CN115305207A - Total synthesis culture medium, preparation method and culture method for Blakeslea trispora - Google Patents

Total synthesis culture medium, preparation method and culture method for Blakeslea trispora Download PDF

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CN115305207A
CN115305207A CN202210768232.6A CN202210768232A CN115305207A CN 115305207 A CN115305207 A CN 115305207A CN 202210768232 A CN202210768232 A CN 202210768232A CN 115305207 A CN115305207 A CN 115305207A
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blakeslea trispora
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卢英华
陈翠雪
段然
陈婷婷
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Xiamen University
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Abstract

The invention discloses a total synthesis culture medium, a preparation method and a culture method for Blakeslea trispora. The total synthesis culture medium comprises a carbon source, a nitrogen source, vitamins, inorganic salts and a thickening agent; the thickening agent is 1-15 g/L of sodium carboxymethylcellulose, the sodium carboxymethylcellulose is simultaneously used as one of carbon source components and the thickening agent which cannot be quickly degraded by the blakeslea trispora, the viscosity of fermentation liquor can be maintained to a certain degree, the growth of the blakeslea trispora is promoted by blending other components, the hyphal form and the hyphal ball form of the blakeslea trispora are adjusted, and the utilization rate of a fermentation system on dissolved oxygen is improved. In addition, the invention provides a culture method of Blakeslea trispora based on the culture medium, which is used for carrying out fermentation culture and carotenoid accumulation, regulates and controls the fermentation process by using a material supplementing mode and combining with thallus growth change, and promotes the accumulation of the carotenoid in the Blakeslea trispora by using a synthetic ester fed-batch substrate.

Description

Total synthesis culture medium, preparation method and culture method for Blakeslea trispora
Technical Field
The invention belongs to the technical field of microorganisms and microbial fermentation, and particularly relates to a fully synthetic culture medium, a preparation method and a culture method for Blakeslea trispora.
Background
Beta-carotene is an orange-yellow terpenoid, the most widely occurring natural pigment in daily life. The fat-soluble compound is an important precursor for synthesizing vitamin A, and belongs to carotenoids such as lycopene, canthaxanthin and astaxanthin. The carotenoid has strong capability of eliminating oxygen free radicals, namely has the characteristic of oxidation resistance, and has obvious effects of cancer prevention, cancer resistance and the like. The application of carotenoids has been gradually expanded from the earliest use as pigments added to foods and feeds to the fields of medicines, daily chemicals and the like, and has become a very important industrial product.
Blakeslea trispora (B.trispora) is a heterozygospora belonging to the family of Choaneidae, and has a high growth rate and can rapidly generate developed hyphae. On a solid culture medium, hyphae can quickly spread, spread and form lawn, and in a liquid culture medium, hypha balls are easy to form.
Blakeslea trispora in submerged liquid culture forms mainly three distinct morphologies, namely discrete mycelia (discrete filaments), clumps (with some aggregation but still visible as loose hyphae) and globes (compact spherical aggregates) (Varzakakou M, roukas T, kotzekidou P, et al. Effect of non-ionic surfactants and beta-one on the morphology of Blakeslea trispora and carotenoid production from chemical engineering once in biological technology,2010,24 (2): 197-214). The growth and morphology of filamentous fungi in submerged culture depends on various operating conditions and factors, including inoculation, medium composition, broth rheology, and culture conditions. Wherein, the rheological property of the fermentation liquor and the stirring intensity in the fermentation tank have great influence on the convection type and the thallus balling state: the shear force received by hyphae is increased due to the increase of the stirring strength, thereby being beneficial to hyphae dispersion, but easily causing hyphae fragmentation; the reduction of stirring strength can cause the reduction of mass transfer efficiency, directly change the composition and rheological property of the culture medium, and can reduce the influence of mechanical stirring on the growth process of hypha.
