CN112553094A - Fermentation method based on yarrowia lipolytica, fermentation broth and application of fermentation broth - Google Patents
Fermentation method based on yarrowia lipolytica, fermentation broth and application of fermentation broth Download PDFInfo
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/60—Feeding-stuffs specially adapted for particular animals for weanlings
Abstract
The application relates to the technical field of microbial fermentation, in particular to a fermentation method based on yarrowia lipolytica, fermentation liquor and application of the fermentation liquor. The fermentation method comprises the following steps: activating yarrowia lipolytica strain to obtain a seed solution; placing the seed liquid in a liquid culture medium to perform stirring fermentation treatment under an acidic condition; wherein the liquid medium comprises: 20-30g/L glucose, 8-16g/L yeast extract, 0.1-0.3g/L anhydrous magnesium sulfate, and 2-3g/L potassium dihydrogen phosphate; during the stirring fermentation treatment, the dissolved oxygen concentration is kept between 20 and 40 percent, and when the dissolved oxygen concentration is lower than 20 percent, glycerol and yeast peptone are added. The fermentation method can reduce the risk of contamination, has high fermentation degree, and can obviously improve the dry weight of the cells of the yarrowia lipolytica in the fermentation product, thereby having good application prospect.
Description
Technical Field
The application belongs to the technical field of microbial fermentation, and particularly relates to a fermentation method based on yarrowia lipolytica, fermentation broth and application of the fermentation broth.
Background
Yarrowia lipolytica (Yarrowia Ipolytica) is an excellent lipase and single cell protein producer. The yeast has the following characteristics: (1) the strain is Regarded as Safe strain by the food and drug administration (GRAS), and has wide application prospect in food, medicine and feed industries; (2) can synthesize a large amount of intracellular protein and can secrete protease and the like; (3) short growth period, easy culture, high thallus protein content and is one excellent feed yeast.
The yarrowia lipolytica expression system is safe and reliable, has no resistance gene and no deficient protease, can economically and efficiently express active molecules, and has industrial development prospect. However, the conventional batch fermentation method adopted by the existing fermentation process of the lipid yarrowia yeast has low fermentation degree and easy bacterial contamination, and is not beneficial to industrial large-scale production.
Therefore, the related art is in need of improvement.
Disclosure of Invention
The application aims to provide a fermentation method based on yarrowia lipolytica, fermentation liquor and application of the fermentation liquor, and aims to solve the technical problems of low fermentation degree and easy bacterial contamination of the existing yarrowia lipolytica fermentation process.
In order to achieve the purpose of the application, the technical scheme adopted by the application is as follows:
in a first aspect, the present application provides a yarrowia lipolytica-based fermentation method comprising the steps of:
activating yarrowia lipolytica strain to obtain a seed solution;
placing the seed liquid in a liquid culture medium to perform stirring fermentation treatment under an acidic condition;
wherein the content of the first and second substances,
the liquid medium comprises: 20-30g/L glucose, 8-16g/L yeast extract, 0.1-0.3g/L anhydrous magnesium sulfate, and 2-3g/L potassium dihydrogen phosphate;
during the stirring fermentation treatment, the dissolved oxygen concentration is kept between 20 and 40 percent, and when the dissolved oxygen concentration is lower than 20 percent, glycerol and yeast peptone are added.
The fermentation method provided by the application is a high-density fermentation method based on yarrowia lipolytica, by selecting a proper culture medium, stirring and fermenting continuously under an acidic condition, the dissolved oxygen concentration is always kept at 20-40% in the stirring and fermenting process, and when the dissolved oxygen concentration is lower than 20%, glycerol and yeast peptone are supplemented, the yeast peptone can supplement energy in the supplement, and the glycerol can inhibit the formation of a large amount of filamentous yarrowia lipolytica, so that the spherical yarrowia lipolytica more suitable for high-density fermentation and target protein production can grow better, and the glycerol can inhibit the growth of other mixed bacteria, thereby reducing the risk of bacterial contamination; the fermentation method has high fermentation degree, can obviously improve the dry weight of the cells of the yarrowia lipolytica in the fermentation product, and has good application prospect.
