CN106399429A - Method for improving yield of adenosine through feeding and mixing nitrogen source - Google Patents
Method for improving yield of adenosine through feeding and mixing nitrogen source Download PDFInfo
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- CN106399429A CN106399429A CN201611024129.1A CN201611024129A CN106399429A CN 106399429 A CN106399429 A CN 106399429A CN 201611024129 A CN201611024129 A CN 201611024129A CN 106399429 A CN106399429 A CN 106399429A
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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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/38—Nucleosides
- C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
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Abstract
The invention belongs to the technical field of microorganism fermentation and in particular relates to a method for improving the yield of adenosine through feeding and mixing a nitrogen source. The method comprises a process including strain slant culture, seed culture and fermentation culture, wherein the fermentation culture comprises a process of starting to feed yeast extract and corn steep liquor powder mixed nitrogen sources at a constant speed when the absorbance OD (optical density) value of thalli reaches to 0.5 to 0.6; the two nitrogen sources are mixed at the weight ratio of 1 to (2+/-0.5). According to the method provided by the invention, the nitrogen sources are supplemented at a constant speed in the middle-late period of the fermentation, so that nutrient substances are provided for growth of the thalli and production of the adenosine through the fermentation, and the activity and stability in the middle-late period of the fermentation are kept; the yield of the fermentation is increased and the conversion rate of glucoside is finally improved.
Description
Technical field
The invention belongs to technical field of microbial fermentation is and in particular to a kind of add the side that mixed nitrogen improves adenosine yield by stream
Method.
Background method
Ribosidoadenine (Adenosine), also referred to as adenosine, trade name AR, chemical name is 9- β-D-RIBOSE base gland
Purine (9- β-D-ribofuranosyl adenine), molecular formula is C10H13N5O4, molecular weight be 267.24.Adenosine is a kind of
Spread all over the endogenous nucleoside of human body cell, myocardium phosphorylated generation adenylic acid can be directly entered, participate in energy metabolism of myocardial, with
When also participate in dilating coronary blood vessel, increase blood flow.Many other systems also to cardiovascular system and human body for the adenosine and tissue
All there is physiological action, be the important intermediate for synthesizing adenosine triphosphate (ATP), adenine, adenylic acid, vidarabine,
Wide DEVELOPMENT PROSPECT is had on medical market.The main production process of adenosine has chemical synthesiss, enzyme process and fermentation method etc..Before
, due to high cost, pollution is big, and large-scale application is somewhat limited for two methods.And fermentation method is from nineteen sixty-eight, Japan
Little western true eight et al. beginnings, experience the effort of a lot of researchers, nowadays have been enter into the large-scale industrial production stage.State's inner gland
Glycosides fermentation starts to walk a little later, compared with abroad, however it remains the problems such as yield is relatively low.With the carrying out of sweat, fermentation culture
In base, nitrogen concentration reduces, especially ucleosides nitrogen source and amino acidses nitrogen source, and strain lacks the necessary material of synthetic product, generation
Thank to stream to change.
Content of the invention
Present invention aim at solving the problems, such as that producing adenosine through zymotechnics conversion ratio is relatively low, a kind of stream that passes through is provided to add mixed nitrogen
Improve adenosine yield and the method for conversion ratio.
For solving above-mentioned technical problem, the technical solution adopted in the present invention is：A kind of stream that passes through adds mixed nitrogen raising adenosine
The method of yield, including the process of strain inclined plane culture, seed culture and fermentation culture, described fermentation culture includes thalline and inhales
When luminosity OD value reaches 0.5～0.6 (650nm dilutes 50 times), start at the uniform velocity stream plus yeast extract and Semen Maydiss starch mixed nitrogen
Process, two kinds of nitrogen sources are with 1:The part by weight mixing of (2 ± 0.5).The method is passed through at the uniform velocity to fill into nitrogen source in fermentation middle and late stage, is
Thalli growth and fermentation are produced glycosides and are provided nutrient substance, maintain vigor and the stability of fermentation middle and late stage, increased fermentation and produce
Amount, finally improves glucosides conversion ratio.
Further:Improving in the method for adenosine yield above by stream plus mixed nitrogen, described strain inclined plane culture refers to
Bacillus subtillis (Bacillus subtilis) SL03 is seeded to activated inclined plane, 28～32 DEG C, cultivates 20～28h.
Described seed culture includes first order seed culture and secondary seed culture；Described first order seed culture refers to inoculation
Ring takes ring slant strains, is seeded to seed culture medium, 28～32 DEG C, cultivates 8～12h, as primary seed solution.