Chinese patent (application No. CN 201511028714.4) discloses a Blakeslea trispora fermentation medium using starch phosphate as a carbon source, chinese patent (application No. CN 201911221401.9) discloses a main composition of a Blakeslea trispora fermentation medium using acidolyzed starch milk in combination with soybean flour, jin et al discloses a fermentation medium containing soybean flour, corn flour (jin, keju, shuya, et al. Engineering a-carotene biosynthesis and gene transduction regulation in Blakeslea trispora with sodium acetate [ J ] Biochemical Engineering Journal,2016, 114. Currently, main Blakeslea trispora fermentation media all use natural raw materials or natural raw material derivatives, provide a Blakeslea trispora viscosity with a certain viscosity component for Blakeslea trispora fermentation medium, and are suitable for a certain nutrient accumulation due to the fact that the ingredients of Blakeslea mycelium are not sufficiently degraded and the accumulation of the Blakeslea nutrient is not enough to be carried out in the fermentation process. Chinese patent (application No. CN 201810028777.7) discloses a method for promoting Blakeslea trispora to synthesize carotenoids by adding sunflower oil containing ethylene, wherein the use of natural oil can increase the viscosity of a fermentation system, the natural oil can be used as an oxygen carrier, but the addition of the oil can not reduce the diameter of bubbles at a distributor of a fermentation tank, so that the utilization rate of air is reduced, and the metabolic characteristics of the used sunflower oil are more favorable for the accumulation of carotenoids in Blakeslea trispora compared with esters with a specific formula.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a total synthesis culture medium, a preparation method and a culture method for Blakeslea trispora, wherein a modified cellulose thickener is used as a culture medium component, synthetic esters with determined components are used as fed-batch substrates, and Blakeslea trispora is subjected to fermentation culture, so that the problems in the background technology are solved.
One of the technical schemes adopted by the invention for solving the technical problems is as follows: provides a fully synthetic culture medium, which comprises a carbon source, a nitrogen source, microorganisms, inorganic salts and a thickening agent; the thickening agent is 1-15 g/L of sodium carboxymethyl cellulose, the sodium carboxymethyl cellulose is also used as one of carbon source components, and the concentration of the carbon source is 40-60 g/L in terms of glucose.
In a preferred embodiment of the present invention, the carbon source comprises: 20-60 g/L of glucose, 2-5 g/L of glycerol, 5-10 g/L of sucrose, 1-3 g/L of sodium acetate, 801-10 g/L of Tween-200.5-5 g/L of Tween-5-10 g/L of methyl palmitate, 5-10 g/L of methyl oleate, 2-5 g/L of ethyl linoleate and 1-15 g/L of sodium carboxymethylcellulose.
In a preferred embodiment of the present invention, the nitrogen source comprises: 5-10 g/L of urea, 2-10 g/L of aspartic acid, 2-5 g/L of sodium glutamate, 2-5 g/L of asparagine, 1-1.5 g/L of histidine, 0.5-1 g/L of proline and 0.1-0.2 g/L of methionine.
In a preferred embodiment of the present invention, the inorganic salt comprises: 0.2 to 0.5g/L of magnesium sulfate, 0.5 to 1g/L of dipotassium hydrogen phosphate, 0.05 to 0.15g/L of ferrous sulfate, 0.05 to 0.1g/L of zinc chloride, 0.02 to 0.06g/L of manganese chloride, 0.02 to 0.08g/L of copper sulfate, 0.1 to 0.2g/L of calcium chloride, 0.02 to 0.08g/L of nickel sulfate and 0.01 to 0.03g/L of cobalt chloride.
In a preferred embodiment of the present invention, the vitamins include: VB 1 0.002-0.01 g/L, 0.002-0.005 g/L, VB 12 0.002-0.005 g/L, biotin 0.003-0.009 g/L.
The second technical scheme adopted by the invention for solving the technical problems is as follows: provides a preparation method of a total synthesis culture medium, which comprises the following steps:
1) Dissolving sodium carboxymethylcellulose in 70% volume of fermentation liquor water, and stirring to make it fully dissolved and swollen to form colorless transparent solution;
2) Dissolving glucose, glycerol, sucrose, sodium acetate, a nitrogen source and inorganic salt by using water accounting for 30% of the volume of the fermentation liquor;
3) Mixing the solutions obtained in the step 1) and the step 2), adjusting the pH value to 6.9-7.5 by using sodium hydroxide, and performing damp-heat sterilization at the temperature of 115-125 ℃ for 20-35 min to obtain a sterilized culture medium;
4) Mixing tween-80, methyl palmitate, methyl oleate, ethyl linoleate and isopropyl myristate, and performing damp-heat sterilization at the temperature of 115-125 ℃ for 20-35 min to obtain an ester fed-batch substrate;
5) Dissolving vitamin components in the water-ethanol mixed solution, and filtering and sterilizing by using a sterile filter membrane of 0.2 mu m;
6) Adjusting the pH value of the sterilized culture medium in the step 3) to 6.9-7.1 by using sterilized phosphoric acid, and adding the vitamin component filtered in the step 5) to prepare the main component of the total synthetic culture medium.
The third technical scheme adopted by the invention for solving the technical problems is as follows: the application of the total synthetic culture medium comprises the main components of the total synthetic culture medium and an ester fed-batch substrate, and is used for culturing Blakeslea trispora, wherein the culturing comprises primary culturing, secondary culturing and fermentation culturing.
