.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The preparation method of the blackcurrant stem cell extract provided by the invention comprises the following steps:
the method comprises the steps of cutting off delignified tender stems from tender branches of annual blackcurrant plants to serve as explants, and cutting off all leaves on the tender stems;
soaking the explant in the step of preparing the implant with 75% alcohol for 5min, sterilizing with 30% sodium hypochlorite for 5min, washing with sterile water for 3 times, shearing into stem tips with the length of 0.2-0.5cm with sterile scissors, and stripping from the stem tips to obtain cambium tissues.
Inoculating the cambium tissue obtained in the step to a culture medium for culturing at the temperature of 23-25 ℃ and the illumination of 1500LX-2500LX, wherein the pH value is 5.2-6.2, and culturing for 14-20d; wherein the culture medium comprises inorganic salt, vitamins, agar and sucrose; wherein the inorganic salt comprises: the inorganic salts include: caCl (CaCl) 2 、KH 2 PO 4 、KNO 3 、MgSO 4 、NH 4 NO 3 、CuSO 4 ·5H 2 O, etc., preferably, the medium is MS medium; the concentration of agar is 5g/L, and the concentration of sucrose is 30g/L; more preferably, in the mediumAlso comprises biochar, 6-benzylaminopurine, 3-indoleacetic acid and L-cystine; wherein, the concentration of 6-benzyl amino purine is 0.8mg/L, the concentration of 3-indoleacetic acid is 0.2mg/L, the concentration of biochar is 1g/L, and the concentration of L-cystine is 5mg/L.
Transferring the upper culture medium and cell clusters to a sterilized empty triangular flask, adding 5% -8% chromic acid solution, controlling the rotation speed to 110rpm, carrying out oscillation treatment for 5-8h, and separating to obtain blackcurrant stem cells;
fifthly, placing the blackcurrant stem cells obtained in the step in a cell subculture medium, culturing in a dark constant-temperature culture table at 23-27 ℃ and a rotating speed of 120rpm, standing the blackcurrant stem cell suspension for 24 hours every 3 days, continuously oscillating and culturing the supernatant of the subculture medium for 4 times, and obtaining a stable blackcurrant stem cell suspension, wherein the cell subculture medium comprises the following components: inorganic salts, vitamins, amino acids, and saccharides, wherein the inorganic salts include: caCl (CaCl) 2 、KH 2 PO 4 、KNO 3 、MgSO 4 、NH 4 NO 3 、CuSO 4 ·5H 2 O, etc., and the saccharide is any one of sucrose, glucose or fructose; preferably, the medium is preferably MS medium, more preferably, the medium further comprises: multiwall carbon nanotubes, salicylic acid, jasmonic acid, 2, 4-dichlorophenoxyacetic acid; wherein the concentration of the multi-wall carbon nano tube is 150mg/L, the concentration of salicylic acid is 75umonl/L, the concentration of jasmonic acid is 75umonl/L, and the concentration of 2, 4-dichlorophenoxyacetic acid is 2umonl/L;
transferring the blackcurrant stem cell suspension obtained in the step (II) to a bioreactor for culturing for 4-7d, adding a fresh cell subculture medium, and continuously culturing for 12d to obtain amplified blackcurrant stem cells;
crushing the amplified blackcurrant stem cells obtained in the step (II) by using an ultrasonic crusher, filtering by using an ultrafiltration membrane, concentrating filtrate in a rotary vacuum evaporator, pre-freezing the concentrated filtrate in a refrigerator at-80 ℃ for 6 hours, transferring the pre-frozen filtrate into a vacuum freeze-drying device at-50 ℃ under the condition of 20Pa of vacuum degree for 48 hours to obtain the blackcurrant stem cell extract.
The invention further provides application of the blackcurrant stem cell extract in preparing cosmetics.
The invention further provides application of the blackcurrant stem cell extract in preparing repairing essence in the field of cosmetics.
