CN108624639A - A kind of preparation process of guanosine - Google Patents
A kind of preparation process of guanosine Download PDFInfo
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- CN108624639A CN108624639A CN201810651148.XA CN201810651148A CN108624639A CN 108624639 A CN108624639 A CN 108624639A CN 201810651148 A CN201810651148 A CN 201810651148A CN 108624639 A CN108624639 A CN 108624639A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
Abstract
The invention belongs to biotechnologies, and in particular to a kind of preparation process of guanosine includes the following steps：（1）By bacillus subtilis（Bacillus subtilis）Bacterial strain activates, and is inoculated on slant medium, is cultivated in constant incubator；（2）Seed culture medium is added in triangular flask, gauze sealing is placed in high-pressure sterilizing pot, sterilizes, the constant temperature in thermostat water bath, inoculation step（1）Strain is cultivated, is cultivated in constant-temperature shaking incubator；（3）Fermentation medium is added in triangular flask, gauze sealing is placed in high-pressure sterilizing pot, sterilizes, the constant temperature in thermostat water bath, inoculation step（2）Gained seed liquor, is cultivated in constant-temperature shaking incubator.Guanosine may be up to 10.6g/L in gained zymotic fluid of the invention, and guanosine yield is high.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of preparation process of guanosine.
Background technology
Guanylic acid also known as 5 '-guanylic acids（Guanosine-5'-monophos phate, GMP）, molecular formula
For：C10H14N5O8P is made of hydroxyl and the phosphoric acid condensation on the 5th carbon atom on nucleotide ribose, is composition nucleic acid
One of five kinds of nucleotide, be a kind of colourless to white crystals or white crystal powder shape, with special similar mushroom
Taste, easily dissolving forms stablizing solution in water, is slightly soluble in organic solvent, in food industry mainly in the form of sodium salt（Guanosine
Acid disodium, Disodium guanylate, GMPNa2）As food additives and the raw material as flavouring.As food additive
Add the guanylic acid of agent that also there is high bioactivity, body growth development can be promoted, body Nutrition and Metabolism is adjusted, improve body
Immunity and adjunct antineoplastic and other effects.
Guanylic acid fermentation process is divided into two methods of direct fermentation and microbe conversion method, and direct fermentation utilizes a fixed number
Microorganism is measured, is fermented to obtain guanylic acid, but the guanylic acid low output that the method obtains using glucose as carbon source, cannot reached
Industrial requirement.At present microbe conversion method with widest method, using certain microorganism, using polysaccharide or glucose as
Carbon source produces guanosine, and biological or chemical method is recycled to be translated into guanylic acid.The item but existing microbe conversion method is fermented
Part is poor, gained guanosine low output.
Invention content
To overcome drawbacks described above, the purpose of the present invention is to provide a kind of preparation processes of guanosine.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of preparation process of guanosine, includes the following steps：
（1）By bacillus subtilis（Bacillus subtilis）Bacterial strain activates, and is inoculated on slant medium, is trained in constant temperature
Support 32-40 DEG C of constant temperature stationary culture 20-30 h in case；
（2）45-55 mL seed culture mediums are added in 500 mL triangular flasks, 6-10 layers of gauze sealing are placed in high-pressure sterilizing pot
In, 120-123 DEG C of sterilizing 10-20 min, constant temperature is inoculated with a ring or two ring steps to 32-40 DEG C in thermostat water bath（1）
Culture strain cultivates 8-16h under the conditions of 32-40 DEG C, 100-150 r/min in constant-temperature shaking incubator；
（3）25-30 mL fermentation mediums are added in 500 mL triangular flasks, 6-10 layers of gauze sealing are placed in high-pressure sterilizing pot
In, 120-123 DEG C of sterilizing 10-20 min, constant temperature is to 32-40 DEG C in thermostat water bath, inoculation step（2）Gained seed liquor,
Inoculum concentration is 6-14%, and under the conditions of 32-40 DEG C, 100-150 r/min, 48-78h is cultivated in constant-temperature shaking incubator.
Preferably, step（1）The slant medium main component and its weight percentage are：Peptone 0.8-
1.2%, sodium chloride 0.3-0.7%, beef extract powder 0.2-0.5%, agar 1.2-1.8%.
