WO2020182183A1 - 一组配对的天然β-胡萝卜素高产菌株及其制备方法和应用 - Google Patents

一组配对的天然β-胡萝卜素高产菌株及其制备方法和应用 Download PDF

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WO2020182183A1
WO2020182183A1 PCT/CN2020/078939 CN2020078939W WO2020182183A1 WO 2020182183 A1 WO2020182183 A1 WO 2020182183A1 CN 2020078939 W CN2020078939 W CN 2020078939W WO 2020182183 A1 WO2020182183 A1 WO 2020182183A1
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strain
strains
fermentation
ionone
trispora
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陈克杰
陈迎迎
刘燕
周旭燕
孙新强
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浙江医药股份有限公司新昌制药厂
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    • 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
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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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    • 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
    • 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
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the invention relates to the technical field of microbial fermentation, in particular to a pair of paired natural ⁇ -carotene high-yielding strains and a preparation method and application thereof.
  • Carotenoids are natural isoprene pigments biosynthesized by certain plants, algae, bacteria and fungi. According to whether they contain oxygen atoms, they can be divided into: (1) Carotenes of pure hydrocarbons. ), such as ⁇ -carotene, ⁇ -carotene, ⁇ -carotene and lycopene, etc.; (2) Xanthophylls containing oxygen atoms, such as astaxanthin, zeaxanthin, lutein and pepper Hongsu etc. Carotenoids have excellent anti-oxidation and coloring properties. The above two important properties give them a wide range of applications in the fields of medicine, food, health care products, and cosmetics.
  • Beta-carotene also known as provitamin A, is an important member of more than 600 carotenoids currently known. It has anti-inflammatory, detoxification, anti-cancer, prevention of cardiovascular diseases, prevention and treatment of cataracts, and liver protection. Medical and biological functions present great commercial value. Recent market research shows that the total ⁇ -carotene market in 2015 was US$436 million, and the market demand in 2017 was 371.56 tons. It is expected to reach US$572 million by the end of 2022, with a compound annual growth rate of 3.5%.
  • beta-carotene sold in the market can be divided into two categories: synthetic beta-carotene and natural beta-carotene according to different sources.
  • the latter includes natural production microorganisms (such as the algae Dunaliella salina).
  • the methods for commercial production of natural ⁇ -carotene mainly use Dunaliella salina and B. trispora.
  • Australia Aquacarotene and BASF (Australia) extract natural ⁇ -carotene from Dunaliella salina and microalgae, respectively.
  • the Vitan Company in Ukraine, Vitatene Company in Spain and Xinchang Pharmaceutical Factory use the fermentation of B. trispora to produce natural ⁇ -carotene. Due to the demanding environmental conditions of the production of the Dunaliella salina method, the production area is restricted, and only a few months of the year can be produced.
  • B. trispora is a natural ⁇ -carrot due to its safe application (B. trispora is a “Generally Recognized As Safe” (GRAS) strain of the US FDA), rapid growth, large hyphae, and high yield. Ideal strain for vegetable production.
  • GRAS Generally Recognized As Safe
  • B. trispora to produce natural ⁇ -carotene is characterized by the fact that the (+) and (-) strains of B. trispora undergo heterozygosity during the mixed culture process, and the (+) strain secretes three Sporic acid as a sex hormone stimulates the (-) strain that synthesizes only a small amount of pigment to accumulate large amounts of ⁇ -carotene when cultured alone.
  • the synthesis of trisporic acid is closely related to the metabolic pathway of ⁇ -carotene. The synthesis route is as follows:
  • US5422247 uses chemical mutagenesis to screen nystatin, lovastatin, ⁇ -ionone and duquinone resistant mutants, and investigates the effects of (+), (-) pairing ratio, kerosene and isoniazid, When 2% kerosene is added, the ⁇ -carotene output of the shake flask reaches 5.8 g ⁇ L -1 .
  • US7252965 designed the mutagenesis of B. trispora strain and the screening method of high-yielding strains, and optimized the cultivation conditions of the mutant strains. By adding ⁇ -ionone and purifying oxygen to enhance the oxygen supply level, the yield reached 9g. ⁇ L -1 .
  • EP1306444B1 adopts a mixture of lecithin and ⁇ -ionone oil in batches, and after 36 hours of fermentation, the pH value is controlled to 6.8 ⁇ 0.1, and the fermentation output reaches 5.5g ⁇ L -1 .
  • Both the US7252965 and EP1306444B1 patents simply add ⁇ -ionone in batches based on the fermentation time. The concentration of ⁇ -ionone is pulsating during the fermentation process, but ⁇ -ionone has certain effects on B. trispora. Toxicity, this method of operation is difficult to keep ⁇ -ionone at an appropriate concentration during the fermentation process or does not exceed the tolerance threshold concentration of B. trispora to ⁇ -ionone.
  • the kerosene proposed by US5422247 has certain carcinogenicity, which is not allowed by law.
  • surfactants such as lecithin, Span 20 and Triton X-100 will produce a large amount of foam, which limits its industrial application.
  • the strategy proposed by Desai et al. to add 1 mg ⁇ L -1 penicillin after 24 hours of fermentation to promote the synthesis of carotenoids in B. trispora, it is even more undesirable because of the issue of antibiotic residues.
  • B. trispora has complex genome characteristics.
  • the combination of vegetable oil and ⁇ -ionone can increase ⁇ -The characteristics of carotene production, the method of obtaining vegetable oil and ⁇ -ionone dual tolerance mutants through mutagenesis breeding technology to screen high-yielding strains is not a bad strategy.
  • the purpose of the present invention is to overcome the above-mentioned shortcomings in the prior art, and to provide a high-concentration vegetable oil with strong metabolic capacity and a high utilization rate, resistance to high-concentration ⁇ -ionone, so as to relieve the inhibition in the synthesis pathway Or high-yielding mutants with feedback effects.
  • the present invention adopts the following technical solutions: a pair of natural ⁇ -carotene high-yielding strains, which belong to the taxonomy of Blakeslea trispora, are heterozygous bacteria, including Blakeslea trispora ( +) and B. trispora(-) are two unisexual strains, the preservation number of the positive strain is CGMCC No. 16491, and the preservation number of the negative strain is CGMCC No. 16492.
  • Another object of the present invention is to provide a method for mutagenesis of B. trispora and screening of double tolerance mutant strains of vegetable oil and ⁇ -ionone to obtain a set of paired natural ⁇ -carotene high-yielding strains.
  • the technical scheme adopted to achieve the purpose of the invention is: starting from the B. trispora (+) strain X ⁇ -15-02 positive-03 and the B. trispora (-) strain X ⁇ -15-04 minus-03
  • the strains are selected by physical and/or chemical mutagenesis, using vegetable oil and ⁇ -ionone as the selection pressure to obtain high-yielding strains.
  • the mutagenesis breeding spore concentration of 10 4 to 10 8 / mL, and most preferably 10 5 to 10 6 / mL.
  • Another object of the present invention is to provide the application of the above-mentioned high-yielding strains in the preparation of natural ⁇ -carotene fermentation broth, and the technical solutions adopted are:
  • Fermentation tank culture the seed liquid of the high-yielding strains of B. trispora (+) and (-) strains are inoculated into a medium containing carbon sources, nitrogen sources, inorganic salts, antioxidants and nutrients, and cultured by stirring and aeration More than 3 days;
  • the pH value is controlled to be 5.5-7.5 with acid and/or alkali solution, and the ⁇ -ionone vegetable oil mixture is continuously added.
  • the matching ratio of the seed solution of the B. trispora (+) strain and the (-) strain is 1:3 ⁇ 1:200, preferably 1:4 ⁇ 1:50, the most It is preferably 1:8 to 1:12; the seed liquid is transplanted into the fermenter at a ratio of 5 to 15% v/v.
  • the fermentation culture temperature is 22 ⁇ 32°C, preferably 26 ⁇ 30°C; the compressed air ventilation ratio is 0.2 ⁇ 2.5vvm, and the best is 0.6 ⁇ 1.6vvm; commercial production scale stirring speed 15-250rpm, tank pressure is 0.01-0.1MPa, the dissolved oxygen is controlled to be greater than 5% during the process, and the optimal dissolved oxygen is controlled to be greater than 10%.
  • the alkali and acid controlling the pH value during the fermentation process are 10-30% by mass ammonia water and 0.1-2mol ⁇ L -1 HCl or phosphoric acid, respectively, and the control time is 10-80h after fermentation Start (optimally 20-40h), to the end of fermentation.
  • carbon sources and nitrogen sources can be added during the fermentation process to supplement nutrition and adjust the pH value during the fermentation process; these carbon sources include glucose, starch and glycerol; nitrogen sources include ammonium Salts such as ammonium chloride and ammonium sulfate, and nitrates such as potassium nitrate and sodium nitrate.
  • step b in the ⁇ -ionone vegetable oil mixture added during the fermentation process, the ⁇ -ionone is dissolved in the vegetable oil at a mass concentration of 5-30% (optimally 8-12%) after a mixture of a vegetable oil [beta] -ionone start additional fermentation time is 20 ⁇ 80h (optimally 30 ⁇ 50h);; ⁇ - ionone accelerated flow rate of the mixture of vegetable oil 0.01 ⁇ 0.5mL ⁇ h -1 ⁇ L - 1 (optimally 0.05-0.2 mL ⁇ h -1 ⁇ L -1 ), the mass concentration of vegetable oil after feeding is maintained at 0.2-4.0% (optimally 1.0-2.0%).
  • the composition of the culture medium is: carbon source 1 to 3%, nitrogen source 2 to 6%, KH 2 PO 4 0.02 to 0.2%, BHT 0.02 to 0.2%, VB 1 0.0001 to 0.001%, Vegetable oil 2-10%, ⁇ -ionone 0.01-0.5%, pH is adjusted to 5.0-9.0 with 2mol ⁇ L -1 NaOH solution before elimination, and the above percentages are all mass percentages.
  • the carbon source is one or more carbohydrates or fatty substances;
  • the nitrogen source is one or more organic nitrogen sources or inorganic nitrogen sources;
  • the vegetable oil is sesame oil, A mixture of one or more of sunflower oil, rapeseed oil, cottonseed oil, peanut oil, and soybean oil.
  • the present invention optimizes the process of dissolved oxygen, pH value, vegetable oil and ⁇ -ionone feeding on the obtained high-yield mutants to prepare natural ⁇ -carotene fermentation broth, and finally realize the unification of high yield, high yield and high production intensity .
