CN116790382B - High-yield lutein mutant chlorella, and preparation method and application thereof - Google Patents
High-yield lutein mutant chlorella, and preparation method and application thereof Download PDFInfo
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- 241000195649 Chlorella <Chlorellales> Species 0.000 title claims abstract description 100
- KBPHJBAIARWVSC-RGZFRNHPSA-N lutein Chemical compound C([C@H](O)CC=1C)C(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\[C@H]1C(C)=C[C@H](O)CC1(C)C KBPHJBAIARWVSC-RGZFRNHPSA-N 0.000 title claims abstract description 82
- 229960005375 lutein Drugs 0.000 title claims abstract description 82
- KBPHJBAIARWVSC-XQIHNALSSA-N trans-lutein Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2C(=CC(O)CC2(C)C)C KBPHJBAIARWVSC-XQIHNALSSA-N 0.000 title claims abstract description 82
- FJHBOVDFOQMZRV-XQIHNALSSA-N xanthophyll Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2C=C(C)C(O)CC2(C)C FJHBOVDFOQMZRV-XQIHNALSSA-N 0.000 title claims abstract description 82
- 235000012680 lutein Nutrition 0.000 title claims abstract description 75
- ORAKUVXRZWMARG-WZLJTJAWSA-N lutein Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CCCC1(C)C)C=CC=C(/C)C=CC2C(=CC(O)CC2(C)C)C ORAKUVXRZWMARG-WZLJTJAWSA-N 0.000 title claims abstract description 75
- 239000001656 lutein Substances 0.000 title claims abstract description 75
- 238000002360 preparation method Methods 0.000 title abstract description 13
- 241000195652 Auxenochlorella pyrenoidosa Species 0.000 claims abstract description 12
- 244000005700 microbiome Species 0.000 claims abstract description 8
- 238000004519 manufacturing process Methods 0.000 claims description 10
- 238000000855 fermentation Methods 0.000 abstract description 27
- 230000004151 fermentation Effects 0.000 abstract description 27
- 230000012010 growth Effects 0.000 abstract description 21
- 239000002028 Biomass Substances 0.000 abstract description 11
- 230000035425 carbon utilization Effects 0.000 abstract description 7
- 230000002349 favourable effect Effects 0.000 abstract description 7
- 238000004321 preservation Methods 0.000 abstract description 6
- 238000002703 mutagenesis Methods 0.000 description 37
- 231100000350 mutagenesis Toxicity 0.000 description 37
- 239000000126 substance Substances 0.000 description 14
- 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 description 13
- 239000008103 glucose Substances 0.000 description 13
- 238000000034 method Methods 0.000 description 10
- 238000012216 screening Methods 0.000 description 7
- 235000008210 xanthophylls Nutrition 0.000 description 7
- 241000195493 Cryptophyta Species 0.000 description 6
- 230000009286 beneficial effect Effects 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 239000000306 component Substances 0.000 description 5
- 229910019614 (NH4)6 Mo7 O24.4H2 O Inorganic materials 0.000 description 4
- 235000007089 Chlorella vulgaris Nutrition 0.000 description 4
- PLUBXMRUUVWRLT-UHFFFAOYSA-N Ethyl methanesulfonate Chemical compound CCOS(C)(=O)=O PLUBXMRUUVWRLT-UHFFFAOYSA-N 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- FIXLYHHVMHXSCP-UHFFFAOYSA-H azane;dihydroxy(dioxo)molybdenum;trioxomolybdenum;tetrahydrate Chemical compound N.N.N.N.N.N.O.O.O.O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O[Mo](O)(=O)=O.O[Mo](O)(=O)=O.O[Mo](O)(=O)=O FIXLYHHVMHXSCP-UHFFFAOYSA-H 0.000 description 4
- 239000004202 carbamide Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 238000009423 ventilation Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 3
- 238000009776 industrial production Methods 0.000 description 3
- 235000005881 Calendula officinalis Nutrition 0.000 description 2
- 240000000785 Tagetes erecta Species 0.000 description 2
- 239000012527 feed solution Substances 0.000 description 2
- 239000006052 feed supplement Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 230000009469 supplementation Effects 0.000 description 2
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 235000007091 Chlorella pyrenoidosa Nutrition 0.000 description 1
- 241000195628 Chlorophyta Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 235000021466 carotenoid Nutrition 0.000 description 1
- 150000001747 carotenoids Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000000576 food coloring agent Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000009569 heterotrophic growth Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 125000000468 ketone group Chemical group 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000004224 protection Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
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- 231100000331 toxic Toxicity 0.000 description 1
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- 230000001131 transforming effect Effects 0.000 description 1
Classifications
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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
- C12N1/125—Unicellular algae isolates
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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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/89—Algae ; Processes using algae
Abstract
The application relates to the technical field of chlorella, in particular to a high-yield lutein-induced chlorella, a preparation method and application thereof; provides a strain of high-yield lutein-producing mutant chlorella which isChlorella pyrenoidosa (Auxenochlorella pyrenoidosa)The preservation number of the strain ZC-1003 is GDMCC No.63502, and the preservation center is the Guangdong province microorganism strain preservation center; the mutagenized chlorella has high biomass density and high growth speed under the condition of no light heterotrophy, can obtain high-content lutein, and is favorable for the industrial wide application of the lutein produced by heterotrophic fermentation of chlorella.
