CN105803033A - Method for inducing freshwater chlorella pyrenoidosa (C. pyrenoidosa) ZF strain to efficiently accumulate carotenoids by utilizing plant growth regulator 6-BA - Google Patents

Abstract

The invention provides a method for inducing a freshwater chlorella pyrenoidosa (C. pyrenoidosa) ZF strain to efficiently accumulate carotenoids by utilizing a plant growth regulator 6-BA. The method is characterized by adopting the following steps: 1) preparing an alga liquid culturing alga cells till the logarithmic phase by adopting 1/4 times concentration of BG11 nutritive salt under the conditions that the temperature is 23+/-2 DEG C, the light intensity is 1900+/-100lx and the light-dark ratio is 12h/12h, adding 1 times concentration of BG11 nutritive salt every other 14+/-1 days and shaking algae 3+/-1 times every day; and 2) accumulating carotenoids: putting 200ml of alga solution in the logarithmic phase in a 300mL conical flask, then adding 6-BA till 0.01+/-0.005mg/L and carrying out culture under the same conditions for 14+/-1 days and inducing carotenoids to be quickly accumulated. The technology is mature, simple and practicable, is low in cost, is efficient and pollution-free and can achieve the effect of obviously increasing the yield of carotenoids of the C. pyrenoidosa ZF strain in a short time.

Description

One utilizes plant growth regulator 6-BA to induce fresh water Chlorella pyrenoidesa ZF algae strain The method of efficient accumulation carotenoid

Technical field

The present invention provides one to utilize plant growth regulator 6-BA induction fresh water Chlorella pyrenoidesa ZF algae plant height effect long-pending The method of tired carotenoid, belongs to biological technical field.

Background technology

Chlorella (Chlorella) is a kind of common aquatic unicellular alga, is in Chlorophyta chlorella section Important genus, ecologicaI distribution is extensive, all has distribution, include about 10 kinds at present in sea water and fresh water, and most common of which is Chlorella vulgaris (C. vulgaris), Chlorella pyrenoidesa (C. pyrenoidosa) and chlorella ellipsoidea (C. Ellipoidea) etc..Chlorella cells is contained within rich in protein (the 50% of dry weight), essential amino acids, polysaccharide, lipid (fat The result of fat acid accumulation), chlorophyll, carotenoid and vitamin etc., at health food, aquaculture, (feedstuff adds Agent), cosmetics, be widely used in medicine and other fields, supply falls short of demand in domestic and international market.Phase national with the U.S., Japan, Israel etc. Ratio, the chlorella industrialization large-scale culture level of relative of China is delayed.

Carotenoid be widely present in animal, higher plant, fungus, algae a class terpenoid natural Pigment.All carotenoid the most all can be by lycopene (1ycopene) basis having carbochain in the middle of 11 conjugated double bonds Structure is derived by oxidation, hydrogenation, dehydrogenation, cyclisation and the rearrangement of carbon skeleton, degraded.Carotenoid has in vivo Highly important physiological function, it is the accessory pigments of photosynthesis of plant, is protecting cells from high light, active oxygen and sensitization The adverse effect of pigment plays an important role.Meanwhile, carotenoid has higher pharmaceutical value, by ingesting class recklessly Radix Raphani element is possible not only to prevent oculopathy and the generation of cardiovascular disease, it is also possible to strengthen the immunologic function of human body, has anticancer, anti- Oxidation, anti-ageing effect of waiting for a long time.Therefore, carotenoid as nutrition, keep healthy, the multifunctional natural pigment such as coloring, have been widely used In industrial circles such as food, medicine, health product, feedstuff, cosmetics.Plant growth regulator can be efficiently under relatively low concentration The regulation and control growth promoter of plant and the kind of secondary metabolites and yield, be widely used in higher plant especially farming On thing produces.Plant growth regulator induction have simple to operate, inductivity is high, speed is fast, cost is low, pollution-free, safety The plurality of advantages such as strong.Microalgae is by the excellent material of plant growth regulator regulation and control secondary metabolism, its simple in construction, Life Cycle Phase is short, heliosensitivity is strong, metabolic process is the most affected by environment, is the most easily detected.