  • the present invention adopts protoplast ultraviolet mutagenesis combined with chemical mutagenesis technology to carry out multiple rounds of mutagenesis breeding on original strains, using the screening pressure of ⁇ -ionone and vegetable oil on the screening plate and combining the pairing of Erlenmeyer (+) and (-) strains
  • the validated strategy obtained high-yield mutant strains with high concentration of ⁇ -ionone and vegetable oil adaptability: (+) strain XC0705 and (-) strain XC0706 combination.
  • High-yield strains have impurities that meet the requirements of industrial production, have better stability, and have a fast oil utilization rate.
  • the ⁇ -carotene output can reach 6.80g ⁇ L -1 on the shaker flask.
  • the production-scale fermentation tanks are used for such things as dissolved oxygen, pH and vegetable oil. With continuous flow control of ⁇ -ionone, the output can reach 8.42g ⁇ L -1 , which has obvious cost advantages in industrial production.
  • Figure 1 is a mutational pedigree diagram of the strains in Example 1 of the present invention.
  • the present invention provides a set of paired natural ⁇ -carotene high-yielding strains, which belong to Blakeslea trispora in taxonomy, belong to the order Mucorales, and the family of Mycomyces, and are heterozygous bacteria, including Blakeslea trispora (+) and B. trispora (-) are two unisexual strains. And it has been preserved, the specific preservation information is as follows:
  • CGMCC China Common Microorganism Collection Management Center
  • the deposit number of the positive strain is CGMCC No. 16491, and the deposit number of the negative strain is CGMCC No. 16492.
  • the enzymolysis solution was filtered with 4 layers of lens cleaning paper to remove the hyphae, centrifuged at 4000 rpm for 10 min, the supernatant was removed and resuspended with isotonic solution, repeated twice to obtain the protoplast suspension, and counted with a hemocytometer to obtain the total number of protoplasts.
  • composition of the seed culture medium is: carbon source 3 to 6%, nitrogen source 1 to 3%, KH 2 PO 4 0.02 to 0.2%, VB 1 0.0001 to 0.001%, using 2mol ⁇ L -1 Adjust the pH value of the NaOH solution to 5.5-7.5.
  • the carbon source may be one or more carbohydrates or fatty substances, such as starch, dextrin, maltose, sucrose, lactose, glucose, molasses, vegetable oil or animal oil; the nitrogen source may be one or more An organic or inorganic nitrogen source, such as ammonium salt (NH 4 Cl, (NH 4 ) 2 SO 4 ), nitrate (NaNO 3 , KNO 3 ), urea, raw/cooked soybean meal, corn steep liquor/dry powder, yeast Flour, yeast extract, cottonseed meal, corn meal.
  • the solid slant medium is a PDA medium, and its preparation method is as follows: peel the potatoes, cut them into cubes, weigh 200 g, add an appropriate amount of water, boil for 20 minutes, filter the clear liquid with 8 layers of gauze, and set the volume to 1L. Add 20g glucose and 20g agar powder, heat and melt, and it is ready for use.
  • Said isotonic solution 0.6mol ⁇ L -1 NaCl solution.
  • the condition with the lethality rate of 95% is selected as the UV mutagenesis condition of protoplasts, that is, the UV irradiation time is 50s.
  • Said resistant plate medium 2-10% vegetable oil, 0.01-0.5% ⁇ -ionone and 0.15-1.5% emulsifier are added to the PDA medium.
  • the vegetable oil can be one or more vegetable oils, such as sesame oil, sunflower oil, rapeseed oil, cottonseed oil, peanut oil, and soybean oil.
  • the emulsifier can be one or more surfactants, especially surfactants with a hydrophilic-lipophilic balance (HLB value) between 6 and 8, such as Tween series, Span series, soybean phospholipids, fatty acids Glycerides, higher fatty acid salts.
  • HLB value hydrophilic-lipophilic balance
  • the preliminary screening of high-yielding mutant strains is carried out by using the screening pressure of ⁇ -ionone and vegetable oil on the resistant plate, combined with the strategy of pairing verification of Erlenmeyer flask (+) and (-) strains.
  • the principle is based on the principle that high-yielding mutant strains must have high concentrations of vegetable oil. Adaptability and high lipase activity, screening of the trisporic acid structural analogue ⁇ -ionone resistant mutants can relieve the feedback inhibition/repression that may exist in the pathway.
  • the process is to coat the spores obtained by regeneration culture after ultraviolet mutagenesis of protoplasts or the spores treated by chemical mutagenesis on a resistant plate with high ⁇ -ionone concentration and high vegetable oil concentration, at a temperature of 22 ⁇ 32°C , Cultivate for 96-192h under the condition of 20-80% humidity. After the bacteria grow on the plate, perform a shake flask preliminary screening. It should be pointed out that due to the luxuriant filamentous fungi of B. trispora, poor mass transfer and agglomeration when cultured in a 96-well plate or test tube, the present invention still uses a triangular flask as a primary screening method.
  • the process is as follows: inoculate the fermentation medium (FM medium, with the colonies of the mutant strain of B. trispora (-) grown on the rod-point resistant plate 25mL/250mL), and then inoculate the spores of the original strain X ⁇ -15-02positive-03 of B. trispora(+) strain with another, stir it in 1-20mL sterile water, and draw 1mL spore liquid into a triangular flask It is paired with the mutant strain of B. trispora(-), and cultured with shaking for 96-144 hours at a temperature of 22-32°C, a rotation speed of 150-350 rpm, and a humidity of 20-80%.
  • the process is as follows: inoculate the colonies of the B. trispora (+) mutant strain grown on a rod-point resistant plate, and stir in 1-20mL sterile water. , Draw 1mL of inoculation FM medium (25mL/250mL), and then inoculate the spores of the original strain X ⁇ -15-04 minus-03 of B.
  • trispora(-) strain with another one, and inoculate it into a triangular flask with trisporum
  • Mutant strains of the pullomyces (+) strain are paired, and cultured for 96-144 hours at a temperature of 22-32°C, a rotation speed of 150-350 rpm, and a humidity of 20-80%.
  • the above-mentioned fermentation broth sample was processed according to the method described in CN 101955455B, and the specific process was as follows: a 2mL pipette was used to suck 1mL fermentation broth into a 15mL polyplastic test tube and placed in a refrigerator at -10°C for 1 hour. Take out the natural dissolution, add 10 mL of water for ultrasonic treatment for 5 min, centrifuge at 4000 rpm for 10 min, discard the supernatant, add 5 mL of absolute ethanol, centrifuge at 4000 rpm for 10 min, and discard the supernatant.
  • strains of B. trispora(-) mutant strains were obtained by preliminary screening, which were nF 2 70, nF 2 779, nF 3 97, nF 3 123, nF 3 127, nF 3 256, nF 3 301 , NF 3 415, nF 3 489, nF 3 554, nF 3 591, nF 3 682, nF 3 745, nF 3 774 and nF 3 810; 12 strains of B.
  • trispora(+) mutant strains were obtained through preliminary screening, They are pF 2 395, pF 2 652, pF 3 29, pF 3 78, pF 3 182, pF 3 240, pF 3 437, pF 3 499, pF 3 570, pF 3 601, pF 3 673, and pF 3 711, respectively.
  • the composition of the fermentation medium is: carbon source 1 to 3%, nitrogen source 2 to 6%, KH 2 PO 4 0.02 to 0.2%, BHT 0.02 to 0.2%, VB 1 0.0001 to 0.001%, Vegetable oil 2-10%, ⁇ -ionone 0.01-0.5%, pH value 5.0-9.0 adjusted with 2mol ⁇ L -1 NaOH solution before elimination.
  • the carbon source may be one or more carbohydrates or fatty substances, such as starch, dextrin, maltose, sucrose, lactose, glucose, molasses, vegetable oil or animal oil;
  • the nitrogen source may be one or more An organic or inorganic nitrogen source, such as ammonium salt (NH 4 Cl, (NH 4 ) 2 SO 4 ), nitrate (NaNO 3 , KNO 3 ), urea, raw/cooked soybean meal, peanut meal, corn syrup /Dry powder, yeast powder, yeast extract, cottonseed meal, corn meal.
  • the vegetable oil can be one or more vegetable oils, such as sesame oil, sunflower oil, rapeseed oil, cottonseed oil, peanut oil, and soybean oil.
  • the purpose of the re-screening is to obtain the best combination of B. trispora (+) and B. trispora (-) mutants, and to make the system have the highest stability.
  • all the mutant strains obtained were coated with PDA medium, cultured at a temperature of 22 ⁇ 32°C and humidity of 20 ⁇ 80% for 96 ⁇ 192h to obtain mature spores, and then 2cm 2 spore colonies were inoculated from the PDA medium to SM Culture medium (25mL/250mL), temperature 22 ⁇ 32°C, humidity 20 ⁇ 80%, 150 ⁇ 350rpm shaking culture for 36 ⁇ 72h.
  • trispora (-) mutant strain obtained by the above conditions were paired in sequence, (-) strain: (+) strain according to 3:1 ⁇ 200: The ratio of 1 is matched, preferably 4:1 ⁇ 50:1, most preferably 8:1 ⁇ 12:1, inoculation ratio 5 ⁇ 15%, inoculation FM medium (25mL/250mL), temperature 22 ⁇ 32°C, Incubate with shaking for 96-144 hours at a humidity of 20-80% and a rotation speed of 150-350 rpm.
  • the processing method of the second-screened fermentation bottle sample is the same as the aforementioned initial-screened fermentation bottle sample processing method, and then the concentration and content of ⁇ -carotene are determined by high performance liquid chromatography (HPLC method).
  • (+) strain pF 3 570 and (-) strain nF 3 123 are respectively named: (+) strain XC0705 and (-) strain XC0706, which are high-yield mutant strains obtained by screening in the present invention.
  • the high-yield mutant strains XC0705 and XC0706 are regulated on the fermenter for dissolved oxygen, pH value, and vegetable oil and ⁇ -ionone flow addition, so as to achieve a unified method of high yield, high yield, and high production intensity.
  • the specific process is As described below:
  • step (b) Seed culture The spore suspensions of (+) strain XC0705 and (-) strain XC0706 obtained in step (a) above were respectively inoculated with SM medium (100mL/1000mL) to control the final concentration of spores at 10 5 ⁇ 10 7 A ⁇ mL -1 , shake culture for 36-72h under the conditions of temperature 22 ⁇ 32°C, humidity 20 ⁇ 80%, rotation speed 150 ⁇ 350rpm.
  • the above-mentioned (+) and (-) seed cultures were respectively inoculated into seed pots for amplification, and the seed pot medium was the same as the first stage SM medium.