Preferably, step（2）The seed culture medium main component and its weight percentage are：Peptone 0.8-
1.2%, beef extract 0.2-0.45%, sodium chloride 0.4-0.7%.
Preferably, step（3）The fermentation medium main component and its weight percentage are：Glucose 8-
15%, yeast powder 1.2-2.8%, corn steep liquor 0.1-0.3%, sodium glutamate 0.8-1.6%, dipotassium hydrogen phosphate 0.1-0.3%, chlorine
Change calcium 0.1-0.3%, calcium carbonate 1.5-2.5%, ammonium sulfate 1.0-3.0%, magnesium sulfate 0.03-0.08%.
Preferably, step（3）The pH of the fermentation medium is 6.8-7.4.
Preferably, step（3）Further include the assay method of guanosine yield later, step is：Draw 0.1 mL of zymotic fluid in
In 10 mL centrifuge tubes, 10 mL are settled to 4 mol/L NaOH, are uniformly mixed, boiling water bath 5 min, 3000 r/min centrifugation 5
Min takes centrifuged supernatant 1mL, and dilution is settled to 100 mL, using high effective liquid chromatography for measuring guanosine yield.
Preferably, the high effective liquid chromatography for measuring condition is：Pillar：C18, mobile phase：Acetonitrile：Water=10:90, stream
Speed：1 mL/min, Detection wavelength：256 nm, column temperature：25 DEG C, 10 uL of sample size.
The positive beneficial effect of the present invention：
1. fermentation method of the present invention prepares guanosine, non-environmental-pollution, raw material sources are extensive, and guanosine content can be high in gained zymotic fluid
Up to 10.6g/L, guanosine yield is high, more in the prior art guanosine output increased 71%.
It is microorganism growth, metabolism 2. fermentation medium glucose of the present invention is the carbon source in fermentation culture medium for microbe
The important sources of energy, concentration of glucose is too low, cannot be bacillus subtilis by enough energy, attenuation degree is low, guanosine
Low output, concentration of glucose is excessively high, can generate inhibiting effect to substrate, influence guanosine fermentation；Yeast powder is that microorganism grows generation
It thanks and the nutriments such as organic nitrogen source, growth factor, vitamin and purine is provided, the adding too much of yeast powder can produce fermentation
Raw feedback inhibition；Corn steep liquor provides organic nitrogen source and growth factor for microorganism；Sodium glutamate contributes in earlier fermentation
Thalline synthesizes, and can promote thalli growth, increase guanosine yield, ferment the later stage, glutamic acid can be closed by the own metabolism of thalline
At if concentration of sodium glutamate is excessively high, the consumption of microorganism initial stage can generate inhibiting effect plus later stage synthesis to fermentation, hinder bird
Glycosides synthesizes；Ammonium sulfate can adjust culture medium osmotic pressure and PH values, and each raw material type and its dosage are suitable in fermentation medium, protect
The utmostly elongation of thalline is demonstrate,proved.
3. the seed culture time of the present invention is suitable, incubation time is too short, is transferred in fermentation medium, it may appear that before seed
Phase is slow-growing, and entire fermentation period extends, common several phenomenons such as time retardation that product initially forms, or even will appear
Fermentation abnormal phenomenon；Spawn incubation overlong time, thalline can the too early self-dissolving in seed culture medium, influence later stage guanosine accumulation.
Fermented and cultured inoculum concentration of the present invention is 6-14%, i.e. seed liquor accounts for the 6-14% of fermentation medium volume, and inoculum concentration is suitable, inoculum concentration
Very few, culture medium is not fully utilized；Inoculum concentration is excessive, and culture medium nutrition is limited, can be accumulated to guanosine and generate inhibiting effect.
Specific implementation mode
With reference to some specific implementation modes, the present invention is further described.
