CN105586367A - Method for conducting fermentative production of citric acid by adding saccharifying enzyme stage by stage based on pH responses - Google Patents
Method for conducting fermentative production of citric acid by adding saccharifying enzyme stage by stage based on pH responses Download PDFInfo
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- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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Abstract
The invention discloses a method for conducting fermentative production of citric acid by adding a saccharifying enzyme stage by stage based on pH responses. The method includes the steps that a starchy material is subjected to pulp mixing, liquefaction and inoculated fermentation to obtain end fermentation liquor; when the pH of the fermentation liquor is reduced to 2.0-3.0 in the fermentative culture process, the saccharifying enzyme is added into the fermentation liquor stage by stage in an equivalent mode every 2-8 h, wherein the total addition quantity of the saccharifying enzyme is 40-1000 U/g residual non-glucose quantity. By means of the method, the problem of saccharifying enzyme activity loss caused in the middle-late stage of citric acid fermentation can be solved, the fermentation period can be effectively shortened, the fermentation conversion rate can be increased, the residual glucose quantity of the end fermentation liquor can be reduced, wastewater discharge can be reduced, and meanwhile the activity of glucosidase secreted by glucose can be offset to reduce non-fermentable glucose. The method has significant economic benefits and environmental benefits.
Description
Technical field
The invention belongs to fermentation engineering field, relate in particular to a kind of method of adding carbohydrase fermentation production of citric acid based on pH response phase.
Background technology
Citric acid (CitricAcid) is a kind of important organic acid with several functions, is widely used in the fields such as food, medicine, chemical industry, is the edible organic acid of output and consumption figure maximum on our times, and global output exceedes 1,700,000 tons. Meanwhile, citric acid has again good biological nature, has huge application potential in new industry fields such as biopolymerization, medicament transport, cell cultivations, and its demand is with annual 5% speed increment.
Citric acid often obtains by liquid submerged fermentation method, still continues to use traditional batch fermentation mode in suitability for industrialized production. Because aspergillus niger has the advantages such as enzyme system is abundant, substrate is extensive, productive rate is high, be the most important bacterial strain of citric acid fermentation, aspergillus niger self can be secreted carbohydrase, generally adopts the mode of saccharification limit, limit fermentation. But produce in citric acid process at fermentation of Aspergillus niger, along with synthetic citric acid constantly accumulates, zymotic fluid pH can significantly decline, and diastatic activity is progressively suppressed, cause in whole fermentation liquid residual sugar higher, fermentation period is longer.
Prior art has been carried out some researchs for the saccharification of Citric Acid Fermentation. The patent of invention of application number 201010513719.7 " a kind of add the method that citric acid is prepared in carbohydrase fermentation " open a kind of after liquefaction, inoculate before, be cooled to 40 ~ 60 DEG C of techniques of adding carbohydrase, its shortcoming is that enzyme-added temperature is too high, and most of carbohydrase inactivation causes carbohydrase utilization rate low; The patent of invention " a kind of method of fermentation production of citric acid " of application number 201110162278.5 discloses after inoculation fermentation, in 36 ~ 38 DEG C of normal temperature control processes, adds carbohydrase; The patent of invention " method of citric acid is prepared in a kind of fermentation " of application number 201210009294.5 discloses at citric acid fermentation bacterial classification adds carbohydrase in postvaccinal 0 ~ 8 hour in fermentation medium.
The guiding theory of above-mentioned several method based on common obtain the small molecular sugar (as glucose) of high concentration by adding carbohydrase at the fermentation initial stage, but and unresolved sweat middle and later periods pH significantly declines, saccharifying enzymic activity loss and cause glucose supplies and the problem such as bacterial strain sugar consumption rate is inharmonious, citric acid synthesis rate slows down, fermentation period is grown partially, residual sugar amount is higher. Bring new problem: the basic body of fermentation initial-stage culture exists glucose simultaneously, the carbohydrase of self secreting can meet the demand of fermentation strain to nutrition, excessive concentrations glucose can suppress the growth of bacterial classification on the contrary, extends fermentation lag phase, is unfavorable for the synthetic fast of citric acid.
Summary of the invention
The object of the present invention is to provide a kind of method of adding carbohydrase fermentation production of citric acid based on pH response phase, the method can solve the problem of citric acid fermentation middle and later periods saccharifying enzymic activity loss, effectively shorten fermentation period, improve fermentation conversion rate, reduce whole zymotic fluid residual sugar amount.
