CN102443611A - Production method of citric acid - Google Patents
Production method of citric acid Download PDFInfo
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- CN102443611A CN102443611A CN2010105119536A CN201010511953A CN102443611A CN 102443611 A CN102443611 A CN 102443611A CN 2010105119536 A CN2010105119536 A CN 2010105119536A CN 201010511953 A CN201010511953 A CN 201010511953A CN 102443611 A CN102443611 A CN 102443611A
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 title claims abstract description 132
- 238000004519 manufacturing process Methods 0.000 title abstract description 19
- 238000000855 fermentation Methods 0.000 claims abstract description 75
- 230000004151 fermentation Effects 0.000 claims abstract description 75
- 125000004432 carbon atom Chemical group C* 0.000 claims abstract description 14
- LPQOADBMXVRBNX-UHFFFAOYSA-N ac1ldcw0 Chemical compound Cl.C1CN(C)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN3CCSC1=C32 LPQOADBMXVRBNX-UHFFFAOYSA-N 0.000 claims description 81
- 239000007788 liquid Substances 0.000 claims description 78
- 238000000034 method Methods 0.000 claims description 69
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 44
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 37
- 239000008103 glucose Substances 0.000 claims description 37
- 239000000463 material Substances 0.000 claims description 25
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- 230000001332 colony forming effect Effects 0.000 claims description 19
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- 230000008569 process Effects 0.000 claims description 10
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 7
- 229910052799 carbon Inorganic materials 0.000 claims description 7
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 6
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 5
- 229910052698 phosphorus Inorganic materials 0.000 claims description 5
- 239000011574 phosphorus Substances 0.000 claims description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 4
- 229920002494 Zein Polymers 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 239000005019 zein Substances 0.000 claims description 4
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- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 3
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 2
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 2
- 229910052757 nitrogen Inorganic materials 0.000 claims description 2
- 241000228245 Aspergillus niger Species 0.000 abstract description 26
- 238000006243 chemical reaction Methods 0.000 abstract description 7
- 238000005265 energy consumption Methods 0.000 abstract description 2
- 150000002772 monosaccharides Chemical class 0.000 abstract 3
- 239000000243 solution Substances 0.000 description 44
- 239000002609 medium Substances 0.000 description 43
- 230000000052 comparative effect Effects 0.000 description 34
- 239000000047 product Substances 0.000 description 29
- 230000009466 transformation Effects 0.000 description 28
- 108090000790 Enzymes Proteins 0.000 description 19
- 102000004190 Enzymes Human genes 0.000 description 19
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- 229940088598 enzyme Drugs 0.000 description 19
- 235000015097 nutrients Nutrition 0.000 description 16
- 238000000926 separation method Methods 0.000 description 12
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- 238000010298 pulverizing process Methods 0.000 description 11
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- 240000008042 Zea mays Species 0.000 description 8
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 8
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 8
- 235000005822 corn Nutrition 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 7
- 239000002253 acid Substances 0.000 description 7
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 4
- 108010059892 Cellulase Proteins 0.000 description 4
- 229930091371 Fructose Natural products 0.000 description 4
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 4
- 239000005715 Fructose Substances 0.000 description 4
- 229940106157 cellulase Drugs 0.000 description 4
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
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- FXLJDRXREUZRIC-BAOOBMCLSA-N (3s,4r,5r)-1,3,4,5,6-pentahydroxyhexan-2-one;hydrate Chemical compound O.OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)CO FXLJDRXREUZRIC-BAOOBMCLSA-N 0.000 description 2
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- 240000003183 Manihot esculenta Species 0.000 description 1
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- 244000061456 Solanum tuberosum Species 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention provides a production method of citric acid, which comprises the step of inoculating Aspergillus niger into a fermentation medium to be fermented under the condition of generating the citric acid to obtain fermentation liquor, and is characterized in that monosaccharide with 6 carbon atoms is added into the fermentation liquor, and the time for adding the monosaccharide with 6 carbon atoms is within the time period from 24 hours after the Aspergillus niger is inoculated into the fermentation medium to 5 hours before the fermentation is completed. According to the invention, monosaccharide is added, the conversion function of Aspergillus niger is fully utilized, and a larger amount of citric acid is obtained on the premise of not changing energy consumption such as equipment, reaction conditions and the like.
Description
Technical field
The present invention relates to a kind of working method of Hydrocerol A.
Background technology
Hydrocerol A is first acid in the organic acid, because the excellent properties of aspects such as physics, chemistry is widely used in industrial circles such as medicine, chemistry, electronics, weaving, oil, leather, building, photography, plastics, casting and pottery.
The working method of Hydrocerol A mainly contains two kinds: a kind of is from the natural fruit juice that contains Hydrocerol A, to extract; Another kind is to produce with fermentation method, and it is main mainly producing Hydrocerol A with the fermentation of Aspergillus niger method in the industry at present.Concrete way is that black mold is inoculated in the fermention medium, contains starchy material enzymolysis product and nitrogenous source in the fermention medium, obtains the solution of Hydrocerol A by fermentation with solid-liquid separation.At present, industrial top fermentation method is produced Hydrocerol A commonly used 3 * 10
5The fermentor tank of L, the starchy material that each fermentation is used is 5.6 * 10
4Kilogram, the Hydrocerol A that final fermentation obtains is 32.5 * 10
3-3.4 * 10
3Kilogram.If want to enhance production capacities, can only increase the volume of fermentor tank, the number of increase fermentor tank or the method for other change existing installations, certainly will cause the very big increase of industrial production cost like this.
