CN107299061A - The culture medium and its cultural method of a kind of itaconic acid producing strain - Google Patents
The culture medium and its cultural method of a kind of itaconic acid producing strain Download PDFInfo
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- CN107299061A CN107299061A CN201710668448.4A CN201710668448A CN107299061A CN 107299061 A CN107299061 A CN 107299061A CN 201710668448 A CN201710668448 A CN 201710668448A CN 107299061 A CN107299061 A CN 107299061A
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- C12P7/44—Polycarboxylic acids
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Abstract
The invention discloses a kind of culture medium of itaconic acid producing strain and its cultural method, the bacterium culture medium is made up of wheat bran, rice husk and malt extract medium, and wherein the mass ratio of wheat bran and rice husk is 1:0.03‑1:0.15, the addition of malt extract medium is 0.02% the 0.1% of dry culture medium (wheat bran and rice husk gross mass), and the moisture of culture medium is 60% 80%.Culture medium sponginess provided by the present invention is good, ponding is few；Simultaneously during mycelial growth, good permeability sheds beneficial to heat in incubation, moisture, promotes the sporiparous ability of wheat bran, improve the spore quality and quantity of wheat bran, it is ensured that the stability of strain.Using the strain of medium culture of the present invention, spore count is up to 1.5 × 1011The dry wheat brans of individual/g, itaconic acid is produced for inoculation fermentation, fermentation culture initial sugar concentration 14.0% 15.0%, and production acid is up to more than 9.76g/100mL, and within fermentation period 48hr, residual sugar is below 0.35%, and conversion rate of products is up to more than 67.30%.
Description
Technical field
The present invention relates to Spawn incubation field, and in particular to a kind of culture medium of itaconic acid producing strain and its culture side
Method.
Background technology
Itaconic acid scientific name methylene-succinic acid, methene butanedioic acid, are a kind of unsaturated binary organic acids.It contains unsaturation
Double bond, with active chemical property, can carry out itself, polymerizeing between other monomers, soluble in water, ethanol etc. is other molten
Agent, can also carry out various additions, esterification, and purposes is quite varied.Itaconic acid is the important source material of chemical synthesis industry, is also
The important source material of Chemical Manufacture；In addition, itaconic acid also has use extensively in the fields such as medicine, cosmetic agent, water treatment agent
On the way.
Itaconic acid international market demand amount is big, and the production method of current itaconic acid has a synthetic method and fermentation method, synthetic method by
It is complicated in cost height, process hazard, it should not industrially use；And fermentation method raw material is easy to get, cost is relatively low, mature technology,
It is the method that industrial production is unanimously used both at home and abroad at present.It is in itaconic acid fermentation production to use aspergillus as fermented bacterium more,
And the preparation of aspergillus wheat bran culture medium typically uses wheat bran, because the feature of wheat bran in itself causes in sterilization process
In, bran mass is easy to stick in bottle wall, easily caking, final influence sterilizing and culture effect, is unfavorable for heat, water during culture
The dredging shed with oxygen divided, influences growth and the wheat bran production spore amount of mycelia, causes the extension of wheat bran incubation time, spore count
Measure Quality Down, wheat bran strain qualification rate low, it is impossible to meet the inoculation demand that large scale fermentation method produces itaconic acid, given birth to realizing
Production serialization brings difficulty, causes the waste of equipment, manpower etc., adds production cost.Therefore, grope a kind of suitable
The strain that culture medium and its cultural method provide abundance for large scale fermentation method production itaconic acid in time is extremely urgent.
The content of the invention
Based on above-mentioned technical problem, the present invention provides a kind of culture medium and its cultural method of itaconic acid producing strain.
The adopted technical solution is that：
A kind of culture medium of itaconic acid producing strain, includes following component：Wheat bran, rice husk and malt extract medium, it is described
The mass ratio of wheat bran and rice husk is 1:0.03-1:0.15, the addition of malt extract medium is wheat bran and rice husk gross mass
0.02%-0.1%.
It is preferred that, one or more of the wheat bran in wheat bran, Fructus Hordei Vulgaris bran, oat bran.
