CN107400644B - Culture medium, application of culture medium in rhodobacter sphaeroides fermentation and preparation method of sorbic acid - Google Patents
Culture medium, application of culture medium in rhodobacter sphaeroides fermentation and preparation method of sorbic acid Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 69
- 230000004151 fermentation Effects 0.000 title claims abstract description 69
- 239000001963 growth medium Substances 0.000 title claims abstract description 67
- 235000010199 sorbic acid Nutrition 0.000 title claims abstract description 54
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 title claims abstract description 53
- 239000004334 sorbic acid Substances 0.000 title claims abstract description 53
- 229940075582 sorbic acid Drugs 0.000 title claims abstract description 53
- 241000191043 Rhodobacter sphaeroides Species 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 238000006243 chemical reaction Methods 0.000 claims abstract description 23
- RCYFOPUXRMOLQM-UHFFFAOYSA-N pyrene-1-carbaldehyde Chemical compound C1=C2C(C=O)=CC=C(C=C3)C2=C2C3=CC=CC2=C1 RCYFOPUXRMOLQM-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000012258 culturing Methods 0.000 claims description 36
- 239000007788 liquid Substances 0.000 claims description 35
- 238000003756 stirring Methods 0.000 claims description 33
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 33
- 239000001888 Peptone Substances 0.000 claims description 26
- 108010080698 Peptones Proteins 0.000 claims description 26
- 229940041514 candida albicans extract Drugs 0.000 claims description 26
- 235000019319 peptone Nutrition 0.000 claims description 26
- 239000000843 powder Substances 0.000 claims description 26
- 239000012138 yeast extract Substances 0.000 claims description 26
- 241001052560 Thallis Species 0.000 claims description 21
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 19
- 239000008103 glucose Substances 0.000 claims description 19
- 230000001580 bacterial effect Effects 0.000 claims description 17
- 238000009423 ventilation Methods 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 12
- 230000004913 activation Effects 0.000 claims description 5
- 230000003213 activating effect Effects 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 3
- 150000001299 aldehydes Chemical class 0.000 claims description 2
- 239000000758 substrate Substances 0.000 claims description 2
- 230000000813 microbial effect Effects 0.000 abstract description 2
- 238000011218 seed culture Methods 0.000 description 32
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 27
- 238000011081 inoculation Methods 0.000 description 20
- 238000004519 manufacturing process Methods 0.000 description 20
- 239000002609 medium Substances 0.000 description 11
- 241000894006 Bacteria Species 0.000 description 8
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 8
- 238000005273 aeration Methods 0.000 description 8
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 8
- 238000004128 high performance liquid chromatography Methods 0.000 description 8
- 238000011534 incubation Methods 0.000 description 8
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 8
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 8
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 8
- 239000013028 medium composition Substances 0.000 description 8
- 239000012898 sample dilution Substances 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 239000011592 zinc chloride Substances 0.000 description 8
- 239000002054 inoculum Substances 0.000 description 7
- 239000011684 sodium molybdate Substances 0.000 description 7
- 235000015393 sodium molybdate Nutrition 0.000 description 7
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 7
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- 235000013379 molasses Nutrition 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 230000008520 organization Effects 0.000 description 4
- WSWCOQWTEOXDQX-UHFFFAOYSA-N 2,4-Hexadienoic acid Chemical compound CC=CC=CC(O)=O WSWCOQWTEOXDQX-UHFFFAOYSA-N 0.000 description 3
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000003755 preservative agent Substances 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 241000193155 Clostridium botulinum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000014443 Pyrus communis Nutrition 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 241001148470 aerobic bacillus Species 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 235000021474 generally recognized As safe (food) Nutrition 0.000 description 1
- 235000021473 generally recognized as safe (food ingredients) Nutrition 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- VUZPPFZMUPKLLV-UHFFFAOYSA-N methane;hydrate Chemical compound C.O VUZPPFZMUPKLLV-UHFFFAOYSA-N 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229960004793 sucrose Drugs 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- 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
- C12N1/20—Bacteria; Culture media therefor
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- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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Abstract
The invention relates to the technical field of microbial fermentation, in particular to a culture medium, application thereof in rhodobacter sphaeroides fermentation and a preparation method of sorbic acid. The culture medium provided by the invention is used for fermenting rhodobacter sphaeroides in a mode of feeding the pyrenal, the pyrenal can be converted into sorbic acid, the conversion rate can reach 74.98% -79.94%, and the concentration in a culture solution can reach 7.55-8.05 g/L. The preparation method provided by the invention is safe and does not cause pollution to the environment, the yield of the obtained sorbic acid is high, and the fermentation time is short.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a culture medium, application thereof in rhodobacter sphaeroides fermentation and a preparation method of sorbic acid.