In a second aspect, the present application provides a fermentation broth obtained by fermentation according to the fermentation method described herein.
The fermentation liquor provided by the application is obtained by fermentation by the fermentation method specific to the application. Therefore, the fermentation liquid has high fermentation degree, less infectious microbe, high dry cell weight of yarrowia lipolytica and good application prospect.
In a third aspect, the present application provides a use of the fermentation broth described herein in pig feed.
The application evaluates the effect of the fermentation broth on weaned piglets by mixing the fermentation broth for feeding and adding drinking water for feeding, and results show that the weight of the experimental piglets is obviously improved, and the piglets do not have diarrhea during the experiment, and have a trend of improving daily feed intake and average daily weight gain of the piglets, so that the fermentation broth has the effects of improving the immunity of the piglets and promoting the growth of the piglets, and can be well used in pig feed.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a schematic flow chart of a yarrowia lipolytica-based fermentation process provided in an embodiment of the present application;
FIG. 2 is a schematic diagram of the result of carbon source optimization in a liquid medium in a fermentation method provided in the examples of the present application;
FIG. 3 is a schematic diagram showing the result of optimizing a nitrogen source in a liquid medium in a fermentation method provided in an embodiment of the present application;
FIG. 4 is a schematic diagram of the results of optimization of inorganic salts in a liquid medium in a fermentation process provided in an embodiment of the present application;
FIG. 5 is an analysis chart of the results of the liquid medium response surface optimization test in the fermentation method provided in the embodiment of the present application.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present application more clearly apparent, the present application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.
In a first aspect, the embodiments of the present application provide a fermentation method based on yarrowia lipolytica, as shown in fig. 1, the fermentation method comprises the following steps:
s01: activating yarrowia lipolytica strain to obtain a seed solution;
s02: placing the seed liquid in a liquid culture medium to perform stirring fermentation treatment under an acidic condition;
wherein the content of the first and second substances,
the liquid medium comprises: 20-30g/L glucose, 8-16g/L yeast extract, 0.1-0.3g/L anhydrous magnesium sulfate, and 2-3g/L potassium dihydrogen phosphate;
during the stirring fermentation treatment, the dissolved oxygen concentration is kept between 20 and 40 percent, and when the dissolved oxygen concentration is lower than 20 percent, glycerol and yeast peptone are added.
The fermentation method provided by the application is a high-density fermentation method based on yarrowia lipolytica, by selecting a proper culture medium, stirring and fermenting continuously under an acidic condition, the dissolved oxygen concentration is always kept at 20-40% in the stirring and fermenting process, and when the dissolved oxygen concentration is lower than 20%, glycerol and yeast peptone are supplemented, the yeast peptone can supplement energy in the supplement, and the glycerol can inhibit the formation of a large amount of filamentous yarrowia lipolytica, so that the spherical yarrowia lipolytica more suitable for high-density fermentation and production of target protein can grow better, and the glycerol can inhibit the growth of other mixed bacteria, thereby reducing the risk of bacterial contamination; the fermentation method has high fermentation degree, can obviously improve the dry weight of the cells of the yarrowia lipolytica in the fermentation product, and has good application prospect.
In step S01, the step of activating the yarrowia lipolytica species to obtain a seed solution includes: streaking the frozen yarrowia lipolytica strain on a plate, then picking out a single colony and coating the single colony on a slant, inoculating the slant onto a yeast extract peptone glucose (YPD) liquid culture medium, and carrying out amplification culture with the inoculation amount of 10% to obtain the seed liquid. Specifically, the strain cryopreserved in a-80 ℃ glycerol tube can be streaked on a plate, followed by culture at 30 ℃ for 24 hours. Subsequently, the culture was inoculated into YPD liquid medium from the slant. In the embodiment of the application, the seed can be expanded and cultured to 1.5L of seed liquid with the inoculation amount of 10%.
