阅读说明：本技术 一种法夫酵母补糖培养基及其进行高密度培养的方法和应用 (Phaffia yeast sugar supplement culture medium, and high-density culture method and application thereof ) 是由 陈克杰 孙新强 杨一恭 陈迎迎 周旭燕 邵东 徐作武 王小平 于 2020-04-21 设计创作，主要内容包括：本发明提供了一种法夫酵母补糖培养基及其进行高密度培养的方法和应用,所述补糖培养基含有利于细胞增殖和虾青素合成的碳源组合,能弥补单一碳源产量低的缺点。所述高密度培养方法用在线活细胞传感仪检测活细胞浓度,通过采用指数补料结合溶氧反馈脉冲补料(DO-STAT补料)的两阶段补糖策略,达到解除克勒勃屈利效应(Crabtree效应)和降低副产物乙醇生成的目的,从而实现法夫酵母细胞的大量增殖和虾青素产量的提高。本发明提供的补糖培养基及高密度培养方法,可大幅提高法夫酵母虾青素产量水平,在100m～(3)发酵罐上虾青素产量为570.0mg/L,适用于商业化生产。(The invention provides a Phaffia rhodozyma saccharide-supplementing culture medium, a high-density culture method and application thereof, wherein the saccharide-supplementing culture medium contains a carbon source combination beneficial to cell proliferation and astaxanthin synthesis, and can make up for the defect of low yield of a single carbon source. The high-density culture method uses an online living cell sensor to detect the concentration of living cells, and achieves the aims of relieving the Crabtree effect and reducing the generation of a byproduct ethanol by adopting a two-stage sugar supplement strategy of combining index supplement with dissolved oxygen feedback pulse supplement (DO-STAT supplement), thereby realizing the mass proliferation of the Phaffia rhodozyma cells and the improvement of the yield of astaxanthin. The sugar supplement culture medium and the high-density culture method provided by the invention can greatly improve the yield level of phaffia rhodozyma astaxanthin at 100m 3 The astaxanthin yield on the fermenter was 570.0mg/L, is suitable for commercial production.)

1. The sugar supplement culture medium for the phaffia rhodozyma is a mixed carbon source consisting of a carbon source which is favorable for cell proliferation and a carbon source which is favorable for astaxanthin synthesis.

2. The carbohydrate supplementation medium of claim 1, wherein the ratio of the concentration of the carbon source that favors cell proliferation to the concentration of the carbon source that favors astaxanthin synthesis is 1: 3-3: 1.

3. the sugar-supplementing culture medium according to claim 1, wherein the concentration of the sugar-supplementing culture medium is 320-800 (g/L).

4. The sugar-supplemented medium of any one of claims 1-3, wherein the carbon source that facilitates cell proliferation comprises molasses, and one or more selected from the group consisting of glucose, maltose, and sucrose; the carbon source favorable for astaxanthin synthesis comprises one or more selected from maltodextrin, starch hydrolysate, glycerol and soluble starch.

5. The sugar-supplemented medium of claim 4, wherein the carbon source that facilitates cell proliferation is a combined carbon source of molasses and glucose.

6. The carbohydrate supplementation medium according to claim 4, wherein the carbon source favoring astaxanthin synthesis is maltodextrin.

7. The sugar-supplementing culture medium according to claim 4, wherein the concentration of the molasses is 20-100 (g/L).

8. A high-density culture method using the sugar supplement culture medium for Phaffia yeast according to any one of claims 1 to 7, the high-density culture method comprising the steps of:

a) solid culture: carrying out plate streaking culture on the frozen glycerol preservation solution of the phaffia rhodozyma to obtain a single colony;

b) seed bottle culture: picking single bacterial colony on a solid plate, and performing primary seed bottle culture and secondary seed bottle culture;

c) seed tank culture: inoculating the seed bottle culture solution into a seed tank, and performing primary seed tank culture or secondary seed tank culture;

d) culturing in a fermentation tank: transferring the culture solution in the seeding tank to a fermentation tank, introducing compressed air, supplementing a carbon source by adopting a two-stage sugar supplementing process, and controlling relevant process parameters such as environment, physiology and the like to obtain the high-yield astaxanthin phaffia rhodozyma culture solution.

9. The method according to claim 8, wherein in step d), the two-stage sugar supplement process comprises the following steps:

I) batch culture: during the period from the beginning of fermentation to the end of consumption of the basic material carbon source and when the dissolved oxygen rises to more than 60-80%, no material is supplemented;

II) exponential feeding: after the dissolved oxygen rises to more than 60-80%, supplementing a sugar-supplementing culture medium according to an index feed supplement, and controlling the specific growth rate mu to be 0.02-0.10 h^-1Changing the feeding rate every 0.5-2.0 h, wherein the feeding rate is calculated according to the following formula:

F＝μV₀ρ_c0e^μt/[Y_X/S(ρ_SF-ρ_S)]wherein:

f is the feed rate (L/h);

μ is the set specific growth rate (h) of the yeast^-1)；

V₀Start the volume of medium in the tank (L) for feeding;

ρ_c0the mass concentration (g/L) of the thalli in the tank is the beginning of material supplementing;

Y_X/Sthe yield (%) of the cells was determined;

ρ_SFthe total sugar mass concentration (g/L) of a fed-batch culture medium;

ρ_Sthe mass concentration (g/L) of total sugar in the tank when the material feeding is started;

t is the exponential feeding time (h);

III) DO-STAT feeding: and (3) after the exponential feeding is carried out until the late stage of the exponential feeding, when 1-10 g/L ethanol is detected in the fermentation liquor, changing the feeding mode into DO-STAT feeding, wherein the dissolved oxygen correlation value of the DO-STAT feeding is 20-60%, sugar feeding is stopped when the dissolved oxygen is lower than a set value, and sugar feeding is started when the dissolved oxygen is higher than the set value.