Background
Sorbic acid is an unsaturated fatty acid known by the english name of Sorbic acid, also known as 2, 4-hexadienoic acid, and 2-propenylacrylic acid. Like other natural fatty acids, sorbic acid participates in metabolic processes in the human body and is digested and absorbed by the human body to produce carbon dioxide and water. Sorbic acid has inhibitory effect on yeast, mold, aerobic bacteria, and filamentous bacteria, and can prevent growth and reproduction of Clostridium botulinum, Staphylococcus aureus, and Salmonella. In terms of safety, sorbic acid is an internationally recognized as safe (GRAS) preservative and has high safety. The food and agriculture organization, the world health organization and the FDA in the United states all give a positive on the safety of the food and agriculture organization and the world health organization. Therefore, sorbic acid and its salts are widely used in the fields of foods, tobaccos and cosmetics as preservatives or preservatives.
At present, the preparation of sorbic acid and salts thereof is mainly completed by a chemical method, but the chemical synthesis process of sorbic acid is very complicated, has high control difficulty, generates a large amount of waste materials and causes serious pollution to the ecological environment. The ubiquitous problems of the production enterprises in China are as follows: the synthesis technology is lagged behind, the process is incomplete and the production cost is high. Therefore, the laggard sorbic acid production process has not been able to meet the expanding sorbic acid dosage. Therefore, further research on a sorbic acid production method, which improves the yield of sorbic acid and reduces environmental pollution, is an urgent problem to be solved.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a culture medium and its application in rhodobacter sphaeroides fermentation and a method for preparing sorbic acid; the culture medium provided by the invention is suitable for growth of rhodobacter sphaeroides, and the suitable fermentation condition fully improves the yield of sorbic acid.
The culture medium provided by the invention comprises water and:
in some embodiments of the invention, the medium comprises water and:
in some embodiments of the invention, the medium comprises water and:
in some embodiments of the invention, the medium comprises water and:
in the present invention, FeCl3、CuSO4、MnSO4、ZnCl2The hydrate or non-hydrate can be used, and when the hydrate is used, the content of the inorganic salt in the culture solution is consistent with the formula of the culture medium, and the content is within the protection scope of the invention.
The culture medium provided by the invention is more suitable for culturing rhodobacter sphaeroides, and experiments show that when other culture media are used for culturing rhodobacter sphaeroides, thalli grow poorly, and conversion of sorbosol into sorbic acid is influenced.
The culture medium provided by the invention is applied to the fermentation preparation of sorbic acid by rhodobacter sphaeroides.
The preparation method of sorbic acid provided by the invention comprises the steps of fermenting rhodobacter sphaeroides by using the culture medium provided by the invention with the sorbosol as a substrate to obtain fermentation liquor containing sorbic acid.
The rhodobacter sphaeroides adopted by the invention is rhodobacter sphaeroides with the preservation number of CGMCC 1.3368.
In the invention, the fermentation comprises a thallus growth step and a sorbosol conversion step;
the thallus growth step comprises: inoculating thalli to the culture medium, wherein the temperature is 28-35 ℃, the stirring speed is 300-500 rpm, the ventilation volume is 3mL/min, and the pH value is 6.8-7.4; culturing to OD of bacterial liquid600The value is 9 to 12.5;
the conversion step of the pyrialdehyde comprises the following steps: to OD600Adding the pyrenal into the bacterial liquid with the value of 9-12.5, stirring at the rotating speed of 300-500 rpm and the air flow of 30 ℃, wherein the air flow is 3-7 mL/min, and the fermentation time is 60-84 h.
The thalli refers to activated seed liquid, and the volume fraction of inoculation is 10-15%; preferably 10%, 13% or 15%.
Culturing until bacterial liquid OD600The value of 9 to 12.5 requires 96 to 120 hours.
In the step of thallus growth, acid liquor is fed in a mode of maintaining the pH value. Specifically, hydrochloric acid solution is fed, and in some examples, hydrochloric acid solution with a concentration of 2M is fed.