In the above step S02, the results of the one-way orthogonal test were analyzed by using glucose as a carbon source, yeast extract as a nitrogen source, and anhydrous magnesium sulfate as an inorganic salt in the liquid medium. As shown in FIG. 2 (in the figure, 1-sucrose; 2-dry oil; 3-sorbitol; 4-glucose; 5-maltose; 6-lactose), sucrose, dry oil, sorbitol, glucose, maltose and lactose are optimized, and glucose is selected as a carbon source; as shown in FIG. 3 (in the figure, 1-yeast extract; 2-tryptone; 3-urea; 4-ammonium chloride; 5-yeast extract + ammonium chloride), yeast extract, tryptone, urea, ammonium chloride, yeast extract-ammonium chloride were optimized, and yeast extract was selected as a nitrogen source; as shown in figure 4 (in the figure, 1-anhydrous magnesium sulfate; 2-zinc sulfate heptahydrate, 3-manganese sulfate monohydrate; 4-ferrous sulfate heptahydrate; 5-anhydrous calcium chloride), anhydrous magnesium sulfate, zinc sulfate heptahydrate, manganese sulfate monohydrate, ferrous sulfate heptahydrate and anhydrous calcium chloride are optimized, and the anhydrous magnesium sulfate is selected as inorganic salt. In FIGS. 2-4, the higher the OD, the more yarrowia lipolytica grows, the better the effect.
In some embodiments, the response surface optimization fermentation process is performed by determining glucose, yeast extract and anhydrous magnesium sulfate as three central points of the response surface optimization process according to the results of the one-factor orthogonal test optimization culture medium, and the optimal conditions are that the glucose concentration is 25.21g/L, the yeast extract concentration is 15g/L, the anhydrous magnesium sulfate concentration is 0.22g/L and the potassium dihydrogen phosphate concentration is 2.5 g/L. Specifically, as shown in fig. 5, the response surface and its contour line of each influence factor are interacted with each other two by two through a regression equation. In addition, the P value of the regression model is 0.0054 < 0.01, indicating that the regression equation is very significant. The mismatching term has a value of 0.1062, is statistically insignificant, i.e., no mismatching factor exists, and the coefficient of determination R of the model291.45%, adjusted R2ADJBy accounting for 80.45%, the equation can account for 80.45% of the response change, so the model can be used instead of a real experiment. After processing the data using Design Expert 8.0.6 software, the optimal conditions for obtaining OD600 for 100mL fermentation culture of yarrowia lipolytica were 25.21g/L glucose concentration, 15g/L yeast extract concentration, and anhydrous magnesium sulfate concentrationThe degree is 0.22g/L, and the potassium dihydrogen phosphate is 2.5 g/L. The preferred liquid medium described above is more effective in fermentation.
In some embodiments, the step of placing the seed liquid in a liquid medium comprises a volume ratio of the seed liquid to the liquid medium of 1: 18-22. The fermentation can be well carried out under the proportion, and the deeply fermented fermentation liquor is obtained. Further, the volume ratio of the seed liquid to the liquid culture medium is 1: 20, for example, 1.5L of the seed liquid obtained by the spread culture is added to a fermenter containing 30L of the above liquid medium, and the high-density fermentation process is carried out.