The fourth technical scheme adopted by the invention for solving the technical problems is as follows: the method for culturing the Blakeslea trispora is provided, the fully synthetic culture medium is used as a fermentation culture medium for fermentation culture, and the method comprises the following steps:
1) Activating strains: inoculating positive and negative strains of Blakeslea trispora on a slope of a commercial PDA culture medium under an aseptic condition, culturing at 25 ℃ in a dark place for 6 days, and washing off spores by using sterile normal saline after the spores grow out to obtain a spore suspension;
2) Directly inoculating spore suspension into fermentation culture medium to make the concentration of negative bacteria spore be 1.2 × 10 based on the volume of fermentation culture medium 5 L, inoculating and culturing the negative bacteria spores for 16-24 h, and then counting the number of the positive bacteria spores by 8 multiplied by 10 3 The culture medium is inoculated to the fermentation culture system at the culture temperature of 22-28 ℃ for 168-268 h, and the material is supplemented according to the fermentation monitoring state (the concentration of residual reducing sugar in the fermentation liquid, the acid-base consumption for adjusting the pH of the fermentation system and the viscosity change of the fermentation liquid are taken as the material supplementing basis); the supplement comprises supplementAdding glucose, sodium carboxymethylcellulose and ester fed-batch substrate.
The fifth technical scheme adopted by the invention for solving the technical problems is as follows: provides a method for culturing Blakeslea trispora, which comprises the following steps:
1) Activating strains: inoculating positive and negative strains of Blakeslea trispora on a commercial PDA culture medium inclined plane under an aseptic condition, culturing for 6 days in a dark place at 25 ℃, and washing off spores by using sterile normal saline after the spores grow out to obtain a spore suspension;
2) First-stage seed culture: counting the number of spores according to the number of negative spores of 1.2 multiplied by 10 in terms of the volume of the primary seed liquid 7 L, inoculating, and culturing at 22-26 ℃ for 16-24 h; then, the number of eumycete spores is 8 multiplied by 10 according to the volume of the first-stage seed liquid 5 L, inoculating the negative bacteria culture solution, and culturing at 20-25 ℃ for 36-48 h;
3) Secondary seed culture: inoculating the first-level seeds into a second-level seed culture medium according to the inoculation amount of 15-25%, and culturing for 12-16 h at 28 ℃;
4) Fermentation culture: inoculating the first-stage or second-stage seeds to a fermentation culture medium according to the inoculation amount of 10-15%, culturing at the temperature of 22-28 ℃ for 168-268 h, and supplementing materials according to the fermentation monitoring state; the fermentation culture medium adopts the fully synthetic culture medium, and the feeding comprises the supplement of glucose, sodium carboxymethylcellulose and an ester fed-batch substrate.
In a preferred embodiment of the present invention, the primary seed culture medium or the secondary seed culture medium comprises the following components: 0.5-3.5 g/L of sodium carboxymethylcellulose, 20-30 g/L of glucose, 2-3 g/L of aspartic acid, 1-2 g/L of asparagine, 3-5 g/L of sodium glutamate and 0.005-0.009 g/L, VB of biotin 1 0.005g/L, and further comprises malt extract 1g/L, and pH is 6.9-7.1.
Compared with the background technology, the technical scheme has the following advantages:
1. compared with the prior art, the fully synthetic culture medium avoids the use of natural materials such as starch, corn flour, soybean flour, corn steep liquor dry powder and the like, does not need to dissolve and gelatinize insoluble powdery materials, can effectively avoid the risk of bacterial contamination caused by incomplete dissolution of the powdery materials and embedding of mixed bacteria, and greatly reduces the situation of mixed bacteria pollution in the fermentation process. Meanwhile, the food thickeners such as sodium carboxymethylcellulose can well simulate the rheological property of a natural culture medium, change the rheological property of fermentation liquor according to the specific fermentation condition, and can well control the growth form and the nodulation form of hyphae under the condition of changing the addition amount and the type of the thickeners. Because the accumulation level of the carotenoid in the Blakeslea trispora is closely related to the nodulation form of hyphae, the process control of the hyphae nodulation of the Blakeslea trispora can be carried out by the total synthesis culture medium, so as to further research the anabolism of the carotenoid in the Blakeslea trispora. The fatty acid methyl ester is used for replacing vegetable oil, so that the composition of fatty acid can be more accurately adjusted, and a more suitable environment is provided for the accumulation of carotenoid.