In the embodiment of the invention, the sorbal is fed until the final concentration of the sorbal in the culture solution is 10 mL/L.
In some embodiments, the step of growing the bacteria is: inoculating thalli to the culture medium, wherein the temperature is 35 ℃, the stirring speed is 300rpm, the ventilation volume is 3mL/min, and the pH value is 7.0; culturing to OD of bacterial liquid600The value is 9 to 12.5;
the conversion step of the pyrialdehyde comprises the following steps: to OD600Adding the pyrialdehyde into the bacterial liquid with the value of 9-12.5, stirring at the rotating speed of 300rpm and the air flow of 30 ℃, wherein the air flow is 7mL/min, and the fermentation time is 72 hours.
In some embodiments, the step of growing the bacteria is: inoculating thalli to the culture medium, wherein the temperature is 30 ℃, the stirring speed is 500rpm, the ventilation volume is 3mL/min, and the pH value is 6.8; culturing to OD of bacterial liquid600The value is 9 to 12.5;
the conversion step of the pyrialdehyde comprises the following steps: to OD600Adding the pyrialdehyde into the bacterial liquid with the value of 9-12.5, stirring at the rotating speed of 400rpm, the temperature of 30 ℃, the ventilation volume of 3mL/min, and the fermentation time of 72 h.
In some embodiments, the step of growing the bacteria is: inoculating thalli to the culture medium, wherein the temperature is 35 ℃, the stirring speed is 300rpm, the ventilation volume is 3mL/min, and the pH value is 7; culturing to OD of bacterial liquid600The value is 9 to 12.5;
the conversion step of the pyrialdehyde comprises the following steps: to OD600Adding the pyrialdehyde into the bacterial liquid with the value of 9-12.5, stirring at 500rpm at 30 ℃, ventilating at 5mL/min, and fermenting for 72 h.
In some embodiments, the step of growing the bacteria is: inoculating thalli to the culture medium, wherein the temperature is 28 ℃, the stirring speed is 400rpm, the ventilation volume is 3mL/min, and the pH value is 7.4; culturing to OD of bacterial liquid600The value is 9 to 12.5;
the conversion step of the pyrialdehyde comprises the following steps: to OD600Adding the bacteria liquid with the value of 9-12.5The pear aldehyde is stirred at the rotating speed of 300rpm and 30 ℃, the ventilation volume is 7mL/min, and the fermentation time is 72 h.
In some embodiments, the step of growing the bacteria is: inoculating thalli to the culture medium, wherein the temperature is 30 ℃, the stirring speed is 500rpm, the ventilation volume is 3mL/min, and the pH value is 6.8; culturing to OD of bacterial liquid600The value is 9 to 12.5;
the conversion step of the pyrialdehyde comprises the following steps: to OD600Adding the pyrialdehyde into the bacterial liquid with the value of 9-12.5, stirring at the rotating speed of 400rpm, the temperature of 30 ℃, the ventilation volume of 3mL/min, and the fermentation time of 72 h.
In some embodiments, the step of growing the bacteria is: inoculating thalli to the culture medium, wherein the temperature is 28 ℃, the stirring speed is 400rpm, the ventilation volume is 3mL/min, and the pH value is 7.4; culturing to OD of bacterial liquid600The value is 9 to 12.5;
the conversion step of the pyrialdehyde comprises the following steps: to OD600Adding the pyrialdehyde into the bacterial liquid with the value of 9-12.5, stirring at 500rpm at 30 ℃, ventilating at 5mL/min, and fermenting for 72 h.
In the invention, before the fermentation, a step of activating seeds is further included, and a culture medium adopted for activating the seeds comprises: water and peptone 15 g/L; 5g/L of yeast extract powder; glucose 5 g/L.
In the invention, the activation temperature is 28-35 ℃, the stirring speed is 150-200 rpm, and the time is 24-48 h.
In the specific embodiment, the activation is divided into two steps, the strain frozen in glycerol is inoculated to an activation culture medium, after culturing for 48 hours at 30 ℃ by a shaking table at 150rpm, the obtained bacterial liquid is inoculated to the activation culture medium again at 35 ℃ and at 150rpm, and the culture time is 36 hours. The inoculum size for the re-inoculation was 1.5%.