It is preferred that, the moisture of the culture medium is 60%-80%.
A kind of cultural method of itaconic acid producing strain, using above-mentioned culture medium, specifically includes following steps：
Step a, the strain amount needed for, by wheat bran and rice husk according to mass ratio 1:0.03-1:0.15 ratio is entered respectively
Row weighs, is sufficiently mixed dry culture medium is made, standby；
Step b, the addition according to dry culture medium 0.02%-0.1%, weigh malt extract medium, first train brewer's wort
Foster base is dissolved in a certain proportion of water, is subsequently poured into dry culture medium and is stirred, mixed repeatedly, is prepared into water content
60%-80% wheat bran culture medium；
Step c, by the wheat bran culture medium obtained by step b by 45-65g/ bottle dispense into 500mL conical flasks, sealed bundle
Prick, high-temp steam sterilizing, sterilising temp is 115-121 DEG C, and sterilization time is 25-30min, sterilizing is cooled down after terminating；
Step d, by the wheat bran culture medium inoculated slant pore after the cooling that sterilized through step c, breaing up culture medium makes spore point
Dissipate uniform, move into culturing room and cultivated 6-10 days under suitable temperature, humidity, periodically break up culture medium during culture, interrupt bacterium
Silk, makes it be laid in bottom of bottle, is cultivated for being covered with whole culture medium to spore, wheat bran strain is ripe, moves into refrigerating chamber storage
It is standby.
It is preferred that, in step c, medium sterilization will fully be broken up and shake up culture medium, then natural cooling while hot after terminating.
It is preferred that, in step d, cultivation temperature is 35-40 DEG C, and culture humidity is 55-75%；Culture medium is broken up during culture
Time respectively be inoculation after 10-14hr, 28-32hr.
The method have the benefit that：
(1) the loose good permeability of culture medium of the present invention, nutrient water are suitable, and heat, moisture can dissipate in time in incubation
Go out, good culture environment is provided for strain, spawn activity is significantly improved, production spore amount is greatly increased, and spore count is reachable
1.5×1011The dry wheat brans of individual/g, greatly meet the inoculation demand that fermentation method mass produces itaconic acid, to realize continuous metaplasia
Production provides reliable guarantee.
(2) the production strain prepared using the present invention, steady quality, fermenting property are good, inoculation fermentation production itaconic acid, production
Acid is up to more than 9.76g/100mL, within fermentation period 48hr, and residual sugar is below 0.35%, conversion rate of products up to 67.30% with
On.Itaconic acid yield is improved, fermentation period is shortened, reduces fermentation residual sugar, is that subsequent extracted process reduces work by force
Degree, has saved production cost, has added economic benefit.
(3) inventive formulation culture medium is cheap is easy to get, without special Spawn incubation equipment, reduces cost throwing
Enter.
(4) operation is simple for cultural method of the present invention, and incubation time is short, and condition of culture is beneficial to control, reduces manpower
The waste of material resources.
Embodiment
The invention provides a kind of culture medium of itaconic acid producing strain and its cultural method, the culture medium is by wheat bran, rice
Shell, malt extract medium composition, the wherein mass ratio of wheat bran and rice husk are 1:0.03-1:0.15, the addition of malt extract medium
Amount is the 0.02%-0.1% of dry culture medium quality, and the moisture of culture medium is 60%-80%.Culture medium of the present invention is dredged
Pine property is good, ponding is few；During mycelial growth, good permeability sheds beneficial to heat in incubation, moisture, promotes bran
Bent sporiparous ability, improves the spore quality and quantity of wheat bran；Cultural method efficiently solves itaconic acid producing strain simultaneously
Wheat bran spore quantity is low, spawn activity is poor, the problem of supply not in time, simple and easy to apply, greatly meets the life of large scale fermentation method
The inoculation demand of itaconic acid is produced, to realize that production serialization provides guarantee, the utilization rate of equipment, manpower etc. is improved, saves
Production cost.
