CN110760468A - Method for improving efficiency of vitamin C precursor 2-keto-L-gulonic acid - Google Patents
Method for improving efficiency of vitamin C precursor 2-keto-L-gulonic acid Download PDFInfo
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Abstract
The invention relates to a method for improving the efficiency of vitamin C precursor 2-keto-L-gulonic acid, which is characterized in that in a fermentation system for producing the 2-keto-L-gulonic acid by fermenting mixed bacteria of large and small bacteria by taking L-sorbose as a substrate, a seed culture medium and a fermentation culture medium used by the fermentation system contain yeast peptone or/and riboflavin. By improving the composition of the culture medium, the growth and acid production capability of the small bacteria can be effectively improved, the fermentation conversion rate is improved, the fermentation period is shortened, the substance residue after the fermentation is finished is reduced, and the quality of the fermentation liquid is improved.
Description
Technical Field
The invention belongs to biological and new medical technology, and particularly relates to a method for improving the efficiency of vitamin C precursor 2-keto-L-gulonic acid.
Background
Vitamin C, also known as L-ascorbic acid, is a water-soluble vitamin, has important physiological functions and has wide application. At present, China is a large country for producing vitamin C, and the capacity accounts for more than 90% of the worldwide vitamin C market. The domestic production method is mainly a two-step fermentation method, the first step of fermentation is to take D-glucose as a raw material, generate D-sorbitol by hydrogenation and catalysis, then add Acetobacter nigrum to oxidize to obtain L-sorbose, which is commonly called alcohol-sugar conversion; the second step of fermentation is to further oxidize the L-sorbose into 2-keto-L-gulonic acid in a mixed fermentation system of gluconobacter oxydans (commonly called small bacteria) and bacillus megaterium (commonly called large bacteria), commonly called saccharic acid conversion. In the second step of mixed bacteria fermentation system, the large bacteria are associated bacteria, and the small bacteria are acid-producing bacteria. The large bacteria release bioactive protein in the growth and metabolism process, and act on acid-producing bacteria to start the growth and acid production of small bacteria. The small bacteria acid production is based on the high specificity of Sorbose Dehydrogenase (SDH) contained in the thallus to L-sorbose, the acid production is a typical enzymatic reaction process, and the activity of the SDH is the key for synthesizing 2-KLG by converting L-sorbose. Therefore, the method has decisive effects on improving the production efficiency of the 2-keto-L-gulonic acid and reducing the production cost by optimizing the growth environment of the thalli and regulating and controlling the metabolism of the auxiliary base of the microbial oxidoreductase.
Disclosure of Invention
The invention aims to provide a method for improving the efficiency of vitamin C precursor 2-keto-L-gulonic acid.
The technical scheme adopted for realizing the aim of the invention is as follows:
a method for improving the efficiency of vitamin C precursor 2-keto-L-gulonic acid is characterized in that in a fermentation system for producing the 2-keto-L-gulonic acid by fermenting mixed bacteria of large and small bacteria by taking L-sorbose as a substrate, a seed culture medium and a fermentation culture medium used by the fermentation system contain yeast peptone or/and riboflavin.
The seed culture medium and the fermentation culture medium do not contain corn steep liquor and yeast extract.
The seed culture medium and the fermentation culture medium comprise the following components:
primary seed culture medium: 1.2-1.5% of sorbose, 0.2-0.5% of glucose, 0.08-0.12% of magnesium sulfate, 0.1-0.2% of urea, 0.05-0.2% of yeast peptone and 0.05-0.1% of calcium carbonate;
secondary seed culture medium: 1.2-1.5% of L-sorbose, 0.2-0.5% of glucose, 0.08-0.12% of magnesium sulfate, 0.1-0.2% of urea, 0.05-0.2% of yeast peptone and 0.05-0.1% of calcium carbonate;
third-level seed culture medium: 1.2-1.5% of L-sorbose, 0.2-0.5% of glucose, 0.08-0.12% of magnesium sulfate, 0.1-0.2% of urea, 0.05-0.2% of yeast peptone and 0.05-0.1% of calcium carbonate;
fermentation medium: 8.0% of L-sorbose, 0.1-0.3% of urea, 0.5-2.0% of yeast peptone, 20-100 ppm of riboflavin, 0.1-0.3% of monopotassium phosphate and 0.01-0.04% of magnesium sulfate.