2. The rheological property of the fermentation liquor is controlled by the thickening agent, so that the property that the nutrient content is positively correlated with the viscosity when powdery materials are used can be avoided, the nutrient composition of a fermentation system is conveniently controlled, and the product accumulation is better realized. The fully synthetic culture medium can well simulate a fermentation culture medium for separating mycelia such as mould and the like by means of viscosity generated by materials such as starch, corn flour, soybean flour, corn steep liquor and the like, is suitable for fermentation culture of Blakeslea trispora, mortierella alpina and other similar filamentous moulds with nodulation behaviors, and provides a foundation for research on the aggregation form of the mould mycelia;
3. the total synthetic culture medium containing the thickening agent is used for fermentation culture of Blakeslea trispora, the content of beta-carotene in mycelium reaches 6.6-7.1 (mg/100 mg), the yield of beta-carotene in a fermentation system reaches 3.32g/L, and a foundation is provided for morphological research of Blakeslea trispora and similar filamentous fungi.
Drawings
FIG. 1 shows the results of the fermentation of the negative bacteria of B.trispora with the addition of different thickeners in example 1;
FIG. 2 shows the hyphal nodulation in example 1, in which a) sodium carboxymethylcellulose b) polyacrylic acid c) hydroxypropylmethylcellulose d) hydroxyethylcellulose e) sodium alginate f) xanthan gum;
FIG. 3 is the effect of different amounts of sodium carboxymethyl cellulose added on the biomass of B.trispora in example 2;
FIG. 4 is a graph showing the effect of different amounts of sodium carboxymethylcellulose added on the production of B.trispora beta-carotene in example 2;
FIG. 5 is a graph showing the effect of different amounts of sodium carboxymethylcellulose added on the beta-carotene content of Blakeslea trispora in example 2;
FIG. 6 is the effect of different amounts of sodium carboxymethyl cellulose added on the viscosity of the fermentation broth in example 2;
FIG. 7 is a graph of the effect of different sodium carboxymethyl cellulose additions on negative mycelium pellet diameters in example 2;
FIG. 8 shows the results of 30L mixed fermentation of positive and negative bacteria in example 3.
Detailed Description
The positive and negative Blakeslea trispora (Blakeslea trispora < + > ) used in the following examples were from ATCC and were numbered ATCC 14271 (+), ATCC 14272 (-).
Example 1
In this example, a fully synthetic medium was prepared as the fermentation medium using different thickeners:
sodium carboxymethylcellulose (MW =250000, DS = 0.7), polyacrylic acid (MW-4000), hydroxypropylmethylcellulose (type I, viscosity: 20000mPa. S), hydroxyethylcellulose (5000-6400mPa. S,25 ℃ C.), sodium alginate, xanthan gum were used as thickeners, and the addition amount was controlled to be 1-15 g/L so that the viscosity of the sterilized medium was 300 mPa. S, and the other components were (g/L): glucose 20, glycerol 5, tween-800.5, aspartic acid 2, sodium glutamate 2, asparagine 2, histidine 1, proline 0.5, methionine 0.1, magnesium sulfate 0.2, dipotassium hydrogen phosphate 1, ferrous sulfate 0.05, zinc chloride 0.05, manganese chloride 0.02, copper sulfate 0.02, calcium chloride 0.1, nickel sulfate 0.02 and cobalt chloride 0.01;
vitamins (g/L): VB 1 0.01, calcium pantothenate 0.002, VB 12 0.002, biotin 0.003;
dissolving the thickening agent in 70mL of water, stirring until the thickening agent is fully dissolved to form a transparent and uniform solution, dissolving other components in 30mL of water, adding the thickening agent solution, uniformly mixing, sterilizing in a 500mL conical flask at 121 ℃ for 20min, cooling, adjusting the pH to 7.0 by using 1mol/L sodium hydroxide solution, filtering the vitamin by using a sterile filter membrane with the diameter of 0.2 mu m, and adding the vitamin into a culture medium.
The method for culturing Blakeslea trispora of the present embodiment comprises the following steps:
inoculating the preserved Blakeslea trispora to a slope of a commercial PDA culture medium, culturing at 25 ℃ in a dark place for 6 days, and washing off spores by using sterile normal saline after the spores grow out;
according to the number of the negative bacteria spores of 1.2 multiplied by 10 5 L inoculating starter culture medium, culturing at 28 deg.C for 240 hr, measuring bacterial biomass, grinding and extracting carotenoid in mycelium with petroleum ether, measuring beta-carotene content by HPLC, and calculating beta-carotene content (mg/100 mg biomass) in dried mycelium.