The technical characteristics of the present invention are exactly to have found a kind of compound wheat bran culture medium to instead of the single wheat of tradition
Bran mass, and found out the appropriate proportioning of each culture medium.Main medium wheat bran therein can be wheat, big
One or more in wheat, oat bran, can be mixed with arbitrary proportion；Rice husk is in addition to providing a small amount of nutrition primarily as training
The raising agent for supporting base is mutually supported with wheat bran, is prevented culture medium from sticking bottle caking after sterilization, is conducive to the heat in incubation
The evaporation of moisture, the circulation of air, and avoid producing excessive ponding, promote the uniform fast-growth of mycelia, improve spore output,
Shorten incubation time；Malt extract medium is in addition to it can provide carbon source nutrition for strain, its abundant trace element can promote bacterium
Sporiparous ability is planted, the vigor performance of spore is improved, so as to effectively increase strain quality.
The mass ratio of wheat bran and rice husk is 1 described in culture medium of the present invention:0.03-1:0.15, rice husk addition it is very few not
Beneficial to the sponginess of whole culture medium, the water imbibition of culture medium can at most be reduced by crossing, and cause to produce excessive ponding, be unfavorable for culture
The supply of base oxygen, influences the normal growth of mycelia.Described malt extract medium, its addition is dry culture medium
0.02%-0.1%, malt extract medium for strain except that can provide part carbon source nutrition, and its abundant trace element can promote
The sporiparous ability of strain, improves the vigor performance of spore.Its addition is crossed does not have the sporogenic effect of promotion, addition at least
Measuring at most can cause culture medium overnutrition, mycelial growth vigorous, and production spore amount declines.The moisture of the culture medium is
60%-80%, water content is very few to influence the normal growth of strain, and the gas permeability of culture medium can at most be influenceed by crossing, therefore, culture
The moisture general control of base is within the above range.
In order to be better understood from the present invention, it is described further with reference to specific embodiment.
Wheat bran of the present invention, rice husk, malt extract medium, slant pore can be obtained by commercial channel purchase
.
Embodiment 1
Step 1, by wheat bran and rice husk according to 1:0.05 mass ratio according to needed for strain amount weighed respectively,
It is sufficiently mixed and dry culture medium is made, it is standby.
Step 2,0.03% according to dry culture medium quality, weigh malt extract medium, are dissolved in a certain proportion of water,
Pour into and stirred, mixed repeatedly in dry culture medium, the wheat bran culture medium of water content 65% is made.
Step 3, by the culture medium obtained by step 2 by 50g/ bottle packing into 500mL conical flasks, sealing wrap up, high temperature
Steam sterilizing, sterilising temp is 121 DEG C, and sterilization time is 25min, and sterilizing is fully broken up and shakes up culture medium while hot after terminating, then
Natural cooling.
Step 4, the culture medium inoculated slant pore by step 3 gained, breaing up culture medium makes spore be uniformly dispersed, and moves into training
Room is supported to cultivate under 37-40 DEG C, 60-75% damp condition；And culture is broken up respectively in 12hr, 28hr respectively after inoculation
Base, interrupts mycelia, fully shakes up culture medium, it is laid in bottom of bottle and continues to cultivate 7 days；It is covered with spore inside and outside whole culture medium,
Wheat bran strain is ripe, moves into 12-15 DEG C of refrigerating chamber and stores for future use.
With the production strain of the present embodiment culture, it is 1.52 × 10 to carry out spore count11The dry wheat brans of individual/g；For being inoculated with clothing
Health acid fermentation is produced, in fermentation medium initial sugar concentration 14.3%, 40 DEG C of fermentation temperature, dissolved oxygen 75%, tank pressure 0.12MPa, hair
Fermented under the conditions of ferment pH4.8, fermentation period 46hr produces acid for 9.78g/100mL, residual sugar 0.29%, conversion rate of products is
67.89%.
Embodiment 2
Step 1, by wheat, oat bran and rice husk according to mass ratio 1:0.07 ratio strain amount needed for is entered respectively
Row weighs, is sufficiently mixed dry culture medium is made, standby；The mass ratio of wheat bran and oat bran is 0.5: 0.5.
