CN105586366A - Method for improving fermentation performance of citric acid on basis of mycelium structure control - Google Patents
Method for improving fermentation performance of citric acid on basis of mycelium structure control Download PDFInfo
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- CN105586366A CN105586366A CN201610135591.2A CN201610135591A CN105586366A CN 105586366 A CN105586366 A CN 105586366A CN 201610135591 A CN201610135591 A CN 201610135591A CN 105586366 A CN105586366 A CN 105586366A
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- C12P7/00—Preparation of oxygen-containing organic compounds
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Abstract
The invention discloses a method for improving the fermentation performance of citric acid on the basis of mycelium structure control. The method includes the steps that mature aspergillus niger spores are prepared into a spore suspension, and the spore suspension is inoculated into a seed culture medium for seed culture; during seed culture, stirring speed is increased, the processes of spore germination and hypha winding are disturbed, and a mature seed solution is obtained; the mature seed solution is inoculated to a fermentation culture medium for fermentation culture, and fermentation ends; the technology of increasing stirring speed for disturbance is started when seed culture is conducted for 6-20 hours. By means of the method, the hypha shape, of a special structure, facilitating citric acid synthesis can be obtained, the fermentation performance is improved, the yield of citric acid is increased, the use quantity of spores can be reduced, production cost is reduced, and the method is simple and easy to implement and has important economic benefits and wide application prospects.
Description
Technical field
The present invention relates to fermentation engineering field, relate in particular to one and send out based on controlling mycelium Structure Improvement citric acidThe method of ferment performance.
Background technology
Citric acid (citricacid), has another name called the acid of Chinese holly edge, is a kind of very important work of utilizing now microorganism fermenting and producingIndustry organic acid, is widely used in the key areas such as food, medicine, chemical industry. The main flow mode of production of citric acid is deep liquidFermentation, bacterial classification used is aspergillus niger (Aspergillusniger); In commercial process, generally adopt second order fermentation sideFirst formula, adopt ripe aspergillus niger spore suspension inoculation seed culture medium to cultivate, and spore, through imbibition, is cultured to 8H starts to sprout, and grows sprout, cultivates 24 ~ 32h, prepares ripe seed, then by ripe seed liquor switching fermented and cultured.Ripe aspergillus niger spore preparation process complexity, generally passes through plate screening, and inclined-plane-eggplant bottle-wheat bran bucket etc. expand step by stepIncubation, preparation process is loaded down with trivial details, more than manufacturing cycle at least needs 30d.
Bulb forms in Citric Acid Fermentation are to be formed by one or several spore physical action in growth course, mycelial big or small quantitative relation is to the success or failure of fermentation. General bulb forms are less, the more many citric acids that are more conducive to of quantityAccumulation. Spore inoculating quantity can significantly affect mycelium size and number, and inoculating spores number is directly proportional to bacterium spheroid quantity, inoculationSpore count deficiency can have a strong impact on ferment effect. Citric acid fermentation is the process of a height oxygen consumption, and oxygen supply is to affect lemonThe conditioning step that lemon acid is synthetic, and the transmission of oxygen is that dissolved oxygen is passed in hyphal cell from solution; Mycelia is wound around fine and closeMycelium pellet, the transmission of oxygen only occurs over just mycelium pellet surface, and the oxygen supply of the loose mycelium pellet of mycelia is abundant, citric acidSynthesis rate is very fast. In addition, the mycelia internal structure of bulb forms also can produce material impact to citric acid fermentation.
But, there is not so far a kind of simple effective method of controlling mycelium pellet mycelial structure, and improve Fermented with thisCan, improve citric acid output.
Summary of the invention
The above-mentioned blank existing for prior art, the object of the present invention is to provide a kind of based on controlling mycelium Structure ImprovementThe method of citric acid fermentation performance, the method can obtain and be conducive to the synthetic ad hoc structure mycelium morphology of citric acid, improves and sends outFerment performance, improves citric acid output.
In order to achieve the above object, the present invention by the following technical solutions: a kind of based on controlling mycelium Structure Improvement lemonThe method of lemon acid fermentation performance, comprises the following steps: be mixed with spore suspension by cultivating ripe aspergillus niger spore, be seeded to kindSub-culture medium carries out seed culture; In seed culture process, improve speed of agitator disturbance spore germination and mycelia winding process,Obtain mature seed liquid; Above-mentioned mature seed liquid is seeded to fermentation medium and carries out fermented and cultured, finish fermentation.
Particularly, the postvaccinal final concentration of described aspergillus niger spore is 20 ~ 800,000/mL.
Particularly, the technique of described raising mixing speed disturbance is carried out 6 ~ 20h in described seed culture and is started, willSpeed of agitator is increased to 800 ~ 1200rpm.