Embodiment 1
A kind of preparation process of guanosine, includes the following steps：
（1）By bacillus subtilis（Bacillus subtilis）Bacterial strain activates, and is inoculated on slant medium, is trained in constant temperature
Support 32 DEG C of 30 h of constant temperature stationary culture in case；
Step（1）The slant medium main component and its weight percentage are：Peptone 0.8%, sodium chloride
0.5%, beef extract powder 0.4%, agar 1.2%；
（2）45 mL seed culture mediums are added in 500 mL triangular flasks, 8 layers of gauze sealing are placed in high-pressure sterilizing pot, 121 DEG C
Sterilize 15 min, and constant temperature is inoculated with a ring step to 40 DEG C in thermostat water bath（1）Cultivate strain, at 40 DEG C, 100 r/min
Under the conditions of, 10h is cultivated in constant-temperature shaking incubator；
Step（2）The seed culture medium main component and its weight percentage are：Peptone 1.0%, beef extract
0.3%, sodium chloride 0.7%；
（3）30 mL fermentation mediums are added in 500 mL triangular flasks, 10 layers of gauze sealing are placed in high-pressure sterilizing pot, 121
DEG C 15 min of sterilizing, constant temperature is to 32 DEG C in thermostat water bath, inoculation step（2）Gained seed liquor, inoculum concentration 6%, 32
DEG C, under the conditions of 100 r/min, 60h is cultivated in constant-temperature shaking incubator；
Step（3）The fermentation medium main component and its weight percentage are：Glucose 15%, yeast powder 1.6%,
Corn steep liquor 0.1%, sodium glutamate 1.4%, dipotassium hydrogen phosphate 0.2%, calcium chloride 0.2%, calcium carbonate 1.5%, ammonium sulfate 1.0%, sulphur
Sour magnesium 0.03%；
Step（3）The pH of the fermentation medium is 6.9；
（4）Determination step（3）The yield of guanosine in zymotic fluid：0.1 mL of zymotic fluid is drawn in 10 mL centrifuge tubes, with 4 mol/
L NaOH are settled to 10 mL, are uniformly mixed, and boiling water bath 5 min, 3000 r/min 5 min of centrifugation take centrifuged supernatant 1
ML, dilution are settled to 100 mL, and using high effective liquid chromatography for measuring guanosine yield, testing result is shown in Table 1；
High effective liquid chromatography for measuring condition：Pillar：C18, mobile phase：Acetonitrile：Water=10:90, flow velocity：1 mL/min detects wave
It is long：256 nm, column temperature：25 DEG C, 10 uL of sample size.
Embodiment 2
A kind of preparation process of guanosine, includes the following steps：
（1）By bacillus subtilis（Bacillus subtilis）Bacterial strain activates, and is inoculated on slant medium, is trained in constant temperature
Support 34 DEG C of constant temperature stationary culture 25h in case；
Step（1）The slant medium main component and its weight percentage are：Peptone 1.0%, sodium chloride
0.7%, beef extract powder 0.3%, agar 1.6%；
（2）50 mL seed culture mediums are added in 500 mL triangular flasks, 6 layers of gauze sealing are placed in high-pressure sterilizing pot, 120 DEG C
Sterilize 20 min, and constant temperature is inoculated with a ring step to 32 DEG C in thermostat water bath（1）Cultivate strain, at 32 DEG C, 110 r/min
Under the conditions of, 8h is cultivated in constant-temperature shaking incubator；
Step（2）The seed culture medium main component and its weight percentage are：Peptone 0.8%, beef extract
0.45%, sodium chloride 0.4%；
（3）25mL fermentation mediums are added in 500 mL triangular flasks, 6 layers of gauze sealing are placed in high-pressure sterilizing pot, 123 DEG C
Sterilize 15 min, and constant temperature is to 36 DEG C in thermostat water bath, inoculation step（2）Gained seed liquor, inoculum concentration 14%, at 36 DEG C,
Under the conditions of 110 r/min, 48h is cultivated in constant-temperature shaking incubator；
Step（3）The fermentation medium main component and its weight percentage are：Glucose 8%, yeast powder 1.2%,
Corn steep liquor 0.3%, sodium glutamate 0.8%, dipotassium hydrogen phosphate 0.1%, calcium chloride 0.1%, calcium carbonate 1.8%, ammonium sulfate 1.5%,
Magnesium sulfate 0.04%；
Step（3）The pH of the fermentation medium is 6.8；
（4）Determination step（3）The yield of guanosine in zymotic fluid：0.1 mL of zymotic fluid is drawn in 10 mL centrifuge tubes, with 4 mol/
L NaOH are settled to 10 mL, are uniformly mixed, and boiling water bath 5 min, 3000 r/min 5 min of centrifugation take centrifuged supernatant 1
ML, dilution are settled to 100 mL, and using high effective liquid chromatography for measuring guanosine yield, testing result is shown in Table 1；
High effective liquid chromatography for measuring condition：Pillar：C18, mobile phase：Acetonitrile：Water=10:90, flow velocity：1 mL/min detects wave
It is long：256 nm, column temperature：25 DEG C, 10 uL of sample size.