Therefore, pressing for a kind of existing installation that do not change of exploitation just can make black mold bring into play the method for its maximum utility, raising Hydrocerol A production capacity.
Summary of the invention
The purpose of this invention is to provide a kind of working method that can effectively improve the Hydrocerol A of Hydrocerol A production capacity.
Contriver of the present invention is through discovering; The lower reason of fermentation of Aspergillus niger method integrated artistic production capacity is that the utilization ratio of black mold is not high at present, and the fermenting process of black mold roughly can be divided into three phases, the one, earlier fermentation; It mainly is the growth phase of black mold; Thalline produces saccharifying enzyme simultaneously, and saccharifying enzyme is converted into the utilizable glucose of black mold with the polysaccharide in the raw material, and glucose is through a series of pathways metabolisms; With conversion of glucose is Hydrocerol A, makes the decline of pH value get into lord ferment period owing to sugar has been synthesized organic acid by the black mold utilization; The 2nd, lord ferment period promptly produces acid phase, and in this stage, Hydrocerol A accumulates in a large number; The 3rd, the fermentation later stage, i.e. in the self-dissolving stage, along with exhausting of nutrient, the tropina enzyme comes to life, and causes the pH value in rising trend again, and this moment, thalline was tending towards self-dissolving and the Metabolic activity termination.In whole fermentation process, after the entering subordinate phase, because the pH value descends, the activity of saccharifying enzyme is suppressed, and causes in the lord ferment period process, and the part black mold does not have enough glucose to transform, and has caused the significant wastage of bioenergy and equipment.
The invention provides a kind of working method of Hydrocerol A; This method is included under the condition that generates Hydrocerol A; Black mold is seeded in the fermention medium ferments, obtain fermented liquid, it is characterized in that; This method also comprises in fermented liquid adds the monose with 6 carbon atoms, and the time of adding the monose with 6 carbon atoms accomplished in the preceding 5 hours time period to fermentation after black mold is inoculated into fermention medium in 24 hours.
The present invention has made full use of the transformation function of black mold, under the prerequisite that does not change energy consumptions such as equipment and reaction conditions through adding monose; Obtained higher Hydrocerol A production capacity; In embodiment 1 and Comparative Examples 2, use the equipment of same 300L model, the starchy material of the same weight of fermenting; Embodiment 1 can obtain 45.69 kilograms of Hydrocerol As, and Comparative Examples 2 only obtains 33.32 kilograms of Hydrocerol As.This shows, compare prior art, the present invention has improved the utilization ratio of integral device, and then has improved the production capacity of integrated artistic.
Embodiment
The invention provides a kind of working method of Hydrocerol A; This method is included under the condition that generates Hydrocerol A; Black mold is seeded in the fermention medium ferments, obtain fermented liquid, it is characterized in that; This method also comprises in fermented liquid adds the monose with 6 carbon atoms, and the time of adding the monose with 6 carbon atoms accomplished in the preceding 5 hours time period to fermentation after black mold is seeded to fermention medium in 24 hours.
Wherein, Said fermention medium is for well known to a person skilled in the art notion; Refer to the nutriment of required confession microorganism growth of microbial fermentation and the manual work preparation of keeping usefulness, generally all contain glucide, nitrogenous substances, inorganic salt (comprising trace element) and VITAMINs and water etc.Said fermented liquid refers to an access the liquid nutrient medium (this liquid nutrient medium also is an alleged fermention medium among the present invention) of microbial strains, products therefrom after cultivation after a while also for well known to a person skilled in the art notion.
According to the present invention; The addition of said monose can change in a big way, considers the transformation function that should make full use of black mold, combines cost factor again; Under the preferable case; The weight ratio of the addition of said monose and fermented liquid total amount is 1: 8-100 further is preferably 1: 10-50 most preferably is 1: 13-28.
Adding monose is in order to make full use of the transformation function of black mold; The time point that adds is very crucial; Too early interpolation has caused the reduction of transformation efficiency on the contrary; Comparative Examples 1 of the present invention was just being added monose in fermented liquid before black mold is inoculated into fermention medium, the transformation efficiency of Hydrocerol A is merely 92.5% in the Comparative Examples 1, and is also lower than the transformation efficiency 93.31% of Hydrocerol A in the Comparative Examples of not adding monose 2.Tracing it to its cause has two aspects, and the first, carbon source is too much in the Comparative Examples 1, and it is too fast to cause monose to be converted into the speed of Hydrocerol A, and the speed that the pH value reduces is also too fast, causes the not enough and thalline early ageing of thalli growth; The second, added monose in the Comparative Examples 1, be easy to generate the flash phenomenon during phase at the high foam of fermentation, thereby cause the loss of raw material.And the interpolation in evening excessively, black mold is to the phase of decaying of growth, and the function that monose is converted into Hydrocerol A dies down, and also is difficult to obtain good effect even add monose.
Therefore; The time that preferably begins to add the monose with 6 carbon atoms is for being seeded to black mold behind the fermention medium 24-56 hour; Further be preferably 40-50 hour, the preferred time that finishes to add the monose with 6 carbon atoms like this can be under the situation that does not change equipment and operating condition for black mold being seeded to behind the fermention medium 60-70 hour; Improve the production capacity of Hydrocerol A, thereby improve the utilization ratio of integral device.