Particularly, the described seed culture time is 24 ~ 32h, obtains mature seed liquid.
Preferably, when described fermentation medium concentration of reduced sugar finishes fermentation lower than 0.5% time.
Particularly, described seed culture medium, fermentation medium are prepared by starchy material, and described starchy material comprises jadeAt least one in ground rice, wheat flour, tapioca starch, sweet potato powder, starch, molasses, wheat.
Compared with prior art, the present invention has following useful technique effect:
The present invention is based on germination process spore assemble different to the sensitiveness of applying shearing force with mycelium pellet winding, by seedBe cultured to the mode that moment (6 ~ 20h) starts mechanical disturbance, disturbance spore germination and mycelia winding process, increase and sproutCollision probability between mycelia, forms the bulb forms that diameter is less, internal structure is loosened more, simultaneously the mycelium meeting of fractureAgain be wound new bacterium spheroid, be conducive to dissolved oxygen and mass transfer, fermenting property is able to remarkable lifting, and (fermentation period shortens, and sends outFerment produces sour amount, fermentation conversion rate and fermentation index and improves); In addition, perturbation action has increased mycelium pellet quantity, can under the same termsReduce spore use amount, reduced production cost, there is important economic benefit. The present invention is simple to operate, has wide answeringUse prospect.
Brief description of the drawings
The morphological feature figure that Fig. 1 is seed liquor mycelium pellet under 50 times of light microscopes, wherein, a: comparative example, b: the present invention is realExecute example 1.
Detailed description of the invention
Below in conjunction with specific embodiment, the invention will be further described.
In the following embodiments, the assay method of total reducing sugar, reduced sugar adopts film titration, and the mensuration of citric acid adoptsThe NaOH titration of concentration 0.1429mol/L, spore counting adopts blood counting chamber; If no special instructions, all adopt this area normalUse equipment and process method.
Raw materials used and reagent is the commercially available universal product if no special instructions.
Embodiment 1
Corn flour mixes according to 1:3 ratio with water, and alpha-amylase addition is 25U/g corn flour, secondary injection liquidChange, wherein a spray temperature is 97 DEG C, and 127 DEG C of secondary injection temperature are qualified through iodine examination, the mixed liquid that obtains liquefying, and partial liquefaction mixes liquidObtain through plate-frame filtering the clear liquid that liquefies; The mixed liquid of liquefaction mixes with water, adds a certain amount of ammonium sulfate preparation seed cultureBase (total reducing sugar 10%, total nitrogen 0.30%); The mixed liquid of liquefaction, liquefaction clear liquid and water mixed preparing fermentation medium (total reducing sugar 16.4%, total nitrogen0.08%); Ripe spore suspension is with 200,000/mL of concentration inoculation seed culture medium, and speed of agitator 600rpm, is cultured to 6hTime, speed of agitator is increased to 800rpm, and disturbance spore germination and mycelia winding process continue to be cultured to 32h and obtain ripeSeed liquor, the fermentation medium of then transferring, when fermentation reducing concentration finishes fermentation lower than 0.5% time. Fermentation period 50h, recordsFermentation and acid 16.02%, glucose acid invert ratio 100.1%.
Embodiment 2
With example 1, corn flour and water mix in proportion, and obtain the mixed liquid of corn flour liquefaction, through sheet frame through secondary injectionFiltration obtains corn flour liquefaction clear liquid, and the mixed liquid of corn flour liquefaction mixes with water and adds a certain amount of ammonium sulfate preparation seed and trainSupport base (total reducing sugar 10%, 0.28%). Sweet potato powder is mixed according to 1:3 ratio with 65 DEG C of water, regulates pH6.0, adds resistance to according to 35U/gAlpha-amylase, through secondary injection liquefaction, qualified through iodine examination, obtain the mixed liquid of Ipomoea batatas liquefaction, obtain Ipomoea batatas liquefaction through plate-frame filteringClear liquid. Corn flour liquefaction clear liquid, Ipomoea batatas liquefaction clear liquid and water according to a certain percentage mixed preparing fermentation medium (total reducing sugar 16.8%,Total nitrogen 0.10%); Ripe spore suspension is with 500,000/mL inoculation seed culture medium, and speed of agitator 600rpm, is cultured to 12hTime, speed of agitator is increased to 900rpm, and disturbance spore germination and mycelia winding process continue to be cultured to 28h and obtain ripeSeed liquor, the fermentation medium of then transferring, when fermentation reducing concentration finishes fermentation lower than 0.5% time. Fermentation period 53h, recordsProduce acid amount 16.9%, glucose acid invert ratio 100.6%.