Embodiment 3
A kind of preparation process of guanosine, includes the following steps：
（1）By bacillus subtilis（Bacillus subtilis）Bacterial strain activates, and is inoculated on slant medium, is trained in constant temperature
Support 40 DEG C of 20 h of constant temperature stationary culture in case；
Step（1）The slant medium main component and its weight percentage are：Peptone 1.2%, sodium chloride 0.3%,
Beef extract powder 0.4%, agar 1.4%；
（2）55 mL seed culture mediums are added in 500 mL triangular flasks, 6 layers of gauze sealing are placed in high-pressure sterilizing pot, 120 DEG C
Sterilize 20min, and constant temperature is inoculated with two ring steps to 36 DEG C in thermostat water bath（1）Cultivate strain, at 36 DEG C, 150r/min items
Under part, 16h is cultivated in constant-temperature shaking incubator；
Step（2）The seed culture medium main component and its weight percentage are：Peptone 1.0%, beef extract
0.2%, sodium chloride 0.6%；
（3）26 mL fermentation mediums are added in 500 mL triangular flasks, 8 layers of gauze sealing are placed in high-pressure sterilizing pot, 121 DEG C
Sterilize 20 min, and constant temperature is to 36 DEG C in thermostat water bath, inoculation step（2）Gained seed liquor, inoculum concentration 8%, at 36 DEG C,
Under the conditions of 120 r/min, 48h is cultivated in constant-temperature shaking incubator；
Step（3）The fermentation medium main component and its weight percentage are：Glucose 13.0%, yeast powder 2.0%,
Corn steep liquor 0.3%, sodium glutamate 1.2%, dipotassium hydrogen phosphate 0.1%, calcium chloride 0.2%, calcium carbonate 2.5%, ammonium sulfate 2.5%, sulfuric acid
Magnesium 0.06%；
Step（3）The pH of the fermentation medium is 7.2；
（4）Determination step（3）The yield of guanosine in zymotic fluid：0.1 mL of zymotic fluid is drawn in 10 mL centrifuge tubes, with 4 mol/
L NaOH are settled to 10 mL, are uniformly mixed, and boiling water bath 5 min, 3000 r/min 5 min of centrifugation take centrifuged supernatant 1
ML, dilution are settled to 100 mL, and using high effective liquid chromatography for measuring guanosine yield, testing result is shown in Table 1；
High effective liquid chromatography for measuring condition：Pillar：C18, mobile phase：Acetonitrile：Water=10:90, flow velocity：1 mL/min detects wave
It is long：256 nm, column temperature：25 DEG C, 10 uL of sample size.