In some embodiments, the dissolved oxygen concentration is maintained in the range of 20% -40% (specifically, 20%, 25%, 28%, 30%, 35%, 40%, etc.) by stirring and pressure during the stirred fermentation process. When the seed liquid is placed in a liquid culture medium to prepare for starting fermentation, the stirring speed can be in the range of 150-200r/min, and the pressure is in the range of 0.05MPa-0.1MPa, so that the dissolved oxygen concentration is kept in the range of 20% -40%. When the dissolved oxygen concentration is lower than the initial fermentation value, the liquid culture medium begins to be further consumed, and the dissolved oxygen generally has the phenomena of rapid reduction and rebound. Therefore, when the dissolved oxygen concentration is less than 20%, glycerol and yeast peptone may be added with continuous stirring and appropriate pressurization to maintain the dissolved oxygen concentration in the range of 20% -40%. Thus, the fermentation degree can be significantly improved by supplementing energy with yeast peptone, promoting better growth of spherical yarrowia lipolytica and inhibiting growth of miscellaneous bacteria with glycerol. Further, glycerol and yeast peptone are added when the dissolved oxygen concentration is below 10% (e.g., 5%, 8%, 10%, etc.).
In some embodiments, the step of adding glycerol and yeast peptone comprises: adding a dissolving solution containing glycerol and yeast peptone into a fermentation tank at the speed of 200-300ml/h (namely adding a composite culture medium formed by glycerol and yeast peptone in a fed-batch manner); wherein, in the dissolved solution, the glycerol is 18-22g/L, and the yeast peptone is 12-17 g/L. Under the above conditions, yarrowia lipolytica can be fermented more stably. In general, 1L (fed-batch rate 250ml/h, 4h completed) of glycerol and yeast peptone solution (glycerol 18-22g/L + yeast peptone 12-17g/L) can be added for energy supplementation every 30L of liquid medium for fermentation.
In one embodiment, the acidic condition is a pH of 5-6. Preferably, the optimum pH is maintained at 5.5.
In one embodiment, the stirring speed of the stirring fermentation treatment is 150-; the specific stirring speed is adjusted according to the dissolved oxygen.
In one embodiment, the fermentation time of the stirring fermentation treatment is 36-48h, and the fermentation temperature of the stirring fermentation treatment is 26-30 ℃.
The yarrowia lipolytica is fermented by YPD culture medium liquid or conventional batch fermentation method, the thallus is generally in the shape of mycelium, the fermentation degree is low and the strain is easy to infect. The method selects a proper culture medium, continuously stirs and ferments under an acidic condition, the dissolved oxygen is stabilized at 20-40% in the stirring and fermenting process, the material is supplemented properly, and the glycerol in the supplemented material can inhibit the formation of a large amount of filamentous yarrowia lipolytica, so that the spherical yarrowia lipolytica which is more suitable for high-density fermentation and can produce target protein can grow, and the glycerol can inhibit the growth of other mixed bacteria, thereby reducing the risk of bacterial contamination; for example, 1.5L of seed liquid after propagation is added into a fermentation tank containing 30L of liquid culture medium, the initial pH value is adjusted to be 5, and when the dissolved oxygen rapidly drops and rebounds during the fermentation process, a composite culture medium formed by glycerol and yeast peptone is fed, specifically, 20g/L of glycerol and 15g/L of yeast peptone can be added for about 1000mL, the pH is controlled to be 5.5, and the dissolved oxygen is adjusted to be 20% -40%.
In a second aspect, embodiments of the present application provide a fermentation broth, which is obtained by fermentation according to the fermentation method described herein.
The fermentation liquor provided by the application is obtained by fermentation by the fermentation method specific to the application. Therefore, the fermentation liquid has high fermentation degree, less infectious microbe, high dry cell weight of yarrowia lipolytica and good application prospect.
Specifically, the dry cell weight (i.e., dry cell weight of yarrowia lipolytica) in the fermentation broth is at least 40 g/L. The dry cell weight of yarrowia lipolytica in fermentation broth after fermentation is finished is obviously improved by adopting the fermentation method, for example, in one embodiment, the dry cell weight of yarrowia lipolytica reaches 45g/L, and the yield is improved by 125% compared with the yield of the conventional fermentation method.
Finally, the embodiment of the application also provides an application, namely the application of the fermentation liquor in the pig feed.