According to the present invention, there is not special qualification for said monose or the addition manner that contains the aqueous solution of monose, can once add, repeatedly add or add continuously.Repeatedly add to make with interpolation continuously that total reducing sugar keeps within the specific limits in the fermented liquid, therefore, addition manner is preferably and repeatedly adds or add continuously.
When addition manner when repeatedly adding; Preferably make repeatedly to add to be evenly distributed on and add in the time period, the timed interval of adjacent twice interpolation is not more than 8 hours, preferably; The timed interval of adjacent twice interpolation is not more than 4 hours; The amount of each monose that adds can be identical or different, and under the preferable case, the amount of each monose that adds is identical.
In order to obtain the higher Hydrocerol A production capacity and the stability of fermentation reaction, said addition manner further is preferably continuous interpolation, and the amount of the monose of said continuous interpolation makes that total reducing sugar remains 2-5 weight % in the said fermented liquid.
According to the present invention, there is not strict requirement for the form of the monose of said interpolation, can and/or contain the aqueous solution of monose for the monose solid.Total reducing sugar keeps improving final Hydrocerol A production capacity within the specific limits in the fermented liquid, and therefore, the concentration of monose is more than the 25 weight % in the preferred aqueous solution that contains monose that adds.
According to the present invention, said monose can be for any monose that contains 6 carbon atoms, like glucose; Fructose; Semi-lactosis etc. are preferably glucose and/or fructose, generate the natural matter monose in the Hydrocerol A process because glucose is fermentation of Aspergillus niger; Consider cost factor simultaneously, said monose is preferably glucose.
According to the present invention, the said aqueous solution that contains monose is the various aqueous solution that contain monose, and preferably, the said aqueous solution that contains monose is glucose mother liquid.Said glucose mother liquid is the known notions of those skilled in the art, when referring to the last crystallization process production of industry glucose, and the glucose solution that is left after the crystallization.Comparing embodiment 1 can be found out with embodiment 2; Embodiment 1 utilizes glucose mother liquid as the monose that adds; Embodiment 2 utilizes pure D/W as the monose that adds; Embodiment 1 and embodiment 2 can access suitable Hydrocerol A production capacity, and utilize glucose mother liquid can more effectively to reduce production costs as the monose that adds, so the present invention preferably uses glucose mother liquid as the monose that adds.
According to method of the present invention; This method also comprises adds nitrogenous source in fermented liquid, the time of adding nitrogenous source accomplished in the preceding 5 hours time period to fermentation after black mold is seeded to fermention medium in 24 hours, and the effect of adding nitrogenous source is in order better to keep the growth of black mold; Therefore under the preferable case; In the nitrogen element, the addition of said nitrogenous source can further be preferably 0.1-0.2 weight % for the 0.033-0.35 weight % of the amount of the monose of said interpolation.
According to the present invention; After black mold is seeded to fermention medium in 24 hours, the concrete time of adding said nitrogenous source is not had special qualification, can be identical or different with the interpolation time of monose; Preferably; The interpolation time of said nitrogenous source is close with the interpolation time of said monose, and more preferably, said nitrogenous source and monose add simultaneously.It should be explicitly made clear at this point that the amount of the nitrogenous source that adds simultaneously and the amount of monose meet above-mentioned nitrogenous source and add the proportionlity between total amount and the monose interpolation total amount.
According to the present invention, said nitrogenous source can comprise organic nitrogen source and/or inorganic nitrogen-sourced as the grow material of required nitrogenous source of black mold for any.Preferably, said nitrogenous source is at least a in urea, ammonium sulfate, an ammonium nitrate, zein and the steeping water.The nutritive ingredient that contains other in the organic nitrogen source, producing Hydrocerol A for fermentation of Aspergillus niger has better promoter action, and therefore more preferably, said nitrogenous source is an organic nitrogen source, like zein and/or steeping water.
Fermenting process is the biochemical reaction process of being participated in by mikrobe with regard to its essence, so the quantity of microorganism cells, state, metabolism situation are to the biosynthesizing important influence of product.The size of cell concentration is to the productive rate important influence of tunning.Cell concentration is big more in theory, and the output of product is also big more, can produce other influences but cell concentration is too high; Consume too fast like nutritive substance; Nutritive ingredient in the fermented liquid takes place significantly to change, the accumulation of toxic substance etc., and these possibly change the pathways metabolism of thalline.Therefore, for citric acid fermentation, inoculum size is 2.4 * 10
4Individual colony-forming unit just can reach best acid production rate, not only more inoculum size can not improve final Hydrocerol A production capacity, can influence the Hydrocerol A production capacity on the contrary.