Description
Technical Field
The application belongs to the technical field of chlorella, and particularly relates to a high-yield lutein-induced chlorella, and a preparation method and application thereof.
Background
Lutein (lutein) is an oxygen-containing carotenoid of formula C 40 H 56 O 2 Contains two ketone rings, which are the main pigments present in the macular area of the human eye. Lutein has better activity in the aspects of vision protection, antioxidation, anticancer, early arteriosclerosis delaying and the like, is also often used as food colorant, and has wide application in the food industry.
At present, lutein products on the market mainly come from marigold. The marigold produces higher plants of lutein, however, the problems of low yield, strong seasonality, occupied cultivated land and the like exist, so that the selling price of lutein is extremely high. And the chlorella with rapid growth is a good source for lutein production. Chlorella is a green algae, and can utilize heterotrophic growth of glucose, can realize stable continuous industrial production of lutein, and is a potential production source.
However, the existing industrial production mode of chlorella mainly adopts photoautotrophic culture, and has the problems of low growth speed, low cell density, easy pollution by micro-organisms (bacteria, toxic algae, protozoa and the like) and the like. The chlorella can be cultivated in a dark heterotrophic way, but the content of the lutein serving as a light-capturing pigment is extremely low, so that the industrial application of the heterotrophic fermentation chlorella in lutein production is limited.
Disclosure of Invention
The application aims to provide a mutagenesis chlorella with high lutein yield, a preparation method and application thereof, and aims to solve the problem of low chlorella lutein under heterotrophic culture conditions in the prior art.
In order to achieve the purposes of the application, the technical scheme adopted by the application is as follows:
in a first aspect, the application provides a high-yield lutein-producing mutant chlorella, which is mutant intoChlorellapvrenoidosa( Auxenochlorellapyrenoidosa)The mutant strain ZC-1003 has a deposit number of GDMCC No.63502 and a deposit center of Guangdong province microorganism strain deposit center.
Further, under the condition of no heterotrophy, the lutein content of the mutagenized chlorella is 7.0-7.5 mg/g.
Further, the lutein content of the mutagenized chlorella was 7.1 mg/g in the absence of heterotrophic conditions.
Further, the specific growth rate of the mutagenized chlorella is 1.3-1.4 d -1 。
Further, under the condition of fermenting and culturing in a fermentation tank for 72 hours, the biomass of the mutagenized chlorella is 84-85 g/L; and the lutein yield is 7.1-7.2 mg/(L.h).
Further, 1.01 g KNO per liter of the fermentation medium of the fermenter 3 、9.3 mg EDTANa 2 、0.62 g NaH 2 PO 4 .H 2 O、0.089 g Na 2 HPO 4 .2H 2 O、0.247 g MgSO 4 .7H 2 O、14.7 mg CaCl 2 .2H 2 O、6.95 mg FeSO 4 .7H 2 O、0.061 mg H 3 BO 3 、0.169 mg MnSO 4 .H 2 O、0.287 mg ZnSO 4 .7H 2 O、0.0025 mg CuSO 4 .5H 2 O、0.01235 mg (NH 4 ) 6 MO 7 O 24 .4H 2 O, 40 g glucose, 5 g urea; and adjusting the pH to 6.0-6.5.