The application evaluates the effect of the fermentation broth on weaned piglets by mixing the fermentation broth for feeding and adding drinking water for feeding, and results show that the weight of the experimental piglets is obviously improved, and the piglets do not have diarrhea during the experiment, and have a trend of improving daily feed intake and average daily weight gain of the piglets, so that the fermentation broth has the effects of improving the immunity of the piglets and promoting the growth of the piglets, and can be well used in pig feed.
Specifically, the fermentation liquor is prepared into a yarrowia lipolytica preparation, the weight of piglets in the test for 30d is obviously improved by feeding the yarrowia lipolytica preparation, and the piglets fed with the yeast preparation during the test have no diarrhea (P < 0.05); the daily feed intake and average daily weight gain of piglets tend to be improved.
The following description will be given with reference to specific examples.
Example 1
A fermentation liquid is obtained by the following fermentation method:
1. activating the seed liquid: streaking yarrowia lipolytica strains preserved by freezing in a glycerol tube at-80 ℃, culturing for 24h at 30 ℃, picking out a single colony, coating a slant, and inoculating the single colony in a YPD liquid culture medium; expanding and culturing to 1.5L of seed liquid with the inoculation amount of 10 percent;
2. and (3) fermentation process: adding the expanded 1.5L seed liquid into a fermentation tank filled with 30L liquid culture medium (components: glucose concentration is 25.21g/L, yeast extract concentration is 15g/L, anhydrous magnesium sulfate concentration is 0.22g/L, potassium dihydrogen phosphate is 2.5g/L) to carry out stirring fermentation, adjusting the initial pH value to be 5, feeding (feeding speed is 250mL/h) about 1000mL (glycerol 20g/L + yeast peptone 15g/L) of a solution of glycerol and yeast peptone after the dissolved oxygen rapidly drops and rebounds in the fermentation process, controlling the pH value to be 5.5, stirring at a rotation speed of 150 and 200r/min, keeping the dissolved oxygen at 20-40% according to the dissolved oxygen adjustment, and controlling the fermentation temperature to be 28 ℃ and the fermentation time to be 40 h.
Example 2
The fermentation liquid obtained by the fermentation method in the example 1 is fed with a mixed feed to evaluate the effects of improving the immunity and promoting the growth of weaned piglets under the non-antibiotic feeding condition. The same batch of healthy Dan series (large X long X large) three-way hybrid weaned piglets with average weight of about 6.7kg are selected for the test, the piglets are divided into 2 treatment groups according to the weight (a control group A is fed with conventional feed; a fermentation liquor group A is fed with the conventional feed and the fermentation liquor of the example 1 is mixed into the feed), the feeding test for 30 days is carried out after the feeding of the feed and the weight measurement, the feces are collected on the 0 th day, the 14 th day and the 27 th day of the test, and 9 healthy pigs are respectively selected for the two groups to collect the feces. The relevant parameters and results are shown in table 1.
TABLE 1 Effect of fermentation broth fed with yarrowia lipolytica on growth Performance of weaned piglets
From table 1 above, it can be seen that:
(1) in terms of average daily food intake: the contrast group A is 435 +/-58 g, the fermentation liquid group A is 468 +/-77 g, and the fermentation liquid group A is improved by 7.58 percent compared with the contrast group A;
(2) in terms of average daily gain: the contrast group A is 302 +/-47 g, the fermentation liquid group A is 324 +/-65 g, and the fermentation liquid group A is improved by 7.28 percent compared with the contrast group A;
(3) in the aspect of material weight ratio: the contrast group A is 1.44 +/-0.02, and the fermentation liquid group A is 1.44 +/-0.03;
(4) aspect of diarrhea: the control group A is 0.01 plus or minus 0.00, and the fermentation liquor group A has no diarrhea condition of 0 plus or minus 0.00(P < 0.05).
The experiment shows that the weight of the piglets is obviously improved by feeding the fermentation liquor for 30 days in the experiment, the daily feed intake and the average daily gain of the piglets are obviously improved, and the piglets fed with the fermentation liquor preparation during the experiment have no diarrhea (P is less than 0.05).