  • the seed tank inoculation ratio is 0.1-15% (v/v), the temperature is 22-32°C, the aeration ratio is 0.2-2.5vvm, the best is 0.6-1.6vvm, the stirring speed is 50-500rpm, the tank pressure 0.01 ⁇ 0.1MPa, the culture time is 16 ⁇ 60h, the second stage seed pot seed culture is obtained.
  • the seed tanking/transplanting ratio is 0.1-15% (v/v), the temperature is 22-32°C, the aeration ratio is 0.2-2.5vvm, the best is 0.6-1.6vvm, and the stirring speed is 30-300rpm ,
  • the tank pressure is 0.01 ⁇ 0.1MPa, the culture time is 16 ⁇ 60h, and enough seed culture is obtained.
  • Fermentation culture The seed culture of (+) strain XC0705 and (-) strain XC0706 obtained in step (b) above should be 1:3 ⁇ 1:200, preferably 1:4 ⁇ 1:50, the most Preferably, the ratio of 1:8 to 1:12 is mixed evenly, and the FM medium is transplanted.
  • the transplanting ratio is 5 to 15% (v/v), the culture temperature is 22 to 32°C, the best is 26 to 30°C, the aeration ratio is 0.2 to 2.5 vvm, and the best is 0.6 to 1.6 vvm, and the small-scale stirring speed is 50 ⁇ 500rpm, commercial production scale stirring speed 15 ⁇ 250rpm, tank pressure 0.01 ⁇ 0.1MPa, the dissolved oxygen is controlled to be greater than 5% in the process, and the optimal dissolved oxygen is controlled to be greater than 10%.
  • the control time is from the start of the fermentation 10 to 80h, and the optimal fermentation is 20-40h until the fermentation End.
  • carbon sources include glucose, starch and glycerol
  • nitrogen sources include ammonium salts such as chloride. Ammonium, ammonium sulfate, nitrates such as potassium nitrate and sodium nitrate.
  • the vegetable oil can be one or more vegetable oils, such as sesame oil, sunflower oil, rapeseed oil, cottonseed oil, peanut oil, and soybean oil.
  • the utilization rate of the strains for vegetable oil during the fermentation process is much stronger than that of the original strains.
  • the utilization rate of vegetable oil in the fermentation process increases as the synthesis rate of ⁇ -carotene increases.
  • the utilization rate of ⁇ -ionone is also closely related to the synthesis rate of ⁇ -carotene. Therefore, maintaining a proper vegetable oil concentration can maintain the ⁇ -ionone in a proper range, and the strain does not appear to be poisoned, which leads to a decrease in bacterial concentration or hindered product synthesis.
  • the fermentation broth treatment and the ⁇ -carotene content determination were carried out as described in the re-screening section.
  • Example 1 This example illustrates the method for obtaining the high-yielding strain of B. trispora
  • SM medium 25mL/250mL
  • the seed bottle was shaken and cultured for 48 hours under the conditions of a temperature of 28° C., a rotation speed of 250 rpm, and a humidity of 40-60% to obtain B. trispora hyphae.
  • the enzymatic hydrolysate was filtered with 4 layers of lens cleaning paper to remove the hyphae, centrifuged at 4000 rpm for 10 min, the supernatant was removed and resuspended in 0.6 mol ⁇ L -1 NaCl isotonic solution, repeated twice to obtain the protoplast suspension, and used a hemocytometer counting protoplasts was diluted to 10 6 ⁇ mL -1.
  • the condition of 95% lethality was selected as the UV mutagenesis condition of protoplasts, that is, the UV irradiation time is 50s.
  • the spores obtained by regeneration culture after ultraviolet mutagenesis of protoplasts were coated on RM medium with the following medium components: soybean oil 10%, ⁇ -ionone 0.08%, soybean phospholipid 0.65%, and other components were the same as PDA medium.
  • the resistant plate is subjected to (+) and (-) strain screening after the plate has grown bacteria under the conditions of a temperature of 28°C and a humidity of 40-60%.
  • the screening operation is to inoculate the rod point (+) starting strain or resistant mutant strain, stir and dilute it in 10 mL sterile water, and transfer 1 mL of spore liquid to the fermentation shake flask to compare with the B. trispora (-) mutant strain Or the starting strains are paired, that is, the starting strains of the (+) and (-) strains are used as the matched strains of the (-) and (+) mutant strains, respectively.
  • (+) Spore pairing ratio is 1:10, inoculate FM medium (25mL/250mL shake flask) containing the following ingredients: cooked soybean meal 3.4%, cottonseed meal 1.6%, corn starch 2.5%, KH 2 PO 4 0.1%, BHT 0.05%, VB 1 0.0002%, soybean oil 5%, ⁇ -ionone 0.02%, adjust pH to 7.5 with 2mol ⁇ L -1 NaOH solution before elimination.
  • the fermentation flask was cultured with shaking for 144h at a temperature of 28°C, a humidity of 40-60%, and 250rpm.
  • the mutant strains were initially screened by measuring the OD 454 of the sample to calculate the carotenoid content. After protoplast mutagenesis, the (+) strain was screened to 4 positive mutant strains, namely: pF 1 113, pF 1 168, pF 1 220, and pF 1 365. (-) Four positive mutant strains were screened, namely: nF 1 90, nF 1 217, nF 1 339, and nF 1 418.
  • (+) and (-) mutant strains were sporulated suspensions, and the dilutions of the (+) strains and the dilutions of the (-) strains were combined in equal amounts to obtain B. trispora (+), (-) Mutant single spore pool.
  • (+) strains were screened to obtain 5 high-yielding mutant strains, namely: pF 2 25, pF 2 221, pF 2 380, pF 2 395, pF 2 652.
  • mutant strains 12 (+) strains and 15 (-) strains of high-yielding mutants (each strain containing 2 high-yielding mutants of the second generation) were coated with PDA medium at a temperature of 28°C and humidity. Cultivate for 120h under 40-60% conditions to obtain mature spores, and then inoculate SM medium (25mL/250mL) with 2cm 2 spore colonies from PDA medium, and culture at 28°C, humidity 40-60%, 250rpm with shaking 48h. The seed cultures of the mutant strains of B. trispora (+) and B.
  • trispora (-) obtained by the above conditions were paired in sequence according to the ratio of (-) strain: (+) strain of 10:1, and press 10
  • The% inoculation ratio was inoculated with FM medium (25mL/250mL), and the temperature was 28°C, humidity was 40-60%, and the rotation speed was 250rpm for shaking culture for 144h.
  • (+) pF 3 570 and (-) nF 3 123 produced at the shake flask level The highest, reaching 6.80g ⁇ L -1 , and the parallelism and stability are good.
  • the (+) strain pF 3 570 and the (-) strain nF 3 123 were named XC0705 and XC0706, respectively, and they were high-yield mutant strains screened in the present invention.
  • Example 2 This example illustrates the passage stability of high-yielding mutant strains
  • the screened (+) strain XC0705 and (-) strain XC0706 were respectively coated with PDA slant medium and cultured at a temperature of 28°C and a humidity of 40-60% for 120 hours to obtain mature spores of (+) and (-) high-yield mutants , Which is the F 1 generation.
  • a 2cm 2 F 1 generation slant spore colony was inoculated with the same SM medium (25mL/250mL) in Example 1, at a temperature of 28°C, a humidity of 40-60%, and a rotation speed of 250rpm for shaking culture for 48h.
  • Example 3 This example illustrates the ability of a high-yielding mutant strain to produce ⁇ -carotene by shaking flask fermentation
  • the high-yielding mutant strain has strong adaptability to vegetable oil and ⁇ -ionone, its lipase activity is stronger, the rate of metabolizing vegetable oil is significantly higher than that of the starting strain, and the feedback inhibition/repression that may exist in the pathway is lifted, so The shake flask further verified the ability of the high-yielding mutant strain to produce ⁇ -carotene by fermentation.
  • Example 2 Was prepared as previously described and inoculated with a spore suspension of single SM media (25mL / 250mL) in Example 1, a concentration of 106 spores / mL, and at a temperature of 28 °C, humidity 40% to 60%, under the conditions at 250rpm Shake culture for 48 hours to obtain (+) and (-) high-yield mutant laboratory seed culture solutions.
  • the other ingredients were the same as in Example 1), and the fermentation flask was cultured at a temperature of 28°C, a humidity of 40-60%, and a rotating speed of 250 rpm for 144 hours.
  • the FM medium contains 8% soybean oil and 0.04% ⁇ -ionone
  • the yield of ⁇ -carotene in the high-yield mutant shake flask can reach 7.22 g/L.
  • the starting strains X ⁇ -15-02 positive -03 and X ⁇ -15-04 negative -03 in the fermentation medium of 5% soybean oil and 0.02% ⁇ -ionone the yield is only 4.83g under the same experimental conditions /L.
  • the control strains are X ⁇ -15-02 plus -03 and X ⁇ -15-04 minus -03; the strains in groups A to D are XC0705 and XC0706.
  • Example 4 This example illustrates the method for producing ⁇ -carotene in a 70L fermentor with a high-yield mutant strain
  • (+) strain XC0705 and (-) strain XC0706 obtained by the screening of high-yield mutant strains were obtained according to the method described in Example 3 to obtain (+) and (-) mutant strain laboratory seed culture solutions.
  • the above-mentioned (+) and (-) seed culture solutions were respectively inoculated with 20L/30L seed pots.
  • the seed pot medium was the same as the SM medium in the first stage.
  • the inoculation ratio of (+) and (-) seed tanks is 0.5% (v/v), the temperature is 28°C, the aeration ratio is 1.0vvm, the tank pressure is 0.05MPa, the stirring speed is 200rpm, and the culture time is 48h. Seed pot seed culture.
  • the second stage seed pot seed culture of (+) strain XC0705 and (-) strain XC0706 obtained above were mixed at a ratio of 1:10, and transplanted in a 70L fermentor at a transplant ratio of 10% (total 4L), and fermented Canned liquid 40L.
  • the FM medium formula of the fermentation tank is: cooked soybean powder 3.4%, cottonseed powder 1.6%, dextrin 2.5%, KH 2 PO 4 0.1%, BHT 0.05%, VB 1 0.0002%, soybean oil 8%, ⁇ -ionone 0.04 %, adjust the pH to 7.5 with 2mol ⁇ L -1 NaOH solution before elimination.
  • the initial conditions are temperature 29.5°C, aeration ratio 0.5vvm, stirring speed 200rpm, tank pressure 0.05MPa, after 20-25h, adjust the aeration ratio to 1.0vvm, and use 25-28% ammonia and 2mol ⁇ L -1 at 40h
  • the pH value of the HCl solution is controlled to be 6.8, and the whole stirring is linked with the dissolved oxygen to maintain the dissolved oxygen at 10-30%.