At present, some are had been disclosed for about preparation, the correlation technique that purifies carotenoid technique, great majority in external For technique improvements such as some fermentation, purification & isolation.Ocean rhodotorula high yield class is utilized as University Of Qingdao discloses one Carotene and copper fermentation culture method (CN201510512641), the purpose of this invention is to provide a strain from marine environment Separate obtain can High Yield of Carotenoid again can the ocean rhodotorula of enriching Cu, and provide it to be capable of high yield carotene And the optimal fermentation culture conditions of enriching Cu, provide theoretical foundation for industrialized production.Concrete method is: training of preferably fermenting Support and basis set become glucose 1%, enzymolysis Semen Maydis powder 1-3%, peptone 2.5%, magnesium sulfate 0.02%, potassium dihydrogen phosphate 0.15%, copper content 150-200mg/L, sodium chloride 20%, remaining be water；Initial pH is 8, and inoculum concentration 3-8%, shaking speed is Cultivate for 50-80r/min, 24-28 DEG C and adjust temperature after 10-15 hour and be 16-20 DEG C and carry out low temperature static gas wave refrigerator, stop stirring, PH is adjusted to 3-5, keeps 2-4 hour；Stairstepping is warmed up to 24-30 DEG C afterwards, and pH is adjusted to 4.6-5.5, according to 2.5% add Dosage adds peptone, and the amount according to 2% adds yeast powder；Shaking speed is 50-100r/min, continues fermentation 10-15h.Excellent Choosing, in culture medium, methionine chela and the copper of culture medium weight 3-5wt% is added in the low temperature static gas wave refrigerator stage.Weihai profit reaches A kind of method (CN104846049A) improving fermentative carotenoid productivity of bio tech ltd's invention, with the red ferment of glue Female bacterium, as fermentative carotenoid strain, is that described rhodotorula mucilaginosa is seeded in culture medium by 2%-10% according to inoculum concentration, institute State culture medium to include: glucose 12-130g/L, yeast powder 2-22g/L, salt 3-12g/L, the pH of this culture medium is at 5.5-7.5 Between, in described culture medium, adding ferrous ion solvent, the addition of described ferrous ion solvent is the most described culture medium Molar concentration between 0.05-0.5mmol/L, fermentation temperature maintains 27-33 DEG C, after fermenting 32-40 hour, shaking table speed Degree 140-200r/min, cools the temperature to 20-25 DEG C and continues fermentation, ferment 24-46 hour, shaking table speed 80-140r/min. Sichuan University of Science & Engineering provides a kind of method utilizing photosynthetic bacteria micro-aerobe fermentation quickly to produce carotenoid (CN104805168A), single for the photosynthetic bacteria (Rhodobacter sphaeroides) of activation colony inoculation is cultivated to MMS In base, aerobic incubated overnight obtains seed；Then by inoculum concentration be 8% the seed of aerobic incubated overnight is inoculated into respectively containing In the culture bottle of MMS culture medium, after inoculation, culture fluid volume is equivalent to the 40% of culture bottle volume, then rotating speed be 200～ 250rpm, temperature are 30～32 DEG C, aerobic fermentation is 0.6-0.8 to OD600 under dark condition；Merge fermentation liquid, make fermentation liquid Volume is equivalent to the 80% of culture bottle volume, then rotating speed be 140～150rpm, temperature be 30-32 DEG C, micro-oxygen under dark condition Ferment 36 hours.China Agricultural University Guo faces upward east etc. and has invented and a kind of improve the method for carotenoid content in tamato fruit (CN104686215A), the method mainly utilizes the melatonin solution of variable concentrations that green ripe stage Fructus Lycopersici esculenti is sprayed process, from And reach to improve the content of carotenoid in Fructus Lycopersici esculenti.

Although above-mentioned relevant purification, the Patents technology preparing carotenoid technique are advanced, but some existence are by a huge sum of money Belonging to the possibility polluted, such as copper, and fermentation technique technique is relative complex, and high to producing equipment requirements, this adds undoubtedly and produces into Originally with Technique Popularizing difficulty.They are not directed to be widely used in the mature technology plant growing of crops agricultural production and adjust The cultivation fast, easy of agent induced growth, the content of the microalgae accumulation carotenoid that yield is high, aquaculture cost is cheap.

Summary of the invention

The present invention provides one to it is an object of the invention to provide one, and technique is simple, speed is fast, cost is low, pollution-free, peace The side utilizing plant growth regulator 6-BA induction fresh water Chlorella pyrenoidesa ZF algae strain efficient accumulation carotenoid of Quan Xingqiang Method.

Its concrete technical scheme is:

The present invention provides one to utilize plant growth regulator 6-BA to induce fresh water Chlorella pyrenoidesa ZF algae strain efficient accumulation class The method of carotene it is characterized in that use following steps:

1) prepare algae solution: under conditions of 23 DEG C ± 2 DEG C, light intensity 1900 ± 100lx lx and Light To Dark Ratio are 12h/12h, use 1/4 times of concentration BG11 culture medium culturing fresh water Chlorella pyrenoidesa ZF algae strain arrives exponential phase in 28 ± 1 days, it is thus achieved that for 6-BA The algae solution of induction, the BG11 nutritive salt that during cultivation, every 14 ± 1 days add 1 times of concentration；

2) accumulation carotenoid: trophophase algae solution 200mL of taking the logarithm is placed in 300mL triangular pyramidal bottle, is subsequently adding 6-BA extremely Working concentration is 0.01 ± 0.005mg/L, and in 1900 ± 100lx, 23 DEG C ± 2 DEG C carry out induction and process 14 ± 1 days, processing stage Every day manually shakes algae 3 ± 1 times, and shaking algae time interval is 5 ± 0.5 hours, inducing carotenoid Rapid Accumulation in algae solution.