Step 2,0.02% according to dry culture medium quality, weigh malt extract medium, are dissolved in a certain proportion of water,
Pour into and stirred, mixed repeatedly in dry culture medium, the wheat bran culture medium of water content 75% is made.
Step 3, by the culture medium obtained by step 2 by 55g/ bottle packing into 500mL conical flasks, sealing wrap up, high temperature
Steam sterilizing, sterilising temp is 120 DEG C, and sterilization time is 30min, and sterilizing is fully broken up and shakes up culture medium while hot after terminating, then
Natural cooling.
Step 4, the culture medium inoculated slant pore by step 3 gained, breaing up culture medium makes spore be uniformly dispersed, and moves into training
Room is supported to cultivate under 38-40 DEG C, 65-75% damp condition；And culture is broken up respectively in 14hr, 30hr respectively after inoculation
Base, interrupts mycelia, fully shakes up culture medium, it is laid in bottom of bottle and continues to cultivate 6 days；It is covered with spore inside and outside whole culture medium,
Wheat bran strain is ripe, moves into 12-15 DEG C of refrigerating chamber and stores for future use.
With the production strain of the present embodiment culture, it is 1.55 × 10 to carry out spore count11The dry wheat brans of individual/g；For being inoculated with clothing
Health acid fermentation is produced, in fermentation culture initial sugar concentration 14.5%, 39 DEG C of fermentation temperature, dissolved oxygen 75%, tank pressure 0.12MPa, hair
Fermented under the conditions of ferment pH4.8, fermentation period 47.5hr, production acid is 9.86g/100mL, residual sugar 0.31%, saccharic acid conversion ratio
For 67.66%.
Embodiment 3
Step 1, by wheat, Fructus Hordei Vulgaris bran and rice husk according to 1:0.09 mass ratio strain amount needed for is carried out respectively
Weigh, be sufficiently mixed dry culture medium is made, it is standby；The mass ratio of wheat bran and Fructus Hordei Vulgaris bran is 0.2: 0.8.
Step 2,0.05% according to dry culture medium quality, weigh malt extract medium, are dissolved in a certain proportion of water,
Pour into and stirred, mixed repeatedly in dry culture medium, the wheat bran culture medium of water content 80% is made.
Step 3, by the culture medium obtained by step 2 by 60g/ bottle packing into 500mL conical flasks, sealing wrap up, high temperature
Steam sterilizing, sterilising temp is 121 DEG C, and sterilization time is 30min, and sterilizing is fully broken up and shakes up culture medium while hot after terminating, then
Natural cooling.
Step 4, the culture medium inoculated slant pore by step 3 gained, breaing up culture medium makes spore be uniformly dispersed, and moves into training
Room is supported to cultivate under 37-40 DEG C, 65-70% damp condition；And culture is broken up respectively in 13hr, 32hr respectively after inoculation
Base, interrupts mycelia, fully shakes up culture medium, it is laid in bottom of bottle and continues to cultivate 8 days；It is covered with spore inside and outside whole culture medium,
Wheat bran strain is ripe, moves into 12-15 DEG C of refrigerating chamber and stores for future use.
With the production strain of the present embodiment culture, it is 1.56 × 10 to carry out spore count11The dry wheat brans of individual/g；For being inoculated with clothing
Health acid fermentation is produced, in fermentation culture initial sugar concentration 14.7%, 39 DEG C of fermentation temperature, dissolved oxygen 80%, tank pressure 0.12MPa, hair
Fermented under the conditions of ferment pH4.82, fermentation period 48hr produces acid for 9.93g/100mL, residual sugar 0.30%, saccharic acid conversion ratio is
67.40%.
Above example is only the embodiment of the present invention, but protection scope of the present invention is not limited thereto,
Any equivalent way or obvious variant that those skilled in the art are made under the guidance of this specification all should be in the present invention
Protection domain in.