The mass accumulation of the carotenoids by the blakeslea trispora requires the co-culture of positive bacteria and negative bacteria, but the synthesis of the carotenoids is mainly carried out in the negative bacteria, the thickening agent species comparison is carried out on the blakeslea trispora in the embodiment, and the results are shown in fig. 1 and 2: the sodium carboxymethylcellulose is used as the components of the fermentation thickening agent and the total synthesis culture medium, the biomass and the yield of beta-carotene in the fermentation result are highest, and are respectively 6.02g/L and 0.0088g/L, the content is 0.146mg/100mg, and the fermentation thickening agent is superior to other thickening agents. FIG. 2 is a photograph of the hyphal aggregation morphology at the end of fermentation, showing that when sodium carboxymethylcellulose is used as the thickener, the hyphal balls are uniform in size, dispersed, smooth in edges, and suitable for hyphal mass transfer and carotenoid accumulation.
Example 2
In the total synthetic medium components of this example, sodium carboxymethylcellulose with different contents was used:
the fermentation medium comprises the following components (g/L): sodium carboxymethylcellulose (MW =250000, DS = 0.7) 0-13.6, and other components are glucose 20, tween-800.5, aspartic acid 2, sodium glutamate 2, asparagine 2, histidine 1, methionine 0.1, magnesium sulfate 0.2, dipotassium hydrogen phosphate 1, ferrous sulfate 0.05, zinc chloride 0.05, manganese chloride 0.02, copper sulfate 0.02, calcium chloride 0.1, nickel sulfate 0.02 and cobalt chloride 0.01;
vitamins (g/L): VB 1 0.01, calcium pantothenate 0.002, VB 12 0.002 and biotin 0.003.
Respectively weighing 1.72 g, 6.87 g and 13.6g of sodium carboxymethylcellulose, dissolving in 70mL of water, stirring until the sodium carboxymethylcellulose is fully dissolved to form a transparent uniform solution, dissolving other components in 30mL of water, adding a thickening agent solution, uniformly mixing, sterilizing in a 500mL conical flask at 121 ℃ for 20min, cooling, adjusting the pH to 7.0 by using 1mol/L sodium hydroxide solution, filtering vitamins by using a sterile filter membrane of 0.2 mu m, and adding the vitamins into a culture medium.
The method for culturing Blakeslea trispora of the present embodiment comprises the following steps:
inoculating the preserved Blakeslea trispora to a slope of a commercial PDA culture medium, culturing at 25 ℃ in a dark place for 6 days, and washing off spores by using sterile normal saline after the spores grow out;
according to the number of the negative bacteria spores of 1.2 multiplied by 10 5 The mycelia were inoculated in a starter culture medium at 28 ℃ for 264 hours, the biomass of the cells was measured, carotenoids in the mycelia were extracted by grinding with petroleum ether on a dry weight basis (DCW), and the content of β -carotene was measured by HPLC to calculate the content of β -carotene in the dried mycelia (mg/100 mg biomass).
As shown in FIGS. 3 to 7, the effect of different amounts of carboxymethylcellulose on biomass increased the yield and content of beta-carotene as the amount of carboxymethylcellulose increased, and the yield of beta-carotene was 0.151g/L in the high addition group, which was 1.45 times that in the low addition group, which was increased in the amount of beta-carotene accumulated in the cells; FIG. 6 shows the viscosity change of the fermentation system during the culture, which shows that carboxymethyl cellulose can affect the viscosity of the fermentation broth within 120 hours and can result in the growth of the cells; FIG. 7 illustrates the change of the diameter of the mycelium pellet with time under different carboxymethyl cellulose addition conditions, the diameter of the mycelium pellet is gradually reduced along with the increase of the viscosity of the initial culture medium, the increase time of the diameter of the mycelium pellet is prolonged under the low viscosity condition, the mycelium pellet is oversized, the accumulation of carotenoid in bacteria is influenced, the increase of the initial viscosity is beneficial to reducing the diameter of the mycelium pellet, the mass transfer level of the mycelium pellet is improved, and the yield of the carotenoid is improved.
Example 3
In the medium composition of this example, sodium carboxymethylcellulose was added as an experimental group, and sodium carboxymethylcellulose was not added as a control group.
The experimental group fermentation medium comprises the following components (g/L): sodium carboxymethylcellulose (MW =250000, DS = 0.7) 7.21, glucose 20, glycerol 2, sucrose 5, sodium acetate 2, tween-801, tween-200.5, urea 5, aspartic acid 2, sodium glutamate 2, asparagine 2, histidine 1, proline 1, methionine 0.2, magnesium sulfate 0.2, dipotassium hydrogen phosphate 1, ferrous sulfate 0.06, zinc chloride 0.06, manganese chloride 0.03, copper sulfate 0.06, calcium chloride 0.1, nickel sulfate 0.05, cobalt chloride 0.01, VB 1 0.01, calcium pantothenate 0.002, VB 12 0.002, biotin 0.006;
the control group fermentation medium is not added except the sodium carboxymethyl cellulose, and the other components are the same as the experimental group.