10. The method of claim 8, wherein the cultivation temperature of the primary seed bottle and the secondary seed bottle is 17-24 ℃, and the rotation speed of the shaker is 150-250 rpm; the first-stage seed bottle culture time is 36-72 hours, and the second-stage seed bottle culture time is 24-48 hours; the seed culture medium contains the following components(g/L): glucose 20, malt extract powder 6, peptone 10, yeast extract powder 6, CaCl₂0.1、KH₂PO₄ 1、MgSO₄·7H₂O0.5 and molinate 0.2; the pH value of the seed culture medium before consumption is 5.0-6.5.

11. The method as claimed in claim 8, wherein the culture solution of the seed bottle is inoculated to the primary seed tank according to the inoculation ratio of 0.25-1.5%, and the culture solution of the primary seed tank is transferred to the secondary seed tank according to the seed transferring ratio of 5-20%; wherein the culture temperature of the seeding tank is 17-24 ℃, and the culture time is 16-64 h; the seed tank culture is carried out in a small test seed tank, preferably 30L, the stirring speed is 150-600 rpm, and the aeration ratio is 0.3-3.0: 1 vvm; the primary seeding tank culture is carried out in a commercial production primary seeding tank, preferably 1m³Stirring at a speed of 50-300 rpm, and an aeration ratio of 0.25-2.5: 1 vvm; the secondary seeding tank culture is carried out in a commercial production secondary seeding tank, preferably 10m³Stirring at a speed of 50-250 rpm, and an aeration ratio of 0.1-2.5: 1 vvm; the pressure of the seed tank culture tank of each scale is 0.02-0.1 MPa.

12. The method according to claim 8 or 9, characterized in that in the step d), the seed transferring proportion of the fermentation tank is 5-20%, the culture temperature is 17-24 ℃, the pH value is controlled to be 4.5-6.0 by ammonia water in the whole process, the dissolved oxygen is controlled to be 20-60%, the tank pressure is 0.02-0.1 MPa, 3-10 g/L of lactic acid is supplemented in 36-60 h and 84-108 h respectively, and the fermentation time is 120-240 h.

13. The method of claim 8, wherein the fermentation medium comprises the following components (g/L): 15-35% of glucose, 3-7% of molasses and (NH)₄)₂SO₄1.8-4.2, 3-7 of yeast extract powder, 3-7 of soybean peptone and KH₂PO₄ 0.64～1.47、Na₂HPO₄·12H₂1.35-3.15% of O, 3-7% of lactic acid and CaCl₂0.06～0.14、MgSO₄·7H₂1.35-3.15% of O, 0.1-0.4% of foam enemy, 0.5-1.5% of trace metal solution and 0.5-1.5% of vitamin solution; what is needed isThe trace metal solution comprises the following components (g/L): h₃BO₃ 2.67、CuSO₄·5H₂O 1.6、KI 0.27、MnCl₂ 2.7、Na₂MoO₄·2H₂O 1.07、ZnSO₄·7H₂O 24、CoCl₂0.8, ferric citrate 24; the vitamin solution comprises the following components in percentage by weight (g/L): calcium pantothenate 5.2, biotin 0.13, inositol 66.67, nicotinic acid 5.2, p-aminobenzoic acid 0.53, VB₆ 2.67、VB₁2.67, riboflavin 5.2; the pH value of the fermentation medium before consumption is 5.0-6.5.

14. The method according to claim 8 or 9, wherein in the step d), the volume of the small-scale fermentation tank is 70L, the stirring speed is 100-700 rpm, and the aeration ratio is 0.3-3.0: 1 vvm; the pilot plant fermentation tank is 10m³Stirring at a speed of 50-250 rpm, and an aeration ratio of 0.2-2.5: 1 vvm; the commercial fermentor was 100m³Stirring at a speed of 15-200 rpm, and a ventilation ratio of 0.1-1.5: 1 vvm.

15. Use of a method according to any one of claims 8 to 14 in the production of phaffia yeast astaxanthin.

Technical Field

The invention relates to the technical field of microbial fermentation, in particular to a Phaffia yeast sugar-supplementing culture medium, a high-density culture method and application thereof.

Background

Astaxanthin (Astaxanthin) is an oxygen-containing carotenoid with the chemical name of 3,3 ' -dihydroxy-beta, beta ' -carotene-4, 4 ' -dione, and has a molecular structure in which the 3 and 3 ' positions of the ionone ring each contain a chiral (or asymmetric) center, so that 3S-3 ' S, 3R-3 ' S and 3R-3 ' R (also called levorotatory, meso-rotatory and dextrorotatory) 3 enantiomers can be formed. Astaxanthin has strong oxidation resistance, the oxidation resistance of the astaxanthin is 10 times that of beta-carotene and is more than hundred times stronger than vitamin E, the astaxanthin is known as super vitamin E, and the astaxanthin plays roles in delaying senility, improving immunity, preventing and treating tumors, cardiovascular diseases, diabetes and the like.