But contriver of the present invention finds under study for action, in the present invention, inoculates the aspergillus niger strain higher than convention amount and has obtained higher Hydrocerol A transformation efficiency and single output on the contrary.In embodiments of the invention 1, inoculated 3.5 * 10
4Individual aspergillus niger strain forms unit, and has added monose and nitrogenous source, and the transformation efficiency of Hydrocerol A and single output are respectively 96.77% and 45.69 kilogram among the embodiment 1; Comparative Examples 2 is inoculated the black mold of same amount with embodiment 1, but does not add monose and nitrogenous source, and the transformation efficiency of Hydrocerol A and single output are respectively 93.31% and 33.32 kilogram in the Comparative Examples 2; The aspergillus niger strain of embodiment 4 inoculation convention amounts has added monose and nitrogenous source, and the transformation efficiency of Hydrocerol A and single output are respectively 95.60% and 42.88 kilogram among the embodiment 4; The aspergillus niger strain of Comparative Examples 3 inoculation convention amounts, but do not add monose and nitrogenous source, the transformation efficiency of Hydrocerol A and single output are respectively 94% and 33.57 kilogram in the Comparative Examples 3; Comparing embodiment 1 can be found out with Comparative Examples 3 with embodiment 4 with Comparative Examples 2 respectively; Under the condition of adding monose and nitrogenous source; No matter whether improve the amount of the black mold of inoculating in the fermentor tank; Can both improve the transformation efficiency and the single output of Hydrocerol A, comparing embodiment 1 can be found out with embodiment 4, under the situation of adding monose and nitrogenous source; The amount that improves the black mold of inoculating in the fermentor tank can improve the transformation efficiency and the single output of Hydrocerol A; But relatively Comparative Examples 2 can be found out with Comparative Examples 3, adds in the absence of monose and nitrogenous source, and the amount that is the black mold inoculated in the simple raising fermentor tank causes the transformation efficiency of Hydrocerol A and the reduction of single output on the contrary.
Therefore, among the present invention, be benchmark with every gram fermention medium, the inoculum size of said black mold can be 1.8 * 10
4-5.3 * 10
4Individual colony-forming unit preferably, is a benchmark with every gram fermention medium, and the inoculum size of said black mold is 2.5 * 10
4-4.0 * 10
4Individual colony-forming unit.
Among the present invention, the condition of said fermentation has no particular limits, and can be the conventional fermentation condition in this area, and for example, the condition of said fermentation can comprise: temperature is 30-40 ℃, is preferably 30-35 ℃; The pH value is 1-7, is preferably 2-4, and air flow is the 0.1-1 volume: volume minute; Be preferably the 0.2-0.8 volume: volume minute; Time is 75-95 hour, is preferably 80-90 hour.
The method of a certain amount of bacterium liquid through cast or coating after will diluting that be defined as of said colony-forming unit lets unicellular being dispersed in one by one on the culture medium flat plate of mikrobe in it, and after waiting to cultivate, each viable cell just forms a bacterium colony.It is the single celled number that contains in every milliliter of bacterium liquid.
Said colony-forming unit can be measured by means commonly known in the art, for example, counts through blood counting chamber.
According to the present invention, the composition of fermention medium there is not special requirement, as long as can be used for the fermention medium of citric acid fermentation.Preferably; It is 13-21 weight % that said fermention medium contains starchy material enzymolysis product carbon source content, and nitrogenous source content is 0.06-0.14 weight %, and phosphorus source content is 0.005-0.07 weight %; Inorganic salt content 0.1-2.6 weight %, water-content are 77-86 weight %.Usually; The starchy material enzymolysis obtains starchy material enzymolysis residue and starchy material enzymatic liquefaction clear liquid; Usually can starchy material enzymatic liquefaction clear liquid be used to prepare fermention medium, also can with starchy material enzymatic liquefaction clear liquid be used to prepare fermention medium after starchy material enzymolysis residue mixes.Starchy material enzymolysis product according to the invention is preferably mixed with water or is not mixed with water by starchy material enzymolysis residue and starchy material enzymatic liquefaction clear liquid and obtains; And further preferred gross weight with said fermention medium is that 100 weight parts are benchmark; The consumption of said starchy material enzymatic liquefaction clear liquid is the 80-95 weight part; The consumption of said starchy material enzymolysis residue is the 0.5-10 weight part, and the consumption of water is the 0-15 weight part.
According to the present invention, said starchy material enzymatic liquefaction clear liquid can prepare through several different methods, for example; Can prepare through following method: starchy material is pulverized, the product after pulverizing is carried out enzymolysis, obtain enzymolysis product; With the enzymolysis product solid-liquid separation; Obtain starchy material enzymatic liquefaction clear liquid and starchy material enzymolysis residue, it is 5-60 weight %, more preferably 30-50 weight % that the condition of said solid-liquid separation makes the solid content of starchy material enzymolysis residue.
According to the present invention; Said starchy material can be the known various raw materials that contain starch that can be used for enzymolysis, fermentative prepn Hydrocerol A of ability, for example, can be selected from corn, potato class (like cassava) and the wheat one or more; Under the preferable case, said starchy material is a corn.
Said enzymolysis step can be accomplished through this area method commonly used, and such as in crushed products, adding microbes producing cellulase and/or enzyme, insulation is accomplished under the growth temperature of microbes producing cellulase and/or the great-hearted temperature of enzyme.Said microbes producing cellulase be can secreting amylase microbes producing cellulase.Said enzyme comprises glycase.
Because microorganism growth can produce by product, the therefore preferred enzyme that directly adds.The consumption of said enzyme is The more the better, from cost consideration, and the dry weight basis of the crushed products after preferably pulverizing with every gram, said diastatic consumption is a 15-50 enzyme activity unit.