Further, the conditions of fermentation culture in the fermenter are as follows: the initial inoculation amount is 2-2.5 g/L, the culture is carried out for 72 hours under the conditions that the culture temperature is 27-28 ℃, the initial rotation speed is 200-220 r/min, the ventilation amount is 0.5-0.7 vvm, and then the feed supplement is carried out, and the culture is carried out for 90-100 hours.
In a second aspect, the application provides a method for preparing high-yield lutein-containing chlorella mutans, comprising the following steps:
s1, providing wild chlorellaChlorella pyrenoidosa；
S2, preparing wild chlorellaChlorella pyrenoidosaSequentially carrying out chemical mutagenesis treatment and plasma mutagenesis treatment, and then carrying out dull plate culture;
s3, selecting white and rapidly growing single algae, purifying, subculturing, and screening to obtain the high-yield lutein-induced chlorella.
Further, in S2, the chemical mutagenesis treatment includes treatment with ethyl methylsulfonate having a concentration of 0.1 to 0.15 mol/L for 4 to 6 hours.
Further, in S2, the plasma mutagenesis treatment includes a treatment with plasma at normal temperature and normal pressure for 60 to 80 seconds.
In a third aspect, the application provides the use of the above-described high lutein-producing mutant chlorella in the production of lutein.
The application provides a high-yield lutein-producing mutant chlorella which isChlorella pyrenoidosa (Auxenochlorella pyrenoidosa)The preservation number of the strain ZC-1003 is No.63502, and the preservation center is the microorganism strain preservation center of Guangdong province; the mutagenized chlorella has high biomass density and high growth speed under the condition of no light heterotrophy, can obtain high-content lutein, and is favorable for the industrial wide application of the lutein produced by heterotrophic fermentation of chlorella.
According to the preparation method of the high-yield lutein-containing mutagenized chlorella ZC-1003, provided by the second aspect of the application, a combined mutagenesis mode of chemical mutagenesis and plasma mutagenesis is utilized, and the mutagenized chlorella ZC-1003 which is high in lutein content and rapid in growth under heterotrophic conditions is obtained through screening, so that the method is favorable for being widely applied to industry.
The mutagenesis chlorella with high lutein yield or the application of the mutagenesis chlorella prepared by the preparation method of the mutagenesis chlorella with high lutein yield in lutein production is favorable for large-area production of algae lutein because the provided mutagenesis chlorella strain ZC-1003 grows fast and has high lutein yield under the culture condition without heterotrophic light.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments or the description of the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram of biomass analysis of a 100L fermentation tank of wild type Chlorella and mutagenized Chlorella provided by an embodiment of the present application.
FIG. 2 is a graph showing the lutein content and yield analysis of wild type Chlorella and mutagenized Chlorella according to the present application.
Description of the embodiments
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the application is further described in detail below with reference to the embodiments. 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 application.
In the present application, the term "and/or" describes an association relationship of an association object, which means that three relationships may exist, for example, a and/or B may mean: a alone, a and B together, and B alone. Wherein A, B may be singular or plural. The character "/" generally indicates that the context-dependent object is an "or" relationship.
In the present application, "at least one" means one or more, and "a plurality" means two or more. "at least one of" or the like means any combination of these items, including any combination of single item(s) or plural items(s). For example, "at least one (individual) of a, b, or c," or "at least one (individual) of a, b, and c," may each represent: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, c may be single or multiple, respectively.
It should be understood that, in various embodiments of the present application, the sequence number of each process described above does not mean that the execution sequence of some or all of the steps may be executed in parallel or executed sequentially, and the execution sequence of each process should be determined by its functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.
The terminology used in the embodiments of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
The weights of the relevant components mentioned in the description of the embodiments of the present application may refer not only to the specific contents of the components, but also to the proportional relationship between the weights of the components, so long as the contents of the relevant components in the description of the embodiments of the present application are scaled up or down within the scope of the disclosure of the embodiments of the present application. Specifically, the mass in the description of the embodiment of the application can be a mass unit which is known in the chemical industry field such as [ mu ] g, mg, g, kg.
The terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated for distinguishing between objects such as substances from each other. For example, a first XX may also be referred to as a second XX, and similarly, a second XX may also be referred to as a first XX, without departing from the scope of embodiments of the application. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature.
