CN105803030A - Method for inducing B12 botryococcus braunii to efficiently accumulate carotenoids by utilizing plant growth regulator EBR - Google Patents

Abstract

The invention provides a method for inducing a botryococcus braunii B12 strain to efficiently accumulate carotenoids by utilizing a plant growth regulator EBR. 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 EBR 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 botryococcus braunii B12 strain in a short time.

Description

A kind of method utilizing plant growth regulator EBR induction B12 Botryococcus braunii efficient accumulation carotenoid

Technical field

The present invention provides a kind of and utilizes the plant growth regulator EBR method inducing Botryococcus braunii B12 algae strain efficient accumulation carotenoid, belongs to biological technical field.

Background technology

Botryococcus braunii (Botryococcusbraunii) is under the jurisdiction of Xanthophyta (Xanthophyta), Xanthophyceae (Xanthophyceae), handle ball Cutleriales (Mischococcales), Wild Vitis species section (Botryococcaceae), Fructus Vitis viniferae Trentepohlia (Botryococcus), is a kind of unicellular microalgae of universal fresh water.Botryococcus braunii contains higher extracellular polysaccharide, fatty acid, especially hydrocarbonaceous amount.In culture, it produces hydrocarbon amount is the 0.3% ~ 76.0% of biomass dry weight, is generally 25% ~ 40%, Natural Samples is up to 86%, being much higher than the hydrocarbonaceous amount (nearly all lower than 1%) of other microorganisms, its product hydrocarbon mean calorie is higher, and the complete ignition energy of 1kg Fructus Vitis viniferae hydrocarbon releases 3.0 × 10⁴～4.2 × 10⁴KJ heat, to Atmospheric CO after burning₂Content is extremely similar to oil without the Nomenclature Composition and Structure of Complexes of the produced hydrocarbon of net increase and Wild Vitis species, and in some oil deposit, almost all organic matter is all that this algae is formed, therefore be otherwise known as " oil algae ", it is expected most to become industry algae kind.

Carotenoid be widely present in animal, higher plant, fungus, algae a class terpenoid natural pigment.All carotenoid in form all can by there being in the middle of 11 conjugated double bonds lycopene (1ycopene) base structure of carbochains be derived by oxidation, hydrogenation, dehydrogenation, cyclisation and the rearrangement of carbon skeleton, degraded.Carotenoid has highly important physiological function in vivo, and it is the accessory pigments of photosynthesis of plant, plays an important role in the adverse effect protecting cells from high light, active oxygen and sensitization pigment.Meanwhile, carotenoid has higher pharmaceutical value, is possible not only to the generation of prevention oculopathy and cardiovascular disease by carotenoid of ingesting, it is also possible to strengthens the immunologic function of human body, has anticancer, antioxidation, anti-ageing effect of waiting for a long time.Therefore, carotenoid, as nutrition, health care, the multifunctional natural pigment such as painted, is widely used to the industrial circles such as food, medicine, health product, feedstuff, cosmetics.Plant growth regulator gets final product the growth promoter of high efficiency regulatory plant and the kind of secondary metabolites and yield under relatively low concentration, has been widely used on higher plant especially production estimation.Plant growth regulator induction has the plurality of advantages such as simple to operate, inductivity is high, speed is fast, cost is low, pollution-free, high safety.Microalgae is by the excellent material of plant growth regulator regulation and control secondary metabolism, and its simple in construction, life cycle are short, heliosensitivity is strong, metabolic process is very easily affected by environment, are also easily detected.