Phaffia rhodozyma (Phaffia rhodozyma), the extract of which is the most productive strain with commercial production potential, was proven to be safe, and the U.S. Food and Drug Administration (FDA) supplemented it for use as an animal feed additive in 2010 and entered the feed additive variety catalog of the Ministry of agriculture in China in 2013. With the expansion of downstream aquaculture scale, there is an increasing demand in the market for Phaffia yeast-derived astaxanthin. However, there are many problems in the production of astaxanthin by Phaffia rhodozyma, and the focus is on the major economic and technical indexes such as production intensity, yield level and raw material cost, which are far from commercial production. Therefore, the key point of realizing industrial production of the Phaffia rhodozyma is to modify and transform a cell metabolic pathway by using a gene recombination technology to change the cell characteristics and integrate a cell baseThe production intensity of the astaxanthin is further improved due to regulation and control, metabolism regulation and biochemical engineering strategies. The related technical research on the fermentation of Phaffia yeast is quite extensive at home and abroad. The patent with publication number CN 108913746A discloses that tomato powder is added into a fermentation medium, the pH value is controlled to be 4.0-6.0 by citric acid and NaOH, the glucose concentration is maintained to be 15-35 g/L, the biomass reaches 65.9g/L, and the astaxanthin content reaches 81.76 mg/L. Xiaoanfeng and the like research the influence of 5 sugar metabolites of ethanol, sodium acetate, sodium lactate, citric acid and glycerol on the astaxanthin production of Phaffia rhodozyma, and the sodium acetate, the sodium lactate and the citric acid are considered to have certain promotion effects on the astaxanthin production, and the maximum astaxanthin concentration reaches 1.54mg/L when the addition amount of lactic acid is 1 g/L. Yu-ICHI YAMANE and the like studied the influence of oxygen content and glucose supply on the primary metabolism of Phaffia rhodozyma and astaxanthin production, and considered that the respiration rate is in positive correlation with the astaxanthin production rate and in negative correlation with the ethanol production rate, and the results that the astaxanthin yield is improved by high C/N ratio and the cell proliferation is inhibited by excessively high glucose concentration were obtained, and on the basis, a two-stage sugar supplement mode that the C/N ratio is controlled to be low in the cell growth phase and the C/N ratio is controlled to be high in the astaxanthin production phase was proposed. Nighui et al report that the automatic fed-batch pH control is more beneficial to the growth of Phaffia rhodozyma cells and the synthesis of astaxanthin than the intermittent pH control, 1m³The biomass production and the astaxanthin production of the pilot plant fermentation tank respectively reach 85.11g/L and 279.96 mg/L. The above-mentioned related studies and techniques, although having a certain effect on yield improvement, have the following problems: 1) the deep influence of Crabtree effect on the growth of the thallus is not fully considered; 2) regulation and control are only carried out from a carbon source, a promoter or an environmental factor and the like of a single factor, and dynamic, multi-scale and global analysis of physiological processes and nutritional requirements is lacked; 3) the feeding formula which accords with the physiological characteristics of the strain is not fully designed, and fine and effective feeding control is carried out;

the exponential feeding is a feedforward control type feeding mode which is expected to harvest thalli in an exponential growth mode, the feeding rate and the operation time are changed in an exponential mode, but dissolved oxygen is difficult to maintain in the later period of the exponential feeding, and carbon overflow is easy to occur to generate ethanol. The DO-STAT method uses a sharp rise in dissolved oxygen as a signal for carbon source feeding, and can control the substrate (carbon source) concentration at a low level close to 0, thereby effectively suppressing the production of numerous metabolic byproducts, but a long culture period affects the production intensity. The astaxanthin is an intracellular product, the yield of the astaxanthin is related to biomass and the astaxanthin content of dry thalli, so that the environment conditions of proper temperature, pH value, dissolved oxygen, conductivity and the like are ensured in the optimal culture medium components and content, a reasonable fed-batch culture process is established by integrating the characteristics of index feed and DO-STAT feed, the Crabtree effect is reduced to limit or reduce the generation of harmful metabolites (ethanol), the aim of high-density culture can be fulfilled, and the production intensity of the astaxanthin is greatly improved.

Disclosure of Invention

The invention aims to solve the technical problem that the sugar supplement culture medium and the high-density culture method of the phaffia rhodozyma can obviously improve the dry weight of cells and further improve the yield of the astaxanthin, realize the unification of high yield, high yield and high production strength of the astaxanthin and are beneficial to commercial production aiming at the defects that the yield and the dry weight of the cells are low in the fermentation production of the astaxanthin by the phaffia rhodozyma at present.

The invention adopts a sugar supplement culture medium and a high-density culture method, detects the content of living cells by an online living cell sensor, removes the Crabtree effect by adopting a sugar supplement strategy combining index feed and DO-STAT feed, reduces the content of ethanol generated by carbon overflow, and realizes the mass proliferation of the Phaffia rhodozyma cells and the improvement of the yield of astaxanthin.

According to one aspect of the present invention, there is provided a saccharide supplement medium for Phaffia rhodozyma, which is a mixed carbon source consisting of a carbon source that favors cell proliferation and a carbon source that favors astaxanthin synthesis.

In a preferred embodiment of the sugar supplement medium for Phaffia yeast of the present invention, the ratio of the concentration of the carbon source that contributes to cell proliferation to the concentration of the carbon source that contributes to astaxanthin synthesis is preferably 1: 3-3: 1.

in a preferred embodiment of the sugar supplement culture medium for Phaffia rhodozyma of the present invention, the concentration of the sugar supplement culture medium is preferably 320-800 (g/L).

In a preferred embodiment of the sugar supplement medium for Phaffia yeast of the present invention, preferably, the carbon source that facilitates cell proliferation comprises molasses and one or more selected from glucose, maltose and sucrose; more preferably, the carbon source that favors cell proliferation is a combined carbon source of molasses and glucose. The carbon source favorable for astaxanthin synthesis comprises one or more selected from maltodextrin, starch hydrolysate, glycerol and soluble starch, and more preferably, the carbon source favorable for astaxanthin synthesis is maltodextrin.

In a preferable technical scheme of the Phaffia rhodozyma sugar-supplementing culture medium, the concentration of the molasses is preferably 20-100 (g/L).

According to another aspect of the present invention, the present invention provides a method for high density culture using a sugar supplement culture medium for Phaffia yeast, comprising the steps of:

a) solid culture: carrying out plate streaking culture on the frozen glycerol preservation solution of the phaffia rhodozyma to obtain a single colony;

b) seed bottle culture: picking single colony on a solid plate, and performing primary (test tube) and secondary seed bottle culture;

c) seed tank culture: inoculating the seed bottle culture solution into a seed tank, and performing primary or secondary seed tank culture;

d) culturing in a fermentation tank: transferring the culture solution in the seeding tank to a fermentation tank, introducing compressed air, supplementing a carbon source by adopting a two-stage sugar supplementing process, and controlling relevant process parameters such as environment, physiology and the like to obtain the high-yield astaxanthin phaffia rhodozyma culture solution.