Dissolving sodium carboxymethylcellulose in 15L of water, stirring to dissolve completely to form transparent uniform solution, dissolving other components with 3L of water, adding thickener solution, mixing, sterilizing at 121 deg.C for 35min in 30L fermentation tank, cooling, adjusting pH to 7.0 with 1mol/L sodium hydroxide solution, filtering vitamins with 0.2 μm sterile filter membrane, and adding into culture medium.
In this embodiment, a 30L fermentation tank is used for mixed fermentation of positive and negative blakeslea trispora, and the specific steps are as follows:
inoculating the preserved positive and negative bacteria of Blakeslea trispora to the slant of a commercial PDA culture medium, culturing at 25 ℃ in a dark place for 6 days, and washing off the spores by using sterile normal saline after the spores grow out;
the primary seed culture medium comprises the following components (g/L): sodium carboxymethylcellulose (MW =120000, DS = 0.7) 1.7, glucose 20, aspartic acid 2, asparagine 2, malt extract 1, sodium glutamate 3, biotin 0.005, vb 1 0.005 and pH 7.1. Will be provided withStirring and dissolving sodium carboxymethylcellulose, adding other components, sterilizing at 121 deg.C for 20min, cooling, adding filtered vitamins, and adding 1.2 × 10 of number of negative bacteria spores 7 L, inoculating, and culturing at 26 ℃ for 20h. Then, the number of spores of the eumycete is 8X 10 5 L inoculating the negative bacteria culture solution, and culturing at 22 deg.C for 36h.
The secondary seed culture medium comprises the following components (g/L): sodium carboxymethylcellulose (MW =120000, DS = 0.7) 1.7, glucose 30, aspartic acid 2, asparagine 2, malt extract 1, sodium glutamate 3, biotin 0.005, vb 1 0.01, pH 7.1. Stirring and dissolving sodium carboxymethylcellulose, adding other components, sterilizing at 121 deg.C for 20min, cooling, adding filtered vitamins, inoculating 25% of first-stage seeds into second-stage seed culture medium, and culturing at 28 deg.C for 16 hr.
Inoculating the second-stage seeds to a fermentation medium according to the inoculation amount of 10%, culturing at 28 ℃ for 48h, then culturing at 25 ℃ for 144h, adjusting the stirring speed and the ventilation amount in the period to ensure that the dissolved oxygen is not less than 10%, and controlling the fermentation pH to be 6.8 by 1mol/L sodium hydroxide.
As a result, as shown in FIG. 8, the content of beta-carotene in the hyphae of the experimental group was 7.11mg/100mg, which was 1.27 times that of the control group.
Example 4
The fermentation medium of this example comprises the following components (g/L): sodium carboxymethylcellulose (MW =250000, DS = 0.7) 7.21, glucose 40, glycerol 5, sucrose 5, sodium acetate 2, tween-801, tween-200.5, urea 8, aspartic acid 6, sodium glutamate 3, asparagine 5, histidine 1, proline 0.5, methionine 0.2, magnesium sulfate 0.2, dipotassium hydrogen phosphate 1, ferrous sulfate 0.08, zinc chloride 0.06, manganese chloride 0.03, copper sulfate 0.06, calcium chloride 0.1, nickel sulfate 0.05, cobalt chloride 0.01, VB 0, and the like 1 0.01, calcium pantothenate 0.002, VB 12 0.002, biotin 0.006;
the ester feed plus substrate comprises the following components and volume fractions (v/v%): methyl palmitate 28, methyl oleate 55, ethyl linoleate 11, isopropyl myristate 5, tween-801, and a fed-batch substrate, mixing, and sterilizing at 121 deg.C for 40min.
Dissolving sodium carboxymethylcellulose in 2.5L water, stirring to dissolve completely to obtain transparent uniform solution, dissolving other components with 0.5L water, adding thickener solution, mixing, sterilizing at 121 deg.C for 40min in 5L fermentation tank, cooling, adjusting pH to 7.0 with 1mol/L sodium hydroxide solution, filtering vitamins with 0.2 μm sterile filter membrane, and adding into culture medium.