  • the yield of ⁇ -carotene reached 7.44g/L.
  • Example 5 This example illustrates the effect of adding physiologically acidic or alkaline carbon sources and nitrogen sources to the fermentation tank
  • the second stage seed pot seed culture of (+) strain XC0705 and (-) strain XC0706 obtained above were mixed at a ratio of 1:10, and transplanted in a 70L fermentor at a transplant ratio of 10% (total 4L), and fermented Canned liquid 40L.
  • the FM medium formula of the fermenter is the same as in Example 4.
  • the initial conditions are temperature 29.5°C, aeration ratio 0.5vvm, stirring speed 200rpm, tank pressure 0.05MPa, after 20-25h, adjust the aeration ratio to 1.0vvm, and use 25-28% ammonia and 2mol ⁇ L -1 at 40h
  • the HCl solution controls the pH at 6.8.
  • other physiologically acidic or alkaline carbon sources and nitrogen sources are added to enhance the nutrient supply during the fermentation process and also adjust the pH value during the fermentation process.
  • Table 3 The experimental results are shown in Table 3:
  • Example 6 This example illustrates the method of increasing the production of ⁇ -carotene by the mutant strain by adding vegetable oil and ⁇ -ionone on the fermentation tank
  • the second stage seed pot seed culture of (+) strain XC0705 and (-) strain XC0706 obtained above were mixed at a ratio of 1:10, and transplanted in a 70L fermentor at a transplant ratio of 10% (total 4L), and fermented Canned liquid 40L.
  • the FM medium formula of the fermenter is the same as in Example 4.
  • the initial conditions are temperature 29.5°C, aeration ratio 0.5vvm, stirring speed 200rpm, tank pressure 0.05MPa, after 20-25h, adjust the aeration ratio to 1.0vvm, and use 25-28% ammonia and 2mol ⁇ L -1 at 40h
  • the phosphoric acid solution controls the pH at 6.8.
  • glycerol and 1.5g/L ammonium sulfate were added to supplement the carbon source and nitrogen source.
  • the soybean oil content of the fermentation broth is detected.
  • the soybean oil mixture of 10% ⁇ -ionone is continuously added at a flow rate of 0.15 mL ⁇ h -1 ⁇ L -1 to maintain The concentration of soybean oil is between 1.0 and 1.5%.
  • the whole process of stirring is linked with dissolved oxygen to maintain 10-30% of dissolved oxygen. After 144h of cultivation, the yield of ⁇ -carotene reached 8.26g/L.
  • Example 7 This example illustrates the ability of a commercial-scale high-yield mutant strain to produce ⁇ -carotene
  • (+) and (-) high-yield mutant laboratory seed culture solutions were prepared.
  • the obtained (+) and (-) seed culture solutions were respectively connected to two 1m 3 (counting volume 0.5m 3 ) seed pots at a ratio of 0.5%.
  • the cultivation temperature of (+) and (-) seed pots is 28°C
  • the aeration ratio is 1.0vvm
  • the pot pressure is 0.05MPa
  • the stirring speed is 150rpm
  • the cultivation time is 48h.
  • the second stage (-) seed pot seed culture obtained above was transferred to 10m 3 (stock volume 5m 3 ) seed pot at a ratio of 10%, at a temperature of 28°C, aeration ratio of 1.0vvm, pot pressure of 0.05MPa, and stirring
  • the rotation speed is 100 rpm and the cultivation time is 30 hours to obtain sufficient (-) seed culture solution.
  • the above-mentioned production-scale seed tank formulas are all SM formulas described in Example 1.
  • the (+) and (-) seed liquids obtained from 1m 3 and 10m 3 seed tanks were simultaneously transplanted into a 50m 3 fermentor at a ratio of 1:10, and the fermenter counted 40m 3 .
  • the FM medium formula of the fermenter is the same as in Example 4.
  • the initial conditions are temperature 29.5°C, compressed air ventilation ratio 0.5vvm, stirring speed 50rpm, tank pressure 0.05MPa, after 20-25h, adjust the aeration ratio to 1.0vvm, and use 10-30% ammonia and 2mol ⁇ L at 40h -1 phosphoric acid solution to control the pH at 6.2.
  • glycerol and 1.5g/L ammonium sulfate were added to supplement the carbon source and nitrogen source.
  • the soybean oil content of the fermentation broth is detected.
  • the soybean oil mixture of 10% ⁇ -ionone is continuously added at a flow rate of 0.20 mL ⁇ h -1 ⁇ L -1 to maintain The concentration of soybean oil is between 2.0 and 2.5%.
  • the total dissolved oxygen is controlled above 10%.
  • the stirring speed is gradually increased (5 rpm each time) to maintain the dissolved oxygen level.
  • the ⁇ -carotene output reaches 8.42 g/L.

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Abstract

提供了一组配对的β-胡萝卜素高产菌株及其制备方法和应用。所述菌株为三孢布拉霉（Blakeslea trispora），包括三孢布拉霉(+)和三孢布拉霉(-)两株单性菌株，其正株保藏号为CGMCC No.16491，负株保藏号为CGMCC No.16492。本发明经原生质体紫外诱变并结合化学诱变技术选育，通过在平板上筛选耐高浓度植物油和抗β-紫罗兰酮的抗性突变株，并在摇瓶上进行(+)、(-)株配对初筛、复筛验证，获得高产菌株。