Embodiment 4
A kind of preparation process of guanosine, includes the following steps：
（1）By bacillus subtilis（Bacillus subtilis）Bacterial strain activates, and is inoculated on slant medium, is trained in constant temperature
Support case in 36 DEG C of constant temperature stationary cultures for 24 hours；
Step（1）The slant medium main component and its weight percentage are：Peptone 1.0%, sodium chloride
0.5%, beef extract powder 0.3%, agar 1.5%；
（2）50 mL seed culture mediums are added in 500 mL triangular flasks, 8 layers of gauze sealing are placed in high-pressure sterilizing pot, 123 DEG C
Sterilize 10 min, and constant temperature is inoculated with a ring step to 38 DEG C in thermostat water bath（1）Cultivate strain, at 38 DEG C, 110 r/min
Under the conditions of, 10h is cultivated in constant-temperature shaking incubator；
Step（2）The seed culture medium main component and its weight percentage are：Peptone 1.0%, beef extract
0.3%, sodium chloride 0.5%；
（3）28 mL fermentation mediums are added in 500 mL triangular flasks, 8 layers of gauze sealing are placed in high-pressure sterilizing pot, 120 DEG C
Sterilize 10 min, and constant temperature is to 36 DEG C in thermostat water bath, inoculation step（2）Gained seed liquor, inoculum concentration 10%, at 36 DEG C,
Under the conditions of 100 r/min, 78h is cultivated in constant-temperature shaking incubator；
Step（3）The fermentation medium main component and its weight percentage are：Glucose 12.0%, yeast powder 1.6%,
Corn steep liquor 0.2%, sodium glutamate 1.0%, dipotassium hydrogen phosphate 0.2%, calcium chloride 0.2%, calcium carbonate 2.0%, ammonium sulfate 2.0%, sulfuric acid
Magnesium 0.05%；
Step（3）The pH of the fermentation medium is 7.2；
（4）Determination step（3）The yield of guanosine in zymotic fluid：0.1 mL of zymotic fluid is drawn in 10 mL centrifuge tubes, with 4 mol/
L NaOH are settled to 10 mL, are uniformly mixed, and boiling water bath 5 min, 3000 r/min 5 min of centrifugation take centrifuged supernatant 1
ML, dilution are settled to 100 mL, and using high effective liquid chromatography for measuring guanosine yield, testing result is shown in Table 1；
High effective liquid chromatography for measuring condition：Pillar：C18, mobile phase：Acetonitrile：Water=10:90, flow velocity：1 mL/min detects wave
It is long：256 nm, column temperature：25 DEG C, 10 uL of sample size.
Embodiment 5
A kind of preparation process of guanosine, includes the following steps：
（1）By bacillus subtilis（Bacillus subtilis）Bacterial strain activates, and is inoculated on slant medium, is trained in constant temperature
Support 36 DEG C of 24 h of constant temperature stationary culture in case；
Step（1）The slant medium main component and its weight percentage are：Peptone 1.0%, sodium chloride
0.5%, beef extract powder 0.3%, agar 1.5%；
（2）50 mL seed culture mediums are added in 500 mL triangular flasks, 8 layers of gauze sealing are placed in high-pressure sterilizing pot, 121 DEG C
Sterilize 15 min, and constant temperature is inoculated with a ring step to 36 DEG C in thermostat water bath（1）Cultivate strain, at 36 DEG C, 100 r/min
Under the conditions of, 12h is cultivated in constant-temperature shaking incubator；
Step（2）The seed culture medium main component and its weight percentage are：Peptone 1.0%, beef extract
0.3%, sodium chloride 0.5%；
（3）27mL fermentation mediums are added in 500 mL triangular flasks, 8 layers of gauze sealing are placed in high-pressure sterilizing pot, 121 DEG C
Sterilize 15 min, and constant temperature is to 36 DEG C in thermostat water bath, inoculation step（2）Gained seed liquor, inoculum concentration 10%, at 36 DEG C,
Under the conditions of 110 r/min, 72h is cultivated in constant-temperature shaking incubator；
Step（3）The fermentation medium main component and its weight percentage are：Glucose 12.0%, yeast powder 1.6%,
Corn steep liquor 0.2%, sodium glutamate 1.0%, dipotassium hydrogen phosphate 0.2%, calcium chloride 0.2%, calcium carbonate 2.0%, ammonium sulfate 2.0%, sulfuric acid
Magnesium 0.05%；
Step（3）The pH of the fermentation medium is 7.0；
（4）Determination step（3）The yield of guanosine in zymotic fluid：0.1 mL of zymotic fluid is drawn in 10 mL centrifuge tubes, with 4 mol/
L NaOH are settled to 10 mL, are uniformly mixed, and boiling water bath 5 min, 3000 r/min 5 min of centrifugation take centrifuged supernatant 1
ML, dilution are settled to 100 mL, and using high effective liquid chromatography for measuring guanosine yield, testing result is shown in Table 1；
High effective liquid chromatography for measuring condition：Pillar：C18, mobile phase：Acetonitrile：Water=10:90, flow velocity：1 mL/min detects wave
It is long：256 nm, column temperature：25 DEG C, 10 uL of sample size.