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
The embodiment discloses a preparation method of a blackcurrant stem cell extract, which comprises the following steps:
the method comprises the steps of cutting off delignified tender stems from tender branches of annual blackcurrant plants to serve as explants, and cutting off all leaves on the tender stems;
soaking the explant in the step of preparing the explant with 75% alcohol for 5min, sterilizing with 30% sodium hypochlorite for 5min, washing with sterile water for 3 times, shearing the explant into stem tips with the length of 0.2-0.5cm with sterile scissors, and stripping out tissues containing cambium with an inoculating knife;
third step obtaining cambium tissues per 5cm 2 Inoculating 1g of tissue at a density on a culture medium for culturing at a temperature of 23 ℃ and an illuminance of 1500LX and a pH value of 5.2-6.2 for 14 days; wherein, the culture medium is based on an MS culture medium, and then inorganic salt, vitamin C60 mg/L, 6-benzylaminopurine 0.8mg/L, 3-indoleacetic acid 0.2mg/L, biochar 1g/L, L-cystine 5mg/L, agar 5g/L and sucrose 30g/L are added; wherein the inorganic salt comprises: 370mg/LCaCl 2 、176mg/L KH 2 PO 4 、1650mg/L KNO 3 、169.25mg/L MgSO 4 、1655mg/LNH 4 NO 3 、0.021mg/L CuSO 4 ·5H 2 O；
Transferring the upper culture medium and cell clusters to a sterilized empty triangular flask, adding 5% chromic acid solution, controlling the rotation speed to 110rpm, oscillating for 5 hours, and separating to obtain blackcurrant stem cells;
fifthly, carrying out the step (IV) on the blackcurrantPlacing stem cells in a cell subculture medium, culturing in a dark constant-temperature culture shaking table at 23 ℃ and 120rpm for 3 days at intervals, standing the blackcurrant stem cell suspension for 24 hours, taking supernatant of the subculture medium, continuously shaking and culturing, and circulating for 4 times to obtain a stable blackcurrant stem cell suspension; wherein the culture medium is based on an MS culture medium, and then inorganic salt, vitamin C60 mg/L, L-cystine 5mg/L and glucose 30g/L, multi-wall carbon nano tube 150mg/L, salicylic acid 75umonl/L, jasmonic acid 75umonl/L and 2, 4-dichlorophenoxyacetic acid 2umonl/L are added; wherein the inorganic salt comprises: 375mg/LCaCl 2 、188mg/L KH 2 PO 4 、1810mg/LKNO 3 、176.28mg/L MgSO 4 、1860mg/LNH 4 NO 3 、0.022mg/L CuSO 4 ·5H 2 O；
Transferring the blackcurrant stem cell suspension obtained in the step (II) to a bioreactor for culturing for 4d, and adding a fresh cell subculture medium until the cell density is 1×10 7 Continuously culturing for 12d at a volume of one ml to obtain amplified blackcurrant stem cells;
crushing the amplified blackcurrant stem cells obtained in the step (II) by using an ultrasonic crusher, filtering by using an ultrafiltration membrane, concentrating filtrate in a rotary vacuum evaporator, pre-freezing the concentrated filtrate in a refrigerator at-80 ℃ for 6 hours, transferring the pre-frozen filtrate into a vacuum freeze-drying device at-50 ℃ under the condition of 20Pa of vacuum degree for 48 hours to obtain the blackcurrant stem cell extract. The total phenol content of the blackcurrant stem cell extract was determined to be 51.47mg/mL by HPLC.
Example 2
The embodiment discloses a preparation method of a blackcurrant stem cell extract, which comprises the following steps:
the method comprises the steps of cutting off delignified tender stems from tender branches of annual blackcurrant plants to serve as explants, and cutting off all leaves on the tender stems;
soaking the explant in the step of preparing the explant with 75% alcohol for 5min, sterilizing with 30% sodium hypochlorite for 5min, washing with sterile water for 3 times, shearing the explant into stem tips with the length of 0.2-0.5cm with sterile scissors, and stripping out tissues containing cambium with an inoculating knife;
third step obtaining cambium tissues per 5cm 2 Inoculating 1g of tissue with density, culturing on a culture medium at 25deg.C under illumination 2500LX at pH of 5.2-6.2 for 20d; wherein, the culture medium is based on an MS culture medium, and then inorganic salt, vitamin C60 mg/L, 6-benzylaminopurine 0.8mg/L, 3-indoleacetic acid 0.2mg/L, biochar 1g/L, L-cystine 5mg/L, agar 5g/L and sucrose 30g/L are added; wherein the inorganic salt comprises: 370mg/LCaCl 2 、176mg/L KH 2 PO 4 、1650mg/L KNO 3 、169.25mg/L MgSO 4 、1655mg/LNH 4 NO 3 、0.021mg/L CuSO 4 ·5H 2 O；
Transferring the upper culture medium and cell clusters together into a sterilized empty triangular flask, adding 8% chromic acid solution, controlling the rotation speed to 110rpm, oscillating for 8 hours, and separating to obtain blackcurrant stem cells;
