CN112481324A - Novel amino acid fermentation sterilization process - Google Patents
Novel amino acid fermentation sterilization process Download PDFInfo
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- CN112481324A CN112481324A CN202011606461.5A CN202011606461A CN112481324A CN 112481324 A CN112481324 A CN 112481324A CN 202011606461 A CN202011606461 A CN 202011606461A CN 112481324 A CN112481324 A CN 112481324A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/14—Glutamic acid; Glutamine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/02—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using physical phenomena
- A61L2/04—Heat
- A61L2/06—Hot gas
- A61L2/07—Steam
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
- A61L2/20—Gaseous substances, e.g. vapours
- A61L2/202—Ozone
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Abstract
The invention belongs to the technical field of amino acid fermentation, and discloses a novel amino acid fermentation sterilization process which comprises two procedures of fermentation tank sterilization and fermentation culture. The process of the invention performs sterilization prevention and control in two stages before and during fermentation, avoids mixed bacteria pollution and shortens non-fermentation time.
Description
Technical Field
The invention belongs to the technical field of amino acid fermentation, and particularly relates to a novel amino acid fermentation sterilization process.
Background
The fermentation industry is a large industry in China, and a plurality of series of products such as enzyme, Annona, nucleoside, antibiotic, vitamin, amino acid, xanthan gum and the like are produced by fermentation at present. Amino acid fermentation is to culture microbes in a culture medium with saccharides and ammonium salts as main raw materials to accumulate specific amino acids. Taking the fermentation of glutamic acid as an example, the strain used is mainly corynebacterium glutamicum, and the factors influencing the acid production efficiency and yield of the fermentation of corynebacterium glutamicum are more, and mainly include the following aspects: 1. under different environmental conditions, microorganisms use different substrate metabolic pathways which are different, the cell metabolic pathways are purposefully modified and reformed, the original metabolic characteristics of cells are changed, and the yield of target products can be improved; 2. the cell membrane permeability of the thallus is improved, the secretion of the glutamic acid is increased, the feedback regulation effect of high-concentration glutamic acid in the thallus is relieved, and the yield of the glutamic acid is improved; 3. the fermentation medium and the fermentation parameters are optimized, so that the proliferation rate of the thalli is improved, and the yield of amino acid is improved; 4. the contamination is avoided, and once the contamination is caused, serious loss can be caused.
In the production of amino acid fermentation industry, the production process is mostly a pure culture process, and an important condition for smooth fermentation production is to ensure the pure culture of microorganisms and to perform the fermentation production under the condition of no mixed bacteria pollution. In actual production, fermentation and bacterial contamination are often caused due to reasons such as unreasonable process design, equipment defects, personnel errors and the like. Since the fermentation production with the mixed bacteria consumes the nutrient substances in the culture medium, the metabolite of the mixed bacteria even influences the growth and metabolism of the producing bacteria, and the fermentation yield is reduced. Once the contamination occurs, the cause of the contamination should be found as soon as possible and corresponding effective measures should be taken to minimize the loss caused by the contamination of fermentation. The glutamic acid fermentation phage contamination rate is reported to be 1% -2%. Therefore, the reduction of the contamination rate is the key point of the fermentation production and is an important means for improving the yield of new products and reducing the product cost of enterprises.
In the fermentative production of amino acids, the so-called infectious bacteria include two types: one is the staining of phage, which is extremely harmful to production, and if large-scale outbreaks occur, the plant is forced to stop production. Although the bacteriophage is extremely harmful, as long as various work of the bacteriophage is prevented from being implemented in place, the infection of the bacteriophage can be basically avoided in the fermentation process, and the other is infectious microbes which are thalli except amino acid producing bacteria. Meanwhile, the invasion of the mixed bacteria is often a precursor of the pollution of the bacteriophage in the pure culture process, so the idea of preventing the bacteria from being infected mainly needs to be established, the management work in production is enhanced, and the bacteria-infecting factors are eliminated in the bud. The contamination reasons in the fermentation process mainly focus on the following aspects: air system, environmental factors, equipment factors, operational factors, and seed factors. Therefore, to overcome the contamination problem, it is necessary to take the measures from the above aspects, store the materials layer by layer, and strictly prevent the contamination of the mixed bacteria. When the fermentation equipment is sterilized, the problems of industrial energy consumption and non-fermentation interval time need to be considered.
Disclosure of Invention
In view of the above, the present invention aims to provide a novel amino acid fermentation sterilization process, which performs sterilization prevention and control in two stages before and during fermentation, thereby avoiding the contamination of mixed bacteria and shortening the non-fermentation time.