Embodiment 3
Wheat flour mixes according to 1:3 ratio with water, and alpha-amylase addition is 28U/g wheat flour, secondary injection liquidChange, wherein a spray temperature is 99 DEG C, and 125 DEG C of secondary injection temperature are qualified through iodine examination, obtains the mixed liquid of wheat flour liquefaction, part liquidChange mixed liquid and obtain wheat flour liquefaction clear liquid through plate-frame filtering; The mixed liquid of liquefaction mixes with water, adds a certain amount of ammonium sulfatePreparation seed culture medium (total reducing sugar 9%, total nitrogen 0.23%); The mixed liquid of wheat flour liquefaction, liquefaction clear liquid mixes according to a certain percentage with waterPreparation fermentation medium (total reducing sugar 16.6%, total nitrogen 0.09%); Ripe spore suspension is with 800,000/mL of concentration inoculation seed cultureBase, speed of agitator 600rpm, while being cultured to 20h, speed of agitator is increased to 1200rpm, and disturbance spore germination and mycelia are wound aroundProcess, continues to be cultured to 26h and obtains ripe seed liquor, and the fermentation medium of then transferring, when fermentation reducing concentration is lower than 0.5%In time, finishes to ferment. Fermentation period 56h, records fermentation and acid 16.7%, glucose acid invert ratio 100.1%.
Embodiment 4
Corn flour mixes according to 1:3.5 ratio with water, and alpha-amylase addition is 26U/g corn flour, secondary injection liquidChange, wherein a spray temperature is 99 DEG C, and 130 DEG C of secondary injection temperature are qualified through iodine examination, the mixed liquid that obtains liquefying, and partial liquefaction mixes liquidObtain through plate-frame filtering the clear liquid that liquefies; The mixed liquid of liquefaction mixes with water, adds a certain amount of ammonium sulfate preparation seed cultureBase (total reducing sugar 11%, total nitrogen 0.25%); The mixed liquid of liquefaction, liquefaction clear liquid and water according to a certain percentage mixed preparing fermentation medium are (totalSugar 16.9%, total nitrogen 0.10%); Ripe spore suspension is with 600,000/mL of concentration inoculation seed culture medium, speed of agitator 600Rpm, while cultivating 8h, speed of agitator is increased to 1000rpm, and disturbance spore germination and mycelia winding process, continue to be cultured to 27hObtain ripe seed liquor, the fermentation medium of then transferring, when fermentation reducing concentration finishes fermentation lower than 0.5% time. Fermentation period59h, records fermentation and acid 16.8%, glucose acid invert ratio 99.4%.
Embodiment 5
Corn flour mixes according to 1:2.5 ratio with water, and alpha-amylase addition is 30U/g corn flour, secondary injection liquidChange, wherein a spray temperature is 98 DEG C, and 127 DEG C of secondary injection temperature are qualified through iodine examination, the mixed liquid that obtains liquefying, and partial liquefaction mixes liquidObtain through plate-frame filtering the clear liquid that liquefies; The mixed liquid of liquefaction mixes with water, adds a certain amount of ammonium sulfate preparation seed cultureBase (total reducing sugar 12%, total nitrogen 0.26%); The mixed liquid of liquefaction, liquefaction clear liquid and water according to a certain percentage mixed preparing fermentation medium are (totalSugar 17.3%, total nitrogen 0.12%); Ripe spore suspension is with 200,000/mL of concentration inoculation seed culture medium, speed of agitator 600Rpm, while cultivating 15h, speed of agitator is increased to 900rpm, and disturbance spore germination and mycelia winding process, continue to be cultured to 24hObtain ripe seed liquor, the fermentation medium of then transferring, when fermentation reducing concentration finishes fermentation lower than 0.5% time. Fermentation period62h, records fermentation and acid 17.1%, glucose acid invert ratio 98.8%.
Comparative example 1(prior art)
Corn flour mixes according to 1:3 ratio with water, and alpha-amylase addition is 30U/g corn flour, secondary injection liquidChange, wherein a spray temperature is 98 DEG C, and 130 DEG C of secondary injection temperature are qualified through iodine examination, the mixed liquid that obtains liquefying, and partial liquefaction mixes liquidObtain through plate-frame filtering the clear liquid that liquefies; The mixed liquid of liquefaction mixes with water, adds a certain amount of ammonium sulfate preparation seed cultureBase (total reducing sugar 12%, total nitrogen 0.25%); The mixed liquid of liquefaction, liquefaction clear liquid and water mixed preparing fermentation medium (total reducing sugar 16.4%, total nitrogen0.08%); Ripe spore suspension is with 200,000/mL of concentration inoculation seed culture medium, and speed of agitator 600rpm, is cultured to 30hObtain ripe seed liquor, the fermentation medium of then transferring, when fermentation reducing concentration finishes fermentation lower than 0.5% time. Fermentation period62h, records fermentation and acid 16.3%, glucose acid invert ratio 96.9%.