In order to reach above object, the present invention by the following technical solutions:
A kind of method of adding carbohydrase fermentation production of citric acid based on pH response phase, comprise by starchy material size mixing, liquefaction, inoculation fermentation, make whole zymotic fluid, also be included in fermented and cultured process, in the time that zymotic fluid pH is down to 2.0 ~ 3.0, the step that equivalent is added carbohydrase stage by stage wherein.
Particularly, described equivalent is stage by stage added at interval of 2 ~ 8h equivalent and adds.
Particularly, the interpolation total amount of described carbohydrase is that the content (difference of remaining total reducing sugar and remaining grape) based on remaining non-glucose in described zymotic fluid is determined, adding total amount is the remaining non-glucose amount of 40 ~ 1000U/g.
Particularly, described starchy material comprises at least one in corn flour, wheat flour, tapioca starch, sweet potato powder, starch, molasses, wheat.
Preferably, the present invention specifically comprises the following steps:
(1) described starchy material powder is mixed according to 1:1.5 ~ 1:4 ratio with water, regulate pH to 5.8 ~ 6.0, then add high temperature α-amylase, described amylase addition is 15 ~ 50U/g powder;
(2) mixed liquor step (2) being obtained is through twice injection, 95 ~ 105 DEG C of injection temperations, and 120 ~ 130 DEG C of secondary injection temperature, qualified through iodine examination, obtain the mixed liquid of liquefaction, more mixed described liquefaction liquid is obtained to liquefaction clear liquid through plate-frame filtering;
(3) the mixed liquid of liquefaction step (2) being obtained mixes with water, adds nitrogenous source, and regulating mixed liquor total reducing sugar is 6 ~ 12%, and total nitrogen is 0.2% ~ 0.4%, obtains seed culture medium;
(4) seed culture medium in aspergillus niger spore suspension inoculation step (3) is cultivated, obtained mature seed liquid;
(5) the mixed liquid of liquefaction, the liquefaction clear liquid that step (2) are obtained mix with water, and regulating the total reducing sugar of mixed liquor is 16 ~ 18%, and total nitrogen is 0.05 ~ 0.15%, obtains fermentation medium;
(6) mature seed liquid step (4) being obtained is seeded in the fermentation medium of step (5) acquisition, cultivates 20 ~ 32h;
(7), in the time that above-mentioned fermentation medium zymotic fluid pH is down to 2.0 ~ 3.0, at interval of 2 ~ 8h equivalent interpolation carbohydrase wherein, adding total amount is the remaining non-glucose amounts of 40 ~ 1000U/g.
If no special instructions, the above percentage is percetage by weight (wt%).
The present invention has following useful technique effect:
The present invention is based on the synthetic feature of fermentation strain physiological property and citric acid, adopt in the time that zymotic fluid pH is down in particular range, according to the non-glucose amount of remnants stage by stage equivalent add carbohydrase, coordinate between glucose supplies and glucose consumption, to reach balance optimization in whole sweat. Can realize on the one hand the efficient utilization of self carbohydrase, guarantee the Fast Growth of earlier fermentation bacterial classification; Timely and appropriate discovery adds the loss that carbohydrase again can effective compensation own vitality on the other hand, collaborative Citric Acid Fermentation, guarantee the Rapid Accumulation at fermentation middle and later periods citric acid, can offset the glucose glycoside enzymatic activity of self secreting simultaneously, reduce the generation of non-sugar fermentation. Application result shows, the present invention can shorten fermentation period, improves fermentation conversion rate, reduces whole zymotic fluid residual sugar amount, reduces discharge of wastewater, reduces waste water CODcr, there is important economic benefit and environmental benefit.
Detailed description of the invention
Below by specific embodiment, the invention will be further described.
In following examples and comparative example, the assay method of total reducing sugar, reduced sugar adopts film titration, and the mensuration of citric acid adopts the NaOH titration of concentration 0.1429mol/L, and spore counting adopts blood counting chamber; If no special instructions, all adopt this area common equipment and process.
Raw materials used and reagent is the commercially available universal product if no special instructions.
Embodiment 1
Corn flour is mixed according to 1:1.5 ratio with water, regulate pH to 5.8, then add high-temperatureα-amylase, addition is 50U/g corn flour; Secondary injection liquefaction, wherein a spray temperature is 105 DEG C, 130 DEG C of secondary injection temperature are qualified through iodine examination, the mixed liquid that obtains liquefying, the mixed liquid of partial liquefaction obtains 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 culture medium (total reducing sugar 6%, total nitrogen 0.25%), by ripe aspergillus niger spore suspension inoculation seed culture medium, while cultivating 8h, improve speed of agitator to 800rpm, cultivate altogether 27h, obtain mature seed liquid; To liquefy and mix liquid, liquefaction clear liquid and water mixed preparing fermentation medium (total reducing sugar 16%, total nitrogen 0.05%); Mature seed liquid inoculation fermentation culture medium is carried out to fermented and cultured; In the time that zymotic fluid pH is down to 3.00, add carbohydrase, adding total amount is the remaining non-glucose amounts of 40U/g, adds carbohydrase 3840U at interval of 4h equivalent, when concentration of reduced sugar finishes fermentation lower than 0.5% time. Record fermentation period 52h, citric acid content 16.1%, residual sugar 1.2%.