Example 3
The effect of improving immunity and promoting growth of weaned piglets is evaluated by adding fermentation liquor into drinking water under the non-antibiotic feeding condition obtained by the fermentation method in the embodiment 1. The test selects 66 healthy Dan series (large X long X large) three-way hybrid weaned piglets which are weaned in the same batch and have the average weight of about 6.7kg, and the piglets are divided into 2 treatment groups according to the weight (a control group B: feeding with conventional feed; a fermentation liquor group B: feeding with conventional feed and the fermentation liquor of example 1 combining drinking water feeding), each group is treated for 3 times, 11 piglets are repeatedly fed for 27 days, the feces are collected on the 0 th day, the 14 th day and the 27 th day of the test, and 9 healthy pigs are respectively selected for collecting the feces in the two groups (3 pigs are selected in each column). The test result shows that: (1) in terms of average daily food intake: the contrast group B is 442g, the fermentation liquid group B is 450g, and the fermentation liquid group B is improved by 1.81 percent compared with the contrast group A;
(2) in terms of average daily gain: 339g of the control group B and 331g of the fermentation liquid group B, wherein the fermentation liquid group B is reduced by 2.36 percent compared with the control group B;
(3) in the aspect of material weight ratio: the contrast group B is 1.30, the fermentation liquid group B is 1.36, and the fermentation liquid group B is improved by 4.62 percent compared with the contrast group B;
(4) and (3) wool color: the fermentation liquor group B (the hair color is 2.12) is better than the control group B (the hair color is 2.00);
(5) and (3) skin color: the fermentation liquid group B (skin color 2.15) is better than the control group B (skin color 1.97);
(6) aspect of diarrhea: there was no difference in the diarrhea index of the two groups (0.002).
According to the production performance indexes, the fermentation liquor and the drinking water are combined to improve the appearance of the piglets, and the average daily feed intake and the feed-weight ratio of the piglets can be improved.
The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (10)
1. A yarrowia lipolytica-based fermentation process comprising the steps of:
activating yarrowia lipolytica strain to obtain a seed solution;
placing the seed liquid in a liquid culture medium to perform stirring fermentation treatment under an acidic condition;
wherein the content of the first and second substances,
the liquid medium comprises: 20-30g/L glucose, 8-16g/L yeast extract, 0.1-0.3g/L anhydrous magnesium sulfate, and 2-3g/L potassium dihydrogen phosphate;
during the stirring fermentation treatment, the dissolved oxygen concentration is kept between 20 and 40 percent, and when the dissolved oxygen concentration is lower than 20 percent, glycerol and yeast peptone are added.
2. The fermentation process of claim 1, wherein the step of activating yarrowia lipolytica species to obtain a seed solution comprises: and (3) streaking and culturing the frozen yarrowia lipolytica strain on a flat plate, then picking out a single colony and coating the single colony on a slant, inoculating the slant to a yeast-leached peptone glucose liquid culture medium, and carrying out propagation culture with the inoculation amount of 10% to obtain the seed liquid.
3. The fermentation process of claim 1, wherein the liquid medium comprises: 25.21g/L glucose, 15g/L yeast extract, 0.22g/L anhydrous magnesium sulfate, 2.5g/L potassium dihydrogen phosphate.
4. The fermentation process of claim 1, wherein the step of placing the seed solution in a liquid medium comprises placing the seed solution in a volume ratio of 1: 18-22.
5. The fermentation process of claim 1, wherein during the agitated fermentation process, glycerol and yeast peptone are added when the dissolved oxygen concentration is less than 10%. .
6. The fermentation process of claim 1, wherein the step of adding glycerol and yeast peptone comprises: adding a lysis solution containing glycerol and yeast peptone at a rate of 200-300 ml/h; wherein, in the dissolved solution, the glycerol is 18-22g/L, and the yeast peptone is 12-17 g/L.