The invention provides a culture medium, which is used for fermenting rhodobacter sphaeroides in a mode of feeding the pyrenal, can convert the pyrenal into sorbitol, and has the conversion rate of 74.98-79.94 percent and the concentration of 7.55-8.05 g/L in a culture solution. The preparation method provided by the invention is safe and does not cause pollution to the environment, the yield of the obtained sorbitol is high, and the fermentation time is short.
Drawings
FIG. 1 shows a chromatogram for measuring the sorbic acid content in example 1, wherein the abscissa is time and the ordinate is height;
FIG. 2 shows a chromatogram for measuring the sorbic acid content in example 2, wherein the abscissa is time and the ordinate is height;
FIG. 3 shows a chromatogram for measuring the sorbic acid content in example 3, wherein the abscissa is time and the ordinate is height;
FIG. 4 shows a chromatogram for measuring the sorbic acid content in example 4, wherein the abscissa is time and the ordinate is height;
FIG. 5 shows a chromatogram for measuring the sorbic acid content in example 5, wherein the abscissa is time and the ordinate is height;
FIG. 6 shows a chromatogram for measuring sorbic acid content in example 6, wherein the abscissa is time and the ordinate is height;
FIG. 7 shows a chromatogram for measuring sorbic acid content in comparative example 1, with time on the abscissa and height on the ordinate;
fig. 8 shows a chromatogram for measuring the sorbic acid content in comparative example 2, wherein the abscissa is time and the ordinate is height.
Detailed Description
The invention provides a culture medium, application thereof in rhodobacter sphaeroides fermentation and a preparation method of sorbic acid, and a person skilled in the art can use the contents to reference the culture medium and appropriately improve process parameters to realize the culture. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention is further illustrated by the following examples:
example 1
Firstly, pretreatment
The rhodobacter sphaeroides preserved by conventional glycerol is inoculated into a triangular flask filled with a seed culture medium according to the inoculation amount of 1 percent, and is cultured for 48 hours at the temperature of 30 ℃ by a shaking table at 150 rpm. The components of the seed culture medium are, by weight volume percentage (g/L), 15g/L of peptone, 5g/L of yeast extract powder, 5g/L of glucose and the balance of water, and the pH value is 7.0.
Second, seed liquid
Aseptically transferring the cultured strain into a triangular flask filled with a seed culture medium according to the inoculation amount of 1%, and culturing for 24h at 30 ℃ by a shaking table at 200 rpm. The components of the seed culture medium are, by weight volume percentage (g/L), 15g/L of peptone, 5g/L of yeast extract powder, 5g/L of glucose and the balance of water, and the pH value is 7.0.
Production and fermentation
1. 6L of fermentation medium was placed in a 10L fermenter, the fermentation medium composition: by weight percentage, peptone: 30g/L, yeast extract powder: 5g/L of molasses 7g/L, FeCl3 45mg/L、CuSO4 16mg/L、MnSO4 94mg/L、ZnCl250mg/L, 40mg/L of sodium molybdate and the balance of water.
2. Inoculating the seed liquid into a fermentation tank according to the inoculation amount of 10 percent;
and (3) growth of thalli: the incubation temperature was 35 ℃ and the stirring rate was 300rpm, with a continuous aeration of 3mL/min of air being maintained. Maintaining pH at 7 during culture, culturing for 120 hr, and culturing at OD600To 11.25.
3. Fermentation production: after culturing the cells for 120h, adding sorbol to make the final concentration 10ml/L, stirring at 30 deg.C and 300rpm, ventilating 7ml/min, fermenting for 72h, centrifuging to collect supernatant, measuring sorbic acid content (by 50 times sample dilution) by high performance liquid chromatography, wherein the sorbic acid content is 7.9g/L (figure 1), and the conversion rate is 78.45%.
Example 2
Firstly, pretreatment
The rhodobacter sphaeroides preserved by conventional glycerol is inoculated into a triangular flask filled with a seed culture medium according to the inoculation amount of 1 percent, and is cultured for 48 hours at the temperature of 30 ℃ by a shaking table at 150 rpm. The components of the seed culture medium are, by weight volume percentage (g/L), 15g/L of peptone, 5g/L of yeast extract powder, 5g/L of glucose and the balance of water, and the pH value is 7.0.