Embodiment 2
Corn flour is mixed according to 1:4 ratio with water, regulate pH to 6.0, then add high temperature α-amylase, addition is 15U/g corn flour; Secondary injection liquefaction, wherein a spray temperature is 95 DEG C, 120 DEG C of secondary injection temperature are qualified through iodine examination, the mixed liquid that obtains liquefying, the mixed liquid of partial liquefaction obtains 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 culture medium (total reducing sugar 10%, total nitrogen 0.30%), by ripe aspergillus niger spore suspension inoculation seed culture medium, cultivates 26h, obtains mature seed liquid; To liquefy and mix liquid, liquefaction clear liquid and water mixed preparing fermentation medium (total reducing sugar 16.8%, total nitrogen 0.08%); Mature seed liquid inoculation fermentation culture medium is carried out to fermented and cultured; In the time that zymotic fluid pH is down to 2.52, add carbohydrase, adding total amount is the remaining non-glucose amounts of 100U/g, adds carbohydrase 16000U at interval of 4h equivalent, when concentration of reduced sugar finishes fermentation lower than 0.5% time. Record fermentation period 53h, citric acid content 16.9%, residual sugar 0.9%.
Embodiment 3
Tapioca starch is mixed according to 1:2.5 ratio with water, regulate pH to 5.9, then add high temperature α-amylase, addition is 25U/g tapioca starch; Secondary injection liquefaction, wherein a spray temperature is 100 DEG C, 125 DEG C of secondary injection temperature are qualified through iodine examination, the mixed liquid that obtains liquefying, the mixed liquid of partial liquefaction obtains 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 culture medium (total reducing sugar 12%, total nitrogen 0.40%), by ripe aspergillus niger spore suspension inoculation seed culture medium, cultivates 30h, obtains mature seed liquid; To liquefy and mix liquid, liquefaction clear liquid and water mixed preparing fermentation medium (total reducing sugar 16.7%, total nitrogen 0.12%); Mature seed liquid inoculation fermentation culture medium is carried out to fermented and cultured; In the time that zymotic fluid pH is down to 2.00, add carbohydrase, adding total amount is the remaining non-glucose amounts of 300U/g, adds carbohydrase 48000U at interval of 8h equivalent, when concentration of reduced sugar finishes fermentation lower than 0.5% time. Recording fermentation period is 55h, citric acid content 16.5%, residual sugar 1.2%.
Embodiment 4
Tapioca starch is mixed according to 1:2.5 ratio with water, regulate pH to 5.8, then add high temperature α-amylase, addition is 25U/g tapioca starch; Secondary injection liquefaction, wherein a spray temperature is 100 DEG C, 125 DEG C of secondary injection temperature are qualified through iodine examination, the mixed liquid that obtains liquefying, the mixed liquid of partial liquefaction obtains 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 culture medium (total reducing sugar 12%, total nitrogen 0.40%), by ripe aspergillus niger spore suspension inoculation seed culture medium, cultivates 32h, obtains mature seed liquid; To liquefy and mix liquid, liquefaction clear liquid and water mixed preparing fermentation medium (total reducing sugar 17.5%, total nitrogen 0.12%); Mature seed liquid inoculation fermentation culture medium is carried out to fermented and cultured; In the time that zymotic fluid pH is down to 2.60, add carbohydrase, adding total amount is the remaining non-glucose amounts of 500U/g, adds carbohydrase 50400U at interval of 6h equivalent, when concentration of reduced sugar finishes fermentation lower than 0.5% time. Recording fermentation period is 63h, citric acid content 17.4%, residual sugar 1.6%.
