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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 PDF

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CN107400644B
CN107400644B CN201710617502.2A CN201710617502A CN107400644B CN 107400644 B CN107400644 B CN 107400644B CN 201710617502 A CN201710617502 A CN 201710617502A CN 107400644 B CN107400644 B CN 107400644B
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culture medium
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sorbic acid
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rhodobacter sphaeroides
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李平顺
姜黎黎
张朔
王宏丽
胡紫嫣
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Elbow Shanghai Biotechnology Co ltd
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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

Culture medium, application of culture medium in rhodobacter sphaeroides fermentation and preparation method of sorbic acid
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:
Figure BDA0001360910680000011
Figure BDA0001360910680000021
in some embodiments of the invention, the medium comprises water and:
Figure BDA0001360910680000022
in some embodiments of the invention, the medium comprises water and:
Figure BDA0001360910680000023
in some embodiments of the invention, the medium comprises water and:
Figure BDA0001360910680000024
Figure BDA0001360910680000031
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:
Figure FDA0002701798570000011
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:
Figure FDA0002701798570000012
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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