The invention researches the composition of the culture medium suitable for the mixed fermentation of the large and small bacteria by the two-step fermentation method of vitamin C in the fermentation process, namely, the fermentation metabolism is regulated and controlled by improving the culture medium in the two-step fermentation, and the saccharic acid conversion efficiency is improved. The method specifically comprises the following steps:
1. the water-soluble yeast peptone is adopted to replace the corn steep liquor and the yeast extract in the prior art. In the existing two-step fermentation of vitamin C, corn steep liquor is used as a main nitrogen source due to the fact that the corn steep liquor is rich in soluble protein and various free amino acids, but the quality of the corn steep liquor is affected by the production area of corn, the variety of corn, the processing technology of manufacturers, the storage mode (conditions, environment, time) and the like, so that the stability and the fermentation level of fermentation production are affected, the color of fermentation mash is deepened due to the appearance of tawny or brown, and the extraction and crystallization of the downstream process are affected. Therefore, the yeast peptone rich in protein, peptides, amino acids, nucleotides and biotin provides comprehensive and balanced nutrition for the mixed bacteria, so that the growth environment of the mixed bacteria is effectively improved, the quality of the bacteria is improved, and the yeast peptone has the advantages of shortening the fermentation period and improving the appearance and color of the fermentation liquor due to good water solubility.
2. Riboflavin was added to the medium. Riboflavin is the reactive group of Flavin Adenine Dinucleotide (FAD). The method takes promotion of L-sorbose dehydrogenase enzyme activity as a target, takes riboflavin which is an active group of FAD as an entry point, and adds the riboflavin into a VC two-step fermentation culture medium, so that a large amount of prosthetic group FAD required by redox can be provided for the small bacteria.
In conclusion, the invention can effectively improve the growth and acid production capability of the small bacteria, improve the fermentation conversion rate, shorten the fermentation period, reduce the substance residue after the fermentation is finished and improve the quality of the fermentation liquid by improving the composition of the culture medium. Experiments prove that when the vitamin C precursor 2-keto-L-gulonic acid is produced by fermentation, the average acid production rate is improved by more than 13%, the fermentation index is improved by more than 5%, the saccharic acid conversion efficiency is improved by more than 2%, the seed culture period is shortened by 2-4 h, the fermentation period is shortened by 5-10 h, and the transmittance of the filter liquor put in a tank is improved by more than 6%.
Detailed Description
The following comparative examples and examples are directed to the second fermentation process of producing vitamin C by fermentation of a currently conventional "two-step fermentation process", i.e., the process of further oxidizing L-sorbose to 2-keto-L-gulonic acid in a mixed fermentation system of Gluconobacter oxydans (commonly called small bacteria) and Bacillus megaterium (commonly called large bacteria).
Comparative examples
1, liquid preparation:
1.1 first-class seeds:
primary seed medium (%): 1.2 parts of L-sorbose, 0.25 part of glucose, 0.17 part of yeast extract, 0.09 part of magnesium sulfate, 0.13 part of urea, 1.0 part of corn steep liquor and 0.08 part of calcium carbonate; sterilizing at a pH of 7.0-7.4 and a temperature of 118-121 ℃ for 15-20 min.
And (3) process control:
the culture temperature is as follows: 28-30 ℃; ventilation volume 1: 1.0-1.5v/v/min and stirring, culture period: 22-28 hr.
1.2 second-level seeds:
secondary seed medium (%):
1.2 parts of L-sorbose, 0.25 part of glucose, 0.17 part of yeast extract, 0.09 part of magnesium sulfate, 0.13 part of urea, 1.0 part of corn steep liquor and 0.08 part of calcium carbonate; sterilizing at a pH of 7.0-7.4 and a temperature of 118-121 ℃ for 15-20 min.
And (3) process control:
inoculation amount: 5 percent; the culture temperature is as follows: 28-30 ℃, ventilation volume 1: 1.0-1.5v/v/min and stirring, wherein the culture period is as follows: 22-28 hr.
1.3 three-stage seeds:
tertiary seed medium (%): 1.5 parts of L-sorbose, 0.25 part of glucose, 0.20 part of yeast extract, 0.09 part of magnesium sulfate, 0.14 part of urea, 1.2 parts of corn steep liquor and 0.08 part of calcium carbonate; sterilizing at the temperature of 118-121 ℃ for 15-20 min at the pH of 7.0-7.4.
And (3) process control:
inoculation amount: 10 percent; the culture temperature is as follows: 28-30 ℃; ventilation volume 1: 1.0-1.5v/v/min and stirring, wherein the culture period is as follows: 18-24 hr.