The first aspect of the embodiment of the application provides a high-yield lutein-producing mutant chlorella, which is used for transforming the chlorella intoChlorella pyrenoidosa (Auxenochlorella pyrenoidosa)The mutant strain ZC-1003 has a deposit number of GDMCC No.63502 and a deposit center of Guangdong province microorganism strain deposit center.
The first aspect of the embodiment of the application provides a high-yield lutein-producing mutant chlorella which isChlorella pyrenoidosa (Auxenochlorella pyrenoidosa)The preservation number of the strain ZC-1003 is No.63502, and the preservation center is the microorganism strain preservation center of Guangdong province; the mutagenized chlorella has high biomass density and high growth speed under the condition of no light heterotrophy, can obtain high-content lutein, and is favorable for the industrial wide application of the lutein produced by heterotrophic fermentation of chlorella.
Because the existing industrial production mode of chlorella mainly comprises photoautotrophic culture, if the culture is carried out in a non-phototrophic culture mode, on one hand, the growth speed of the chlorella can be influenced, and on the other hand, the content of the light-capturing color lutein is very low, so that the method is not beneficial to fermenting the chlorella to produce lutein in the non-phototrophic culture mode.
The application combines the mutagenesis method, and the mutagenesis chlorella ZC-1003 with the preservation number of No.63502 is obtained by screening, the growth speed of the mutagenesis chlorella ZC-1003 under the condition of No heterotrophy can be close to that of wild chlorella, and the lutein can be produced at high yield, thereby being beneficial to industrial application.
In some embodiments, under the condition of no heterotrophy, the lutein content of the mutagenized chlorella is 7.0-7.5 mg/g; the lutein content of wild type chlorella is 3.8 mg/g; the lutein content of the mutagenized chlorella is 1.8-1.9 times of that of wild chlorella, and the lutein content is high.
In some embodiments, the lutein content of the mutagenized Chlorella includes, but is not limited to, 7.0 mg/g, 7.1 mg/g, 7.2 mg/g, 7.3 mg/g, 7.4 mg/g, 7.5 mg/g.
In one embodiment, the xanthophyll content of the mutagenized Chlorella is 7.1 mg/g in the absence of heterotrophic conditions.
In some embodiments, the specific growth rate of the mutagenized chlorella is 1.3-1.4 d -1 . The specific growth rate of wild type chlorella is 1.0. 1.0 d -1 . It can be seen that the growth rate of the mutagenized chlorella is 30% -40% faster than that of the wild chlorella.
In some embodiments, the specific growth rate of Chlorella mutagenized strain ZC-1003 includes, but is not limited to, 1.3 d -1 、1.32 d -1 、1.34 d -1 、1.36 d -1 、1.38 d -1 、1.4 d -1 。
In some embodiments, under the condition of fermentation culture in a fermentation tank for 72 hours, the biomass of the mutagenized chlorella is 84-85 g/L; and the lutein yield is 7.1-7.2 mg/(L.h). Wherein, the wild type chlorella reaches more than 90g/L, which takes 5 days; moreover, the lutein yield is 2.85 mg/(L.h), and the growth speed of the chlorella mutagenized strain ZC-1003 is high, so that the lutein yield is high.
In some embodiments, the biomass of the mutagenized Chlorella under conditions of the fermenter for 72 hours of fermentation culture includes, but is not limited to, 84 g/L, 84.1 g/L, 84.2 g/L, 84.3 g/L, 84.4g/L, 84.5 g/L, 84.6 g/L, 84.7 g/L, 84.8 g/L, 84.9 g/L, 85 g/L.
In some embodiments, lutein yields include, but are not limited to, 7.1 mg/(l.h), 7.11 mg/(l.h), 7.12 mg/(l.h), 7.13 mg/(l.h), 7.14 mg/(l.h), 7.15 mg/(l.h), 7.16 mg/(l.h), 7.17 mg/(l.h), 7.18 mg/(l.h), 7.19 mg/(l.h), 7.2 mg/(l.h) under conditions of fermenter fermentation culture for 72 hours.
In some embodiments, 1.01 g KNO per liter of fermenter broth 3 、9.3 mg EDTANa 2 、0.62 g NaH 2 PO 4 .H 2 O、0.089 g Na 2 HPO 4 .2H 2 O、0.247 g MgSO 4 .7H 2 O、14.7 mg CaCl 2 .2H 2 O、6.95 mg FeSO 4 .7H 2 O、0.061 mg H 3 BO 3 、0.169 mg MnSO 4 .H 2 O、0.287 mg ZnSO 4 .7H 2 O、0.0025 mg CuSO 4 .5H 2 O、0.01235 mg (NH 4 ) 6 MO 7 O 24 .4H 2 O, 40 g glucose, 5 g urea; and adjusting the pH to 6.0-6.5.