In a preferred embodiment of the method of the present invention, preferably, the solid culture comprises: freezing the glycerol preservation solution of the phaffia rhodozyma preserved in a refrigerator at the temperature of minus 80 ℃, dipping the thawing solution by using an inoculating loop after thawing, and carrying out plate streaking at the culture temperature of 17-24 ℃ for culturing for 4-8 days to grow larger single colonies.

Here, solid culture is a prior art, including: a) plate streaking and frozen glycerol storage are conventional methods in the field of microorganisms; b) the solid medium formulation is a reported formulation (see document [1 ]); c) the culture temperature range is a temperature generally reported in the literature (see document [1 ]: zhumingjun, Puyuwu, Wuhaizhen, etc. influence of different carbon sources and their concentrations on the culture of Phaffia rhodozyma [ J ]. proceedings of the university of southern China (Nature science edition), 2002,30(04): 77-80). Wherein, the solid culture medium formula (g/L): glucose 10, yeast extract 3, peptone 5, wort 3, agar 20, pH 5.0.

In a preferred embodiment of the method of the present invention, preferably, the seed bottle culture comprises: when the first-level seed bottle is used for culturing, 3-5 single colonies which are relatively red in color and relatively large in colony size are dipped by an inoculating ring, and a seed culture medium (a 10mL/50mL test tube) is inoculated. And (3) when the secondary seed bottle is cultured, taking the primary seed bottle culture solution according to the inoculation proportion of 5-20% to inoculate a seed culture medium for the second time (22.5mL/250mL triangular flask). In the first-stage seed bottle culture and the second-stage seed bottle culture, the culture temperature is 17-24 ℃, the rotating speed of a shaking table is 150-250 rpm, the culture time of the first-stage seed bottle is 36-72 hours, and the culture time of the second-stage seed bottle is 24-48 hours. The seed culture medium contains the following components (g/L): glucose 20, malt extract powder 6, peptone 10, yeast extract powder 6, CaCl₂0.1、KH₂PO₄1、MgSO₄·7H₂O0.5 and molinate 0.2. The pH value of the seed culture medium before consumption is 5.0-6.5.

Here, the operation and culture conditions in seed bottle culture are prior art, including: a) dipping 3-5 single colonies, and inoculating a seed culture medium for conventional operation of microorganisms; b) seed flask culture adopts secondary culture as a reported method (see document [2 ]: YANNE Y, HIGASHIDA K, HAKASHIMADA Y, et al, infection of oxidative and glucose on primary metabolism and activity production by Phaffia rhodozyma in Batch and fed-Batch cultures: kinetic and stoichiometric analysis [ J ]. Appl Environ Microbiol 1997,63(11):4471 and 4478).

However, the seed culture medium of the present invention is an improvement over the prior art.

Document [3](Xiaoanfeng Nihui Lijun, Lilisjun et al repeated batch and repeated fed-batch fermentation of astaxanthin produced by Phaffia rhodozyma [ J]Bioengineering journal, 2011,27(04): 598-: glucose 20, (NH)₄)₂SO₄ 5、KH₂PO₄ 1、MgSO₄·7H₂O 0.5、CaCl₂0.1 and yeast extract 3.

The invention uses 5g/L (NH) of nitrogen source₄)₂SO₄And 3g/L yeast extract is improved into 6g/L malt extract powder, 10g/L peptone and 6g/L yeast extract powder, and the foam killer plays a defoaming role and is a conventional component in the fermentation industry.

In a preferred embodiment of the method of the present invention, the seeding tank culture preferably comprises: inoculating the seed bottle culture solution to a first-stage seed tank according to an inoculation proportion of 0.25-1.5%, and transferring the culture solution of the first-stage seed tank to a second-stage seed tank according to a seed transferring proportion of 5-20%. The culture temperature of the seeding tank is 17-24 ℃, and the culture time is 16-64 h. The seed tank culture is carried out in a small test seed tank, preferably 30L, the stirring speed is 150-600 rpm, and the aeration ratio is 0.3-3.0: 1 vvm. The primary seeding tank culture is carried out in a commercial production primary seeding tank, preferably 1m³Stirring at a speed of 50-300 rpm, and an aeration ratio of 0.25-2.5: 1 vvm. The secondary seeding tank culture is carried out in a commercial production secondary seeding tank, preferably 10m³Stirring at a speed of 50-250 rpm, and an aeration ratio of 0.1-2.5: 1 vvm. The pressure of the seed tank culture tank of each scale is 0.02-0.1 MPa. The seeding tank culture medium is synchronous with the seed culture medium in the step b).

No specific report is found in related documents about seeding tank culture: a) the culture scale of the fermentation tank of Phaffia yeast is reported to be relatively small, mainly using a 5L glass tank as a main fermentation tank, and the corresponding seed culture is provided by a triangular flask (see, for example, U.S. Pat. No. 4, 7432076, 2 and document [2 ]). b) In the report on pilot scale seeding tank culture, US 5356810 was cultured in a seeding tank by inoculating 1L of seed culture into a 100L fermentor (containing 30L of YM medium) and culturing at 20-22 ℃ until the dry weight of the cells became 1 g/L.

The production performance of a Phaffia yeast astaxanthin high-producing strain [ J ] in literature [4] (Nichou, Honghun, Xiaoanfeng, etc.; Productivity of bioengineering, 2011,27(07): 1065-: the inoculation amount is 2%, the temperature is 22 ℃, the pH value is 6.0, the dissolved oxygen is 30-50%, and the culture time is 72 h.