Embodiment 6
A kind of preparation process of guanosine, includes the following steps：
（1）By bacillus subtilis（Bacillus subtilis）Bacterial strain activates, and is inoculated on slant medium, is trained in constant temperature
Support 32 DEG C of 26 h of constant temperature stationary culture in case；
Step（1）The slant medium main component and its weight percentage are：Peptone 1.0%, sodium chloride
0.6%, beef extract powder 0.5%, agar 1.4%；
（2）50 mL seed culture mediums are added in 500 mL triangular flasks, 10 layers of gauze sealing are placed in high-pressure sterilizing pot, 121
DEG C sterilizing 10 min, in thermostat water bath constant temperature to 36 DEG C, be inoculated with a ring step（1）Cultivate strain, at 36 DEG C, 120 r/
Under the conditions of min, 10h is cultivated in constant-temperature shaking incubator；
Step（2）The seed culture medium main component and its weight percentage are：Peptone 0.8%, beef extract
0.4%, sodium chloride 0.5%；
（3）28 mL fermentation mediums are added in 500 mL triangular flasks, 10 layers of gauze sealing are placed in high-pressure sterilizing pot, 121
DEG C 15 min of sterilizing, constant temperature is to 40 DEG C in thermostat water bath, inoculation step（2）Gained seed liquor, inoculum concentration 12%, 36
DEG C, under the conditions of 140 r/min, 72h is cultivated in constant-temperature shaking incubator；
Step（3）The fermentation medium main component and its weight percentage are：Glucose 10%, yeast powder 1.8% are beautiful
Rice ＆ peanut milk 0.2%, sodium glutamate 1.6%, dipotassium hydrogen phosphate 0.3%, calcium chloride 0.2%, calcium carbonate 2.2%, ammonium sulfate 1.8%, sulfuric acid
Magnesium 0.07%；
Step（3）The pH of the fermentation medium is 7.4；
（4）Determination step（3）The yield of guanosine in zymotic fluid：0.1 mL of zymotic fluid is drawn in 10 mL centrifuge tubes, with 4 mol/
L NaOH are settled to 10 mL, are uniformly mixed, and boiling water bath 5 min, 3000 r/min 5 min of centrifugation take centrifuged supernatant 1
ML, dilution are settled to 100 mL, and using high effective liquid chromatography for measuring guanosine yield, testing result is shown in Table 1；
High effective liquid chromatography for measuring condition：Pillar：C18, mobile phase：Acetonitrile：Water=10:90, flow velocity：1 mL/min detects wave
It is long：256 nm, column temperature：25 DEG C, 10 uL of sample size.
Embodiment 7
A kind of preparation process of guanosine, includes the following steps：
（1）By bacillus subtilis（Bacillus subtilis）Bacterial strain activates, and is inoculated on slant medium, is trained in constant temperature
Support 38 DEG C of 20 h of constant temperature stationary culture in case；
Step（1）The slant medium main component and its weight percentage are：Peptone 1.0%, sodium chloride
0.5%, beef extract powder 0.3%, agar 1.8%；
（2）50mL seed culture mediums are added in 500 mL triangular flasks, 8 layers of gauze sealing are placed in high-pressure sterilizing pot, 122 DEG C
Sterilize 15 min, and constant temperature is inoculated with two ring steps to 32 DEG C in thermostat water bath（1）Cultivate strain, at 32 DEG C, 140 r/min
Under the conditions of, 15h is cultivated in constant-temperature shaking incubator；
Step（2）The seed culture medium main component and its weight percentage are：Peptone 1.2%, beef extract
0.3%, sodium chloride 0.4%；
（3）30mL fermentation mediums are added in 500 mL triangular flasks, 8 layers of gauze sealing are placed in high-pressure sterilizing pot, 122 DEG C
Sterilize 15 min, and constant temperature is to 36 DEG C in thermostat water bath, inoculation step（2）Gained seed liquor, inoculum concentration 12%, at 36 DEG C,
Under the conditions of 120 r/min, 72h is cultivated in constant-temperature shaking incubator；
Step（3）The fermentation medium main component and its weight percentage are：Glucose 10.0%, yeast powder 2.2%,
Corn steep liquor 0.2%, sodium glutamate 1.0%, dipotassium hydrogen phosphate 0.2%, calcium chloride 0.2%, calcium carbonate 2.0%, ammonium sulfate 3.0%, sulfuric acid
Magnesium 0.05%；
Step（3）The pH of the fermentation medium is 7.0；
（4）Determination step（3）The yield of guanosine in zymotic fluid：0.1 mL of zymotic fluid is drawn in 10 mL centrifuge tubes, with 4 mol/
L NaOH are settled to 10 mL, are uniformly mixed, and boiling water bath 5 min, 3000 r/min 5 min of centrifugation take centrifuged supernatant 1
ML, dilution are settled to 100 mL, and using high effective liquid chromatography for measuring guanosine yield, testing result is shown in Table 1；
High effective liquid chromatography for measuring condition：Pillar：C18, mobile phase：Acetonitrile：Water=10:90, flow velocity：1 mL/min detects wave
It is long：256 nm, column temperature：25 DEG C, 10 uL of sample size.