In order to achieve the above object, the present invention provides the following technical solutions:
a novel amino acid fermentation and sterilization process comprises two procedures of fermentation tank sterilization and fermentation culture.
Further, the fermenter sterilization is:
(1) the following raw materials are taken according to weight percentage: 12% of glucose, 3% of corn steep liquor, 0.5% of urea, 0.1% of monopotassium phosphate, 0.02% of ferrous sulfate heptahydrate and 0.02% of magnesium sulfate heptahydrate;
(2) sequentially adding the above raw materials into a fermentation tank, adding one fifth of water consumption of the formula, stirring, heating to 121 deg.C, and steam sterilizing at the temperature for 15 min;
(3) placing the rest formula in a liquid storage tank, carrying out ozone sterilization at normal temperature for 30min, and then decomposing residual ozone in water through an ozone catalytic decomposer; then the mixture is thrown into a fermentation tank, mixed evenly and enters a fermentation stage.
Further, the fermentation culture is as follows:
inoculating corynebacterium glutamicum seed liquid into a fermentation tank containing a fermentation medium in an inoculation amount of 8%, wherein the fermentation time is 48 h; the fermentation time is divided into two stages, the first stage is 12h, the fermentation temperature is 32 ℃, the ventilation volume is 0.3vvm, and the pH is controlled to be 3.5 by feeding ammonia water; the second stage is 36h, the fermentation temperature is 32 ℃, and the ventilation volume is 0.4 vvm; when the second stage begins, adding inositol and dimethylformamide into the fermentation medium, wherein the addition amounts are 0.6 percent and 0.3 percent respectively, and simultaneously carrying out ultrasonic treatment, wherein the ultrasonic frequency is 28 kHz, the ultrasonic power density is 100W/L, and the ultrasonic treatment time is 1 h; in the second stage, ammonia water is fed in automatically to control the pH value to be 5.0, and glucose solution with the concentration of 100g/L is fed in to control the residual sugar to be not less than 1.0%; after the second stage is completed, the fermentation broth is collected.
Preferably, the first and second electrodes are formed of a metal,
the preparation method of the corynebacterium glutamicum seed liquid comprises the following steps: inoculating corynebacterium glutamicum into a seed culture medium, and performing shake culture at 32 ℃ and 100rpm for 12h to obtain corynebacterium glutamicum seed liquid.
More preferably still, the first and second liquid crystal compositions are,
the corynebacterium glutamicum seed culture medium comprises the following components: 6% of glucose, 3% of corn steep liquor, 0.2% of potassium dihydrogen phosphate, 0.02% of magnesium sulfate heptahydrate and 0.01% of manganese sulfate monohydrate, and sterilizing for 15min at 121 ℃.
The beneficial effects of the invention mainly comprise the following aspects:
researches show that main mixed bacteria in the fermentation process of the glutamic acid are lactic acid bacteria, the optimum pH of the lactic acid bacteria is 5.5-6.0, when the pH is reduced below 4, the proliferation of the lactic acid bacteria is obviously slowed down, and the growth of the lactic acid bacteria is almost stopped, but the influence of the corynebacterium glutamicum is not large.
The yeast is difficult to kill, the sterilization time needs to be greatly prolonged, the energy consumption is improved, the non-fermentation time is prolonged, most of liquid is sterilized by ozone, the sterilization energy consumption is reduced, the consumption of cooling water is reduced, and the cooling time is greatly shortened.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the technical solutions in the present application will be clearly and completely described below with reference to specific embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
In actual operation, the mixed bacteria can be generated before and during fermentation, and the types of the mixed bacteria are mainly as follows:
TABLE 1 species of infectious microbes
| Species of miscellaneous bacteria | Analysis of cause of contamination |
| Lactic acid bacteria | Seed with bacterium |
| Yeast | Incomplete sterilization of culture medium or long standing time |
A novel amino acid fermentation sterilization process comprises the following steps:
and (3) sterilizing a fermentation tank:
1) the following raw materials are taken according to weight percentage: 12% of glucose, 3% of corn steep liquor, 0.5% of urea, 0.1% of monopotassium phosphate, 0.02% of ferrous sulfate heptahydrate and 0.02% of magnesium sulfate heptahydrate;
2) sequentially adding the above raw materials into a fermentation tank, adding one fifth of water consumption of the formula, stirring, heating to 121 deg.C, and steam sterilizing at the temperature for 15 min;
3) placing the rest formula in a liquid storage tank, carrying out ozone sterilization at normal temperature for 30min, and then decomposing residual ozone in water through an ozone catalytic decomposer; then the mixture is thrown into a fermentation tank, mixed evenly and enters a fermentation stage.