Being defined as of the enzyme activity unit of enzyme according to the invention: be 6.0 in the pH value, temperature is that 1 minute is converted into the required enzyme amount of reducing sugar with 1 milligram of starch is an enzyme activity unit under 70 ℃ the condition.
The temperature of said enzymolysis can in very large range change, and is preferably 70-105 ℃, more preferably 80-95 ℃.The longer the better on the time theory of said enzymolysis, considers plant factor, and the time of preferred said enzymolysis is 90-150 minute, more preferably 100-120 minute.The pH value of said enzymolysis can in very large range change, and is preferably 5.0-7.0, and more preferably the pH value is 5.4-5.7.
Glycase is meant the general name of class of enzymes that can the starch-splitting glycosidic link, and said glycase generally comprises AMS, beta-amylase, saccharifying enzyme and isoamylase.
According to the present invention, preferably use AMS and/or isoamylase.
According to the present invention, the method and apparatus of said solid-liquid separation is conventionally known to one of skill in the art, for example, and pressure filter or whizzer.
Said black mold can adopt conventional method inoculation, for example, in being seeded to fermention medium before, said black mold is handled through seed culture, afterwards the seed liquor that obtains is joined in the fermention medium.The degree of black mold seed culture can be measured through sampling sediments microscope inspection, acid test and pH and observe the growth of black mold, when pH 2.0-2.5, acidity 0.5-2.0%, bacterium ball size evenly, mycelia is sturdy stops to cultivate when stretching out.
Under the preferable case, the method that said seed culture is handled comprises: black mold is seeded in the black mold nutrient solution cultivates, contain the Semen Maydis powder of 10-17 weight % in the said black mold nutrient solution, the concentration of black mold is 3 * 10 in the black mold nutrient solution of inoculation back
5-4 * 10
5Individual/milliliter.
According to the present invention, the preparation method of said black mold nutrient solution has no particular limits, as long as the nutrient solution that obtains can be applicable to the cultivation of black mold.
According to the present invention; The culture condition of said black mold can in very large range change; For example said culture condition can comprise: the temperature of cultivation can be 25-45 ℃; The pH value can be 1-7, and air flow can be the 0.05-0.5 volume: volume minute, and the time of cultivation can be 45-65 hour; Under the preferred situation, said culture condition can comprise: the temperature of cultivation can be 30-40 ℃, and the pH value can be 2-4, and air flow can be the 0.1-0.3 volume: volume minute, the time of said cultivation can be 50-60 hour.
Term " air flow " is generally with ventilation expression recently, and usually recently to represent (V/Vmin) through the volume of air of unit volume nutrient solution in the PM, for example ventilation is than being 1: 0.1-1, the abbreviation air flow is the 0.01-1 volume: volume minute.
The equipment of said cultivation is conventionally known to one of skill in the art, for example, can use fermentor tank to cultivate.
The tunning Hydrocerol A for preparing according to method of the present invention can be used conventional method, separate and refining according to the requirement of different Industrial products, such as neutralization, acidolysis, decolouring, concentrate, crystallization, packing.
The present invention will be described in more detail below in conjunction with embodiment.
Embodiment 1
Present embodiment is used to explain the working method of Hydrocerol A provided by the invention.
(1) 56 kg corn that will gather in the crops are stewing in hot water tank profit, are 15 weight % until the water cut of corn, pulverize then, obtain average particle diameter and be 400 microns pulverizing after product.
(2) product after will pulverizing is sized mixing by the concentration of 25 weight %; With respect to the product after every gram pulverizing, add glycase (Novozymes Company, the AMS of 20 enzyme activity units; Equal glycase for this reason in the embodiment of the invention); Getting into injector, is enzymolysis 100 minutes under 5.5 the condition at 85 ℃, pH, obtains enzymolysis product A1.
(3) with enzymolysis product A1 through carrying out press filtration with the fluid pressure type plate-and-frame filter press, isolate enzymatic liquefaction clear liquid and enzymolysis filter residue, wherein, the water cut of enzymolysis residue is 50%.
(4) preparation fermention medium; With joining in the fermentor tank of 300L after 170 kilograms above-mentioned enzymatic liquefaction clear liquids, 2.0 kilograms the water sterilization of enzymolysis residue and 13 kilograms, obtain fermention medium B1, wherein carbon source content is 16 weight %; Nitrogenous source content is 0.095 weight %; Phosphorus source content is 0.06 weight %, inorganic salt content 0.4 weight %, and water-content is 83.4%.
(5) with the part enzymatic liquefaction liquid in the step (2), 10 weight % of thin up to total reducing sugar obtain nutrient solution; Nutrient solution is dropped into seeding tank, be heated to 121 ℃ of sterilizations, keep after 30 minutes fast cooling to 36 ℃; Insert aspergillus niger strain (black mold T01; Tianjin industrial microorganism institute, equal aspergillus niger strain for this reason in the embodiment of the invention, inoculum size is: every gram enzymatic liquefaction liquid 3 * 10
5Individual colony-forming unit), at 36 ℃, 0.4 volume: carry out spawn culture under the aeration condition of volume minute; Measure through sampling sediments microscope inspection, acid test and pH the growth of black mold observed, when pH 2.0, acidity 1%, bacterium ball size evenly, mycelia is sturdy when stretching out, stop to cultivate.