7. A fermentation process according to any one of claims 1 to 6, wherein the acidic conditions are conditions at a pH of from 5 to 6; and/or the presence of a gas in the gas,
the stirring speed of the stirring fermentation treatment is 150-; and/or the presence of a gas in the gas,
the fermentation time of the stirring fermentation treatment is 36-48 h; and/or the presence of a gas in the gas,
the fermentation temperature of the stirring fermentation treatment is 26-30 ℃.
8. A fermentation broth obtained by fermentation using the fermentation method according to any one of claims 1 to 7.
9. The fermentation broth of claim 8, wherein the dry weight of cells in the fermentation broth is at least 40 g/L.
10. Use of a fermentation broth according to claim 8 or 9 in pig feed.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114958631A (en) * | 2022-05-16 | 2022-08-30 | 万华化学(四川)有限公司 | Method for producing single-cell protein by using heavy-phase lactic acid |
| CN115505736A (en) * | 2022-09-23 | 2022-12-23 | 中南大学 | Method for bioleaching ionic rare earth ore in neutral or partial neutral environment |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005100578A2 (en) * | 2004-04-19 | 2005-10-27 | Csir | Methods for obtaining optically active epoxides and vicinal diols from meso-epoxides |
| CN104711201A (en) * | 2013-12-17 | 2015-06-17 | 成都医学院 | Yarrowia lipolytica and use thereof |
| CN105368728A (en) * | 2015-12-16 | 2016-03-02 | 江南大学 | High-glucose-oxidase-yield yarrowia lipolytica |
| WO2018210867A1 (en) * | 2017-05-18 | 2018-11-22 | Institut National De La Recherche Agronomique | Inducible promoter for gene expression and synthetic biology |
| CN109136116A (en) * | 2017-06-16 | 2019-01-04 | 上海吉态来生物技术有限公司 | A method of culture dimorphism microorganism |
-
2020
- 2020-12-23 CN CN202011545586.1A patent/CN112553094A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005100578A2 (en) * | 2004-04-19 | 2005-10-27 | Csir | Methods for obtaining optically active epoxides and vicinal diols from meso-epoxides |
| CN104711201A (en) * | 2013-12-17 | 2015-06-17 | 成都医学院 | Yarrowia lipolytica and use thereof |
| CN105368728A (en) * | 2015-12-16 | 2016-03-02 | 江南大学 | High-glucose-oxidase-yield yarrowia lipolytica |
| WO2018210867A1 (en) * | 2017-05-18 | 2018-11-22 | Institut National De La Recherche Agronomique | Inducible promoter for gene expression and synthetic biology |
| CN109136116A (en) * | 2017-06-16 | 2019-01-04 | 上海吉态来生物技术有限公司 | A method of culture dimorphism microorganism |
Non-Patent Citations (4)
| Title |
|---|
| JIE-WAN KIM 等: "High Cell Density Culture of Yarrowialipolytica Using a One-Step Feeding Process", 《BIOTECHNOLOGY PROGRESS》 * |
| LV PJ 等: "Dissolved-oxygen feedback control fermentation for enhancing β-carotene in engineered Yarrowia lipolytica", 《SCI REP》 * |
| 亢新鑫 等: "溶氧对耶氏解脂酵母油脂积累和柠檬酸分泌的影响", 《食品工业科技》 * |
| 汤飞飞: "母猪妊娠后期和仔猪日粮添加酵母培养物对母猪生产性能、仔猪肠道健康及免疫机能的影响", 《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114958631A (en) * | 2022-05-16 | 2022-08-30 | 万华化学(四川)有限公司 | Method for producing single-cell protein by using heavy-phase lactic acid |
| CN115505736A (en) * | 2022-09-23 | 2022-12-23 | 中南大学 | Method for bioleaching ionic rare earth ore in neutral or partial neutral environment |
| CN115505736B (en) * | 2022-09-23 | 2024-04-19 | 中南大学 | Method for bioleaching ionic rare earth ore in neutral or near-neutral environment |
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