Fermentation culture:
inoculating Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13761 into seed culture medium, and shake culturing at 32 deg.C and 100rpm for 12 hr to obtain Corynebacterium glutamicum seed solution; the corynebacterium glutamicum seed culture medium comprises the following components in percentage by mass: 6% of glucose, 3% of corn steep liquor, 0.2% of potassium dihydrogen phosphate, 0.02% of magnesium sulfate heptahydrate and 0.01% of manganese sulfate monohydrate, and sterilizing for 15min at 121 ℃;
inoculating the seed liquid into a fermentation tank containing a fermentation culture medium by 8 percent of inoculation amount, wherein the fermentation time is 48 hours; the fermentation time is divided into two stages, the first stage is 12h, the fermentation temperature is 32 ℃, the ventilation volume is 0.3vvm, and the pH is controlled to be 3.5 by feeding ammonia water; the second stage is 36h, the fermentation temperature is 32 ℃, and the ventilation volume is 0.4 vvm; when the second stage begins, adding inositol and dimethylformamide into the fermentation medium, wherein the addition amounts are 0.6 percent and 0.3 percent respectively, and simultaneously carrying out ultrasonic treatment, wherein the ultrasonic frequency is 28 kHz, the ultrasonic power density is 100W/L, and the ultrasonic treatment time is 1 h; in the second stage, ammonia water is fed in automatically to control the pH value to be 5.0, and glucose solution with the concentration of 100g/L is fed in to control the residual sugar to be not less than 1.0%; after the second stage is completed, the fermentation broth is collected.
Example 2
A novel amino acid fermentation sterilization process comprises the following steps:
and (3) sterilizing a fermentation tank:
the following raw materials are taken according to weight percentage: 12% of glucose, 3% of corn steep liquor, 0.5% of urea, 0.1% of monopotassium phosphate, 0.02% of ferrous sulfate heptahydrate and 0.02% of magnesium sulfate heptahydrate;
sequentially adding the above raw materials into a fermentation tank, adding one fourth of water consumption of the formula, stirring, heating to 121 deg.C, and steam sterilizing at the temperature for 20 min;
placing the rest formula in a liquid storage tank, carrying out normal-temperature ozone sterilization for 40min, and then decomposing residual ozone in water through an ozone catalytic decomposer; then the mixture is thrown into a fermentation tank, mixed evenly and enters a fermentation stage.
Fermentation culture:
inoculating Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13761 into seed culture medium, and shake culturing at 32 deg.C and 100rpm for 12 hr to obtain Corynebacterium glutamicum seed solution; the corynebacterium glutamicum seed culture medium comprises the following components in percentage by mass: 6% of glucose, 3% of corn steep liquor, 0.2% of potassium dihydrogen phosphate, 0.02% of magnesium sulfate heptahydrate and 0.01% of manganese sulfate monohydrate, and sterilizing for 15min at 121 ℃;
inoculating the seed liquid into a fermentation tank containing a fermentation culture medium by 8 percent of inoculation amount, wherein the fermentation time is 48 hours; the fermentation time is divided into two stages, the first stage is 12h, the fermentation temperature is 32 ℃, the ventilation volume is 0.3vvm, and the pH is controlled to be 3.5 by feeding ammonia water; the second stage is 36h, the fermentation temperature is 32 ℃, and the ventilation volume is 0.4 vvm; when the second stage begins, adding inositol and dimethylformamide into the fermentation medium, wherein the addition amounts are 0.6 percent and 0.3 percent respectively, and simultaneously carrying out ultrasonic treatment, wherein the ultrasonic frequency is 28 kHz, the ultrasonic power density is 100W/L, and the ultrasonic treatment time is 1 h; in the second stage, ammonia water is fed in automatically to control the pH value to be 5.0, and glucose solution with the concentration of 100g/L is fed in to control the residual sugar to be not less than 1.0%; after the second stage is completed, the fermentation broth is collected.
Comparative example 1
And (3) sterilizing a fermentation tank:
the following raw materials are taken according to weight percentage: 12% of glucose, 3% of corn steep liquor, 0.5% of urea, 0.1% of monopotassium phosphate, 0.02% of ferrous sulfate heptahydrate and 0.02% of magnesium sulfate heptahydrate;
adding the above raw materials into a fermentation tank in sequence, adding formula water consumption, stirring, heating to 121 deg.C, steam sterilizing at the temperature for 15min, cooling to fermentation temperature, and allowing fermentation.