2 fermentation
2.1 fermentation Medium (%): 8.0 parts of L-sorbose, 0.14 part of urea, 4.0 parts of corn steep liquor, 0.15 part of monopotassium phosphate and 0.015 part of magnesium sulfate; sterilizing at a pH of 7.0-7.4 and a temperature of 118-121 ℃ for 15-20 min.
2.2 fermentation control:
inoculation amount: 25 percent; the culture temperature is as follows: 28-30 ℃; ventilation volume 1: 0.5-1.2 v/v/min, stirring and supplementing materials, wherein the dissolved oxygen is ensured to be more than 20% in the fermentation process; and (3) culture period: and (5) putting the pot for 45 hours.
2.3 process feed: (carbon source and alkali liquor are supplemented in the fermentation process)
Carbon source supplement: namely, the L-sorbose is supplemented, and is a product of VC one-step fermentation of sorbitol, and is used for supplying the growth and proliferation of gluconobacter oxydans and producing 2-keto-L-gulonic acid. During the fermentation process, feeding L-sorbose, controlling 12-25 mg/ml of sorbose in the fermentation liquor, finishing feeding the sorbose 4-8 h before tank placing, and keeping residual sugar in the tank placing to be lower than 0.6 mg/ml.
Alkali liquor supplement: namely, 20-25% of sterile sodium carbonate solution is supplemented to be used as a pH regulator of the culture solution in the VC two-step fermentation process. The pH value of the fermentation liquor is controlled to be 7.0-7.5 according to the fermentation period gradient.
2.4 tank placing parameters:
the tank placing period is as follows: 45 hr; content of gulonic acid: 126.7 mg/ml; residual sugar: 0.42 mg/ml; transmittance of filtrate: 85.7 percent; average acid production rate: 2.816 mg/ml.h; the fermentation conversion rate is 90.12%; the fermentation index is 2.837 g/L.h.
Example 1
1, liquid preparation:
1.1 first-class seeds:
primary seed medium (%): 1.2 parts of L-sorbose, 0.25 part of glucose, 0.085 part of magnesium sulfate, 0.15 part of urea, 0.08 part of yeast peptone and 0.08 part of calcium carbonate; sterilizing at a pH of 7.0-7.4 and a temperature of 118-121 ℃ for 15-20 min.
And (3) process control:
the culture temperature is as follows: 28-30 ℃, ventilation volume 1: 1.0-1.5v/v/min and stirring, wherein the culture period is as follows: 20-24 hr.
1.2 second-level seeds:
secondary seed medium (%): 1.2 parts of L-sorbose, 0.3 part of glucose, 0.12 part of magnesium sulfate, 0.17 part of urea, 0.012 part of yeast peptone and 0.1 part of calcium carbonate; sterilizing at a pH of 7.0-7.4 and a temperature of 118-121 ℃ for 15-20 min.
And (3) process control:
inoculation amount: 3-5%; the culture temperature is as follows: 28-30 ℃; ventilation volume 1: 1.0-1.5v/v/min and stirring, wherein the culture period is as follows: 20-24 hr.
1.3 three-stage seeds:
tertiary seed medium (%): 1.5 parts of L-sorbose, 0.5 part of glucose, 0.12 part of magnesium sulfate, 0.17 part of urea, 0.12 part of yeast peptone and 0.1 part of calcium carbonate; sterilizing at a pH of 7.0-7.4 and a temperature of 118-121 ℃ for 15-20 min.
And (3) process control:
inoculation amount: 8-10%; the culture temperature is as follows: 28-30 ℃; ventilation volume 1: 1.0-1.5v/v/min and stirring, wherein the culture period is as follows: 16-22 h.
2, fermentation:
2.1 Medium (%): 8.0 parts of L-sorbose, 0.15 part of urea, 0.5 part of yeast peptone, 30ppm of riboflavin, 0.15 part of monopotassium phosphate and 0.025 part of magnesium sulfate; sterilizing at a pH of 7.0-7.4 and a temperature of 118-121 ℃ for 15-20 min.
2.2, process control:
inoculation amount: 18 percent; the culture temperature is as follows: 28-30 ℃; ventilation volume 1: 0.5-1.2 v/v/min, stirring and supplementing materials; the dissolved oxygen is ensured to be more than 20% in the fermentation process, and the culture period is as follows: placing in a jar for 40 hr.