In some embodiments, the medium has a pH of 6.1.
In some embodiments, the conditions of the fermenter fermentation culture are as follows: the initial inoculation amount is 2-2.5 g/L, the culture is carried out for 72 hours under the conditions that the culture temperature is 27-28 ℃, the initial rotation speed is 200-220 r/min, the ventilation amount is 0.5-0.7 vvm, and then the feed supplement is carried out, and the culture is carried out for 90-100 hours.
In some embodiments, the conditions of the fermenter fermentation culture are as follows: the initial inoculum size was 2g/L, and the culture was carried out at 27℃for 72 hours at an initial rotation speed of 200 rpm and a ventilation of 0.5 vvm, followed by feeding and culturing for 90 hours.
In some embodiments, the feeding comprises glucose supplementation, wherein the glucose index feeding is supplemented according to the following formula:
wherein V is F.S Feed rate (mL/h); mu is the specific growth rate h-1; v (V) 0 Is the fermentation volume (L); c (C) 0 Is the initial cell concentration (g/L); t is fermentation time, and Fs is glucose concentration (g/L) in the glucose feed solution.
The second aspect of the embodiment of the application provides a preparation method of high-yield lutein-containing chlorella mutans, which comprises the following steps:
s01 providing wild ChlorellaChlorella pyrenoidosa；
S02, wild ChlorellaChlorella pyrenoidosaSequentially carrying out chemical mutagenesis treatment and plasma mutagenesis treatment, and then carrying out dull plate culture;
s03, selecting white and rapidly growing single algae, purifying, subculturing, and screening to obtain the high-yield lutein-induced chlorella.
According to the preparation method of the high-yield lutein-containing mutant chlorella ZC-1003 provided by the second aspect of the embodiment of the application, a combined mutagenesis mode of chemical mutagenesis and plasma mutagenesis is utilized, and the mutant chlorella ZC-1003 with high lutein content and rapid growth under heterotrophic conditions is obtained through screening, so that the method is favorable for being widely applied to industry.
In step S01, wild type Chlorella is providedChlorella pyrenoidosaIs 1.0X10 g 7 ~1.2×10 7 And each mL.
In step S02, wild-type chlorella Chlorella pyrenoidosa is subjected to chemical mutagenesis treatment and plasma mutagenesis treatment in this order, and then subjected to dull-plate culture.
In some embodiments, the chemical mutagenesis treatment comprises treatment with ethyl methylsulfonate at a concentration of 0.1 to 0.15 mol/L for 4 to 6 hours.
In some embodiments, the chemical mutagenesis treatment comprises treatment with ethyl methylsulfonate at a concentration of 0.1 mol/L for 4 hours. The treatment is carried out by adopting a chemical reagent with low concentration, so that the cells are damaged to a certain extent, but the damage is not too serious; ensuring that the cells are subjected to subsequent plasma mutagenesis treatment. If the concentration is too high and the treatment time is too long, the mortality is too high and the mutation fails.
In some embodiments, the plasma mutagenesis treatment comprises treating with plasma at room temperature and pressure for 60-80 seconds. Wherein, the plasma is a gas substance with more charge in an ionization state, but the total amount is 0, and the plasma (active ion) is used for treating cells, thereby being beneficial to mutating the cells.
In some embodiments, the plasma mutagenesis treatment comprises treating with a plasma at ambient temperature and pressure for 60 seconds.
In some embodiments, the conditions for flat-plate cultivation are 24-25℃for 7 days.
After detection, after chemical mutagenesis treatment and plasma mutagenesis treatment are sequentially carried out, the lethality of chlorella is 99.0%.
White and large colonies were selected after 7 days of flat plate culture without light, purified and passaged and transferred to liquid culture.
In the step S03, white and rapidly growing single algae are selected for purification treatment and subculture, and then screening is carried out to obtain the high-yield lutein-induced chlorella.
The third aspect of the embodiment of the application provides the above-mentioned high-lutein-yield chlorella mutabilis or the application of the above-mentioned high-lutein-yield chlorella mutabilis prepared by the above-mentioned preparation method of the above-mentioned high-lutein-yield chlorella mutabilis in lutein production.