The steps of this example are as follows:
inoculating the preserved positive and negative bacteria of Blakeslea trispora to the slant of a commercial PDA culture medium, culturing at 25 ℃ in a dark place for 6 days, and washing off the spores by using sterile normal saline after the spores grow out;
the primary seed culture medium comprises the following components (g/L): sodium carboxymethylcellulose (MW =120000, DS = 0.7) 1.7, glucose 20, aspartic acid 2, asparagine 2, malt extract 1, sodium glutamate 3, biotin 0.005, vb10.005, ph 7.1. Dissolving sodium carboxymethylcellulose under stirring, adding other components, sterilizing at 121 deg.C for 20min, cooling, adding filtered vitamins, and regulating the number of spores of negative bacteria to 1.2 × 10 7 L, inoculating, and culturing at 26 ℃ for 30h. Then, the negative bacteria culture medium was inoculated at 8X 105/L of the number of positive spores, and cultured at 22 ℃ for 48 hours.
The secondary seed culture medium comprises the following components (g/L): sodium carboxymethylcellulose (MW =250000, DS = 0.7) 1.7, glucose 30, aspartic acid 2, asparagine 2, malt extract 1, sodium glutamate 3, biotin 0.005, vb10.01, ph 7.1. Stirring and dissolving sodium carboxymethylcellulose, adding other components, sterilizing at 121 deg.C for 20min, cooling, adding filtered vitamins, inoculating 25% of the first-stage seeds in the second-stage seed culture medium, and culturing at 28 deg.C for 12 hr.
Inoculating the secondary seeds to a fermentation medium according to the inoculation amount of 15%, culturing at 28 ℃ for 48h, then culturing at 25 ℃ for 168h, adjusting the stirring speed and ventilation amount to ensure that the dissolved oxygen is not less than 10%, and controlling the fermentation pH to be 6.6 by 1mol/L sodium hydroxide. At fermentation time of 48 h: when the initial sugar is consumed to 5g/L, glucose is supplemented once to enable the residual sugar of the fermentation liquor to reach 20g/L, and the fermentation is maintained until the end of the fermentation; at the moment, the pH begins to rise slightly, the pH is judged to be in the stage of converting primary metabolism into secondary metabolism, meanwhile, ester substrates begin to be fed in, and feeding is carried out in portions according to the addition amount of 15mL/d until the fermentation is finished. When the fermentation is carried out for 72 hours, 500mL of water is supplemented at a time.
Collecting fermentation liquor to obtain thallus, wherein the measured dry weight is 50.31g/L, and the unit of beta-carotene in the fermentation liquor is 3.32g/L. Therefore, the preparation method can reach higher beta-carotene fermentation units and is suitable for industrial production.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. A fully synthetic medium, comprising: comprises a carbon source, a nitrogen source, microorganisms, inorganic salts and a thickening agent; wherein the thickening agent is 1-15 g/L of sodium carboxymethyl cellulose, the sodium carboxymethyl cellulose is also used as one of the carbon source components, and the concentration of the carbon source is 40-60 g/L in terms of glucose.
2. A fully synthetic medium according to claim 1, wherein: the carbon source comprises: 20-60 g/L of glucose, 2-5 g/L of glycerol, 5-10 g/L of sucrose, 1-3 g/L of sodium acetate, 801-10 g/L of Tween-200.5-5 g/L of Tween-5-10 g/L of methyl palmitate, 5-10 g/L of methyl oleate, 2-5 g/L of ethyl linoleate and 1-15 g/L of sodium carboxymethylcellulose.
3. A fully synthetic medium according to claim 1, wherein: the nitrogen source comprises: 5-10 g/L of urea, 2-10 g/L of aspartic acid, 2-5 g/L of sodium glutamate, 2-5 g/L of asparagine, 1-1.5 g/L of histidine, 0.5-1 g/L of proline and 0.1-0.2 g/L of methionine.
4. A fully synthetic medium according to claim 1, wherein: the inorganic salt includes: 0.2 to 0.5g/L of magnesium sulfate, 0.5 to 1g/L of dipotassium hydrogen phosphate, 0.05 to 0.15g/L of ferrous sulfate, 0.05 to 0.1g/L of zinc chloride, 0.02 to 0.06g/L of manganese chloride, 0.02 to 0.08g/L of copper sulfate, 0.1 to 0.2g/L of calcium chloride, 0.02 to 0.08g/L of nickel sulfate and 0.01 to 0.03g/L of cobalt chloride.
5. A fully synthetic medium according to claim 1, wherein: the vitamins include: VB 1 0.002-0.01 g/L, 0.002-0.005 g/L, VB 12 0.002-0.005 g/L, biotin 0.003-0.009 g/L.