In summary, the pilot-scale seeding tank culture of the present invention differs from the prior art in that: a) the seeding tank inoculation ratio described by the invention is 0.25-1.5%, which is different from the prior art report; b) the culture time of the seeding tank is 16-64 h, which is different from the prior art; c) the ventilation capacity and the stirring speed of the seeding tank described by the invention are not reported in the prior art; d) the invention adopts secondary seed tank culture, and has no report; e) the invention describes the pot pressure control and has not been reported.

In the preferable technical scheme of the method, preferably, in the step d), the seed transferring proportion of the fermentation tank is 5-20%, the culture temperature is 17-24 ℃, the pH value is controlled to be 4.5-6.0 by ammonia water in the whole process, the dissolved oxygen is controlled to be 20-60%, the tank pressure is 0.02-0.1 MPa, 3-10 g/L of lactic acid is supplemented in 36-60 h and 84-108 h respectively, and the fermentation time is 120-240 h.

Compared with the prior art about the seed transferring proportion of a fermentation tank, the fermentation temperature, the pH value, the dissolved oxygen, the tank pressure and the lactic acid supplementing process, a) the pH value, the fermentation temperature and the dissolved oxygen described by the invention and the culture pH value control mode of the Phaffia rhodozyma disclosed in US 7432076B 2 are NH₄Compared with the prior art, the process that the pH value of OH solution and/or NaOH solution is controlled to be 4.5-7.0, the fermentation temperature is 15-24 ℃, and the Dissolved Oxygen (DO) is 10-90%; b) proportion of seed transfer in fermenters described in the present invention and literature [2]]The 10% comparison of the single seed transfer ratio disclosed in US 7432076B 2 is different from the prior art reports; c) the tank pressure control of the fermentation tank described by the invention is not reported in the prior art; d) lactic acid supplementation process described in the present invention, and literature [5 ]](Nile, Nihui, Xiaoanfeng, etc.. Effect of sodium lactate on astaxanthin production by Phaffia yeast [ J]Food and fermentation industries 2010,36(8):106-](Shenningyan, Xiaoanfeng, Chua Huiyongong, et al sodium lactate promotes analysis of the anabolic flux of phaffia yeast astaxanthin [ J ]]Microbiological information 2015,42(4):634-645) reported that lactic acid increased astaxanthin production, 72h and 96h addition of sodium lactate reduced astaxanthin volume yield, and that the conclusion or process comparison of glucose and sodium lactate mixed supplementation was an improvement over the prior art.

In a preferred embodiment of the method of the present invention, preferably, in step d), the lab-scale fermentor is cultured, preferably 70L, with a stirring speed of 100-700 rpm,the ventilation ratio is 0.3-3.0: 1 vvm; the pilot fermentation tank is preferably 10m³Stirring at a speed of 50-250 rpm, and an aeration ratio of 0.2-2.5: 1 vvm; the commercial fermenter culture is preferably 100m³Stirring at a speed of 15-200 rpm, and a ventilation ratio of 0.1-1.5: 1 vvm.

Regarding the aeration and agitation process, the dissolved oxygen control described in the present invention is to maintain the dissolved oxygen level by controlling the aeration ratio, the agitation speed and the tank pressure, which is different from the prior art reports in comparison with the fixed agitation and fixed aeration disclosed in US 2003/0049241 a 1.

In a preferred embodiment of the method of the present invention, preferably, in step d), the two-stage sugar supplementation process comprises the following steps:

I) batch culture: during the period from the beginning of fermentation to the end of consumption of the basic material carbon source and when the dissolved oxygen rises to more than 60-80%, no material is supplemented;

II) exponential feeding: after the dissolved oxygen rises to more than 60-80%, supplementing a sugar-supplementing culture medium according to an index feed supplement, and controlling the specific growth rate mu to be 0.02-0.10 h^-1Changing the feeding rate every 0.5-2.0 h, wherein the feeding rate is calculated according to the following formula:

F＝μV₀ρ_c0e^μt/[Y_X/S(ρ_SF-ρ_S)]wherein:

f is the feed rate (L/h);

μ is the set specific growth rate (h) of the yeast^-1)；

V₀Start the volume of medium in the tank (L) for feeding;

ρ_c0the mass concentration (g/L) of the thalli in the tank is the beginning of material supplementing;

Y_X/Sthe yield (%) of the cells was determined;

ρ_SFthe total sugar mass concentration (g/L) of a fed-batch culture medium;

ρ_Sthe mass concentration (g/L) of total sugar in the tank when the material feeding is started;

t is the exponential feeding time (h);

III) DO-STAT feeding: and (3) after the exponential feeding is carried out until the late stage of the exponential feeding, when 1-10 g/L ethanol is detected in the fermentation liquor, changing the feeding mode into DO-STAT feeding, wherein the dissolved oxygen correlation value of the DO-STAT feeding is 20-60%, sugar feeding is stopped when the dissolved oxygen is lower than a set value, and sugar feeding is started when the dissolved oxygen is higher than the set value.

Regarding the sugar supplement process, a) the influence of the US 7432076B 2, the document [6] (Jianxinglong, Hongqinglin, Chuajinong, etc.. on astaxanthin production by two Phaffia yeast strains [ J ]. microbiological report, 2013,40(11):1996 + 2004) was compared only to the influence of fed-batch, exponential, DO-STAT and pH-STAT feeds on Phaffia yeast fermentation; b) a two-stage sugar supplement strategy based on carbon-nitrogen chemical calculation composition analysis is reported in the literature [2], and a two-stage sugar supplement strategy based on carbon-nitrogen chemical calculation composition analysis is reported in the literature [7] (ruminants, epilogies, Liuyongsheng, and the like). feed strategy optimization for astaxanthin production by fermentation of Phaffia yeast based on a kinetic model is reported in the bioengineering science [ J ]. 2008,24(11): 1937-.

In conclusion, the two-stage sugar-supplementing process for index feed supplement-DO-STAT feed supplement disclosed by the invention can be used for removing ethanol generation, improving the bacterial quantity and the product yield, has obvious technical advantages and is different from the reports in the prior art.