The application of the high-yield xanthophyll-containing chlorella vulgaris mutagenesis or the chlorella vulgaris mutagenesis prepared by the preparation method of the high-yield xanthophyll-containing chlorella vulgaris mutagenesis provided by the embodiment of the application in xanthophyll production is beneficial to the large-area production of the algal xanthophyll because the provided chlorella vulgaris mutagenesis strain ZC-1003 grows fast and has high xanthophyll yield under the culture condition without heterotrophic light.
The following description is made with reference to specific embodiments.
Example 1
High-yield lutein-containing mutant chlorella and preparation method thereof
The preparation method of the mutagenized chlorella with high lutein yield comprises the following steps:
providing a concentration of 1X 10 7 Wild chlorella/mLChlorella pyrenoidosa；
Wild type ChlorellaChlorella pyrenoidosaSequentially carrying out chemical mutagenesis treatment and plasma mutagenesis treatment, and then carrying out dull plate culture; wherein the chemical mutagenesis treatment comprises the use of a concentration of 01 mol/L ethyl methylsulfonate for 4 hours, wherein the plasma mutagenesis treatment comprises the steps of adopting plasma for treatment for 60 seconds at normal temperature and normal pressure, and culturing for 7 days at 25 ℃ without flat plate culture;
and (3) selecting white and rapidly growing single algae, purifying, subculturing, and screening to obtain the high-yield lutein-containing mutant chlorella.
Wherein the obtained chlorella mutans is chlorella mutans strain ZC-1003 with the preservation number of No.63502, and is a chlorella mutans strain with high lutein yield.
Example 2
100L fermentation tank enlarged culture of high-yield lutein-induced chlorella
The method comprises the following steps:
the chlorella mutant strain ZC-1003 of accession No.63502 obtained in example 1 was subjected to fermentation tank expansion culture, and the medium components of the fermentation tank fermentation culture included: 1.01 g KNO per liter of culture medium 3 、9.3 mg EDTANa 2 、0.62 g NaH 2 PO 4 .H 2 O、0.089 g Na 2 HPO 4 .2H 2 O、0.247 g MgSO 4 .7H 2 O、14.7 mg CaCl 2 .2H 2 O、6.95 mg FeSO 4 .7H 2 O、0.061 mg H 3 BO 3 、0.169 mg MnSO 4 .H 2 O、0.287 mg ZnSO 4 .7H 2 O、0.0025 mg CuSO 4 .5H 2 O、0.01235 mg (NH 4 ) 6 MO 7 O 24 .4H 2 O, 40 g glucose, 5 g urea; and the pH was adjusted to 6.1.
The conditions of fermentation culture in the fermenter are as follows: the initial inoculation amount is 2g/L, the culture is carried out for 72 hours under the conditions that the culture temperature is 28 ℃, the initial rotating speed is 200 revolutions per minute and the ventilation amount is 0.5 vvm, and then the feed is fed, and the culture is carried out for 90 hours; wherein the feeding comprises glucose supplementation, and the glucose index feeding is supplemented according to the following formula:
wherein V is F.S Feed rate (mL/h); mu is the specific growth rate h-1; v (V) 0 Is the fermentation volume (L); c (C) 0 Is the initial cell concentration (g/L); t is fermentation time, and Fs is glucose concentration (g/L) in the glucose feed solution.
Comparative example 1
100L fermentation tank enlarged culture of wild chlorella
The culture medium components of the initial culture medium fermentation tank for fermentation culture comprise: 1.01 g KNO per liter of culture medium 3 、9.3 mg EDTANa 2 、0.62 g NaH 2 PO 4 .H 2 O、0.089 g Na 2 HPO 4 .2H 2 O、0.247 g MgSO 4 .7H 2 O、14.7 mg CaCl 2 .2H 2 O、6.95 mg FeSO 4 .7H 2 O、0.061 mg H 3 BO 3 、0.169 mg MnSO 4 .H 2 O、0.287 mg ZnSO 4 .7H 2 O、0.0025 mg CuSO 4 .5H 2 O、0.01235 mg (NH 4 ) 6 MO 7 O 24 .4H 2 O, 40 g glucose, 5 g urea; and the pH was adjusted to 6.1.