Described secondary seed culture refers to for primary seed solution to access two grades of kinds by percent by volume with 0.05% inoculum concentration
Sub- culture medium, cultivates 6～7h, obtains secondary seed solution.
Described fermentation culture refers to send out secondary seed solution to equipped with sterilizing by the subcultivation amount subcultivation of percent by volume 10～15%
In the fermentation tank of ferment culture medium, fermentation temperature is 36～37 DEG C, and sweat maintains pH6.6～6.8, dissolved oxygen by Feeding ammonia water
Maintain more than 30%, incubation time 36～42h.
Further：Improving in the method for adenosine yield above by stream plus mixed nitrogen, the inclined-plane of described seed culture
Medium component is in terms of g/L：Glucose 2～3, peptone 8～10, Carnis Bovis seu Bubali cream 8～10, yeast powder 3～5, NaCl 1.5～
2.5, agar 20～30, pH 7.0～7.2；
Described primary-seed medium composition is in terms of g/L：Glucose 20～30, yeast powder 5～8, KH2PO40.5～1.0,
MgSO4·7H2O 0.5～0.8, guanine 0.01～0.015, monosodium glutamate 3～8, carbamide 1～2, Semen Maydis pulp 10～20mL/L, pH
7.0～7.2；
Described secondary seed medium composition is in terms of g/L：Glucose 20～30, KH2PO40.5～1.0, MgSO4·7H2O 0.5
～0.8, guanine 0.002～0.005, Semen Maydis pulp 10～20mL/L, pH 6.6～6.8；
Described fermentation medium components are in terms of g/L：Glucose 130～150, yeast powder 20～30, K2HPO42～3, (NH4)2SO410～15, MgSO4·7H2O 1～2,0.01～0.015, MnSO40.003～0.006, Dried Corn Steep Liquor Powder 10～15.
Compared with prior art, the present invention, with Bacillus subtillis as fermentation strain, enters one to raw material ratio and process feed supplement
Step optimize, on 50L fermentation tank in batches stream plus mixed nitrogen, there is provided the nitrogen source required for bacterial metabolism, simultaneously so that
Middle and late stage spawn activity improves.Advantages of the present invention and having the beneficial effects that：By adding yeast extract, Semen Maydiss in fermentation middle and late stage stream
Slurry mixed nitrogen, there is provided the nitrogen source required for thalline, improves the strong of middle and late stage spawn activity and thalline cometabolism
So that adenosine output increased is to 37g/L, conversion ratio improves to 24% degree.Fermentation period foreshortens to 38h.
Specific embodiment
The analysis method that in following embodiment, tunning adenosine content is adopted is as follows：In fermentation liquid, adenosine content adopts paper
Chromatography separation determination.Fermentation liquid through centrifuge 4000rpm, 15min, gained supernatant separated with paper chromatography after
Ultraviolet absorption value (A is surveyed at 260nm260).The chromatographic solution proportioning of described paper chromatography is, ammonium sulfate：Ammonia：Water is equal to (1～1.5):
2～(3:7～8).Described ammonium sulfate concentrations >=99%, the concentration of ammonia is 28%～29%.Accurately weigh the standard substance of adenosine,
Deionized water is configured to the standard solution that mass concentration is 1g/L；Fermented supernatant fluid sample deionized water to be measured is diluted
To suitable concentration, carry out paper chromatography detection together with standard substance.
Embodiment 1
Slant culture based component includes (in terms of g/L)：Glucose 2.5, peptone 8, Carnis Bovis seu Bubali cream 8, yeast powder 4, NaCl 2, agar
25, pH 7.0～7.2；.
Primary-seed medium composition includes (in terms of g/L)：Glucose 2.5, yeast powder 5, KH2PO40.5, MgSO4·
7H2O 0.8, guanine 0.03, monosodium glutamate 3, carbamide 1, pH 7.0～7.2；
Secondary seed medium composition includes (in terms of g/L)：Glucose 30, KH2PO40.7, MgSO4·7H2O 0.8, guanine
0.003, Semen Maydis pulp 20mL/L, pH 6.6～6.8；
Fermentation medium components include (in terms of g/L)：Glucose 145, yeast powder 20, K2HPO42, MgSO4·7H2O 2,
Guanine 0.015, MnSO40.005, Dried Corn Steep Liquor Powder 12.