Description

一组配对的天然β-胡萝卜素高产菌株及其制备方法和应用 技术领域
本发明涉及微生物发酵技术领域，具体地说是一组配对的天然β-胡萝卜素高产菌株及其制备方法和应用。
背景技术
类胡萝卜素(carotenoids)是某些植物、藻类、细菌和真菌等生物合成的异戊二烯类天然色素，根据是否含有氧原子可划分为：(1)纯碳氢化合物的胡萝卜素族(carotenes)，如α-胡萝卜素、β-胡萝卜素、γ-胡萝卜素和番茄红素等；(2)含有氧原子的叶黄素族(xanthophylls)，如虾青素、玉米黄质、叶黄素和辣椒红素等。类胡萝卜素具有优越的抗氧化性和着色性，上述2点重要的特性赋予它们在医药、食品、保健品及化妆品等领域的广泛应用。
β-胡萝卜素，也称为维生素A原，是目前已知的600多种类胡萝卜素的重要一员，具有抗炎、解毒、抗癌、预防心血管疾病、防治白内障和保护肝脏等多方面的医学和生物学功能，呈现巨大的商业价值。最近的市场调研显示，2015年β-胡萝卜素市场总额为4.36亿美元，2017年市场需求量为371.56吨，预计到2022年底将达到5.72亿美元，复合年增长率为3.5％。
目前，市场上销售的β-胡萝卜素根据来源的不同，可分为合成β-胡萝卜素和天然β-胡萝卜素两大类，后者除了从植物提取外还包括自然生产微生物(如藻类Dunaliella salina，Dunaliella bardawi，Chlorella sp.、真菌Blakeslea trispora，Choanephora cucurbitarum，Xanthophyllomyces dendrorhous、细菌Flavobacterium，Corynebacterium，Mycobacterium和红酵母Rhodotorula等)及基因工程菌株(主要是Saccharomyces cerevisiae、Escherichia coli和Yarrowia lipolytica)来源。虽然，在2014年合成β-胡萝卜素占整个β-胡萝卜素市场总额的70％以上，但随着人们对合成产物的敏感及对天然来源色素的偏爱，天然β-胡萝卜素的市场需求也相应增长。目前，商业化生产天然β-胡萝卜素的方法主要是利用盐藻和三孢布拉霉，例如澳大利亚的Aquacarotene公司和BASF(澳大利亚)分别从杜氏盐藻和微藻中提取天然β-胡萝卜素，而乌克兰的Vitan公司、西班牙的Vitatene公司和新昌制药厂则采用三孢布拉霉发酵生产天然β-胡萝卜素。盐藻法由于生产环境条件要求苛刻，产地受到限制，一年中只有几个月可以生产。三孢布拉霉由于应用安全(三孢布拉霉为美国FDA“一 般公认为安全”(Generally Recognized As Safe,GRAS)菌株)、生长迅速、菌丝量大、产量高，是天然β-胡萝卜素生产的理想菌株。
运用三孢布拉霉生产天然β-胡萝卜素，其显著特点是：三孢布拉霉的(+)、(-)株在混合培养过程中进行异宗接合作用，(+)株分泌的三孢酸作为性激素刺激单独培养时仅合成微量色素的(-)株大量积累β-胡萝卜素。研究表明，在接合过程中β-胡萝卜素生物合成基因的转录、能量代谢、细胞壁的合成以及调节过程，在某种程度上由三孢酸的调节而受到显著的诱导。同时，三孢酸的合成与β-胡萝卜素的代谢途径密切相关，合成路线如下所示：
Figure PCTCN2020078939-appb-000001
上述合成路线清楚地描述了三孢布拉霉中从香叶基焦磷酸香叶酯到三孢酸C的合成途径及两者的关系。虽然，三孢酸在三孢布拉霉两性接合过程中占有关键的作用，但目前未有市售的三孢酸，需要从发酵液中分离提取，这个过程涉及三氯甲烷等毒性化合物。作为代替，三孢酸的结构类似物，如β-紫罗兰酮、α-紫罗兰酮和维生素A等，被报道具有促进β-胡萝卜素合成的作用。
在三孢布拉霉发酵工艺研究方面，学术界和产业界围绕菌株诱变和选育、培养基优化、促进因子筛选、发酵过程控制等方面进行了全方位、多尺度的考察。早在1958年，Ciegle等就发现了外源植物油(尤其是富含油酸和亚油酸的植物油)可显著促进三孢布拉霉β-胡萝卜素的合成。US5422247通过化学诱变，筛选制霉菌素、洛伐他汀、β-紫罗兰酮和杜醌 抗性突变株，并对(+)、(-)株配对比例、煤油及异烟肼的影响进行考察，在添加2％煤油时摇瓶β-胡萝卜素产量达到5.8g·L -1。US7252965设计了三孢布拉霉菌株的诱变及高产菌株的筛选方法，并就突变株的培养条件进行优化，通过添加β-紫罗兰酮和通纯氧强化供氧水平等措施，其产量达到9g·L -1。EP1306444B1采取添加卵磷脂、批加β-紫罗兰酮油混合液，并在发酵36h后控制pH值为6.8±0.1，发酵产量达到5.5g·L -1。US7252965和EP1306444B1两个专利均单纯根据发酵时间采取批加的方式补加β-紫罗兰酮，β-紫罗兰酮浓度在发酵过程中呈脉冲性，但β-紫罗兰酮对三孢布拉霉有一定的毒性，该操作方式难以使β-紫罗兰酮在发酵过程中始终处于合适的浓度或不超过三孢布拉霉对β-紫罗兰酮的耐受阈值浓度。而US5422247提出的煤油具有一定的致癌性，法规上不允许。此外，发酵过程中由于剧烈的搅拌和通风作用，添加诸如卵磷脂、Span 20和Triton X-100这样的表面活性剂会产生大量的泡沫，限制了其工业化应用。至于Desai等提出的在发酵24h后添加1mg·L -1青霉素，从而促进三孢布拉霉中类胡萝卜素合成的策略，因涉及抗生素残留问题更是不可取。
综上所述，报道的关于三孢布拉霉合成β-胡萝卜素的化学促进剂种类和工艺控制策略繁多，但是这些方法往往存在诸多缺陷，离工业化尚有差距。而三孢布拉霉作为多核丝状真菌，具有复杂的基因组特征，在目前尚未具备相应的分子操作手段直接获得三孢布拉霉高产菌株的前提下，结合植物油和β-紫罗兰酮能够提高β-胡萝卜素产量的特征，通过诱变育种技术获得植物油和β-紫罗兰酮双重耐受突变株从而筛选高产菌株的方法，未尝不是个好策略。
发明内容
本发明的目的在于克服上述现有技术中的缺陷，提供一种对高浓度植物油具有较强代谢能力和较高利用速率、对高浓度β-紫罗兰酮形成抗性从而解除合成途径中存在的阻遏或反馈作用的高产突变株。
为此，本发明采用如下的技术方案：一组配对的天然β-胡萝卜素高产菌株，其生物分类学上属于三孢布拉霉Blakeslea trispora，为异宗接合菌，包括三孢布拉霉(+)和三孢布拉霉(-)两株单性菌株，其正株保藏号为CGMCC No.16491，负株保藏号为CGMCC No.16492。
本发明的另一目的是提供一种三孢布拉霉诱变、植物油和β-紫罗兰酮双重耐受突变株筛选的方法，获得一组配对的天然β-胡萝卜素高产菌株。为实现该发明目的所采用的技术方案为：以三孢布拉霉(+)株Xβ-15-02正-03和三孢布拉霉(-)株Xβ-15-04负-03为出发菌株，用物理和/或化学诱变选育的方法，以植物油和β-紫罗兰酮作为筛选压力，获得高产 菌株。
进一步地，所述的诱变选育孢子浓度为10 4～10 8个/mL，最优选为10 5～10 6个/mL。
本发明的再一目的是提供上述高产菌株在制备天然β-胡萝卜素发酵液的应用，其采用的技术方案为：
a.发酵罐培养：高产菌株三孢布拉霉(+)株和(-)株的种子液接种到含有碳源、氮源、无机盐、抗氧化剂和营养素的培养基中，经搅拌通气培养3天以上；
b.发酵过程中用酸和/或碱溶液控制pH值为5.5～7.5，同时，连续流加β-紫罗兰酮植物油混合液。
作为上述应用的补充，步骤a中，三孢布拉霉(+)株和(-)株的种子液配对比例为1:3～1:200，较优为1:4～1:50，最优为1:8～1:12；种子液按5～15％v/v比例移种发酵罐。
作为上述应用的补充，步骤a中，发酵培养温度为22～32℃，最优为26～30℃；压缩空气通气比为0.2～2.5vvm，最优为0.6～1.6vvm；商业生产规模搅拌转速15～250rpm，罐压为0.01～0.1MPa，过程中控制溶氧大于5％，最优溶氧控制大于10％。
作为上述应用的补充，步骤b中，发酵过程中控制pH值的碱和酸分别为10～30％质量浓度的氨水和0.1～2mol·L -1HCl或磷酸，控制时间为发酵10～80h后开始(最优为20～40h)，到发酵结束。
作为上述应用的补充，发酵过程中还可补入其它生理酸性或碱性碳源、氮源用于补充营养和调节发酵过程中pH值；这些碳源包括葡萄糖、淀粉和甘油；氮源包括铵盐如氯化铵、硫酸铵，硝酸盐如硝酸钾、硝酸钠。
作为上述应用的补充，步骤b中，发酵过程中补入的β-紫罗兰酮植物油混合液，其β-紫罗兰酮以5～30％(最优为8～12％)的质量浓度溶解于植物油中；β-紫罗兰酮植物油混合液开始补加时间为发酵20～80h(最优为30～50h)后；β-紫罗兰酮植物油混合液的流加速率为0.01～0.5mL·h -1·L -1(最优为0.05～0.2mL·h -1·L -1)，补料后植物油的质量浓度维持在0.2～4.0％(最优为1.0～2.0％)。
作为上述应用的补充，所述培养基的成份为：碳源1～3％、氮源2～6％、KH 2PO 4 0.02～0.2％、BHT 0.02～0.2％、VB 1 0.0001～0.001％、植物油2～10％、β-紫罗兰酮0.01～0.5％，消前用2mol·L -1NaOH溶液调pH值为5.0～9.0，上述的百分比均为质量百分比。
作为上述应用的补充，所述的碳源为一种或多种碳水化合物或脂肪类物质；所述的氮源为一种或多种有机氮源或无机氮源；所述的植物油为芝麻油、葵花籽油、菜籽油、棉籽油、花生油、大豆油中的一种或多种的混合物。
本发明对获得的高产突变株进行溶氧、pH值及植物油和β-紫罗兰酮流加等工艺优化，制得天然β-胡萝卜素发酵液，最终实现高产量、高产率、高生产强度的统一。
本发明具有的有益效果如下：
本发明采用原生质体紫外诱变结合化学诱变技术，对原始菌株进行多轮诱变育种，利用筛选平板上β-紫罗兰酮和植物油的筛选压力并结合三角瓶(+)、(-)株配对验证的策略，获得了具备高浓度β-紫罗兰酮和植物油适应能力的高产突变株：(+)株XC0705和(-)株XC0706组合。高产菌株杂质符合工业生产要求，稳定性较佳，油脂利用速率快，β-胡萝卜素产量在摇瓶上达到6.80g·L -1，在生产规模发酵罐上进行诸如溶氧、pH值及植物油和β-紫罗兰酮的连续流加控制，产量可达8.42g·L -1，在工业化生产中具有明显的成本优势。
附图说明
图1为本发明实施例1中菌株的诱变谱系图。
具体实施方式
下面通过实施例的方式进一步详细阐述本发明，但实施例不是对本发明的限制。下列实施例中未标明具体条件的实验方法，按照常规的方法和条件，或按商品说明书选择。
本发明提供一组配对的天然β-胡萝卜素高产菌株，其生物分类学上属于三孢布拉霉Blakeslea trispora，属于毛霉目、笄霉科，为异宗接合菌，包括三孢布拉霉(+)和三孢布拉霉(-)两株单性菌株。并对其进行了保藏，具体的保藏信息如下：