In a preferred embodiment of the method of the invention, the fermentation medium preferably contains the following components (g/L): 15-35% of glucose, 3-7% of molasses and (NH)₄)₂SO₄1.8-4.2, 3-7 of yeast extract powder, 3-7 of soybean peptone and KH₂PO₄ 0.64～1.47、Na₂HPO₄·12H₂1.35-3.15% of O, 3-7% of lactic acid and CaCl₂ 0.06～0.14、MgSO₄·7H₂1.35-3.15% of O, 0.1-0.4% of foam enemy, 0.5-1.5% of trace metal solution and 0.5-1.5% of vitamin solution. The trace metal solution comprises the following components in percentage by weight (g/L): h₃BO₃ 2.67、CuSO₄·5H₂O 1.6、KI 0.27、MnCl₂ 2.7、Na₂MoO₄·2H₂O 1.07、ZnSO₄·7H₂O 24、CoCl₂0.8 and ferric citrate 24. The vitamin solution comprises the following components in percentage by weight (g/L): calcium pantothenate 5.2, biotin 0.13, inositol 66.67, nicotinic acid 5.2, p-aminobenzoic acid 0.53, VB₆ 2.67、VB₁2.67 and riboflavin 5.2. The pH value of the fermentation medium before consumption is 5.0-6.5. Most preferably, the fermentation medium contains the following components (g/L): glucose 25, molasses 5, (NH)₄)₂SO₄3. Yeast extract powder 5, soybean peptone 5, KH₂PO₄ 1.05、Na₂HPO₄·12H₂O2.25, lactic acid 5, CaCl₂0.1、MgSO₄·7H₂O2.25, molinate 0.2, trace metal solution 1 and vitamin solution 1. The metal elements, vitamins and pH are as described above.

As regards the fermentation medium, the composition and vitamin components of the batch fermentation medium are reported in the document [7] (CANNIZARO C, RHIELM, MARISON I, et al. on-line monitoring of Phaffia rhodozyma fed-batch process with in situ discrete Raman spectroscopy [ J ]. Biotechnol Bioeng,2003,83(6): 668) and the trace metal solution and vitamin solution components disclosed in the present invention are improvements over the prior art.

According to a further aspect of the invention, the invention provides the use of a method for high density culture of phaffia yeast astaxanthin production according to a phaffia yeast glucose-supplementing medium. The Phaffia strain suitable for the method of the invention is not particularly limited, and can be a naturally screened strain, or a mutant strain of traditional chemical and physical mutagenesis or an engineering bacterium modified by applying a genetic engineering method.

The invention has the advantages that: the optimized sugar supplement culture medium is beneficial to the growth of thalli and the improvement of titer, has low raw material price, is suitable for the high-density culture of the Phaffia yeast, and has the cost competitive advantage of commercial application. The sugar supplementing strategy combining index feed supplement and DO-STAT feed supplement can reduce the Crabtree effect, reduce the ethanol content of fermentation byproducts, and is beneficial to cell proliferation and the improvement of the astaxanthin yield. At 100m³The high-density culture of thallus and the improvement of astaxanthin yield are realized on a fermentation tank, and the culture quantity is 100m³The yield of the astaxanthin in the fermentation tank is 570.0mg/L, which is beneficial to the industrialized production of the astaxanthin.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods in the following examples, which are not specified under specific conditions, were selected according to the conventional methods and conditions, or according to the instructions. All percentages and parts are by weight unless otherwise indicated.

The following examples, the main instrumentation used: BIOTECH-30JS, Shanghai Baoxing Biometrics engineering Co., Ltd; LiLUS SP-70L fermenter, Baiteren (Biotron) of Korea; 1m³Seed tank, 10m³Seed tanks, Jiangsu remote Dewell Equipment science and technology Co., Ltd; 100m³Fermenter, Wuxi Dongshan petrochemical Equipment, Inc.; agilent model 1200 High Performance Liquid Chromatograph (HPLC), Agilent technologies, usa; biomass System type on-line living cell monitor, Inc., Bonnes, Hamilton, Switzerland.

In the following examples, the astaxanthin production was measured by High Performance Liquid Chromatography (HPLC) and the specific method was:

A) cell disruption and acetone extraction: taking 1mL fermentation liquid sample, centrifuging at 12000rpm for 3min, removing supernatant, washing the precipitate with distilled water, adding 2mL DMSO preheated to 75 ℃ into thalli, immediately and violently oscillating, breaking the wall at 50 ℃ for 5min, adding acetone for extraction, oscillating and shaking up, centrifuging at 12000rpm for 3min, extracting for multiple times until the hypha is white, combining the extracts, and fixing the volume to a certain volume;

B) high performance liquid detection of astaxanthin: column Dikma Diamonsil^TM-C18, 250 × 4.6mm i.d., 5 μm; the column temperature is 25 ℃; mobile phase: methanol with the flow rate of 1 mL/min; detection wavelength: 478 nm; sample introduction amount: 10 mu L of the solution;

in the following examples, the cell concentration was measured: properly diluting the fermentation liquor, measuring the absorbance at the wavelength of 600nm, and determining the dry cell weight according to the ratio of the optical density to the dry cell weight (g/L) of 2.50 +/-0.10;

the following examples, total sugar/reducing sugar assay: measured according to the film reagent method.

Example 1

Solid culture: freezing the Phaffia rhodozyma glycerol preservation solution preserved in a refrigerator at the temperature of minus 80 ℃, dipping the thawed Phaffia rhodozyma glycerol preservation solution in an inoculating loop, carrying out plate streaking, and culturing for 4-8 days at the temperature of 20.5 ℃ to grow a large single colony. The plate culture medium is YM culture medium (g/L): glucose 10, peptone 5, malt extract powder 3, yeast extract powder 3 and agar 20, and the pH value before consumption is 5.0.