With Bacillus subtillis as starting strain, it is seeded to inclined-plane and is activated, 32 DEG C of culture 24h, scrape inclined-plane with inoculating loop
Strain is seeded to primary-seed medium, 31.5 DEG C, and 200rpm cultivates 11h；It is connected to two grades of kinds of 30L tank by 0.05% inoculum concentration
31.5 DEG C of sub- culture medium, PH 6.6～6.7, cultivates 7.5h, moves in 50L fermentation tank with 10% inoculum concentration, 36 DEG C, dissolved oxygen is tieed up
Hold more than 30%, sweat maintains pH6.6 by Feeding ammonia water, when fermenting to OD 0.6, start at the uniform velocity stream plus 15g/L and mix
Close nitrogen source solution, wherein yeast extract 5g/L, Dried Corn Steep Liquor Powder 10g/L, maintain fermentation ends, ferment 40h, adenosine yield reaches
37g/L, conversion ratio reaches 24%.
Embodiment 2
Slant culture based component includes (in terms of g/L)：Glucose 2.5, peptone 8, Carnis Bovis seu Bubali cream 8, yeast powder 4, NaCl 2, agar
25, pH 7.0～7.2；.
Primary-seed medium composition includes (in terms of g/L)：Glucose 2.5, yeast powder 5, KH2PO40.5, MgSO4·7H2O
0.8, guanine 0.03, monosodium glutamate 3, carbamide 1, pH 7.0～7.2；
Secondary seed medium composition includes (in terms of g/L)：Glucose 30, KH2PO40.7, MgSO4·7H2O 0.8, guanine
0.003, Semen Maydis pulp 20mL/L, pH 6.6～6.8；
Fermentation medium components include (in terms of g/L)：Glucose 145, yeast powder 20, K2HPO42, MgSO4·7H2O 2,
Guanine 0.015, MnSO40.005, Dried Corn Steep Liquor Powder 12.
With Bacillus subtillis as starting strain, it is seeded to inclined-plane and is activated, 32 DEG C of culture 24h, scrape inclined-plane with inoculating loop
Strain is seeded to primary-seed medium, 31.5 DEG C, and 200rpm cultivates 11h；It is connected to two grades of kinds of 30L tank by 0.05% inoculum concentration
31.5 DEG C of sub- culture medium, PH 6.6～6.7, cultivates 7.5h, moves in 50L fermentation tank with 10% inoculum concentration, 36 DEG C, dissolved oxygen is tieed up
Hold more than 30%, sweat maintains pH6.6 by Feeding ammonia water, when fermenting to OD 0.6, start at the uniform velocity stream plus 15g/L and mix
Close nitrogen source solution, wherein yeast extract 10g/L, Dried Corn Steep Liquor Powder 5g/L, maintain fermentation ends, ferment 40h, produces glycosides 34.1g/L,
Saccharic acid conversion ratio 24%.
Be can be seen that by embodiment 1 and embodiment 2：Stream plus the difference of nitrogen source ratio, impact strain generates the energy of secondary metabolite
Power, impact yield and conversion ratio.Yeast class material is mainly quick-acting nitrogen sources, provides a large amount of nucleosides materials for adenosine strain, beautiful
Rice ＆ peanut milk powder is slow effect nitrogen source, provides a large amount of amino acidses materials and trace element for strain.Fermentation middle and late stage is mainly strain life
Become the period of secondary metabolite, this period Semen Maydis pulp powder etc. imitates the effect maximum that nitrogen source plays late, can advantageously promote strain
Produce adenosine, when the nitrogen source Semen Maydiss starch ratio that therefore in the middle of embodiment, stream adds is higher, the vigor of its strain is stronger, and yield is relatively
Greatly, conversion ratio is higher.
Claims (6)
1. a kind of method improving adenosine yield by stream plus mixed nitrogen, including strain inclined plane culture, seed culture and fermentation
Culture process it is characterised in that：When described fermentation culture includes thalline absorbance OD value and reaches 0.5～0.6, start at the uniform velocity
Stream plus the process of yeast extract and Semen Maydiss starch mixed nitrogen, two kinds of nitrogen sources are with 1:The part by weight mixing of (2 ± 0.5).
2. according to claim 1 by stream plus mixed nitrogen improve adenosine yield method it is characterised in that：Described
Strain inclined plane culture refers to for Bacillus subtillis (Bacillus subtilis) SL03 to be seeded to activated inclined plane, 28～32 DEG C,
Culture 20～28h.