保藏机构：中国普通微生物菌种保藏管理中心(CGMCC)
保藏日期：2018年10月25日
保藏编号：正株保藏号为CGMCC No.16491，负株保藏号为CGMCC No.16492。
本发明所述的一组配对的天然β-胡萝卜素高产菌株及其商业化制备方法，具体过程如下所述：
1原生质体紫外诱变
原生质体的紫外诱变针对出发菌株三孢布拉霉(+)株Xβ-15-02正-03和三孢布拉霉(-)株Xβ-15-04负-03开展。对于原生质体的制备，参照专利CN105802988A报道的方法进行，然后对获得的原生质体进行紫外诱变并再生，具体程序如下所示：
1.1原生质体制备
分别从出发菌株三孢布拉霉(+)株Xβ-15-02正-03和三孢布拉霉(-)株Xβ-15-04负-03的PDA斜面培养基取2cm 2孢子接种子培养基(SM培养基，25mL/250mL)，温度22～32℃、转速150～350rpm、湿度20～80％条件下震荡培养36～72h获得三孢布拉霉菌丝。 吸取10mL上述培养液，4000rpm，离心10min，弃上清液后用等渗溶液洗涤2～3次。按每300mg菌体加入1mL等渗混合酶液(2％溶菌酶+3％纤维素酶+3％蜗牛酶，调pH值为6.0，过滤除菌)，置于摇床上温度28℃、转速75rpm条件下暗处酶解，每隔30min取样显微镜观察，确定酶解时间为12h。最后，酶解液用4层擦镜纸过滤除去菌丝，4000rpm离心10min，去除上清并用等渗溶液重悬，重复2次得原生质体悬浮液，并用血球计数板计数，得原生质体总数。
所述的种子培养基(SM培养基)成份为：碳源3～6％，氮源1～3％，KH 2PO 4 0.02～0.2％，VB 1 0.0001～0.001％，用2mol·L -1NaOH溶液调pH值为5.5～7.5。其中所述碳源可以是一种或多种碳水化合物或脂肪类物质，如淀粉、糊精、麦芽糖、蔗糖、乳糖、葡萄糖、糖蜜、植物油或动物油；所述的氮源可以是一种或多种有机氮源或无机氮源，如铵盐(NH 4Cl、(NH 4) 2SO 4)、硝酸盐(NaNO 3、KNO 3)、尿素、生/熟黄豆粉、玉米浆/干粉、酵母粉、酵母抽提物、棉籽粉、玉米粉。
所述的固体斜面培养基为PDA培养基，其制备方法为，将土豆去皮，切成丁块，称取200g加入适量水中，煮沸20min，用8层纱布过滤清液，定容至1L，加入20g葡萄糖，琼脂粉20g，加热融化后即成待用。
所述的等渗溶液：0.6mol·L -1NaCl溶液。
1.2原生质体紫外诱变
将步骤1.1制备的三孢布拉霉(+)、(-)株原生质体悬浮液稀释至约10 6个·mL -1，取5mL置于直径为6cm的无菌培养皿中，距离15W紫外灯30cm，用磁力搅拌器轻搅悬浮液，分别用紫外线照射0s、10s、20s、30s、40s、50s和60s。照射后进行适当稀释，取0.1mL悬浮液涂再生平板，黑暗闭光条件下温度22～32℃、湿度20～80％培养96～192h，待平板长出菌落后，计菌落数。
将获得的各诱变时间原生质体悬浮液稀释到一定浓度后，吸取0.1mL涂布于再生平板，得原生质体再生菌落数。用无菌水涨破原生质体，以同样的方法涂布再生平板，得对照组再生菌落数。
根据以下公式计算原生质体再生率：
Figure PCTCN2020078939-appb-000002
又根据以下公式计算紫外诱变各时间的原生质体致死率：
Figure PCTCN2020078939-appb-000003
式中：
z—原生质体再生率，
x—紫外线处理时间(s)。
本发明选择致死率为95％的条件作为原生质体紫外诱变条件，即紫外照射时间为50s。
2化学诱变
针对原生质体紫外诱变筛选获得的突变株开展2轮化学诱变。具体程序如下所示：
2.1突变株单孢子库悬浮液的制备
将三孢布拉霉(+)、(-)突变株(包括1代原生质体紫外诱变突变株或2代EMS突变株)分别在PDA培养基上培养，获得(+)、(-)突变株成熟的孢子。然后，在每支试管中加入10mL 0.1％TritonX-100溶液，用小铲刮下孢子，震荡、搅匀，再将其倒入含有玻璃珠的小三角瓶中，置于摇床上转速200rpm打散10min。接着用孔径为20μm的尼龙膜过滤，然后用0.1mol·L -1pH7.0磷酸钠缓冲液稀释至10 4～10 8个·mL -1，最优为10 5～10 6个·mL -1，吸取2ml，分别等量合并各(+)株稀释液和各(-)株稀释液，获得诱变所需的三孢布拉霉(+)、(-)株单孢子库悬浮液。
2.2EMS诱变处理
在50mL三角瓶中加入4mL由步骤2.1获得的单孢子库悬浮液，并加入16mLpH7.0磷酸钠缓冲液，再加入0.2mL EMS，于温度28℃下，100rpm震荡60min。处理液8000rpm离心10min获得诱变孢子，用生理盐水重悬浮，重复3次，取处理液0.1mL涂布抗性平板。
所述的抗性平板培养基(RM培养基)：PDA培养基中加入植物油2～10％，β-紫罗兰酮0.01～0.5％和乳化剂0.15～1.5％。所述的植物油可以是一种或多种植物油，如芝麻油、葵花籽油、菜籽油、棉籽油、花生油、大豆油。所述的乳化剂可以是一种或多种表面活性剂，尤其指亲水亲油平衡值(HLB值)在6～8之间的表面活性剂，如Tween系列、Span系列、大豆磷脂、脂肪酸甘油酯、高级脂肪酸盐。
3高产突变株的初筛
高产突变株的初筛是利用抗性平板上β-紫罗兰酮和植物油的筛选压力并结合三角瓶(+)、(-)株配对验证的策略进行，其原理基于高产突变株必然具备高浓度植物油适应能力和高脂肪酶活性，筛选三孢酸结构类似物β-紫罗兰酮的抗性突变株可解除途径上可能存在 的反馈抑制/阻遏。其过程是，将原生质体紫外诱变后经再生培养获得的孢子或经化学诱变处理的孢子涂布于具有高β-紫罗兰酮浓度和高植物油浓度的抗性平板，于温度22～32℃、湿度20～80％条件下培养96～192h，待平板长出菌落后，再进行摇瓶初筛。需要指出的是，由于三孢布拉霉丝状真菌菌丝茂盛，在96孔板或试管中培养时传质较差且易发生结块，因此本发明仍然采用三角瓶作为初筛的手段。
对于三孢布拉霉(-)株的初筛，过程如下：用接种棒点抗性平板上长出的三孢布拉霉(-)株突变株菌落，接种发酵培养基(FM培养基，25mL/250mL)，再用另一接种棒点三孢布拉霉(+)株原始菌株Xβ-15-02正-03孢子，于1～20mL无菌水中搅匀，吸取1mL孢子液到三角瓶中与三孢布拉霉(-)株突变株配对，于温度22～32℃，转速150～350rpm、湿度20～80％条件下震荡培养96～144h。
对于三孢布拉霉(+)株的初筛，过程如下：用接种棒点抗性平板上长出的三孢布拉霉(+)株突变株菌落，于1～20mL无菌水中搅匀，吸取1mL接种FM培养基(25mL/250mL)，再用另一接种棒点三孢布拉霉(-)株原始菌株Xβ-15-04负-03孢子，接种到三角瓶中与三孢布拉霉(+)株突变株配对，于温度22～32℃，转速150～350rpm、湿度20～80％条件下震荡培养96～144h。
上述发酵液样品按照CN 101955455B所述方法进行处理，具体过程为：用2mL移液管吸取1mL发酵液于15mL聚塑试管中，置于-10℃冰箱冷冻1h。取出自然溶解，加入10mL水超声处理5min，4000rpm离心10min，弃上清液，加入5mL无水乙醇，4000rpm离心10min，弃上清。再加入5mL乙酸乙酯65℃浸泡25min，最后用25mL容量瓶定容。对获得的上述溶液用乙酸乙酯再稀释10～100倍，过滤获得处理样。用可见分光光度计在454nm处检测样品吸光度OD 454。根据以下公式计算类胡萝卜素含量，以此反映β-胡萝卜素的含量：
Figure PCTCN2020078939-appb-000004
式中：
C—类胡萝卜素含量(g·L -1)，
OD 454—454 nm处吸光值，
F—稀释倍数，
2500—类胡萝卜素的平均消光系数
Figure PCTCN2020078939-appb-000005
具体的筛选流程如实施例1中的图1所示。按照上述流程，初筛获得三孢布拉霉(-)株 突变株15株，分别为nF 270、nF 2779、nF 397、nF 3123、nF 3127、nF 3256、nF 3301、nF 3415、nF 3489、nF 3554、nF 3591、nF 3682、nF 3745、nF 3774和nF 3810；初筛获得三孢布拉霉(+)株突变株12株，分别为pF 2395、pF 2652、pF 329、pF 378、pF 3182、pF 3240、pF 3437、pF 3499、pF 3570、pF 3601、pF 3673和pF 3711。
所述的发酵培养基(FM培养基)成份为：碳源1～3％、氮源2～6％、KH 2PO 4 0.02～0.2％、BHT 0.02～0.2％、VB 1 0.0001～0.001％、植物油2～10％、β-紫罗兰酮0.01～0.5％，消前用2mol·L -1NaOH溶液调pH值5.0～9.0。其中所述碳源可以是一种或多种碳水化合物或脂肪类物质，如淀粉、糊精、麦芽糖、蔗糖、乳糖、葡萄糖、糖蜜、植物油或动物油；所述的氮源可以是一种或多种有机氮源或无机氮源，如铵盐(NH 4Cl、(NH 4) 2SO 4)、硝酸盐(NaNO 3、KNO 3)、尿素、生/熟黄豆粉、花生饼粉、玉米浆/干粉、酵母粉、酵母抽提物、棉籽粉、玉米粉。所述的植物油可以是一种或多种植物油，如芝麻油、葵花籽油、菜籽油、棉籽油、花生油、大豆油。
4高产突变株的复筛
复筛的目的是获得最佳的三孢布拉霉(+)株和三孢布拉霉(-)株突变株搭配，并使该体系具有最高的稳定性。首先，将获得的所有突变株分别涂布PDA培养基，温度22～32℃、湿度20～80％条件下培养96～192h获得成熟孢子，再分别从PDA培养基上取2cm 2孢子菌落接种SM培养基(25mL/250mL)，温度22～32℃、湿度20～80％、150～350rpm条件下震荡培养36～72h。将上述条件获得的三孢布拉霉(+)株和三孢布拉霉(-)株突变株种子培养液依次两两配对，(-)株：(+)株按照3:1～200:1的比例配对，较优为4:1～50:1，最优为8:1～12:1，接种比例5～15％，接种FM培养基(25mL/250mL)，温度22～32℃、湿度20～80％、转速150～350rpm条件下震荡培养96～144h。
复筛发酵瓶样品处理方法与前述初筛发酵瓶样品处理方法相同，然后用高效液相色谱法(HPLC法)测定β-胡萝卜素的浓度和含量。
在所有180对三孢布拉霉(+)株和三孢布拉霉(-)株突变株配对组合中，(+)株pF 3570和(-)株nF 3123组合在摇瓶水平产量最高，达到6.80g·L -1，且平行性和稳定性较好。将(+)株pF 3570和(-)株nF 3123分别命名为：(+)株XC0705和(-)株XC0706，为本发明筛选获得的高产突变株。