Embodiment 5
Tapioca starch is mixed according to 1:3.5 ratio with water, regulate pH to 5.9, then add high temperature α-amylase, addition is 30U/g tapioca starch; Secondary injection liquefaction, wherein a spray temperature is 98 DEG C, 127 DEG C of secondary injection temperature are qualified through iodine examination, obtain the mixed liquid of cassava liquefaction, then obtain cassava liquefaction clear liquid through plate-frame filtering. Corn flour is mixed according to 1:2.5 ratio with water, regulate pH to 5.8, then add high temperature α-amylase, addition is 20U/g corn flour; Secondary injection liquefaction, wherein a spray temperature is 105 DEG C, and 125 DEG C of secondary injection temperature are qualified through iodine examination, obtain the mixed liquid of corn liquefaction, and the mixed liquid of part corn liquefaction obtains corn liquefaction clear liquid through plate-frame filtering. Mixed corn liquefaction liquid is mixed with water, add a certain amount of ammonium sulfate preparation seed culture medium (total reducing sugar 8%, total nitrogen 0.28%), by ripe aspergillus niger spore suspension inoculation seed culture medium, cultivate 29h, obtain mature seed liquid; By mixed corn liquefaction liquid, corn liquefaction clear liquid, cassava liquefaction clear liquid and water mixed preparing fermentation medium (total reducing sugar 18.0%, total nitrogen 0.15%); Mature seed liquid inoculation fermentation culture medium is carried out to fermented and cultured; In the time that zymotic fluid pH is down to 2.15, add carbohydrase, adding total amount is the remaining non-glucose amounts of 1000U/g, adds carbohydrase 36800U at interval of 3h equivalent, when concentration of reduced sugar finishes fermentation lower than 0.5% time. Recording fermentation period is 67h, citric acid content 17.7%, residual sugar 2.2%.
Embodiment 6
Corn flour is mixed according to 1:3.0 ratio with water, regulate pH to 5.8, then add high temperature α-amylase, addition is 34U/g corn flour; Secondary injection liquefaction, wherein a spray temperature is 96 DEG C, 123 DEG C of secondary injection temperature are qualified through iodine examination, obtain the mixed liquid of corn liquefaction, then obtain corn liquefaction clear liquid through plate-frame filtering. Wheat flour is mixed according to 1:2.0 ratio with water, regulate pH to 6.0, then add high temperature α-amylase, addition is 23U/g wheat flour; Secondary injection liquefaction, wherein a spray temperature is 95 DEG C, 128 DEG C of secondary injection temperature are qualified through iodine examination, obtain the mixed liquid of wheat liquefaction, then obtain wheat liquefaction clear liquid through plate-frame filtering. Mixed corn liquefaction liquid is mixed with water, add a certain amount of ammonium sulfate preparation seed culture medium (total reducing sugar 10%, total nitrogen 0.32%), by ripe aspergillus niger spore suspension inoculation seed culture medium, cultivate 28h, obtain mature seed liquid; By mixed corn liquefaction liquid, corn liquefaction clear liquid, wheat liquefaction clear liquid and water mixed preparing fermentation medium (total reducing sugar 17.2%, total nitrogen 0.09%); Mature seed liquid inoculation fermentation culture medium is carried out to fermented and cultured; In the time that zymotic fluid pH is down to 2.05, add carbohydrase, add carbohydrase in the time that zymotic fluid pH is down to 2.05, adding total amount is the remaining non-glucose amounts of 70U/g, adds carbohydrase 5600U at interval of 5h equivalent, when concentration of reduced sugar finishes fermentation lower than 0.5% time. Recording fermentation period is 63h, citric acid content 17.1%, residual sugar 1.3%.
Comparative example (prior art)
Corn flour is mixed according to 1:2.5 ratio with water, regulate pH to 5.8, then add high temperature α-amylase, addition is 40U/g corn flour; Secondary injection liquefaction, wherein a spray temperature is 98 DEG C, 130 DEG C of secondary injection temperature are qualified through iodine examination, the mixed liquid that obtains liquefying, the mixed liquid of partial liquefaction obtains 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 culture medium (total reducing sugar 12%, total nitrogen 0.25%), and ripe aspergillus niger spore suspension inoculation seed culture medium is cultivated to 28h, obtains mature seed liquid; To liquefy and mix liquid, liquefaction clear liquid and water mixed preparing fermentation medium (total reducing sugar 16.6%, total nitrogen 0.85%); Mature seed liquid inoculation fermentation culture medium is carried out to fermented and cultured, when concentration of reduced sugar finishes fermentation lower than 0.5% time. Record fermentation period 64h, citric acid content 16.2%, residual sugar 2.8%.
Every embodiment 1 ~ 6 test index and comparative example are compared, and result sees table.