Every embodiment 1 ~ 5 test index and comparative example are compared, and result sees table.
Table 1 embodiment 1 ~ 5 and comparative example indices comparing result
From table 1 data, based on sprouting, spore is different to the sensitiveness of applying shearing force from mycelium pellet, and embodiment 1 ~ 5 passes throughImprove speed of agitator disturbance spore germination and mycelia winding process, can obviously shorten fermentation period, significantly improve fermentation and acid amount,Fermentation conversion rate and fermentation index.
Embodiment 1 and comparative example mature seed liquid mycelium pellet be in 50 times of lower morphological features of light microscope as shown in Figure 1: withComparative example (141 μ m) compare, embodiment 1 mycelium pellet diameter less (m), mycelium pellet structure is more loose for 112 μ, be beneficial to dissolved oxygen withMass transfer, fermenting property is able to remarkable lifting.
The above is only the preferred embodiment of the present invention, the invention is not restricted to above embodiment. Be appreciated that abilityThe oher improvements and changes that field technique personnel directly derive or associate without departing from the spirit and concept in the present invention,Within all should thinking and being included in protection scope of the present invention.
Claims (8)
1. the method based on controlling mycelium Structure Improvement citric acid fermentation performance, is characterized in that comprising: will cultivate intoRipe aspergillus niger spore is mixed with spore suspension, is seeded to seed culture medium and carries out seed culture; In seed culture process, carryHigh speed of agitator disturbance spore germination and mycelia winding process, obtain mature seed liquid; Above-mentioned mature seed liquid is seeded to and is sent outFerment culture medium carries out fermented and cultured, finishes fermentation.
2. the method for improving according to claim 1 citric acid fermentation performance, is characterized in that: described aspergillus niger spore inoculationAfter final concentration be 20 ~ 800,000/mL.
3. the method for improving according to claim 1 citric acid fermentation performance, is characterized in that: described raising mixing speed is disturbedMoving technique is carried out 6 ~ 20h in described seed culture and is started.
4. the method for improving according to claim 1 citric acid fermentation performance, is characterized in that: described speed of agitator is increased to800~1200rpm。
5. the method for improving according to claim 1 citric acid fermentation performance, is characterized in that: described kind
Sub-incubation time is 24 ~ 32h, obtains mature seed liquid.
6. the method for improving according to claim 1 citric acid fermentation performance, is characterized in that: when described fermentation medium alsoRaw sugar concentration finishes fermentation lower than 0.5% time.
7. the method for improving according to claim 1 citric acid fermentation performance, is characterized in that: described seed culture medium, send outFerment culture medium is prepared by starchy material.
8. the method for improving according to claim 7 citric acid fermentation performance, is characterized in that: described starchy material comprisesAt least one in corn flour, wheat flour, tapioca starch, sweet potato powder, starch, molasses, wheat.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107815421A (en) * | 2017-12-08 | 2018-03-20 | 江苏国信协联能源有限公司 | A kind of aspergillus niger seed culture and its method for preparing citric acid |
| CN112921058A (en) * | 2019-12-05 | 2021-06-08 | 中粮生物科技股份有限公司 | Method for producing citric acid by fermenting aspergillus niger |
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| CN102864082A (en) * | 2012-09-19 | 2013-01-09 | 中粮生物化学（安徽）股份有限公司 | Method for culturing citric acid fermenting seeds and method for preparing citric acid by fermenting |
| CN104099253A (en) * | 2014-07-11 | 2014-10-15 | 江南大学 | Citric acid aspergillus niger seed continuous culture method based on mycelium pellet dispersion technology |
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2016
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102864082A (en) * | 2012-09-19 | 2013-01-09 | 中粮生物化学（安徽）股份有限公司 | Method for culturing citric acid fermenting seeds and method for preparing citric acid by fermenting |
| CN104099253A (en) * | 2014-07-11 | 2014-10-15 | 江南大学 | Citric acid aspergillus niger seed continuous culture method based on mycelium pellet dispersion technology |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107815421A (en) * | 2017-12-08 | 2018-03-20 | 江苏国信协联能源有限公司 | A kind of aspergillus niger seed culture and its method for preparing citric acid |
| CN107815421B (en) * | 2017-12-08 | 2020-06-05 | 江苏国信协联能源有限公司 | Aspergillus niger seed culture and citric acid preparation method |
| CN112921058A (en) * | 2019-12-05 | 2021-06-08 | 中粮生物科技股份有限公司 | Method for producing citric acid by fermenting aspergillus niger |
| CN112921058B (en) * | 2019-12-05 | 2023-01-13 | 中粮生物科技股份有限公司 | Method for producing citric acid by fermenting aspergillus niger |
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