Compared with prior art, its advantage is the present invention:

1, simple, raw material fresh water Chlorella pyrenoidesa ZF algae strain cell is easily cultivated, and the cycle is short, low cost, strong stress resistance, Plant growth regulator inductive technology maturation, instant effect, pollution-free, production technology is simple, yield is high；

2, the production efficiency of the fresh water Chlorella pyrenoidesa ZF intracellular carotenoid of algae strain can be significantly improved in the short time, research Showing, after induction process, fresh water Chlorella pyrenoidesa ZF algae strain cell class Hu Luosu content is up to the 15.78% of frustule dry weight (be in blank group frustule 3.1 times of carotenoid content), yield are up to 42.45mg/L algae solution.

Detailed description of the invention

Embodiment 1, employing following steps:

1) prepare algae solution: under conditions of 22 DEG C, light intensity 2000lx and Light To Dark Ratio are 12h/12h, use 1/4 times of concentration BG11 Culture medium culturing fresh water Chlorella pyrenoidesa ZF algae strain arrives exponential phase in 27 days, it is thus achieved that for the algae solution of 6-BA induction, cultivates Every 14 days of period added the BG11 nutritive salt of 1 times of concentration；

2) accumulation carotenoid: trophophase algae solution 200mL of taking the logarithm is placed in 300mL triangular pyramidal bottle, is subsequently adding Beijing rope The 6-BA that Lai Bao Science and Technology Ltd. produces is 0.01mg/L to working concentration, and at 2000lx, 24 DEG C carry out induction and process 14 My god, processing stage every day manually shake algae 3 times, shaking algae time interval is 5 hours, quickly long-pending in algae solution of inducing carotenoid Tired.

Embodiment 2, employing following steps:

1) prepare algae solution: under conditions of 24 DEG C, light intensity 1800x and Light To Dark Ratio are 12h/12h, use 1/4 times of concentration BG11 training Support base cultivation fresh water Chlorella pyrenoidesa ZF algae strain and arrive exponential phase in 28 days, it is thus achieved that for the algae solution of 6-BA induction, culture period Between within every 14 days, add the BG11 nutritive salt of 1 times of concentration；

2) accumulation carotenoid: trophophase algae solution 200mL of taking the logarithm is placed in 300mL triangular pyramidal bottle, is subsequently adding north

The 6-BA that Jing Suolaibao Science and Technology Ltd. produces is 0.0095mg/L to working concentration, induces in 1800x 23 DEG C Process 15 days, processing stage every day manually shake algae 3 times, shake algae time interval 5 hours, fast in algae solution of inducing carotenoid Speed accumulation.

Experiment detection:

1) enrichment frustule: 8000-9000 rpm, 4 DEG C centrifugal 5-8 minute, obtains bath mud, then is done-40 DEG C of freezings by algae mud Dry algae powder；

2) transfer in the acetone soln of 90% under dark condition, after 4 DEG C of extracting 24h, measure 663.6,646.6 and 470 nm Extinction value；

3) according to below equation computational analysis:

4) result of calculation is:

Embodiment 1 gained class Hu Luosu content be the 15.72%(of frustule dry weight be carotenoid in blank group frustule 3.0 times of content) yield is up to 42.24mg/L algae solution；

Embodiment 2 gained class Hu Luosu content is class in blank group frustule up to the 15.78%(of frustule dry weight respectively 3.1 times of carotene carotene content), yield is 42.45mg/L algae solution.

Claims (1)

1. one kind utilizes plant growth regulator 6-BA induction fresh water Chlorella pyrenoidesa ZF algae strain efficient accumulation carotenoid Method, it is characterised in that employing following steps:

1) prepare algae solution: under conditions of 23 DEG C ± 2 DEG C, light intensity 1900 ± 100lx and Light To Dark Ratio are 12h/12h, use 1/4 Times concentration BG11 culture medium culturing fresh water Chlorella pyrenoidesa ZF algae strain cell 28 ± 1 days is to exponential phase, it is thus achieved that for 6- The algae solution of BA induction, the BG11 nutritive salt that during cultivation, every 14 ± 1 days add 1 times of concentration；

2) accumulation carotenoid: trophophase algae solution 200mL of taking the logarithm is placed in 300mL triangular pyramidal bottle, is subsequently adding 6-BA extremely Working concentration is 0.01 ± 0.005mg/L, and in 1900 ± 100lx, 23 DEG C ± 2 DEG C carry out induction and process 14 ± 1 days, processing stage Every day manually shakes algae 3 ± 1 times, and shaking algae time interval is 5 ± 0.5 hours, inducing carotenoid Rapid Accumulation in algae solution.