Second, seed liquid
Aseptically transferring the cultured strain into a triangular flask filled with a seed culture medium according to the inoculation amount of 1%, and culturing for 24h at 30 ℃ by a shaking table at 200 rpm. The components of the seed culture medium are, by weight volume percentage (g/L), 15g/L of peptone, 5g/L of yeast extract powder, 5g/L of glucose and the balance of water, and the pH value is 7.0.
Production and fermentation
1. 6L of fermentation medium was placed in a 10L fermenter, the fermentation medium composition: by weight percentage, peptone: 20g/L, yeast extract powder: 25g/L of molasses 1g/L, FeCl3 27mg/L、CuSO410mg/L、MnSO4 56mg/L、ZnCl230mg/L, 24mg/L of sodium molybdate and the balance of water.
2. Inoculating the seed liquid into a fermentation tank according to the inoculation amount of 15 percent;
and (3) growth of thalli: the incubation temperature was 30 ℃ and the stirring rate was 500rpm, with a continuous aeration of 3mL/min of air being maintained. Maintaining pH at 6.8 during culture, culturing for 108 hr, and culturing at OD600Up to 10.35.
3. Fermentation production: after the thalli are cultured for 108h, the sorbosol is added to make the final concentration of the sorbosol be 10ml/L, the temperature is 30 ℃, the stirring revolution is 400rpm, the ventilation rate is 3ml/min, the fermentation is 72h, the supernatant is collected by centrifugation, the sorbic acid content is measured by high performance liquid chromatography (sample dilution is 50 times), the sorbic acid content is 7.8g/L (figure 2), and the conversion rate is 77.46%.
Example 3
Firstly, pretreatment
The rhodobacter sphaeroides preserved by conventional glycerol is inoculated into a triangular flask filled with a seed culture medium according to the inoculation amount of 1 percent, and is cultured for 48 hours at the temperature of 30 ℃ by a shaking table at 150 rpm. The components of the seed culture medium are, by weight volume percentage (g/L), 15g/L of peptone, 5g/L of yeast extract powder, 5g/L of glucose and the balance of water, and the pH value is 7.0.
Second, seed liquid
Aseptically transferring the cultured strain into a triangular flask filled with a seed culture medium according to the inoculum size of 1.5%, and culturing for 36h at 35 ℃ by a shaking table at 150 rpm. The components of the seed culture medium are, by weight volume percentage (g/L), 15g/L of peptone, 5g/L of yeast extract powder, 5g/L of glucose and the balance of water, and the pH value is 7.0.
Production and fermentation
1. 6L of fermentation medium was placed in a 10L fermenter, the fermentation medium composition: by weight percentage, peptone: 10g/L, yeast extract powder: 12g/L of molasses 15g/L, FeCl3 9mg/L、CuSO43mg/L、MnSO4 19mg/L、ZnCl210mg/L, 8mg/L of sodium molybdate and the balance of water.
2. Inoculating the seed liquid into a fermentation tank according to the inoculation amount of 13 percent;
and (3) growth of thalli: the incubation temperature was 35 ℃ and the stirring rate was 300rpm, with a continuous aeration of 3mL/min of air being maintained. Maintaining pH at 7 during culture, culturing for 120 hr, and culturing at OD600Up to 12.13.
3. Fermentation production: after culturing the cells for 120h, adding sorbol to make the final concentration 10ml/L, stirring at 30 deg.C and 500rpm, ventilating at 5ml/min, fermenting for 72h, centrifuging to collect supernatant, measuring sorbic acid content (by 50 times sample dilution) by high performance liquid chromatography, wherein the sorbic acid content is 8.05g/L (figure 3), and the conversion rate is 79.94%. The conversion rate of the embodiment is proved to be obviously better than that of other embodiments, the effect has statistical significance, and p is less than 0.05.
Example 4
Firstly, pretreatment
The rhodobacter sphaeroides preserved by conventional glycerol is inoculated into a triangular flask filled with a seed culture medium according to the inoculation amount of 1 percent, and is cultured for 48 hours at the temperature of 30 ℃ by a shaking table at 150 rpm. The components of the seed culture medium are, by weight volume percentage (g/L), 15g/L of peptone, 5g/L of yeast extract powder, 5g/L of glucose and the balance of water, and the pH value is 7.0.