Table 1 embodiment 1 ~ 6 and the every test index comparing result of comparative example
As can be seen from Table 1, embodiment 1 ~ 6 responds equivalent stage by stage in the specific pH scope of zymotic fluid (pH2.0 ~ 3.0) and adds carbohydrase, can guarantee the Fast Growth of earlier fermentation bacterial classification, can guarantee again the Rapid Accumulation at fermentation middle and later periods citric acid, shorten fermentation period, improve fermentation conversion rate and fermentation index, effectively reduce whole zymotic fluid residual sugar amount.
The above is only the preferred embodiment of the present invention, the invention is not restricted to above embodiment. Be appreciated that the oher improvements and changes that those skilled in the art 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 (6)
1. one kind is added the method for carbohydrase fermentation production of citric acid based on pH response phase, comprise by starchy material size mixing, liquefaction, inoculation fermentation, make whole zymotic fluid, it is characterized in that: be also included in fermented and cultured process, in the time that zymotic fluid pH is down to 2.0 ~ 3.0, the step that equivalent is added carbohydrase stage by stage wherein.
2. the method for producing according to claim 1 citric acid, is characterized in that: described equivalent is stage by stage added at interval of 2 ~ 8h equivalent adds.
3. the method for producing according to claim 1 citric acid, is characterized in that: the interpolation total amount of described carbohydrase is that the content (difference of remaining total reducing sugar and remaining grape) based on remaining non-glucose in described zymotic fluid is determined.
4. the method for producing according to claim 3 citric acid, is characterized in that: the interpolation total amount of described carbohydrase is the remaining non-glucose amount of 40 ~ 1000U/g.
5. the method for producing according to claim 1 citric acid, is characterized in that: described starchy material comprises at least one in corn flour, wheat flour, tapioca starch, sweet potato powder, starch, molasses, wheat.
6. the method for producing citric acid described in claim 1 ~ 5 any one, is characterized in that specifically comprising the following steps:
(1) described starchy material powder is mixed according to 1:1.5 ~ 1:4 ratio with water, regulate pH to 5.8 ~ 6.0, then add high temperature α-amylase, described amylase addition is 15 ~ 50U/g powder;
(2) mixed liquor step (2) being obtained is through twice injection, 95 ~ 105 DEG C of injection temperations, and 120 ~ 130 DEG C of secondary injection temperature, qualified through iodine examination, obtain the mixed liquid of liquefaction, more mixed described liquefaction liquid is obtained to liquefaction clear liquid through plate-frame filtering;
(3) the mixed liquid of liquefaction step (2) being obtained mixes with water, adds nitrogenous source, and regulating mixed liquor total reducing sugar is 6 ~ 12%, and total nitrogen is 0.2% ~ 0.4%, obtains seed culture medium;
(4) seed culture medium in aspergillus niger spore suspension inoculation step (3) is cultivated, obtained mature seed liquid;
(5) the mixed liquid of liquefaction, the liquefaction clear liquid that step (2) are obtained mix with water, and regulating the total reducing sugar of mixed liquor is 16 ~ 18%, and total nitrogen is 0.05 ~ 0.15%, obtains fermentation medium;
(6) mature seed liquid step (4) being obtained is seeded in the fermentation medium of step (5) acquisition, cultivates 20 ~ 32h;
(7), in the time that above-mentioned fermentation medium zymotic fluid pH is down to 2.0 ~ 3.0, at interval of 2 ~ 8h equivalent interpolation carbohydrase wherein, adding total amount is the remaining non-glucose amounts of 40 ~ 1000U/g;
(8) when above-mentioned fermentation medium concentration of reduced sugar finishes fermentation lower than 0.5% time;
If no special instructions, the above percentage is percetage by weight (wt%).
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| CN109022504A (en) * | 2018-08-17 | 2018-12-18 | 江南大学 | A method of citric acid is produced using electrodialysis process anaerobic digestion solution |
| CN111172204A (en) * | 2020-03-13 | 2020-05-19 | 合肥五粮泰生物科技有限公司 | Preparation method for improving citric acid fermentation efficiency |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN109022504A (en) * | 2018-08-17 | 2018-12-18 | 江南大学 | A method of citric acid is produced using electrodialysis process anaerobic digestion solution |
| CN109022504B (en) * | 2018-08-17 | 2020-07-07 | 江南大学 | Method for producing citric acid by treating anaerobic digestion solution through electrodialysis |
| CN111172204A (en) * | 2020-03-13 | 2020-05-19 | 合肥五粮泰生物科技有限公司 | Preparation method for improving citric acid fermentation efficiency |
| CN111172204B (en) * | 2020-03-13 | 2023-01-24 | 合肥五粮泰生物科技有限公司 | Preparation method for improving citric acid fermentation efficiency |
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Application publication date: 20160518 |