2.3 process feed: (carbon source and alkali liquor are supplemented in the fermentation process)
Carbon source supplement: namely supplementing the L-sorbose. The L-sorbose is a product of VC one-step fermentation sorbitol, and is used for supplying the growth and proliferation of gluconobacter oxydans and producing 2-keto-L-gulonic acid. In the fermentation process, adding sorbose in a fed-batch mode, controlling 15-35 mg/ml of sorbose in the fermentation liquor, finishing the fed-batch of sorbose 4-8 hours before tank placing, and enabling the residual sugar in the tank placing to be lower than 0.6 mg/ml.
Alkali liquor supplement: namely, 20-25% of sterile sodium carbonate solution is supplemented to be used as a pH regulator of the culture solution in the VC two-step fermentation process. The pH value of the fermentation liquor is controlled to be 7.0-7.5 according to the fermentation period gradient.
2.4 tank placing parameters:
the tank placing period is as follows: 40 h; the content of the releasing acid is 127.26 mg/ml; residual sugar is less than or equal to 0.6 mg/ml; transmittance of filtrate: 92.3 percent; fermentation index: 2.986 g/L.h; fermentation conversion rate: 92.5 percent; the average acid production rate is 3.182mg/ml.
Example 2
1, liquid preparation:
1.1 first-class seeds:
primary seed medium (%): 1.5 parts of L-sorbose, 0.2 part of glucose, 0.08 part of magnesium sulfate, 0.1 part of urea, 0.07 part of yeast peptone and 0.05 part of calcium carbonate; sterilizing at a pH of 7.0-7.4 and a temperature of 118-121 ℃ for 15-20 min.
And (3) process control:
the culture temperature is as follows: 28-30 ℃, ventilation volume 1: 1.0-1.5v/v/min and stirring, wherein the culture period is as follows: 19-28 hr.
1.2 second-level seeds:
secondary seed medium (%): 1.2 parts of L-sorbose, 0.50 part of glucose, 0.12 part of magnesium sulfate, 0.15 part of urea, 0.12 part of yeast peptone and 0.08 part of calcium carbonate; sterilizing at a pH of 7.0-7.4 and a temperature of 118-121 ℃ for 15-20 min.
And (3) process control:
inoculation amount: 3-5%; the culture temperature is as follows: 28-30 ℃; ventilation volume 1: 1.0-1.5v/v/min and stirring, wherein the culture period is as follows: 19-28 hr.
1.3 three-stage seeds:
tertiary seed medium (%): 1.5 parts of L-sorbose, 0.35 part of glucose, 0.10 part of magnesium sulfate, 0.18 part of urea, 0.2 part of yeast peptone and 0.05 part of calcium carbonate; sterilizing at a pH of 7.0-7.4 and a temperature of 118-121 ℃ for 15-20 min.
And (3) process control:
inoculation amount: 8-10%; the culture temperature is as follows: 28-30 ℃; ventilation volume 1: 1.0-1.5v/v/min and stirring, wherein the culture period is as follows: 16-24 h.
2, fermentation:
2.1 fermentation Medium (%): 8.0 parts of L-sorbose, 0.12 part of urea, 1.15 parts of yeast peptone, 80ppm parts of riboflavin, 0.18 part of potassium dihydrogen phosphate and 0.025 part of magnesium sulfate; sterilizing at a pH of 7.0-7.4 and a temperature of 118-121 ℃ for 15-20 min.
2.2, process control:
inoculation amount: 30 percent; the culture temperature is as follows: 28-30 ℃; ventilation volume 1: 0.5-1.2 v/v/min, stirring and supplementing materials; the dissolved oxygen is ensured to be more than 20% in the fermentation process, and the culture period is as follows: placing in a jar for 40 hr.
2.3 process feed: the feed was made as in example 1.
2.4 tank placing parameters:
the tank placing period is as follows: 40 hr; content of gulonic acid: 132.27mg/ml, residual sugar 0.42mg/ml, filtrate transmittance: 91.7 percent; average acid production rate: 3.307 mg/ml.h; the fermentation conversion rate is 93.1%; the fermentation index is 3.112 g/L.h.
Example 3
1, liquid preparation:
1.1 first-class seeds:
primary seed medium (%): 1.4 parts of L-sorbose, 0.35 part of glucose, 0.08 part of magnesium sulfate, 0.2 part of urea, 0.06 part of yeast peptone and 0.08 part of calcium carbonate; sterilizing at a pH of 7.0-7.4 and a temperature of 118-121 ℃ for 15-20 min.