Second, seed liquid
Aseptically transferring the cultured strain into a triangular flask filled with a seed culture medium according to the inoculum size of 1.5%, and culturing for 36h at 35 ℃ by a shaking table at 150 rpm. The components of the seed culture medium are, by weight volume percentage (g/L), 15g/L of peptone, 5g/L of yeast extract powder, 5g/L of glucose and the balance of water, and the pH value is 7.0.
Production and fermentation
1. 6L of fermentation medium was placed in a 10L fermenter, the fermentation medium composition: by weight percentage, peptone: 20g/L, yeast extract powder: 25g/L of molasses 1g/L, FeCl3 27mg/L、CuSO410mg/L、MnSO4 56mg/L、ZnCl230mg/L, 24mg/L of sodium molybdate and the balance of water.
2. Inoculating the seed liquid into a fermentation tank according to the inoculation amount of 15 percent;
and (3) growth of thalli: the incubation temperature was 28 ℃ and the stirring rate was 400rpm, with a continuous aeration of 3mL/min of air being maintained. Maintaining pH at 7.4 during culture, culturing for 96 hr, and adjusting OD600Reaching 9.96.
3. Fermentation production: after the thalli are cultured for 96h, the sorbosol is added to make the final concentration of the sorbosol be 10ml/L, the temperature is 30 ℃, the stirring revolution is 300rpm, the ventilation rate is 7ml/min, the fermentation is 72h, the supernatant is collected by centrifugation, the sorbic acid content is measured by high performance liquid chromatography (sample dilution is 50 times), the sorbic acid content is 7.55g/L (figure 4), and the conversion rate is 74.98%.
Example 5
Firstly, pretreatment
The rhodobacter sphaeroides preserved by conventional glycerol is inoculated into a triangular flask filled with a seed culture medium according to the inoculation amount of 1 percent, and is cultured for 48 hours at the temperature of 30 ℃ by a shaking table at 150 rpm. The components of the seed culture medium are, by weight volume percentage (g/L), 15g/L of peptone, 5g/L of yeast extract powder, 5g/L of glucose and the balance of water, and the pH value is 7.0.
Second, seed liquid
The strain obtained by the culture is aseptically transferred into a triangular flask filled with a seed culture medium according to the inoculum size of 2 percent, and is cultured for 48 hours by a shaking table at 28 ℃ and 180 rpm. The components of the seed culture medium are, by weight volume percentage (g/L), 15g/L of peptone, 5g/L of yeast extract powder, 5g/L of glucose and the balance of water, and the pH value is 7.0.
Production and fermentation
1. 6L of fermentation medium was placed in a 10L fermenter, the fermentation medium composition: by weight percentage, peptone: 10g/L, yeast extract powder: 12g/L of molasses 15g/L, FeCl3 9mg/L、CuSO43mg/L、MnSO4 19mg/L、ZnCl210mg/L, 8mg/L of sodium molybdate and the balance of water.
2. Inoculating the seed liquid into a fermentation tank according to the inoculation amount of 13 percent;
and (3) growth of thalli: the incubation temperature was 30 ℃ and the stirring rate was 500rpm, with a continuous aeration of 3mL/min of air being maintained. Maintaining pH at 6.8 during culture, culturing for 108 hr, and culturing at OD600Up to 10.48.
3. Fermentation production: after the thalli are cultured for 108h, the sorbosol is added to make the final concentration of the sorbosol be 10ml/L, the temperature is 30 ℃, the stirring revolution is 400rpm, the ventilation rate is 3ml/min, the fermentation is carried out for 72h, the supernatant is collected by centrifugation, the sorbic acid content is measured by high performance liquid chromatography (sample dilution is 50 times), the sorbic acid content is 7.70g/L (figure 5), and the conversion rate is 76.47%.
Example 6
Firstly, pretreatment
The rhodobacter sphaeroides preserved by conventional glycerol is inoculated into a triangular flask filled with a seed culture medium according to the inoculation amount of 1 percent, and is cultured for 48 hours at the temperature of 30 ℃ by a shaking table at 150 rpm. The components of the seed culture medium are, by weight volume percentage (g/L), 15g/L of peptone, 5g/L of yeast extract powder, 5g/L of glucose and the balance of water, and the pH value is 7.0.
Second, seed liquid
The strain obtained by the culture is aseptically transferred into a triangular flask filled with a seed culture medium according to the inoculum size of 2 percent, and is cultured for 48 hours by a shaking table at 28 ℃ and 180 rpm. The components of the seed culture medium are, by weight volume percentage (g/L), 15g/L of peptone, 5g/L of yeast extract powder, 5g/L of glucose and the balance of water, and the pH value is 7.0.