Fermentation culture:
inoculating Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13761 into seed culture medium, and shake culturing at 32 deg.C and 100rpm for 12 hr to obtain Corynebacterium glutamicum seed solution; the corynebacterium glutamicum seed culture medium comprises the following components in percentage by mass: 6% of glucose, 3% of corn steep liquor, 0.2% of potassium dihydrogen phosphate, 0.02% of magnesium sulfate heptahydrate and 0.01% of manganese sulfate monohydrate, and sterilizing for 15min at 121 ℃;
inoculating the seed liquid into a fermentation tank containing a fermentation culture medium by 8 percent of inoculation amount, wherein the fermentation time is 48 hours; the fermentation time is divided into two stages, the first stage is 12h, the fermentation temperature is 32 ℃, the ventilation volume is 0.3vvm, and the pH is controlled to be 3.5 by feeding ammonia water; the second stage is 36h, the fermentation temperature is 32 ℃, and the ventilation volume is 0.4 vvm; when the second stage begins, adding inositol and dimethylformamide into the fermentation medium, wherein the addition amounts are 0.6 percent and 0.3 percent respectively, and simultaneously carrying out ultrasonic treatment, wherein the ultrasonic frequency is 28 kHz, the ultrasonic power density is 100W/L, and the ultrasonic treatment time is 1 h; in the second stage, ammonia water is fed in automatically to control the pH value to be 5.0, and glucose solution with the concentration of 100g/L is fed in to control the residual sugar to be not less than 1.0%; after the second stage is completed, the fermentation broth is collected.
The foregoing list is only illustrative of the preferred embodiments of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (5)
1. A novel amino acid fermentation and sterilization process comprises two procedures of fermentation tank sterilization and fermentation culture.
2. The process of claim 1, wherein the fermentor sterilization is:
(1) the following raw materials are taken according to weight percentage: 12% of glucose, 3% of corn steep liquor, 0.5% of urea, 0.1% of monopotassium phosphate, 0.02% of ferrous sulfate heptahydrate and 0.02% of magnesium sulfate heptahydrate;
(2) sequentially adding the above raw materials into a fermentation tank, adding one fifth of water consumption of the formula, stirring, heating to 121 deg.C, and steam sterilizing at the temperature for 15 min;
(3) placing the rest formula in a liquid storage tank, carrying out ozone sterilization at normal temperature for 30min, and then decomposing residual ozone in water through an ozone catalytic decomposer; then the mixture is thrown into a fermentation tank, mixed evenly and enters a fermentation stage.
3. The process of claim 1, wherein the fermentation culture is:
inoculating corynebacterium glutamicum seed liquid into a fermentation tank containing a fermentation medium in an inoculation amount of 8%, wherein the fermentation time is 48 h; the fermentation time is divided into two stages, the first stage is 12h, the fermentation temperature is 32 ℃, the ventilation volume is 0.3vvm, and the pH is controlled to be 3.5 by feeding ammonia water; the second stage is 36h, the fermentation temperature is 32 ℃, and the ventilation volume is 0.4 vvm; when the second stage begins, adding inositol and dimethylformamide into the fermentation medium, wherein the addition amounts are 0.6 percent and 0.3 percent respectively, and simultaneously carrying out ultrasonic treatment, wherein the ultrasonic frequency is 28 kHz, the ultrasonic power density is 100W/L, and the ultrasonic treatment time is 1 h; in the second stage, ammonia water is fed in automatically to control the pH value to be 5.0, and glucose solution with the concentration of 100g/L is fed in to control the residual sugar to be not less than 1.0%; after the second stage is completed, the fermentation broth is collected.
4. The process of claim 2, wherein the corynebacterium glutamicum seed solution is prepared by the following steps: inoculating corynebacterium glutamicum into a seed culture medium, and performing shake culture at 32 ℃ and 100rpm for 12h to obtain corynebacterium glutamicum seed liquid.
5. The process of claim 4, wherein the Corynebacterium glutamicum seed medium comprises the following components: 6% of glucose, 3% of corn steep liquor, 0.2% of potassium dihydrogen phosphate, 0.02% of magnesium sulfate heptahydrate and 0.01% of manganese sulfate monohydrate, and sterilizing for 15min at 121 ℃.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109371072A (en) * | 2018-10-18 | 2019-02-22 | 许传高 | A kind of technique for reducing glutamic acid fermentation microbiological contamination and improving fermentation efficiency |
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