6. The method for preparing a total synthesis culture medium according to any one of claims 1 to 5, wherein: the method comprises the following steps:
1) Dissolving sodium carboxymethylcellulose in 70% volume of fermentation liquor water, and stirring to make it fully dissolved and swollen to form colorless transparent solution;
2) Dissolving glucose, glycerol, sucrose, sodium acetate, a nitrogen source and inorganic salt by using water accounting for 30% of the volume of the fermentation liquor;
3) Mixing the solutions obtained in the step 1) and the step 2), adjusting the pH value to 6.9-7.5 by using sodium hydroxide, and performing damp-heat sterilization at the temperature of 115-125 ℃ for 20-35 min to obtain a sterilized culture medium;
4) Mixing tween-80, methyl palmitate, methyl oleate, ethyl linoleate and isopropyl myristate, and performing damp-heat sterilization at the temperature of 115-125 ℃ for 20-35 min to obtain an ester fed-batch substrate;
5) Dissolving vitamin components in the water-ethanol mixed solution, and filtering and sterilizing by using a sterile filter membrane with the diameter of 0.2 mu m;
6) Adjusting the pH value of the sterilized culture medium in the step 3) to 6.9-7.1 by using sterilized phosphoric acid, and adding the vitamin component filtered in the step 5) to prepare the main component of the total synthetic culture medium.
7. Use of a fully synthetic medium prepared according to the method of claim 6, wherein: the method comprises the main components of a total synthetic culture medium and an ester fed-batch substrate, and is used for culturing Blakeslea trispora, wherein the culturing comprises primary culturing, secondary culturing and fermentation culturing.
8. A method for culturing Blakeslea trispora is characterized by comprising the following steps: a fully synthetic medium according to any one of claims 1 to 5 as a fermentation medium for fermentative culture comprising the steps of:
1) Activating strains: inoculating positive and negative strains of Blakeslea trispora on a slope of a commercial PDA culture medium under an aseptic condition, culturing at 25 ℃ in a dark place for 6 days, and washing off spores by using sterile normal saline after the spores grow out to obtain a spore suspension;
2) Directly inoculating spore suspension into fermentation culture medium to make the concentration of negative bacteria spore be 1.2 × 10 based on the volume of fermentation culture medium 5 L, inoculating and culturing the negative bacteria spores for 16-24 h, and then, taking the number of the positive bacteria spores as 8 multiplied by 10 3 L, inoculating the mixture to the fermentation culture system, culturing at the temperature of 22-28 ℃ for 168-268 h, and supplementing materials according to the fermentation monitoring state; the feeding comprises the supplement of glucose, sodium carboxymethylcellulose and an ester fed-batch substrate.
9. A method for culturing Blakeslea trispora is characterized in that: the method comprises the following steps:
1) Activating strains: inoculating positive and negative strains of Blakeslea trispora on a slope of a commercial PDA culture medium under an aseptic condition, culturing at 25 ℃ in a dark place for 6 days, and washing off spores by using sterile normal saline after the spores grow out to obtain a spore suspension;
2) First-stage seed culture: counting spores according to the number of negative bacteria spores of 1.2 multiplied by 10 in terms of the volume of the primary seed liquid 7 L, inoculating, and culturing at 22-26 ℃ for 16-24 h; then, the number of eumycete spores is 8 multiplied by 10 according to the volume of the first-stage seed liquid 5 L, inoculating the negative bacteria culture solution, and culturing at 20-25 ℃ for 36-48 h;
3) Secondary seed culture: inoculating the first-stage seeds into a second-stage seed culture medium according to the inoculation amount of 15-25%, and culturing for 12-16 h at 28 ℃;
4) Fermentation culture: inoculating the first-stage or second-stage seeds to a fermentation culture medium according to the inoculation amount of 10-15%, culturing at the temperature of 22-28 ℃ for 168-268 h, and supplementing materials according to the fermentation monitoring state; the fermentation culture medium adopts a total synthesis culture medium as described in any one of claims 1 to 5, and the feeding comprises the supplement of glucose, sodium carboxymethyl cellulose and ester fed-batch substrate.
10. The method for culturing Blakeslea trispora according to claim 9, wherein: the primary seed culture medium or the secondary seed culture medium comprises the following components in percentage by weight: 0.5 to 3.5g/L of sodium carboxymethylcellulose, 20 to 30g/L of glucose, 2 to 3g/L of aspartic acid, 1 to 2g/L of asparagine, 3 to 5g/L of sodium glutamate and 0.005 to 0.009g/L, VB of biotin 1 0.005g/L, and further comprises malt extract 1g/L, and pH is 6.9-7.1.
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