Production and fermentation
1. 6L of fermentation medium was placed in a 10L fermenter, the fermentation medium composition: by weight percentage, peptone: 30g/L, yeast extract powder: 5g/L of molasses 7g/L, FeCl3 45mg/L、CuSO4 16mg/L、MnSO4 94mg/L、ZnCl250mg/L, 40mg/L of sodium molybdate and the balance of water.
2. Inoculating the seed liquid into a fermentation tank according to the inoculation amount of 10 percent;
thallusGrowing: the incubation temperature was 28 ℃ and the stirring rate was 400rpm, with a continuous aeration of 3mL/min of air being maintained. Maintaining pH at 7.4 during culture, culturing for 96 hr, and adjusting OD600Up to 10.01.
3. Fermentation production: after the thalli are cultured for 96h, the sorbosol is added to make the final concentration of the sorbosol be 10ml/L, the temperature is 30 ℃, the stirring revolution is 500rpm, the ventilation rate is 5ml/min, the fermentation is 72h, the supernatant is collected by centrifugation, the sorbic acid content is measured by high performance liquid chromatography (sample dilution is 50 times), the sorbic acid content is 7.65g/L (figure 6), and the conversion rate is 75.97%.
Comparative example 1
Firstly, pretreatment
The rhodobacter sphaeroides preserved by conventional glycerol is inoculated into a triangular flask filled with a seed culture medium according to the inoculation amount of 1 percent, and is cultured for 48 hours at the temperature of 30 ℃ by a shaking table at 150 rpm. The components of the seed culture medium are, by weight volume percentage (g/L), 15g/L of peptone, 5g/L of yeast extract powder, 5g/L of glucose and the balance of water, and the pH value is 7.0.
Second, seed liquid
Aseptically transferring the cultured strain into a triangular flask filled with a seed culture medium according to the inoculum size of 1.5%, and culturing for 36h at 35 ℃ by a shaking table at 150 rpm. The components of the seed culture medium are, by weight volume percentage (g/L), 15g/L of peptone, 5g/L of yeast extract powder, 5g/L of glucose and the balance of water, and the pH value is 7.0.
Production and fermentation
1. 6L of fermentation medium was placed in a 10L fermenter, the fermentation medium composition: by weight percentage, peptone: 10g/L, yeast extract powder: 12g/L, 15g/L of cane sugar and the balance of water.
2. Inoculating the seed liquid into a fermentation tank according to the inoculation amount of 13 percent;
and (3) growth of thalli: the incubation temperature was 35 ℃ and the stirring rate was 300rpm, with a continuous aeration of 3mL/min of air being maintained. Maintaining pH at 7 during culture, culturing for 120 hr, and culturing at OD600To 8.9.
3. Fermentation production: after culturing the cells for 120h, adding sorbol to make the final concentration 10ml/L, stirring at 30 deg.C and 500rpm, ventilating at 5ml/min, fermenting for 72h, centrifuging to collect supernatant, measuring sorbic acid content (by 50 times sample dilution) by high performance liquid chromatography, wherein the sorbic acid content is 4.8g/L (FIG. 7), and the conversion rate is 47.67%.
Comparative example 2
Firstly, pretreatment
The rhodobacter sphaeroides preserved by conventional glycerol is inoculated into a triangular flask filled with a seed culture medium according to the inoculation amount of 1 percent, and is cultured for 48 hours at the temperature of 30 ℃ by a shaking table at 150 rpm. The components of the seed culture medium are, by weight volume percentage (g/L), 15g/L of peptone, 5g/L of yeast extract powder, 5g/L of glucose and the balance of water, and the pH value is 7.0.
Second, seed liquid
Aseptically transferring the cultured strain into a triangular flask filled with a seed culture medium according to the inoculum size of 1.5%, and culturing for 36h at 35 ℃ by a shaking table at 150 rpm. The components of the seed culture medium are, by weight volume percentage (g/L), 15g/L of peptone, 5g/L of yeast extract powder, 5g/L of glucose and the balance of water, and the pH value is 7.0.