Embodiment 8
A kind of preparation process of guanosine, includes the following steps：
（1）By bacillus subtilis（Bacillus subtilis）Bacterial strain activates, and is inoculated on slant medium, is trained in constant temperature
Support 38 DEG C of 24 h of constant temperature stationary culture in case；
Step（1）The slant medium main component and its weight percentage are：Peptone 1.2%, sodium chloride 0.7%,
Beef extract powder 0.2%, agar 1.5%；
（2）55 mL seed culture mediums are added in 500 mL triangular flasks, 6 layers of gauze sealing are placed in high-pressure sterilizing pot, 121 DEG C
Sterilize 15 min, and constant temperature is inoculated with two ring steps to 36 DEG C in thermostat water bath（1）Cultivate strain, at 36 DEG C, 100 r/min
Under the conditions of, 8h is cultivated in constant-temperature shaking incubator；
Step（2）The seed culture medium main component and its weight percentage are：Peptone 1.0%, beef extract 0.3%,
Sodium chloride 0.5%；
（3）27 mL fermentation mediums are added in 500 mL triangular flasks, 8 layers of gauze sealing are placed in high-pressure sterilizing pot, 121 DEG C
Sterilize 15 min, and constant temperature is to 40 DEG C in thermostat water bath, inoculation step（2）Gained seed liquor, inoculum concentration 10%, at 40 DEG C,
Under the conditions of 150 r/min, 48h is cultivated in constant-temperature shaking incubator；
Step（3）The fermentation medium main component and its weight percentage are：Glucose 8%, yeast powder 2.8%,
Corn steep liquor 0.1%, sodium glutamate 1.0%, dipotassium hydrogen phosphate 0.2%, calcium chloride 0.3%, calcium carbonate 1.6%, ammonium sulfate 1.5%,
Magnesium sulfate 0.08%；
Step（3）The pH of the fermentation medium is 7.0；
（4）Determination step（3）The yield of guanosine in zymotic fluid：0.1 mL of zymotic fluid is drawn in 10 mL centrifuge tubes, with 4 mol/
L NaOH are settled to 10 mL, are uniformly mixed, and boiling water bath 5 min, 3000 r/min 5 min of centrifugation take centrifuged supernatant 1
ML, dilution are settled to 100 mL, and using high effective liquid chromatography for measuring guanosine yield, testing result is shown in Table 1；
High effective liquid chromatography for measuring condition：Pillar：C18, mobile phase：Acetonitrile：Water=10:90, flow velocity：1 mL/min detects wave
It is long：256 nm, column temperature：25 DEG C, 10 uL of sample size.
Comparative example 1
The preparation process of the present embodiment guanosine is substantially the same manner as Example 5, and something in common does not repeat, and somewhat different is：Step
（3）The fermentation medium main component and its weight percentage are：Glucose 6%, yeast powder 3.0%, corn steep liquor
0.4%, sodium glutamate 2.0%, dipotassium hydrogen phosphate 0.2%, calcium chloride 0.3%, calcium carbonate 1.6%, ammonium sulfate 0.5%, magnesium sulfate
0.1%。
Comparative example 2
The preparation process of the present embodiment guanosine is substantially the same manner as Example 5, and something in common does not repeat, and somewhat different is：It is described
Fermentation medium pH be 6.6.