本发明所述的对高产突变株XC0705和XC0706在发酵罐上进行溶氧、pH值及植物油和β-紫罗兰酮流加的调控，实现高产量、高产率、高生产强度统一的方法，具体过程如下所述：
(a)固体培养：将高产突变株(+)株XC0705和(-)株XC0706分别划线于PDA培养基，在温度22～32℃、湿度20～80％条件下培养96～192h，获得(+)株XC0705和(-)株XC0706成熟孢子。然后在每支试管中加入10mL 0.1％TritonX-100溶液，用小铲刮下孢子，震荡、搅匀，再将其倒入含有玻璃珠的小三角瓶中，置于摇床上转速200rpm打散10min，接着用孔径为20μm的尼龙膜过滤，并用血球计数板显微计数。
(b)种子培养：将上述步骤(a)获得的(+)株XC0705和(-)株XC0706孢子悬液分别接种SM培养基(100mL/1000mL)，使孢子终浓度控制在10 5～10 7个·mL -1，在温度22～32℃、湿度20～80％、转速150～350rpm的条件下震荡培养36～72h。为了获得足够的种子培养物，将上述(+)、(-)株种子培养物分别接种种子罐扩增，种子罐培养基同第1阶段的SM培养基。在小试中，种子罐接种比例为0.1～15％(v/v)，温度22～32℃，通气比为0.2～2.5vvm，最优为0.6～1.6vvm，搅拌转速50～500rpm，罐压0.01～0.1MPa，培养时间16～60h，获得第2阶段的种子罐种子培养物。在商业生产规模，种子罐接/移种比例为0.1～15％(v/v)，温度22～32℃，通气比为0.2～2.5vvm，最优为0.6～1.6vvm，搅拌转速30～300rpm，罐压0.01～0.1MPa，培养时间16～60h，获得足够的种子培养物。
(c)发酵培养：将上述步骤(b)获得的(+)株XC0705和(-)株XC0706种子罐种子培养物按照1:3～1:200，较优1:4～1:50，最优为1:8～1:12的比例混合均匀，移种FM培养基。移种比例为5～15％(v/v)，培养温度22～32℃，最优为26～30℃，通气比例0.2～2.5vvm，最优为0.6～1.6vvm，小试规模搅拌转速50～500rpm，商业生产规模搅拌转速15～250rpm，罐压0.01～0.1MPa，过程中控制溶氧大于5％，最优溶氧控制大于10％。发酵过程中用10～30％质量浓度的氨水和0.1～2mol·L -1HCl或磷酸控制pH值为5.5～7.5，控制时间为发酵10～80h开始，最优为发酵20～40h，到发酵结束。在发酵过程中，还可以补入其它生理酸性或碱性碳源、氮源用于补充营养和调节发酵过程中pH值，这些碳源包括葡萄糖、淀粉和甘油，氮源包括铵盐如氯化铵、硫酸铵，硝酸盐如硝酸钾、硝酸钠。同时，补入β-紫罗兰酮的植物油混合液，浓度为5～30％，最佳为8～12％，开始流加时间为发酵20～80h，最优为30～50h，流加速率为0.01～0.5mL·h -1·L -1，最优为0.05～0.2mL·h -1·L -1，补料后植物油浓度维持在0.2～4.0％，最优为1.0～2.0％。所述的植物油可以是一种或多种植物油，如芝麻油、葵花籽油、菜籽油、棉籽油、花生油、大豆油。
由于筛选的高产菌株对植物油和β-紫罗兰酮具有双重耐受性，且脂肪酶活性提高，故在发酵过程中菌株对植物油的利用速率较原始出发菌株大大加强。此外，发酵过程中植物油利用速率随着β-胡萝卜素合成速率增强而提高，β-紫罗兰酮作为促进剂，其利用速率亦 与β-胡萝卜素合成速率密切相关。因此，维持合适的植物油浓度可维持β-紫罗兰酮处于合适的范围，菌株未出现中毒而导致菌浓下降或产物合成受阻的情况。发酵液处理及β-胡萝卜素含量测定按照复筛部分所述进行。
实施例1本实施例说明三孢布拉霉高产菌株获得的方法
以三孢布拉霉(+)株Xβ-15-02正-03和(-)株Xβ-15-04负-03作为出发菌株，分别经过多轮原生质体紫外诱变或化学诱变(EMS诱变)，再经过初筛和复筛，(-)株获得15株与(+)株出发菌株Xβ-15-02正-03配对产量较高的突变株，分别为nF 270、nF 2779、nF 397、nF 3123、nF 3127、nF 3256、nF 3301、nF 3415、nF 3489、nF 3554、nF 3591、nF 3682、nF 3745、nF 3774和nF 3810；(+)株获得12株与(-)株出发菌株Xβ-15-04负-03配对产量较高的突变株，分别为pF 2395、pF 2652、pF 329、pF 378、pF 3182、pF 3240、pF 3437、pF 3499、pF 3570、pF 3601、pF 3673和pF 3711。上述菌株的诱变谱系如图1所示：
原生质体的制备过程中，先从PDA斜面培养基取2cm 2孢子菌落接种含有如下成分的SM培养基(25mL/250mL)：熟黄豆粉3.0％、糊精3.2％、KH 2PO 4 0.1％、VB 1 0.0002％，消前用2mol·L -1NaOH溶液调pH值6.8。种子瓶于温度28℃、转速250rpm、湿度40～60％的条件下震荡培养48h，获得三孢布拉霉菌丝。吸取10mL上述培养液，4000rpm，离心10min，弃上清液后用等渗溶液洗涤2～3次。按每300mg菌体加入1mL等渗混合酶液(2％溶菌酶+3％纤维素酶+3％蜗牛酶，调pH值为6.0，过滤除菌)，置于摇床上28℃，75rpm条件下暗处酶解，每隔30min取样显微镜观察，确定酶解时间为12h。最后，酶解液用4层擦镜纸过滤除去菌丝，4000rpm离心10min，去除上清并用0.6mol·L -1NaCl等渗溶液重悬，重复2次得原生质体悬浮液，并用血球计数板计数，稀释原生质体至10 6个·mL -1。
取5mL上述获得的原生质体稀释液置于直径为6cm的无菌培养皿中，距离15W紫外灯30cm，用磁力搅拌器轻搅悬浮液进行紫外诱变。处理后适当稀释取0.1mL悬浮液涂再生平板，黑暗闭光条件下28℃培养，待平板长出菌落后，计菌落数。根据前述提到的原生质体再生率、原生质体致死率公式，选择致死率为95％的条件作为原生质体紫外诱变条件，即紫外照射时间为50s。
将原生质体紫外诱变后经再生培养获得的孢子涂布于具有如下培养基成分的RM培养基：大豆油10％、β-紫罗兰酮0.08％、大豆磷脂0.65％，其它成分同PDA培养基。抗性平板于温度28℃、湿度40～60％条件下，待平板长出菌落后进行(+)、(-)株初筛。
初筛时，挑选抗性平板上生长良好、孢子茂盛的菌株。筛选操作是用接种棒点(+)株出发菌株或抗性突变株，于10mL无菌水中搅匀、稀释，吸取1mL孢子液到发酵摇瓶中 与三孢布拉霉(-)株突变株或出发菌株配对，即以(+)、(-)株的出发菌株分别作为(-)、(+)株突变株的配对菌株。(+)株：(-)株孢子配对比例为1:10，接种含有如下成分的FM培养基(25mL/250mL摇瓶)：熟黄豆粉3.4％、棉籽粉1.6％、玉米淀粉2.5％、KH 2PO 4 0.1％、BHT 0.05％、VB 1 0.0002％、大豆油5％、β-紫罗兰酮0.02％，消前用2mol·L -1NaOH溶液调pH值7.5。发酵瓶于温度28℃、湿度40～60％、250rpm条件震荡培养144h。
因三孢布拉霉中β-胡萝卜素占类胡萝卜素90～95％以上，故通过测定样品OD 454计算类胡萝卜素含量来初筛突变株。经过原生质体诱变，(+)株筛到4株正突变株，分别为：pF 1113、pF 1168、pF 1220、pF 1365。(-)株筛到4株正突变株，分别为：nF 190、nF 1217、nF 1339、nF 1418。
将获得的上述(+)、(-)突变株制孢子悬液，等量合并各(+)株稀释液和各(-)株稀释液，获得三孢布拉霉(+)、(-)突变株单孢子库。按照前述EMS诱变处理方法、本实施例初筛方法，(+)株筛选获得5株高产突变株，分别为：pF 225、pF 2221、pF 2380、pF 2395pF 2652。(-)株初筛到7株高产突变株，分别为：nF 214、nF 270、nF 2143、nF 2196、nF 2342、nF 2531、nF 2779。再对获得的突变株建库，按照前述EMS诱变处理方法、本实施例初筛方法，(-)株获得13株与(+)株出发菌株Xβ-15-02正-03配对产量较高的突变株，(+)株获得10株与(-)株出发菌株Xβ-15-04负-03配对产量较高的突变株。
突变株的复筛过程，将获得的12株(+)株和15株(-)株高产突变株(各菌株含2代高产突变株2株)分别涂布PDA培养基，温度28℃、湿度40～60％条件下培养120h，获得成熟孢子，再分别从PDA培养基上取2cm 2孢子菌落接种SM培养基(25mL/250mL)，温度28℃、湿度40～60％、250rpm条件下震荡培养48h。将上述条件获得的三孢布拉霉(+)和三孢布拉霉(-)突变株种子培养液按照(-)株：(+)株为10：1的比例依次两两配对，按10％接种比例接FM培养基(25mL/250mL)，温度28℃、湿度40～60％、转速250rpm条件下震荡培养144h。
在所有180对三孢布拉霉(+)株和三孢布拉霉(-)株突变株配对组合中，(+)株pF 3570和(-)株nF 3123组合在摇瓶水平产量最高，达到6.80g·L -1，且平行性和稳定性较好。将(+)株pF 3570和(-)株nF 3123分别命名为XC0705和XC0706，为本发明筛选获得的高产突变株。
实施例2本实施例说明高产突变株的传代稳定性
将筛选获得的(+)株XC0705和(-)株XC0706分别涂布PDA斜面培养基，温度28℃、湿度40～60％条件下培养120h，获得(+)、(-)高产突变株成熟孢子，即为F 1代。取2cm 2 F 1代斜面孢子菌落接种同实施例1中SM培养基(25mL/250mL)，温度28℃、湿度40～60％、转速250rpm条件下震荡培养48h。将获得的(+)、(-)突变株种子培养液按照1:10的比例混合，吸取2.5mL(接种比例10％)接种同实施例1中FM培养基(25mL/250mL)，温度28℃、湿度40～60％、转速250rpm条件下震荡培养144h。
将XC0705和XC0706的F 1代孢子制备孢子悬浮液，分别吸取0.1mL涂布PDA斜面培养基，经上述同样的步骤和培养条件，获得F 2代菌株生产能力，依次进行，考察F 3～F 5代菌株的生产能力。由表1结果得出，本发明获得的高产菌株经过5次传代，β-胡萝卜素产量仍然保持在6.67g/L，比较稳定。
表1 高产突变株的传代稳定性
代数 β-胡萝卜素产量(g/L)
F 1 6.80
F 2 6.75
F 3 6.64
F 4 6.78
F 5 6.67
实施例3本实施例说明高产突变株摇瓶发酵产β-胡萝卜素的能力
由于高产突变株对植物油和β-紫罗兰酮具有较强的适应性，因此其脂肪酶活性较强，代谢植物油的速率较出发菌株显著加强，途径上可能存在的反馈抑制/阻遏被解除，故在摇瓶上进一步验证高产突变株摇瓶发酵产β-胡萝卜素的能力。