And (3) process control:
the culture temperature is as follows: 28-30 ℃, ventilation volume 1: 1.0-1.5v/v/min and stirring, wherein the culture period is as follows: 19-28 hr.
1.2 second-level seeds:
secondary seed medium (%): 1.2 parts of L-sorbose, 0.35 part of glucose, 0.08 part of magnesium sulfate, 0.15 part of urea, 0.12 part of yeast peptone and 0.08 part of calcium carbonate; sterilizing at a pH of 7.0-7.4 and a temperature of 118-121 ℃ for 15-20 min.
And (3) process control:
inoculation amount: 3-5%; the culture temperature is as follows: 28-30 ℃; ventilation volume 1: 1.0-1.5v/v/min and stirring, wherein the culture period is as follows: 19-28 hr.
1.3 three-stage seeds:
tertiary seed medium (%): 1.5 parts of L-sorbose, 0.5 part of glucose, 0.12 part of magnesium sulfate, 0.2 part of urea, 0.15 part of yeast peptone and 0.1 part of calcium carbonate; sterilizing at a pH of 7.0-7.4 and a temperature of 118-121 ℃ for 15-20 min.
And (3) process control:
inoculation amount: 8-10%; the culture temperature is as follows: 28-30 ℃; ventilation volume 1: 1.0-1.5v/v/min and stirring, wherein the culture period is as follows: 16-24 h.
2, fermentation:
2.1 fermentation Medium (%): 8.0 parts of L-sorbose, 0.22 part of urea, 1.25 parts of yeast peptone, 100ppm parts of riboflavin, 0.25 part of monopotassium phosphate and 0.033 part of magnesium sulfate; sterilizing at a pH of 7.0-7.4 and a temperature of 118-121 ℃ for 15-20 min.
2.2, process control:
inoculation amount: 20 percent; the culture temperature is as follows: 28-30 ℃; ventilation volume 1: 0.5-1.2 v/v/min, stirring and supplementing materials; the dissolved oxygen is ensured to be more than 20% in the fermentation process, and the culture period is as follows: placing in a jar for 40 hr.
2.3 process feed: the same as in example 1.
2.4 tank placing parameters:
the tank placing period is as follows: 35 hr; content of gulonic acid: 118.97mg/ml, residual sugar 0.33mg/ml, filtrate transmittance: 90.9 percent; average acid production rate: 3.399 mg/ml.h; the fermentation conversion rate is 94.7%; the fermentation index is 3.087 g/L.h.
The foregoing is only a preferred embodiment of the present invention, and the present invention can provide a conception mode, and the accompanying bacteria of different genera can be adjusted accordingly on the premise of the conception, and the adjustment should be regarded as the protection scope of the present invention.
Claims (3)
1. A method for improving the efficiency of vitamin C precursor 2-keto-L-gulonic acid is characterized in that in a fermentation system for producing the 2-keto-L-gulonic acid by fermenting mixed bacteria of large and small bacteria by using L-sorbose as a substrate, a seed culture medium and a fermentation culture medium used for culturing the strains contain yeast peptone or/and riboflavin.
2. The method for improving the efficiency of 2-keto-L-gulonic acid as a vitamin C precursor according to claim 1, wherein the seed medium and the fermentation medium are free of corn steep liquor and yeast extract.
3. The method for improving the efficiency of 2-keto-L-gulonic acid, a vitamin C precursor, according to claim 1 or 2, wherein the seed medium and the fermentation medium consist of:
primary seed culture medium: 1.2-1.5% of sorbose, 0.2-0.5% of glucose, 0.08-0.12% of magnesium sulfate, 0.1-0.2% of urea, 0.05-0.2% of yeast peptone and 0.05-0.1% of calcium carbonate;
secondary seed culture medium: 1.2-1.5% of L-sorbose, 0.2-0.5% of glucose, 0.08-0.12% of magnesium sulfate, 0.1-0.2% of urea, 0.05-0.2% of yeast peptone and 0.05-0.1% of calcium carbonate;
third-level seed culture medium: 1.2-1.5% of L-sorbose, 0.2-0.5% of glucose, 0.08-0.12% of magnesium sulfate, 0.1-0.2% of urea, 0.05-0.2% of yeast peptone and 0.05-0.1% of calcium carbonate;
fermentation medium: 8.0% of L-sorbose, 0.1-0.3% of urea, 0.5-2.0% of yeast peptone, 20-100 ppm of riboflavin, 0.1-0.3% of monopotassium phosphate and 0.01-0.04% of magnesium sulfate.
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