At present, having been disclosed for some about preparation, the correlation technique purifying carotenoid technique in external, great majority are for technique improvements such as some fermentation, purification & isolation.Ocean rhodotorula High Yield of Carotenoid and copper fermentation culture method (CN201510512641) is utilized as University Of Qingdao discloses one, the purpose of this invention be in that to provide a strain separate from marine environment obtain can High Yield of Carotenoid again can the ocean rhodotorula of enriching Cu, and provide it to be capable of the best fermentation culture conditions of high yield carotene enriching Cu, provide theoretical foundation for industrialized production.Concrete method is: preferred fermentation medium consist of 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%, all the other be water；Initial pH is 8, inoculum concentration 3-8%, and shaking speed is 50-80r/min, 24-28 DEG C and cultivates and adjust temperature after 10-15 hour and be 16-20 DEG C and carry out low temperature static gas wave refrigerator, and stopping 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, and the addition according to 2.5% adds peptone, and the amount according to 2% adds yeast powder；Shaking speed is 50-100r/min, continues fermentation 10-15h.Preferably, in the low temperature static gas wave refrigerator stage to the methionine chela and the copper that add culture medium weight 3-5wt% in culture medium.nullA kind of method (CN104846049A) improving fermentative carotenoid productivity of Li Da bio tech ltd, Weihai invention，Using Rhodotorula mucilaginose as fermentative carotenoid strain，It is that described rhodotorula mucilaginosa is seeded in culture medium by 2%-10% according to inoculum concentration，Described culture medium includes: glucose 12-130g/L、Yeast powder 2-22g/L、Salt 3-12g/L，The pH of this culture medium is between 5.5-7.5，Ferrous ion solvent is added in described culture medium，The addition of described ferrous ion solvent is that the molar concentration of relatively described culture medium is between 0.05-0.5mmol/L，Fermentation temperature maintains 27-33 DEG C，After fermenting 32-40 hour，Shaking table speed 140-200r/min，Cool the temperature to 20-25 DEG C and continue fermentation，Ferment 24-46 hour，Shaking table speed 80-140r/min.Sichuan University of Science & Engineering provides a kind of method (CN104805168A) utilizing photosynthetic bacteria micro-aerobe fermentation quickly to produce carotenoid, by in single for the photosynthetic bacteria (Rhodobactersphaeroides) of activation colony inoculation to MMS culture medium, aerobic incubated overnight obtains seed；Then it is 8% the seed of aerobic incubated overnight is inoculated in the culture bottle containing MMS culture medium respectively by inoculum concentration, after inoculation, culture fluid volume is equivalent to the 40% of culture bottle volume, then rotating speed to be 200～250rpm, temperature be 30～32 DEG C, aerobic fermentation is 0.6-0.8 to OD600 under dark condition；Merge fermentation liquid, make fermentating liquid volume be equivalent to the 80% of culture bottle volume, then in rotating speed to be 140～150rpm, temperature be 30-32 DEG C, micro-aerobe fermentation 36 hours under dark condition.China Agricultural University Guo faces upward east etc. and has invented a kind of method (CN104686215A) improving carotenoid content in tamato fruit, the method mainly utilizes the melatonin solution of variable concentrations that green ripe stage Fructus Lycopersici esculenti is sprayed process, thus reaching to improve the content of carotenoid in Fructus Lycopersici esculenti.

Although above-mentioned relevant purification, the Patents technology preparing carotenoid technique are advanced, but the possibility that some existence are contaminated by heavy metals, such as copper, and fermentation technique technique relative complex, high to producing equipment requirements, this adds production cost and Technique Popularizing difficulty undoubtedly.They are not directed to be widely used in the content of the microalgae accumulation carotenoid that the mature technology plant growing of crops agricultural production is adjusted that induced growth is fast, easily cultivated, yield is high, aquaculture cost is cheap.

Summary of the invention

The present invention provide a kind of it is an object of the invention to provide that a kind of technique is simple, speed is fast, cost is low, pollution-free, high safety utilize the plant growth regulator EBR method inducing Botryococcus braunii B12 algae strain efficient accumulation carotenoid.

Its concrete technical scheme is:

The present invention provides a kind of and utilizes the plant growth regulator EBR method inducing Botryococcus braunii B12 algae strain efficient accumulation carotenoid to it is characterized in that adopting following steps:

1) algae solution is prepared: when 23 DEG C ± 2 DEG C, light intensity 1900 ± 100lxlx and Light To Dark Ratio are 12h/12h, 1/4 times of concentration BG11 culture medium culturing Botryococcus braunii B12 algae strain is adopted to arrive exponential phase in 28 ± 1 days, obtain the algae solution for EBR induction, the BG11 nutritive salt that during cultivation, every 14 ± 1 days add 1 times of concentration；