本实施例设计了不同的大豆油和β-紫罗兰酮浓度组合，验证高产突变株摇瓶发酵产β-胡萝卜素的能力。将筛选获得的(+)株XC0705和(-)株XC0706涂布PDA斜面培养基，温度28℃、湿度40～60％条件下培养120h，获得(+)、(-)高产突变株成熟孢子。按前述方法制备单孢子悬液并接种同实施例1中的SM培养基(25mL/250mL)，使孢子浓度达到10 6个/mL，在温度28℃、湿度40～60％、转速250rpm条件下震荡培养48h，获得(+)、(-)高产突变株实验室种子培养液。将获得的(+)、(-)株种子培养液按照1:10的比例混合，吸取2.5mL(接种比例10％)接种FM培养基(各组大豆油和β-紫罗兰酮浓度见表2，其它成份同实施例1)，发酵瓶于温度28℃、湿度40～60％、转速250rpm条件震荡培养144h。在FM培养基中含8％大豆油和0.04％β-紫罗兰酮时，高产突变株摇瓶中β-胡萝卜素产量可达7.22g/L。作为对照，出发菌株Xβ-15-02正-03和Xβ-15-04负-03在5％大豆油和0.02％β-紫罗兰酮发酵培养基中，在同样的实验条件下产量仅为4.83g/L。
表2 高产突变株摇瓶发酵产β-胡萝卜素能力的验证
项目 # FM培养基大豆油、β-紫罗兰酮浓度 & β-胡萝卜素产量(g/L)
对照组 5％大豆油+0.02％β-紫罗兰酮 4.83
A组 5％大豆油+0.02％β-紫罗兰酮 6.82
B组 8％大豆油+0.02％β-紫罗兰酮 6.98
C组 5％大豆油+0.04％β-紫罗兰酮 7.07
D组 8％大豆油+0.04％β-紫罗兰酮 7.22
#：对照组菌株为Xβ-15-02正-03和Xβ-15-04负-03；A～D组菌株为XC0705和XC0706。
&：各组大豆油和β-紫罗兰酮浓度见表，其它成份同实施例1所述FM培养基保持不变。
实施例4本实施例说明高产突变株70L发酵罐产β-胡萝卜素的方法
将筛选获得的高产突变株(+)株XC0705和(-)株XC0706按照实施例3所述方法获得(+)、(-)突变株实验室种子培养液。为了获得足够的种子培养物，将上述(+)、(-)株种子培养液分别接种20L/30L种子罐，种子罐培养基同第1阶段的SM培养基。(+)、(-)株种子罐接种比例均为0.5％(v/v)，温度28℃，通气比为1.0vvm，罐压0.05MPa，搅拌转速200rpm，培养时间48h，获得第2阶段的种子罐种子培养物。
将上述获得的(+)株XC0705和(-)株XC0706第2阶段种子罐种子培养物按照1:10的比例混合，并按照10％(共4L)的移种比例移种70L发酵罐，发酵罐装液40L。发酵罐FM培养基配方为：熟黄豆粉3.4％、棉籽粉1.6％、糊精2.5％、KH 2PO 4 0.1％、BHT 0.05％、VB 1 0.0002％、大豆油8％、β-紫罗兰酮0.04％，消前用2mol·L -1NaOH溶液调pH值7.5。发酵过程中，初始条件为温度29.5℃、通气比0.5vvm、搅拌转速200rpm、罐压0.05MPa，20～25h后调通气比为1.0vvm，40h时用25～28％氨水和2mol·L -1HCl溶液控制pH值为6.8，全程搅拌与溶氧联动，维持溶氧10～30％。经过144h培养，β-胡萝卜素产量达到7.44g/L。
实施例5本实施例说明在发酵罐上补加生理酸性或碱性碳源、氮源的影响
按照实施例4所述方法制备(+)、(-)突变株第1阶段和第2阶段的种子培养液。
将上述获得的(+)株XC0705和(-)株XC0706第2阶段种子罐种子培养物按照1:10的比例混合，并按照10％(共4L)的移种比例移种70L发酵罐，发酵罐装液40L。发酵罐FM培养基配方同实施例4。发酵过程中，初始条件为温度29.5℃、通气比0.5vvm、搅拌转速200rpm、罐压0.05MPa，20～25h后调通气比为1.0vvm，40h时用25～28％氨水和2mol·L -1HCl溶液控制pH值在6.8。发酵过程中补加其它生理酸性或碱性碳源、氮源用于强化发酵过程中的营养供给，同时也调节发酵过程中的pH值。实验结果如表3所示：
表3 发酵罐上补加生理酸性或碱性碳源、氮源的影响
项目 FM培养基大豆油、β-紫罗兰酮浓度 β-胡萝卜素产量(g/L)
对照组 不补加生理酸性或碱性碳源、氮源 7.44
A组 36h补加1％葡萄糖+36h补加1.5g/L硫酸铵 7.59
B组 36h补加1％葡萄糖+36h补加1.5g/L硝酸钾 7.06
C组 36h补加1％甘油+36h补加1.5g/L硫酸铵 7.70
D组 36h补加1％甘油+36h补加1.5g/L硝酸钾 7.35
实施例6本实施例说明通过在发酵罐上流加植物油和β-紫罗兰酮提高突变株产β-胡萝卜素的方法
按照实施例4所述方法制备(+)、(-)突变株第1阶段和第2阶段的种子培养液。
将上述获得的(+)株XC0705和(-)株XC0706第2阶段种子罐种子培养物按照1:10的比例混合，并按照10％(共4L)的移种比例移种70L发酵罐，发酵罐装液40L。发酵罐FM培养基配方同实施例4。发酵过程中，初始条件为温度29.5℃、通气比0.5vvm、搅拌转速200rpm、罐压0.05MPa，20～25h后调通气比为1.0vvm，40h时用25～28％氨水和2mol·L -1磷酸溶液控制pH值在6.8。在36h分别补加1.0％甘油和1.5g/L硫酸铵补充碳源和氮源。发酵过程中检测发酵液大豆油含量，当大豆油含量低于1.0％时以0.15mL·h -1·L -1的流加速率连续补加10％β-紫罗兰酮的大豆油混合液，维持大豆油浓度在1.0～1.5％之间。全程搅拌与溶氧联动，维持溶氧10～30％。经过144h培养，β-胡萝卜素产量达到8.26g/L。
实施例7本实施例说明商业生产规模高产突变株产β-胡萝卜素的能力
按照实施例4所述方法制备(+)、(-)高产突变株实验室种子培养液。将获得的(+)、(-)株种子培养液按0.5％的比例分别接2只1m 3(计料体积0.5m 3)种子罐。(+)、(-)株种子罐培养温度28℃，通气比为1.0vvm，罐压0.05MPa，搅拌转速150rpm，培养时间48h。将上述获得的第2阶段(-)株种子罐种子培养物按10％的比例移10m 3(计料体积5m 3)种子罐，在温度28℃，通气比1.0vvm，罐压0.05MPa，搅拌转速100rpm，培养时间30h获得足够的(-)株种子培养液。上述生产规模种子罐配方均为实施例1所述SM配方。
将1m 3和10m 3种子罐获得的(+)和(-)株种子液按照1:10的比例同时移种50m 3发酵罐，发酵罐计料40m 3。发酵罐FM培养基配方同实施例4。发酵过程中，初始条件为温度29.5℃、压缩空气通气比0.5vvm、搅拌转速50rpm、罐压0.05MPa，20～25h后调通气比为1.0vvm，40h时用10～30％氨水和2mol·L -1磷酸溶液控制pH值在6.2。在36h分别补加1.0％甘油和1.5g/L硫酸铵补充碳源和氮源。发酵过程中检测发酵液大豆油含量，当大豆油含量低于1.0％时以0.20mL·h -1·L -1的流加速率连续补加10％β-紫罗兰酮的大豆油混合液，维持大豆油浓度在2.0～2.5％之间。全程溶氧控制在10％以上，当溶氧低于10％时逐步提高搅拌转速(每次提高5rpm)维持溶氧水平，经过120h培养，β-胡萝卜素产量达到 8.42g/L。
上述实施方式已经对本发明的一些细节进行了描述，但是不能理解为对本发明的限制，本领域的技术人员在不脱离本发明的原理和宗旨的情况下在本发明的范围内可以对其进行变化、修改、替换和变型。

Claims (11)

  1. 一组配对的天然β-胡萝卜素高产菌株，其特征在于，其生物分类学上属于三孢布拉霉Blakeslea trispora，为异宗接合菌，包括三孢布拉霉(+)和三孢布拉霉(-)两株单性菌株，其正株保藏号为CGMCC No.16491，负株保藏号为CGMCC No.16492。
  2. 权利要求1所述天然β-胡萝卜素高产菌株的制备方法，其特征在于，以三孢布拉霉(+)株Xβ-15-02正-03和三孢布拉霉(-)株Xβ-15-04负-03为出发菌株，用物理和/或化学诱变选育的方法，以植物油和β-紫罗兰酮作为筛选压力，获得高产菌株。
  3. 根据权利要求2所述的制备方法，其特征在于，所述的诱变选育孢子浓度为10 4～10 8个/mL。
  4. 权利要求1-3任一项所述高产菌株在商业化制备天然β-胡萝卜素发酵液的应用，其特征在于，包括如下步骤：
    a.发酵罐培养：高产菌株三孢布拉霉(+)株和(-)株的种子液接种到含有碳源、氮源、无机盐、抗氧化剂和营养素的培养基中，经搅拌通气培养3天以上；
    b.发酵过程中用酸和/或碱溶液控制pH值为5.5～7.5，同时，连续流加β-紫罗兰酮植物油混合液。
  5. 根据权利要求4所述的应用，其特征在于，步骤a中，三孢布拉霉(+)株和(-)株的种子液配对比例为1:3～1:200；种子液按5～15％v/v比例移种发酵罐。
  6. 根据权利要求4所述的应用，其特征在于，步骤a中，发酵培养温度为22～32℃；压缩空气通气比为0.2～2.5vvm；商业生产规模搅拌转速15～250rpm，罐压为0.01～0.1MPa，过程中控制溶氧大于5％。
  7. 根据权利要求4所述的应用，其特征在于，步骤b中，发酵过程中控制pH值的碱和酸分别为10～30％质量浓度的氨水和0.1～2mol·L -1 HCl或磷酸，控制时间为发酵10～80h后开始，到发酵结束。
  8. 根据权利要求4所述的应用，其特征在于，发酵过程中还补入其它生理酸性或碱性碳源、氮源，该碳源包括葡萄糖、淀粉和甘油，该氮源包括氯化铵、硫酸铵、硝酸钾和硝酸钠，用于补充营养和调节发酵过程中pH值。
  9. 根据权利要求4所述的应用，其特征在于，步骤b中，发酵过程中补入的β-紫罗兰酮植物油混合液，其β-紫罗兰酮以5～30％的质量浓度溶解于植物油中；β-紫罗兰酮植物油混合液开始补加时间为发酵20～80h后；β-紫罗兰酮植物油混合液的流加速率为0.01～0.5 mL·h -1·L -1，补料后植物油的质量浓度维持在0.2～4.0％。
  10. 根据权利要求4所述的应用，其特征在于，所述培养基的成份为：碳源1～3％、氮源2～6％、KH 2PO 4 0.02～0.2％、BHT 0.02～0.2％、VB 1 0.0001～0.001％、植物油2～10％、β-紫罗兰酮0.01～0.5％，消前用2mol·L -1NaOH溶液调pH值为5.0～9.0，上述的百分比均为质量百分比。
  11. 根据权利要求4所述的应用，其特征在于，所述的碳源为一种或多种碳水化合物或脂肪类物质；所述的氮源为一种或多种有机氮源或无机氮源；所述的植物油为芝麻油、葵花籽油、菜籽油、棉籽油、花生油、大豆油中的一种或多种的混合物。
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