fifthly, placing the blackcurrant stem cells obtained in the step in a cell subculture medium, culturing in a dark constant-temperature culture table at 27 ℃ and a rotating speed of 120rpm, standing the blackcurrant stem cell suspension for 24 hours every 3 days, and continuously oscillating and culturing the supernatant of the subculture medium for 4 times to obtain a stable blackcurrant stem cell suspension; wherein the culture medium is based on an MS culture medium, and then inorganic salt, vitamin C60 mg/L, L-cystine 5mg/L and fructose 30g/L, multi-wall carbon nano tube 150mg/L, salicylic acid 75umonl/L, jasmonic acid 75umonl/L and 2, 4-dichlorophenoxyacetic acid 2umonl/L are added; wherein the inorganic salt comprises: 375mg/LCaCl 2 、188mg/L KH 2 PO 4 、1810mg/LKNO 3 、176.28mg/L MgSO 4 、1860mg/LNH 4 NO 3 、0.022mg/L CuSO 4 ·5H 2 O；
Transferring the blackcurrant stem cell suspension obtained in the step (II) to a bioreactor for culturing for 7d, and adding fresh cell subculture medium to a cell density of 1×10 7 Continuously culturing for 12d at a volume of one ml to obtain amplified blackcurrant stem cells;
crushing the amplified blackcurrant stem cells obtained in the step (II) by using an ultrasonic crusher, filtering by using an ultrafiltration membrane, concentrating filtrate in a rotary vacuum evaporator, pre-freezing the concentrated filtrate in a refrigerator at-80 ℃ for 6 hours, transferring the pre-frozen filtrate into a vacuum freeze-drying device at-50 ℃ under the condition of 20Pa of vacuum degree for 48 hours to obtain the blackcurrant stem cell extract. The total phenol content of the blackcurrant stem cell extract was determined to be 51.26mg/mL by HPLC.
Example 3
The embodiment discloses a preparation method of a blackcurrant stem cell extract, which comprises the following steps:
the method comprises the steps of cutting off delignified tender stems from tender branches of annual blackcurrant plants to serve as explants, and cutting off all leaves on the tender stems;
soaking the explant in the step of preparing the explant with 75% alcohol for 5min, sterilizing with 30% sodium hypochlorite for 5min, washing with sterile water for 3 times, shearing the explant into stem tips with the length of 0.2-0.5cm with sterile scissors, and stripping out tissues containing cambium with an inoculating knife;
third step obtaining cambium tissues per 5cm 2 Inoculating 1g of tissue at a density on a culture medium for culturing at 24 ℃ under the conditions of illumination 2000LX and pH value of 5.2-6.2 for 18 days; wherein, the culture medium is based on an MS culture medium, and then inorganic salt, vitamin C60 mg/L, 6-benzylaminopurine 0.8mg/L, 3-indoleacetic acid 0.2mg/L, biochar 1g/L, L-cystine 5mg/L, agar 5g/L and sucrose 30g/L are added; wherein the inorganic salt comprises: 370mg/LCaCl 2 、176mg/L KH 2 PO 4 、1650mg/L KNO 3 、169.25mg/L MgSO 4 、1655mg/LNH 4 NO 3 、0.021mg/L CuSO 4 ·5H 2 O；
Transferring the upper culture medium and cell clusters together into a sterilized empty triangular flask, adding 7% chromic acid solution, controlling the rotation speed to 110rpm, oscillating for 7 hours, and separating to obtain blackcurrant stem cells;
fifthly, placing the blackcurrant stem cells obtained in the step in a cell subculture medium, culturing in a dark constant-temperature culture table at 26 ℃ and 120rpm, and culturing for 3 days at intervals to obtain blackcurrant stem cell suspensionStanding for 24h, and then taking the supernatant of the secondary culture medium to continue shaking culture, and circulating for 4 times to obtain a stable blackcurrant stem cell suspension; wherein the culture medium is based on an MS culture medium, and then inorganic salt, vitamin C60 mg/L, L-cystine 5mg/L and sucrose 30g/L, multi-wall carbon nano tube 150mg/L, salicylic acid 75umonl/L, jasmonic acid 75umonl/L and 2, 4-dichlorophenoxyacetic acid 2umonl/L are added; wherein the inorganic salt comprises: 375mg/LCaCl 2 、188mg/L KH 2 PO 4 、1810mg/LKNO 3 、176.28mg/L MgSO 4 、1860mg/LNH 4 NO 3 、0.022mg/L CuSO 4 ·5H 2 O；
Transferring the blackcurrant stem cell suspension obtained in the step (II) to a bioreactor for culturing for 6d, and adding a fresh cell subculture medium until the cell density is 1 multiplied by 10 7 Continuously culturing for 12d at a volume of one ml to obtain amplified blackcurrant stem cells;
crushing the amplified blackcurrant stem cells obtained in the step (II) by using an ultrasonic crusher, filtering by using an ultrafiltration membrane, concentrating filtrate in a rotary vacuum evaporator, pre-freezing the concentrated filtrate in a refrigerator at-80 ℃ for 6 hours, transferring the pre-frozen filtrate into a vacuum freeze-drying device at-50 ℃ under the condition of 20Pa of vacuum degree for 48 hours to obtain the blackcurrant stem cell extract. The total phenol content of the blackcurrant stem cell extract was 51.49mg/mL as determined by HPLC.