Seed bottle culture: dipping 3-5 single colonies with red colors and large colonies by using an inoculating loop, inoculating a seed culture medium (10mL/50mL test tube), and performing shake culture for 36-72 h at 20.5 ℃ and 200 rpm. Inoculating the mature primary seed bottle culture solution into a secondary seed culture medium (22.5mL/250mL triangular flask) according to the proportion of 10 percent, and performing shake culture at the temperature of 20.5 ℃ and the speed of 200rpm for 24-48 h to obtain the mature seed bottle culture solution. The seed culture medium is (g/L): glucose 20, malt extract powder 6, peptone 10, yeast extract powder 6, CaCl₂ 0.1、KH₂PO₄ 1、MgSO₄·7H₂0.5 percent of O, 0.2 percent of natural enemy, and the pH value is adjusted to 5.0-6.5 before disinfection.

30L of seed tank culture: inoculating the culture solution of the secondary mature seed bottle to the seed culture medium (20L/30L seed tank) of the same seed bottle according to the inoculation ratio of 1.0%, stirring at the temperature of 20.5 ℃ and the rotation speed of 350rpm, and adjusting the ventilation volume ratio to be 2: culturing for 16-64 h under the conditions of 1vvm and 0.06MPa of tank pressure.

Culturing in a 70L fermentation tank: transferring a fermentation tank (30L/70L fermentation tank) according to the seed transferring proportion of 12%, wherein the culture temperature is 20.5 ℃, and the aeration ratio is 0.3-3.0: 1vvm and a tank pressure of 0.06MPa, controlling the pH value to be 5.0 by using 25-28% ammonia water in the whole process, linking the stirring rotation speed with dissolved oxygen, controlling the stirring rotation speed to be 40%, and supplementing 8g/L lactic acid in 48h and 96h respectively (adjusting the pH value to be 4.5-6.0 according to the volume of fermentation liquor). When the carbon source of the basic material is exhausted and the dissolved oxygen begins to rise to 60-80%, feeding a sugar supplementing culture medium (the feeding rate is changed every 1 h), and setting the specific growth rate to be 0.06h^-1. And when the living cell detector indicates that the sugar supplement enters the late stage of exponential feeding and detects that the ethanol content is 1-10 g/L, setting a sugar supplement pump to be interlocked with the dissolved oxygen value, wherein the correlation value is 40%, and carrying out DO-STAT mode sugar supplement. Wherein, the fermentation basal medium (g/L): glucose 25, molasses 5, (NH)₄)₂SO₄3. Yeast extract powder 5, soybean peptone 5, KH₂PO₄1.05、Na₂HPO₄·12H₂O2.25, lactic acid 5, CaCl₂ 0.1、MgSO₄·7H₂O2.25, 0.2 of natural enemy, 1 of trace metal solution, 1 of vitamin solution and 5.0-6.5 of pH value before consumption. The trace metal solution comprises the following components in percentage by weight (g/L): h₃BO₃ 2.67、CuSO₄·5H₂O 1.6、KI 0.27、MnCl₂ 2.7、Na₂MoO₄·2H₂O 1.07、ZnSO₄·7H₂O 24、CoCl₂0.8, ferric citrate 24; the vitamin solution comprises the following components (g/L): calcium pantothenate 5.2, biotin 0.13, inositol 66.67, nicotinic acid 5.2, p-aminobenzoic acid 0.53, VB₆ 2.67、VB₁2.67, riboflavin 5.2; sugar supplement medium (g/L): glucose 175, maltodextrin 175, molasses 50.

And (3) detecting total sugar, reducing sugar, amino nitrogen, thallus absorbance, astaxanthin content, ethanol content and the like simultaneously in the fermentation process, culturing for 185h, wherein no obvious Crabtree effect occurs in the fermentation process, no obvious ethanol accumulation is detected, the thallus dry weight reaches 100.8g/L, and the astaxanthin yield reaches 561.4 mg/L.

Example 2

Solid culture, seed bottle culture and seed tank culture: the same as in example 1.

Culturing in a 70L fermentation tank: when the carbon source of the basic material is exhausted and the dissolved oxygen begins to rise to 60-80%, feeding a sugar supplementing culture medium (the feeding rate is changed every 1 h), and setting the specific growth rate to be 0.06h^-1. Sugar supplement medium (g/L): maltose 130, molasses 20, soluble starch 150. The DO-STAT mode glucose supplementation stage is provided with a glucose supplementation pump interlocked with the dissolved oxygen value, and the correlation value is 40%. The fermentation medium and other process control conditions were the same as in example 1.

And (3) detecting total sugar, reducing sugar, amino nitrogen, thallus absorbance, astaxanthin content, ethanol content and the like simultaneously in the fermentation process, culturing for 185h, wherein no obvious Crabtree effect occurs in the fermentation process, no obvious ethanol accumulation is detected, the thallus dry weight reaches 101.6g/L, and the astaxanthin yield reaches 550.2 mg/L.

Example 3

Solid culture, seed bottle culture and seed tank culture: the same as in example 1.

Culturing in a 70L fermentation tank: when the basic material carbon source is exhausted and the dissolved oxygen begins to rise to 60-80%, feeding a sugar supplementing culture medium (the feeding rate is changed every 1 h), and setting the specific growth rate to 0.02h^-1. Sugar supplement medium (g/L): 100 parts of cane sugar, 100 parts of molasses, 300 parts of starch hydrolysate and 300 parts of glycerol. The DO-STAT mode glucose supplementation stage is provided with a glucose supplementation pump interlocked with the dissolved oxygen value, and the correlation value is 60%. The fermentation medium and other process control conditions were the same as in example 1.

And (3) detecting total sugar, reducing sugar, amino nitrogen, thallus absorbance, astaxanthin content, ethanol content and the like simultaneously in the fermentation process, culturing for 240h, wherein no obvious Crabtree effect occurs in the fermentation process, no obvious ethanol accumulation is detected, the thallus dry weight reaches 98.5g/L, and the astaxanthin yield reaches 546.1 mg/L.