2) accumulation carotenoid: the trophophase algae solution 200mL that takes the logarithm is placed in 300mL triangular pyramidal bottle, being subsequently adding EBR to working concentration is 0.01 ± 0.005mg/L, in 1900 ± 100lx, 23 DEG C ± 2 DEG C carry out induction and process 14 ± 1 days, processing stage every day manually shake algae 3 ± 1 times, shaking algae 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 Botryococcus braunii B12 algae strain cell is easily cultivated, and the cycle is short, and cost is low, strong stress resistance, and plant growth regulator inductive technology maturation, instant effect, pollution-free, production technology is simple, yield is high；

2, the production efficiency of carotenoid in Botryococcus braunii B12 algae strain cell can be significantly improved in the short time, research shows, induction process after Botryococcus braunii B12 algae strain cell class Hu Luosu content up to the 4.21%(of frustule dry weight be in blank group frustule 1.13 times of carotenoid content), yield is 38.96mg/L algae solution.

Detailed description of the invention

Embodiment 1, adopts following steps:

1) algae solution is prepared: when 22 DEG C, light intensity 2000lx and Light To Dark Ratio are 12h/12h, 1/4 times of concentration BG11 culture medium culturing Botryococcus braunii B12 algae strain is adopted to arrive exponential phase in 27 days, obtain the algae solution for EBR induction, the BG11 nutritive salt that during cultivation, every 14 days add 1 times of concentration；

2) accumulation carotenoid: the trophophase algae solution 200mL that takes the logarithm is placed in 300mL triangular pyramidal bottle, the EBR being subsequently adding the production of Beijing Suo Laibao Science and Technology Ltd. is 0.01mg/L to working concentration, at 2000lx, 24 DEG C carry out induction and process 14 days, processing stage every day manually shake algae 3 times, shaking algae interval is 5 hours, inducing carotenoid Rapid Accumulation in algae solution.

Embodiment 2, adopts following steps:

1) algae solution is prepared: when 24 DEG C, light intensity 1800x and Light To Dark Ratio are 12h/12h, 1/4 times of concentration BG11 culture medium culturing Botryococcus braunii B12 algae strain is adopted to arrive exponential phase in 28 days, obtain the algae solution for EBR induction, the BG11 nutritive salt that during cultivation, every 14 days add 1 times of concentration；

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

The EBR that Jing Suolaibao Science and Technology Ltd. produces is 0.0095mg/L to working concentration, carries out induction process in 1800x23 DEG C

15 days, processing stage every day manually shake algae 3 times, shake algae interval 5 hours, inducing carotenoid Rapid Accumulation in algae solution.

Experiment detection:

1) enrichment frustule: 8000-9000rpm, 4 DEG C are centrifugal 5-8 minute, obtain bath mud, then by algae mud at-40 DEG C of lyophilization algae powders；

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

3) according to below equation computational analysis:

4) result of calculation is:

Embodiment 1 gained class Hu Luosu content is the 4.18%(of frustule dry weight be in blank group frustule 1.11 times of carotenoid content), yield is up to 38.59mg/L algae solution；

Embodiment 2 gained class Hu Luosu content respectively up to the 4.21%(of frustule dry weight be in blank group frustule 1.13 times of carotenoid content), yield is 38.96mg/L algae solution.

Claims (1)

1. one kind utilizes the plant growth regulator EBR method inducing Botryococcus braunii B12 algae strain efficient accumulation carotenoid, it is characterised in that adopt following steps:

1) algae solution is prepared: when 23 DEG C ± 2 DEG C, light intensity 1900 ± 100lx and Light To Dark Ratio are 12h/12h, 1/4 times of concentration BG11 culture medium culturing Botryococcus braunii B12 algae strain cell is adopted to arrive exponential phase in 28 ± 1 days, obtain the algae solution for EBR induction, the BG11 nutritive salt that during cultivation, every 14 ± 1 days add 1 times of concentration；

2) accumulation carotenoid: the trophophase algae solution 200mL that takes the logarithm is placed in 300mL triangular pyramidal bottle, being subsequently adding EBR to working concentration is 0.01 ± 0.005mg/L, in 1900 ± 100lx, 23 DEG C ± 2 DEG C carry out induction and process 14 ± 1 days, processing stage every day manually shake algae 3 ± 1 times, shaking algae interval is 5 ± 0.5 hours, inducing carotenoid Rapid Accumulation in algae solution.