Comparative example 1
A preparation method of blackcurrant extract comprises the following steps: crushing blackcurrants, performing enzymolysis and ultrafiltration membrane filtration, concentrating filtrate in a rotary vacuum evaporator, pre-freezing the concentrated filtrate in a refrigerator at-80 ℃ for 6 hours, transferring to a blackcurrants extract obtained by vacuum freeze drying for 48 hours under the conditions of the temperature of-50 ℃ and the vacuum degree of 20Pa, and determining that the total phenol content in the blackcurrants extract is 38.95mg/mL by adopting an HPLC method.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (8)
1. A method for preparing a blackcurrant stem cell extract, comprising the steps of:
the method comprises the steps of cutting off delignified tender stems from tender branches of annual blackcurrant plants to serve as explants, and cutting off all leaves on the tender stems;
soaking the explant in the step (A) with 75% alcohol for 5min, sterilizing with 30% sodium hypochlorite for 5min, washing with sterile water for 3 times, shearing with sterile scissors to form a stem tip with the length of 0.2-0.5cm, and stripping from the stem tip to obtain cambium tissue;
thirdly, inoculating the cambium tissue obtained in the step II on a culture medium for culture;
transferring the upper culture medium and cell clusters to a sterilized empty triangular flask, adding 5% -8% chromic acid solution, controlling the rotation speed to 110rpm, carrying out oscillation treatment for 5-8h, and separating to obtain blackcurrant stem cells;
fifthly, placing the obtained blackcurrant stem cells in a cell subculture medium, and culturing to obtain blackcurrant stem cell suspension;
transferring the blackcurrant stem cell suspension obtained in the step (II) to a bioreactor for culturing for 4-7d, adding a fresh cell subculture medium, and continuously culturing for 12d to obtain amplified blackcurrant stem cells;
and crushing, filtering, concentrating and freeze-drying the amplified blackcurrant stem cells obtained in the step (II) to obtain the blackcurrant stem cell extract.
2. The method of claim 1, wherein the culture medium comprises inorganic salts, vitamins, agar, sucrose, biochar, 6-benzyl amino purine, 3-indoleacetic acid, and L-cystine.
3. The method of claim 1, wherein the cell subculture medium of step d comprises: inorganic salts, vitamins, amino acids, saccharides, multi-walled carbon nanotubes, salicylic acid, jasmonic acid and 2, 4-dichlorophenoxyacetic acid.
4. The method for preparing a blackcurrant stem cell extract according to claim 3, wherein the saccharide is any one of sucrose, glucose or fructose.
5. The method of claim 1, wherein the culturing conditions in step ii) are: culturing at 23-25deg.C under illumination of 1500LX-2500LX and pH of 5.2-6.2 for 14-20d.
6. The method for preparing blackcurrant stem cell extract as claimed in claim 1, wherein the cell subculture process in the step of bathing is carried out in a dark constant temperature culture shaker at a temperature of 23-27 ℃, the rotation speed is 120rpm, every 3 days of culture, the blackcurrant stem cell suspension is kept stand for 24 hours, the supernatant of the secondary culture medium is continuously cultured in a shaking way, and the circulation is carried out for 4 times, so as to obtain the blackcurrant stem cell suspension.
7. The method of claim 1, wherein the step of crushing, filtering, concentrating and freeze-drying the blackcurrant stem cells comprises the steps of: crushing cells of the blackcurrant stem cells by using an ultrasonic breaker, filtering by using an ultrafiltration membrane, concentrating filtrate in a rotary vacuum evaporator, pre-freezing the concentrated filtrate in a refrigerator at-80 ℃ for 6 hours, transferring the filtrate into a vacuum freeze-drying under the condition of the temperature of-50 ℃ and the vacuum degree of 20Pa for 48 hours to obtain the blackcurrant stem cell extract.
8. A blackcurrant stem cell extract obtained by a method of preparing a blackcurrant stem cell extract according to any one of claims 1-7.
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