Claims (6)
1. a kind of culture medium of itaconic acid producing strain, it is characterised in that include following component：Wheat bran, rice husk and brewer's wort culture
Base, the mass ratio of the wheat bran and rice husk is 1:0.03-1:0.15, the addition of malt extract medium is wheat bran and the total matter of rice husk
The 0.02%-0.1% of amount.
2. a kind of culture medium of itaconic acid producing strain according to claim 1, it is characterised in that：The wheat bran is selected from small
One or more in wheat bran skin, Fructus Hordei Vulgaris bran, oat bran.
3. a kind of culture medium of itaconic acid producing strain according to claim 1, it is characterised in that：The water of the culture medium
It is 60%-80% to divide content.
4. a kind of cultural method of itaconic acid producing strain, using the culture as described in any claim in claim 1-3
Base, it is characterised in that comprise the following steps：
Step a, the strain amount needed for, by wheat bran and rice husk according to mass ratio 1:0.03-1:0.15 ratio is claimed respectively
Measure, be sufficiently mixed dry culture medium is made, it is standby；
Step b, the addition according to dry culture medium 0.02%-0.1%, weigh malt extract medium, first by malt extract medium
It is dissolved in a certain proportion of water, is subsequently poured into dry culture medium and is stirred, mixed repeatedly, is prepared into water content 60%-
80% wheat bran culture medium；
Step c, by the wheat bran culture medium obtained by step b by 45-65g/ bottle packing into 500mL conical flasks, sealing wrapping, height
Warm steam sterilizing, sterilising temp is 115-121 DEG C, and sterilization time is 25-30min, and sterilizing is cooled down after terminating；
Step d, by the wheat bran culture medium inoculated slant pore after the cooling that sterilized through step c, breaing up culture medium makes spore scattered equal
It is even, move into culturing room and cultivated 6-10 days under suitable temperature, humidity, periodically break up culture medium during culture, interrupt mycelia, make
It is laid in bottom of bottle, is cultivated for being covered with whole culture medium to spore, wheat bran strain is ripe, moves into refrigerating chamber and stores for future use.
5. a kind of cultural method of itaconic acid producing strain according to claim 4, it is characterised in that：In step c, culture
Base sterilizing will fully break up and shake up culture medium, then natural cooling while hot after terminating.
6. a kind of cultural method of itaconic acid producing strain according to claim 4, it is characterised in that：In step d, culture
Temperature is 35-40 DEG C, and culture humidity is 55-75%；Broken up during culture time of culture medium be respectively 10-14hr after inoculation,
28-32hr。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110938662A (en) * | 2019-12-12 | 2020-03-31 | 青岛科海生物有限公司 | Production method for improving itaconic acid yield by using bran |
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|---|---|---|---|---|
| CN101717309A (en) * | 2009-11-26 | 2010-06-02 | 武汉生物工程学院 | Culture medium for straw rotting edible fungi solid strain and method for preparing solid strain |
| CN101914450A (en) * | 2010-05-19 | 2010-12-15 | 浙江省农业科学院 | Fungus agent for biologically dewatering compost and preparation method thereof |
| CN102839132A (en) * | 2012-09-21 | 2012-12-26 | 中粮生物化学（安徽）股份有限公司 | Aspergillus niger moldy bran culture medium and preparation method thereof, and Aspergillus niger moldy bran culture method |
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2017
- 2017-08-08 CN CN201710668448.4A patent/CN107299061A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101717309A (en) * | 2009-11-26 | 2010-06-02 | 武汉生物工程学院 | Culture medium for straw rotting edible fungi solid strain and method for preparing solid strain |
| CN101914450A (en) * | 2010-05-19 | 2010-12-15 | 浙江省农业科学院 | Fungus agent for biologically dewatering compost and preparation method thereof |
| CN102839132A (en) * | 2012-09-21 | 2012-12-26 | 中粮生物化学（安徽）股份有限公司 | Aspergillus niger moldy bran culture medium and preparation method thereof, and Aspergillus niger moldy bran culture method |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110938662A (en) * | 2019-12-12 | 2020-03-31 | 青岛科海生物有限公司 | Production method for improving itaconic acid yield by using bran |
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