Example 4

Solid culture, seed bottle culture and seed tank culture: the same as in example 1.

Culturing in a 70L fermentation tank: when the basic material carbon source is exhausted and the dissolved oxygen begins to rise to 60-80%, feeding a sugar supplementing culture medium (the feeding rate is changed every 1 h), and setting the specific growth rate to 0.1h^-1. Sugar supplement medium (g/L): glucose 250, maltose 250, molasses 100, maltodextrin 200. The DO-STAT mode glucose supplementation stage is provided with a glucose supplementation pump interlocked with the dissolved oxygen value, and the correlation value is 20%. The fermentation medium and other process control conditions were the same as in example 1.

And (3) detecting total sugar, reducing sugar, amino nitrogen, thallus absorbance, astaxanthin content, ethanol content and the like simultaneously in the fermentation process, culturing for 180 hours, wherein no obvious Crabtree effect occurs in the fermentation process, no obvious ethanol accumulation is detected, the thallus dry weight reaches 97.9g/L, and the astaxanthin yield reaches 557.8 mg/L.

Comparative example 1

The difference between the comparative example 1 and the example 1 is that in 70L fermentation tank culture, the traditional sugar supplement process of feeding back and adjusting sugar supplement rate by using a reducing sugar-total sugar detection result is adopted in the whole process, the total sugar concentration is maintained at 5-15 g/L, and other process conditions are completely the same. When the culture time is 180 hours, obvious ethanol accumulation (the content is 1.25 percent) is detected in the fermentation process, the dry weight of the thallus reaches 85.7g/L, and the yield of the astaxanthin reaches 357.2 mg/L.

Comparative example 2

The difference between the comparative example 2 and the example 1 is that in the 70L fermentation tank culture, after 1-10 g/L ethanol is detected by exponential feeding in the logarithmic phase, the traditional sugar feeding process of feeding sugar rate is adjusted by feeding back the detection result of reducing sugar and total sugar, the total sugar concentration is maintained at 5-15 g/L, and other process conditions are completely the same. When the culture time is 180 hours, obvious ethanol accumulation (the content is 1.02 percent) is detected in the fermentation process, the dry weight of the thallus reaches 94.4g/L, and the yield of the astaxanthin reaches 489.1 mg/L.

Comparative example 3

The difference between the comparative example 3 and the example 1 is that in the 70L fermentation tank culture, when the dissolved oxygen rises back to 60-80% after the basic material carbon source is consumed and the carbon source begins to be supplemented, a DO-STAT sugar supplement mode is adopted, a sugar supplement pump is connected with the dissolved oxygen value, sugar supplement is stopped when the dissolved oxygen is lower than 30%, sugar supplement is started when the dissolved oxygen is higher than 30%, and other process conditions are completely the same. When the culture time is 240 hours, no obvious Crabtree effect occurs in the fermentation process, no obvious ethanol accumulation is detected, the dry weight of the thallus reaches 71.6g/L, and the yield of the astaxanthin reaches 339.0 mg/L.

Example 5:

solid culture and seed bottle culture: the same as in example 1.

Seed tank culture: inoculating the second-stage mature seed bottle culture solution to the seed culture medium (0.5 m) of the same seed bottle according to the inoculation ratio of 1.0%³/1m³Seeding tank) at 20.5 + -0.2 deg.C, stirring speed of 200rpm, aeration ratio of 2.0: culturing for 16-64 h under the conditions of 1vvm and 0.06MPa of tank pressure; transferring the culture solution of the first-stage mature seeding tank to a second-stage seeding tank (5 m) according to the seed transferring proportion of 9.09%³/10m³Seeding tank) at 20.5 + -0.2 deg.C, stirring speed of 200rpm, aeration ratio of 2.0: culturing for 16-64 h under the conditions of 1vvm and 0.06MPa of tank pressure.

100m³Culturing in a fermentation tank: transferring the secondary seed tank to the fermentation tank (35 m) according to the seed transferring ratio of 13.5%³/100m³Fermenter), the culture temperature is 20.5 + -0.2 deg.C, and the aeration ratio is 0.1-1.5: 1vvm, the tank pressure is 0.06MPa, and the pH value is controlled to be 5.0 by 25 to 28 percent ammonia water in the whole process. The initial stirring speed is 40rpm, when the dissolved oxygen is lower than 20%, the stirring speed is adjusted up to 10-30 rpm until the maximum power of the fermentation tank, the dissolved oxygen is controlled within the range of 20-60%, and 8g/L lactic acid is supplemented in 48h and 96h respectively (the pH value is adjusted to be 4.5-6.0). When the carbon source of the basic material is exhausted and the dissolved oxygen begins to rise to 60-80%, feeding a sugar supplementing culture medium (the feeding rate is changed every 1 h), and setting the specific growth rate to be 0.06h^-1. And when the living cell detector indicates that the sugar-supplementing rate is in the late stage of exponential feeding and the ethanol content is 1-10 g/L, setting the sugar-supplementing rate and dissolved oxygen interlocking, wherein the correlation value is 40%, and carrying out DO-STAT mode sugar supplementing. Wherein the components of the fermentation basal medium, the trace metal solution, the vitamin solution, the sugar supplement medium and the supplement medium are the same as those in example 1.

And (3) detecting total sugar, reducing sugar, amino nitrogen, thallus absorbance, astaxanthin content, ethanol content and the like simultaneously in the fermentation process, culturing for 185h, wherein no obvious Crabtree effect occurs in the fermentation process, no obvious ethanol accumulation is detected, the thallus dry weight reaches 101.4g/L, and the astaxanthin yield reaches 570.0 mg/L.

It should be noted that the above summary and the detailed description are intended to demonstrate the practical application of the technical solutions provided by the present invention, and should not be construed as limiting the scope of the present invention. Various modifications, equivalent substitutions, or improvements may be made by those skilled in the art within the spirit and principles of the invention.