Comparative example 3
The preparation process of the present embodiment guanosine is substantially the same manner as Example 5, and something in common does not repeat, and somewhat different is：Step
（3）Fermentation temperature is 30 DEG C.
Comparative example 4
The preparation process of the present embodiment guanosine is substantially the same manner as Example 5, and something in common does not repeat, and somewhat different is：Step
（2）7h is cultivated in constant-temperature shaking incubator.
The yield testing result of 1 1-8 of the embodiment of the present invention of table and comparative example 1-4 guanosines
As shown in Table 1, guanosine preparation process guanosine yield of the present invention may be up to 10.6g/L, and guanosine yield is high, compared with comparative example
1 improves 71%.
Claims (7)
1. a kind of preparation process of guanosine, which is characterized in that include the following steps：
（1）By bacillus subtilis（Bacillus subtilis）Bacterial strain activates, and is inoculated on slant medium, is trained in constant temperature
Support 32-40 DEG C of constant temperature stationary culture 20-30 h in case；
（2）45-55 mL seed culture mediums are added in 500 mL triangular flasks, 6-10 layers of gauze sealing are placed in high-pressure sterilizing pot
In, 120-123 DEG C of sterilizing 10-20min, constant temperature is inoculated with a ring or two ring steps to 32-40 DEG C in thermostat water bath（1）
Culture strain cultivates 8-16h under the conditions of 32-40 DEG C, 100-150 r/min in constant-temperature shaking incubator；
（3）25-30 mL fermentation mediums are added in 500 mL triangular flasks, 6-10 layers of gauze sealing are placed in high-pressure sterilizing pot
In, 120-123 DEG C of sterilizing 10-20min, constant temperature is to 32-40 DEG C in thermostat water bath, inoculation step（2）Gained seed liquor, connects
Kind amount is that 6-14% cultivates 48-78h under the conditions of 32-40 DEG C, 100-150 r/min in constant-temperature shaking incubator.
2. the preparation process of guanosine according to claim 1, which is characterized in that step（1）The slant medium master
The ingredient and its weight percentage is wanted to be：Peptone 0.8-1.2%, sodium chloride 0.3-0.7%, beef extract powder 0.2-0.5%, fine jade
Fat 1.2-1.8%.
3. the preparation process of guanosine according to claim 1, which is characterized in that step（2）The seed culture medium master
The ingredient and its weight percentage is wanted to be：Peptone 0.8-1.2%, beef extract 0.2-0.45%, sodium chloride 0.4-0.7%.
4. the preparation process of guanosine according to claim 1, which is characterized in that step（3）The fermentation medium master
The ingredient and its weight percentage is wanted to be：Glucose 8-15%, yeast powder 1.2-2.8%, corn steep liquor 0.1-0.3%, glutamic acid
Sodium 0.8-1.6%, dipotassium hydrogen phosphate 0.1-0.3%, calcium chloride 0.1-0.3%, calcium carbonate 1.5-2.5%, ammonium sulfate 1.0-
3.0%, magnesium sulfate 0.03-0.08%.
5. the preparation process of guanosine according to claim 1, which is characterized in that step（3）The fermentation medium pH
For 6.8-7.4.
6. the preparation process of guanosine according to claim 1, which is characterized in that step（3）Further include guanosine yield later
Assay method, step is：0.1 mL of zymotic fluid is drawn in 10 mL centrifuge tubes, 10 mL are settled to 4 mol/L NaOH,
It is uniformly mixed, boiling water bath 5 min, 3000 r/min centrifuge 5 min, and 1 mL of centrifuged supernatant, dilution is taken to be settled to 100
ML, using high effective liquid chromatography for measuring guanosine yield.
7. the preparation process of guanosine according to claim 6, which is characterized in that the high effective liquid chromatography for measuring condition
For：Pillar：C18, mobile phase：Acetonitrile：Water=10:90, flow velocity：1 mL/min, Detection wavelength：256 nm, column temperature：25 DEG C, sample introduction
Measure 10 uL.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112592946A (en) * | 2020-12-31 | 2021-04-02 | 河南巨龙生物工程股份有限公司 | Environment-friendly high-yield guanosine production process method |
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