(6) aspergillus niger strain of step (5) being cultivated joins in the fermentor tank of step (4) and begins fermentation, and detects the total reducing sugar in the fermented liquid during the fermentation, and inoculum size is: 3.5 * 104 colony-forming units of every gram fermention medium; Fermentation condition comprises that temperature is 35 ℃, and the pH value is 3, and air flow is 0.4 volume: volume minute;, fermentation (begins the fermentor tank to calculate when proceeding to the 45th hour from black mold is inoculated into; Fermentation time in the embodiments of the invention all so calculates), adopt the mode of adding continuously, add 28 kilograms interpolation solution evenly; Said interpolation solution is that urea concentration is the glucose mother liquid of 0.16 weight %; Wherein the concentration of glucose is 50 weight %, when fermentation proceeds to the 65th hour, stops to add, and the total sugar content that adds fermented liquid in the process of adding solution is 3.5 weight %; Carry out solid-liquid separation when fermentation proceeds to the 80th hour, obtain citric acid solution.
Concentration (abbreviation acidity) according to gained citric acid solution among the GB 1987-2007 standard detection embodiment 1; Calculate the transformation efficiency and the single output of Hydrocerol A; Weight * 100% of the volume/total reducing sugar of the concentration of transformation efficiency (%)=citric acid solution (abbreviation acidity) * citric acid solution; The volume of the concentration * citric acid solution of the single output=citric acid solution of Hydrocerol A, the result is as shown in table 1.
Comparative Examples 1
Produce Hydrocerol A according to the described method of embodiment 1, different is, the glucose with embodiment 1 same amount in the step (6) joins in the fermentor tank in fermentation with urea before, and promptly step (6) is:
The interpolation solution that in fermentor tank, adds 28 kilograms; Said interpolation solution is that urea concentration is the glucose mother liquid of 0.16 weight %; Wherein the concentration of glucose is 50 weight %; The aspergillus niger strain that step (5) is cultivated joins in the fermentor tank of step (4) and begins fermentation, and detects the total reducing sugar in the fermented liquid, and inoculum size is: every gram fermention medium 3.5 * 10
4Individual colony-forming unit, fermentation condition comprise that temperature is 35 ℃, and the pH value is 3, and air flow is 0.4 volume: volume minute, and when proceeding to the 80th hour, fermentation carries out solid-liquid separation, obtain citric acid solution.
Comparative Examples 2
Produce Hydrocerol A according to the described method of embodiment 1, different is, in step (6), does not add sugar and urea, but adds 28 kilograms fermention medium B1, and promptly the step of Comparative Examples 2 (6) is:
The aspergillus niger strain that step (5) is cultivated joins in the fermentor tank of step (4) and begins fermentation, and detects the total reducing sugar in the fermented liquid, and inoculum size is: every gram fermention medium 3.5 * 10
4Individual colony-forming unit, fermentation condition comprise that temperature is 35 ℃, and the pH value is 3; Air flow is 0.4 volume: volume minute; When fermentation proceeds to the 45th hour, adopt the mode of adding continuously, add 28 kilograms fermention medium B1 evenly; Carry out solid-liquid separation when fermentation proceeds to the 80th hour, obtain citric acid solution.
Embodiment 2
Present embodiment is used to explain the working method of Hydrocerol A provided by the invention.
(1) 56 kilograms corn will gathering in the crops is stewing in hot water tank profit, is 20 weight % until the water cut of corn, pulverizes then, obtains average particle diameter and be 800 microns pulverizing after product.
(2) product after will pulverizing is sized mixing by the concentration of 25 weight %, and the product after pulverizing with respect to every gram adds the glycase of 45 enzyme activity units, gets into injector, is enzymolysis 90 minutes under 5 the condition at 90 ℃, pH, obtains enzymolysis product A2.
(3) with enzymolysis product A2 through carrying out press filtration with the fluid pressure type plate-and-frame filter press, isolate enzymatic liquefaction clear liquid and enzymolysis filter residue, wherein, the water cut of enzymolysis residue is 30%.
(4) preparation fermention medium joins in the fermentor tank after specifically consisting of 160 kilograms enzymatic liquefaction clear liquid, 9 kilograms the water sterilization of enzymolysis residue and 16 kilograms, obtains fermention medium B2.Wherein carbon source content is 18.6 weight %, and nitrogenous source content is 0.1 weight %, and phosphorus source content is 0.055 weight %, inorganic salt content 0.17 weight %, and water-content is 81%
(5) with the part enzymatic liquefaction liquid that obtains in the step (2), thin up to total reducing sugar 10% obtains nutrient solution; Nutrient solution is dropped into seeding tank, drop into seeding tank, add urea; The add-on of urea is 0.35% of a nutrient solution gross weight; Be heated to 120 ℃ of sterilizations, keep after 20 minutes fast cooling to 36 ℃, (inoculum size is: every gram enzymatic liquefaction clear liquid 3.5 * 10 to insert aspergillus niger strain
5Individual colony-forming unit), at 36 ℃, 0.4 volume: carry out spawn culture under the aeration condition of volume minute; Measure through sampling sediments microscope inspection, acid test and pH the growth of black mold observed, when pH 2.0, acidity 1%, bacterium ball size evenly, mycelia is sturdy when stretching out, stop to cultivate.