3. according to claim 2 by stream plus mixed nitrogen improve adenosine yield method it is characterised in that：Described
Seed culture includes first order seed culture and secondary seed culture；Described first order seed culture refers to take a ring oblique with inoculating loop
Face strain, is seeded to seed culture medium, 28～32 DEG C, cultivates 8～12h, as primary seed solution.
4. according to claim 3 by stream plus mixed nitrogen improve adenosine yield method it is characterised in that：Described
Secondary seed culture refers to for primary seed solution to access secondary seed medium, training by percent by volume with 0.05% inoculum concentration
Support 6～7h, obtain secondary seed solution.
5. according to claim 4 by stream plus mixed nitrogen improve adenosine yield method it is characterised in that：Described
Ferment culture refer to by secondary seed solution by percent by volume 10～15% subcultivation amount subcultivation to equipped with sterilization fermentation culture medium
In fermentation tank, fermentation temperature is 36～37 DEG C, and sweat maintains pH6.6～6.8 by Feeding ammonia water, and dissolved oxygen maintains 30%
More than, incubation time 36～42h.
6. according to claim 5 by stream plus mixed nitrogen improve adenosine yield method it is characterised in that：Described kind
The slant culture based component of son culture is in terms of g/L：Glucose 2～3, peptone 8～10, Carnis Bovis seu Bubali cream 8～10, yeast powder 3～
5, NaCl 1.5～2.5, agar 20～30, pH 7.0～7.2；
Described primary-seed medium composition is in terms of g/L：Glucose 20～30, yeast powder 5～8, KH2PO40.5～1.0,
MgSO4·7H2O 0.5～0.8, guanine 0.01～0.015, monosodium glutamate 3～8, carbamide 1～2, Semen Maydis pulp 10～20mL/L, pH
7.0～7.2；
Described secondary seed medium composition is in terms of g/L：Glucose 20～30, KH2PO40.5～1.0, MgSO4·7H2O 0.5
～0.8, guanine 0.002～0.005, Semen Maydis pulp 10～20mL/L, pH 6.6～6.8；
Described fermentation medium components are in terms of g/L：Glucose 130～150, yeast powder 20～30, K2HPO42～3, (NH4)2SO4
10～15, MgSO4·7H2O 1～2,0.01～0.015, MnSO40.003～0.006, Dried Corn Steep Liquor Powder 10～15.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111254172A (en) * | 2019-12-10 | 2020-06-09 | 新疆阜丰生物科技有限公司 | Method for producing adenosine by fermentation |
| CN111876462A (en) * | 2020-07-21 | 2020-11-03 | 伊犁川宁生物技术有限公司 | Fermentation method of cephalosporin C |
| CN112301071A (en) * | 2020-11-04 | 2021-02-02 | 赤峰蒙广生物科技有限公司 | Method for producing adenine by fermentation method |
| CN112322508A (en) * | 2020-12-29 | 2021-02-05 | 青岛润达生物科技有限公司 | Ganoderma lucidum mycelium culture method for improving content of ganoderma lucidum polysaccharide |
| CN114426996A (en) * | 2021-11-22 | 2022-05-03 | 南京师范大学 | Method for producing adenosine by fermenting cordyceps sinensis |
| CN114891656A (en) * | 2021-12-31 | 2022-08-12 | 杭州中美华东制药有限公司 | Bacillus subtilis and application thereof in adenosine production by fermentation |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111254172A (en) * | 2019-12-10 | 2020-06-09 | 新疆阜丰生物科技有限公司 | Method for producing adenosine by fermentation |
| CN111876462A (en) * | 2020-07-21 | 2020-11-03 | 伊犁川宁生物技术有限公司 | Fermentation method of cephalosporin C |
| CN112301071A (en) * | 2020-11-04 | 2021-02-02 | 赤峰蒙广生物科技有限公司 | Method for producing adenine by fermentation method |
| CN112322508A (en) * | 2020-12-29 | 2021-02-05 | 青岛润达生物科技有限公司 | Ganoderma lucidum mycelium culture method for improving content of ganoderma lucidum polysaccharide |
| CN114426996A (en) * | 2021-11-22 | 2022-05-03 | 南京师范大学 | Method for producing adenosine by fermenting cordyceps sinensis |
| CN114426996B (en) * | 2021-11-22 | 2024-04-12 | 南京师范大学 | Method for producing adenosine by cordyceps fermentation |
| CN114891656A (en) * | 2021-12-31 | 2022-08-12 | 杭州中美华东制药有限公司 | Bacillus subtilis and application thereof in adenosine production by fermentation |
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Application publication date: 20170215 |