The culture conditions are as follows: the culture temperature is 28 ℃, the initial rotating speed is 200 revolutions per minute, the aeration rate is 0.5 vvm, and the initial inoculation amount is 120h under the condition of 2g/L, and the culture is carried out by feeding the feed.
Performance testing and results analysis
The following properties were measured for the mutagenized chlorella obtained by the 100L fermenter enlargement culture of the xanthophyll-rich mutagenized chlorella of example 2 and the wild-type chlorella obtained by the 100L fermenter enlargement culture of the wild-type chlorella of comparative example 1, respectively
Biomass (one)
As shown in FIG. 1, the highest biomass of the chlorella mutabilis obtained in the example 2 can reach 84.4g/L within 72 hours; the highest biomass of the wild-type Chlorella obtained in comparative example 1 at 120 hours was 90g/L.
(II) lutein content
As shown in FIG. 2, the lutein content of the chlorella mutabilis obtained in example 2 at 72 hours is 6.1mg/g; lutein yield 7.15 mg/(l.h); the wild type Chlorella obtained in comparative example 1 had a lutein content of 3.8mg/g at 120 hours and a lutein yield of 2.85 mg/(L.h).
(III) specific growth Rate
The specific growth rate of the mutant Chlorella obtained in example 2 was 1.30. 1.30 d -1 The method comprises the steps of carrying out a first treatment on the surface of the The specific growth rate of wild type Chlorella obtained in comparative example 1 was 1d -1 。
In summary, the embodiment of the application provides a high-yield lutein-producing mutant chlorella which isChlorella pyrenoidosa (Auxenochlorella pyrenoidosa)The preservation number of the strain ZC-1003 is No.63502, and the preservation center is the microorganism strain preservation center of Guangdong province; the mutagenized chlorella has high biomass density and high growth speed under the condition of no light heterotrophy, can obtain high-content lutein, and is favorable for the industrial wide application of the lutein produced by heterotrophic fermentation of chlorella.
The foregoing description of the preferred embodiments of the application is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the application.
Claims (2)
1. The mutagenized chlorella with high lutein yield is characterized in that the mutagenized chlorella isChlorella pyrenoidosa( Auxenochlorella pyrenoidosa)The mutant strain ZC-1003 has a deposit number of GDMCC No.63502 and a deposit center of Guangdong province microorganism strain deposit center.
2. Use of the high lutein-yielding mutant chlorella in accordance with claim 1 for the production of lutein.
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| CN101555454A (en) * | 2009-05-15 | 2009-10-14 | 华南理工大学 | Method for synchronously improving biomass and lutein of heterotrophic chlorella |
| CN102094061A (en) * | 2010-12-01 | 2011-06-15 | 华东理工大学 | Method for producing lutein from microalgae |
| CN102888347A (en) * | 2011-07-22 | 2013-01-23 | 中国科学院烟台海岸带研究所 | Chlorella mutant strain and application thereof |
| CN102976992A (en) * | 2012-12-14 | 2013-03-20 | 国家海洋局第三海洋研究所 | Method for extracting lutein from chlorella |
| CN106520558A (en) * | 2016-12-01 | 2017-03-22 | 中国科学院昆明植物研究所 | Mutant chlorella strain capable of producing zeaxanthine and beta-carotene and culturing method thereof |
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| ES2374079T3 (en) * | 2006-01-12 | 2012-02-13 | Cognis Ip Management Gmbh | PROCEDURES FOR OBTAINING LUTEINE FROM ALGAS. |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101555454A (en) * | 2009-05-15 | 2009-10-14 | 华南理工大学 | Method for synchronously improving biomass and lutein of heterotrophic chlorella |
| CN102094061A (en) * | 2010-12-01 | 2011-06-15 | 华东理工大学 | Method for producing lutein from microalgae |
| CN102888347A (en) * | 2011-07-22 | 2013-01-23 | 中国科学院烟台海岸带研究所 | Chlorella mutant strain and application thereof |
| CN102976992A (en) * | 2012-12-14 | 2013-03-20 | 国家海洋局第三海洋研究所 | Method for extracting lutein from chlorella |
| CN106520558A (en) * | 2016-12-01 | 2017-03-22 | 中国科学院昆明植物研究所 | Mutant chlorella strain capable of producing zeaxanthine and beta-carotene and culturing method thereof |
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