(6) aspergillus niger strain of step (5) being cultivated joins in the fermentor tank of step (4) and begins fermentation, and detects the total reducing sugar in the fermented liquid during the fermentation, and inoculum size is: every gram fermention medium 2.5 * 10
4Individual colony-forming unit, fermentation condition comprise that temperature is 30 ℃, and the pH value is 2; Air flow is 1 volume: volume minute, when fermentation proceeds to the 40th hour, adopt the mode of adding continuously; The interpolation solution that adds 30 kilograms evenly, said interpolation solution are that urea concentration is the D/W (promptly add and have only urea in the solution, glucose and water) of 0.125 weight %; Wherein the concentration of glucose is 30 weight %, when fermentation proceeds to the 60th hour, stops to add, and the total sugar content that adds fermented liquid in the process of adding solution is 4.5 weight %; Carry out solid-liquid separation when fermentation proceeds to the 83rd hour, obtain citric acid solution.
Embodiment 3
Present embodiment is used to explain the working method of Hydrocerol A provided by the invention.
(1) 56 kg corn that will gather in the crops are stewing in hot water tank profit, are 10 weight % until the water cut of corn, pulverize then, obtain average particle diameter and be 500 microns pulverizing after product.
(2) product after will pulverizing is sized mixing by the concentration of 25 weight %, and the product after pulverizing with respect to every gram adds the glycase of 30 enzyme activity units, gets into injector, is enzymolysis 120 minutes under 6 the condition at 80 ℃, pH, obtains enzymolysis product A3.
(3) with enzymolysis product A3 through carrying out press filtration with the fluid pressure type plate-and-frame filter press, isolate enzymatic liquefaction clear liquid and enzymolysis filter residue, wherein, the water cut of enzymolysis residue is 15%.
(4) the preparation fermention medium specifically consists of 155 kilograms enzymatic liquefaction clear liquid, 12 kilograms enzymolysis residue and 18 kilograms water, joins after the sterilization in the fermentor tank, obtains fermention medium B3.Wherein carbon source content is 14.75 weight %, and nitrogenous source content is 0.12 weight %, and phosphorus source content is 0.015 weight %, inorganic salt content 2.4 weight %, and water-content is 82.7%.
(5) with the part enzymatic liquefaction clear liquid in the step (3), thin up to total reducing sugar 10% drops into seeding tank, obtains nutrient solution; Nutrient solution is dropped into seeding tank; Add urea, the add-on of urea is 0.35% of a seed tank culture liquid gross weight, is heated to 120 ℃ of sterilizations; Keep after 20 minutes fast cooling to 36 ℃, (inoculum size is: every gram enzymatic liquefaction clear liquid 4 * 10 to insert aspergillus niger strain
5Individual colony-forming unit), at 36 ℃, 0.4 volume: carry out spawn culture under the aeration condition of volume minute; Measure through sampling sediments microscope inspection, acid test and pH the growth of black mold observed, when pH 2.0, acidity 1%, bacterium ball size evenly, mycelia is sturdy when stretching out, stop to cultivate.
(6) aspergillus niger strain of step (5) being cultivated joins in the fermentor tank of step (4) and begins fermentation, and detects the total reducing sugar in the fermented liquid during the fermentation, and inoculum size is: every gram fermention medium 4 * 10
4Individual colony-forming unit, fermentation condition comprise that temperature is 32 ℃, and the pH value is 4; Air flow is 0.1 volume: volume minute, when fermentation proceeds to the 50th hour, adopt the mode of adding continuously; The interpolation solution that adds 18 kilograms evenly, said interpolation solution are that urea concentration is the glucose mother liquid of 0.088 weight %, and wherein the concentration of glucose is 40 weight %;, fermentation stops to add when proceeding to the 70th hour; The total sugar content that adds fermented liquid in the process of adding solution is 2.5 weight %, carries out solid-liquid separation when fermentation proceeds to the 88th hour, obtains citric acid solution.
Embodiment 4
Present embodiment is used to explain the working method of Hydrocerol A provided by the invention.
Produce Hydrocerol A according to embodiment 1 described method, different is that the inoculum size of black mold is in the step (6): every gram fermention medium 2.4 * 10
4Individual colony-forming unit.
Comparative Examples 3
Produce Hydrocerol As according to the described methods of embodiment 4, different is, step is not added sugar and urea in (6), and promptly the step of Comparative Examples 3 (6) is:
The aspergillus niger strain that step (5) is cultivated joins in the fermentor tank of step (4) and begins fermentation, and detects the total reducing sugar in the fermented liquid, and inoculum size is: every gram fermention medium 2.4 * 10
4Individual colony-forming unit, fermentation condition comprise that temperature is 35 ℃, and the pH value is 3, and air flow is 0.4 volume: volume minute, and ferment and carry out solid-liquid separation after 88 hours, obtain citric acid solution.
Embodiment 5
Present embodiment is used to explain the working method of Hydrocerol A provided by the invention.
Produce Hydrocerol As according to the described methods of embodiment 2, different is, in the step (6), the concentration of adding 30 kilograms is that the concentration of 30 kilograms of the fructose water solution replacements of 30 weight % is the D/W of 30 weight %.
Embodiment 6
Present embodiment is used to explain the working method of Hydrocerol A provided by the invention.