Production and fermentation
1. 6L of fermentation medium was placed in a 10L fermenter, the fermentation medium composition: by weight percentage, peptone: 10g/L, yeast extract powder: 12g/L glucose 15g/L, FeCl3 9mg/L、CuSO43mg/L、MnSO4 19mg/L、ZnCl210mg/L, 8mg/L of sodium molybdate and the balance of water.
2. Inoculating the seed liquid into a fermentation tank according to the inoculation amount of 13 percent;
and (3) growth of thalli: the incubation temperature was 35 ℃ and the stirring rate was 300rpm, with a continuous aeration of 3mL/min of air being maintained. Maintaining pH at 7 during culture, culturing for 120 hr, and culturing at OD600Reaching 9.2.
3. Fermentation production: after culturing the cells for 120h, adding sorbol to make the final concentration 10ml/L, stirring at 30 deg.C and 500rpm, ventilating at 5ml/min, fermenting for 72h, centrifuging to collect supernatant, measuring sorbic acid content (by 50 times sample dilution) by HPLC, and making the sorbic acid content 6.05g/L (FIG. 8), with a conversion rate of 60.08%.
The results of examples 1-6 and comparative examples 1-2 show that the conversion rate of sorbic acid prepared by the methods provided by examples 1-6 is higher than that of comparative examples 1-2, the effect is statistically significant, and p is less than 0.05. In each example, the conversion rate of example 3 is significantly better than that of the other examples, the effect is statistically significant, and p is less than 0.05.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (6)
1. The application of the culture medium in the fermentation preparation of sorbic acid by rhodobacter sphaeroides with the preservation number of CGMCC 1.3368; the culture medium consists of water and the following components:
2. a method for preparing sorbic acid is characterized in that the sorbic acid-containing fermentation liquor is obtained by using the pyrenal as a substrate and fermenting rhodobacter sphaeroides with the culture medium preservation number of CGMCC 1.3368; the culture medium consists of water and the following components:
3. the method according to claim 2, wherein the fermentation comprises a step of growing thallus and a step of converting sorbosol;
the thallus growth step comprises: inoculating the thalli to the culture medium, wherein the temperature is 28-35 ℃, the stirring speed is 300-500 rpm, the ventilation volume is 3mL/min, and the pH value is 6.8-7.4; culturing to OD of bacterial liquid600The value is 9 to 12.5;
the conversion step of the pyrialdehyde comprises the following steps: to OD600Adding the pyrialdehyde into the bacterial liquid with the value of 9-12.5, and stirring at the rotating speed of 300-500rpm, 30 ℃, the ventilation volume of 3-7 mL/min and the fermentation time of 60-84 h.
4. The method according to claim 3, wherein the final concentration of the aldehyde in the culture medium is 10 mL/L.
5. The method of claim 2, further comprising a step of activating seeds before the fermentation, wherein the culture medium used for activating the seeds comprises: water and peptone 15 g/L; 5g/L of yeast extract powder; glucose 5 g/L.
6. The preparation method according to claim 5, wherein the activation temperature is 28-35 ℃, the stirring speed is 150-200 rpm, and the time is 24-48 h.
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| CN1986774A (en) * | 2005-12-23 | 2007-06-27 | 王栋 | Industrial preparing process, fermented liquid and use of spheroidal erythrocyte |
| CN101348770A (en) * | 2007-07-18 | 2009-01-21 | 烟台天泰生物工程有限公司 | Rhodobacter sphaeroides, microbial preparation containing viable bacteria or zymocyte liquid of Rhodobacter sphaeroides and use of Rhodobacter sphaeroides |
| CN101928689A (en) * | 2010-07-13 | 2010-12-29 | 王栋 | Method for industrially preparing Rhodobacter sphaeroides, fermentation liquor of Rhodobacter sphaeroides and application |
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| CN1986774A (en) * | 2005-12-23 | 2007-06-27 | 王栋 | Industrial preparing process, fermented liquid and use of spheroidal erythrocyte |
| CN101348770A (en) * | 2007-07-18 | 2009-01-21 | 烟台天泰生物工程有限公司 | Rhodobacter sphaeroides, microbial preparation containing viable bacteria or zymocyte liquid of Rhodobacter sphaeroides and use of Rhodobacter sphaeroides |
| CN101928689A (en) * | 2010-07-13 | 2010-12-29 | 王栋 | Method for industrially preparing Rhodobacter sphaeroides, fermentation liquor of Rhodobacter sphaeroides and application |
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