Produce Hydrocerol A according to the described method of embodiment 1, different is, step is not added urea in (6), and promptly the step of embodiment 6 (6) is:
The aspergillus niger strain that step (5) is cultivated joins in the fermentor tank of step (4) and begins fermentation, and detects the total reducing sugar in the fermented liquid, and inoculum size is: every gram fermention medium 3.5 * 10
4Individual colony-forming unit, fermentation condition comprise that temperature is 35 ℃, and the pH value is 3; Air flow is 0.4 volume: volume minute, when fermentation proceeds to the 45th hour, adopt the mode of adding continuously; The interpolation solution that adds 28 kilograms evenly, said interpolation solution are that glucose concn is the glucose mother liquid of 50 weight %, when fermentation proceeds to the 65th hour, stop to add; Carry out solid-liquid separation when fermentation proceeds to the 80th hour, obtain citric acid solution.
Embodiment 7
Present embodiment is used to explain the working method of Hydrocerol A provided by the invention.
Produce Hydrocerol A according to the described method of embodiment 1, different is, monose and nitrogenous source in the step (6) add several times, and promptly the step of embodiment 7 (6) is:
The aspergillus niger strain that step (5) is cultivated joins in the fermentor tank of step (4) and begins fermentation, and detects the total reducing sugar in the fermented liquid during the fermentation, and inoculum size is: every gram fermention medium 3.5 * 10
4Individual colony-forming unit, fermentation condition comprise that temperature is 35 ℃, and the pH value is 3; Air flow is 0.4 volume: volume minute, and, fermentation divides to add 28 kilograms of glucose mother liquids 6 times when proceeding to the 45th hour, and wherein the concentration of glucose is 50 weight %; Added once in average per 4 hours; In 10 minutes after each interpolation glucose solids, 0.15 kilogram of concentration of disposable interpolation is 20% zein, when fermentation proceeds to the 65th hour, stops to add; When fermentation proceeds to the 80th hour, carry out solid-liquid separation, obtain citric acid solution.
Table 1
| Numbering | Fermenting acidity % | Fermentation conversion rate (%) | The single output (kg) of Hydrocerol A |
| Embodiment 1 | 18.65 | 96.77 | 45.69 |
| Comparative Examples 1 | 15.60 | 92.50 | 38.22 |
| Comparative Examples 2 | 13.60 | 93.31 | 33.32 |
| Embodiment 2 | 18.55 | 96.23 | 45.45 |
| Embodiment 3 | 18.50 | 95.96 | 45.33 |
| Embodiment 4 | 17.50 | 95.60 | 42.88 |
| Comparative Examples 3 | 13.70 | 94.00 | 33.57 |
| Embodiment 5 | 18.40 | 95.43 | 45.08 |
| Embodiment 6 | 15.70 | 94.50 | 38.47 |
| Embodiment 7 | 18.30 | 95.89 | 44.84 |
Data from last table 1 can be found out; Production methods of citric acid provided by the invention; Under the prerequisite that does not change equipment and reaction conditions, higher product acidacidity, transformation efficiency and Hydrocerol A single output have been obtained, under constant situation of each production cycle of producing; The raising of Hydrocerol A single output has improved the utilization ratio of integral device undoubtedly, has improved the single output of integrated artistic.
Comparative Examples 1 is just added glucose and urea in the fermentation beginning; Than embodiment 1; The product acidacidity and the transformation efficiency of Comparative Examples 1 differ greatly, even are lower than the Comparative Examples 2 of not adding glucose and urea, explain and select proper time point adding glucose and urea can realize the present invention.
In the embodiments of the invention 1, inoculated 3 * 10
4Individual aspergillus niger strain forms unit, and has added monose and nitrogenous source, and the transformation efficiency of Hydrocerol A and single output are respectively 96.77% and 45.69 kilogram among the embodiment 1; The black mold of Comparative Examples 2 embodiment 1 inoculation same amount, but monose and nitrogenous source do not added, and the transformation efficiency of Hydrocerol A and single output are respectively 93.31% and 33.32 kilogram in the Comparative Examples 2; The aspergillus niger strain of embodiment 4 inoculation convention amounts, and added monose and nitrogenous source, the transformation efficiency of Hydrocerol A and single output are respectively 95.60% and 42.88 kilogram among the embodiment 4; The aspergillus niger strain of Comparative Examples 3 inoculation convention amounts, but do not add monose and nitrogenous source, the transformation efficiency of Hydrocerol A and single output are respectively 94% and 33.57 kilogram in the Comparative Examples 3; Comparing embodiment 1 can be found out with Comparative Examples 3 with embodiment 4 with Comparative Examples 2 respectively; Under the condition of adding monose and nitrogenous source; No matter whether improve the amount of the black mold of inoculating in the fermentor tank; Can both improve the transformation efficiency and the single output of Hydrocerol A, comparing embodiment 1 can be found out with embodiment 4, under the situation of adding monose and nitrogenous source; The amount that improves the black mold of inoculating in the fermentor tank can improve the transformation efficiency and the single output of Hydrocerol A; But relatively Comparative Examples 2 can be found out with Comparative Examples 3, adds in the absence of monose and nitrogenous source, and the amount that is the black mold inoculated in the simple raising fermentor tank causes the transformation efficiency of Hydrocerol A and the reduction of single output on the contrary.This shows that under the situation of adding monose and nitrogenous source, suitably raising is inoculated into that the amount of black mold is a preferred implementation of the present invention in the fermentor tank.
The sugar that adds among the embodiment 5 is fructose water solution, compares with embodiment 1, and the transformation efficiency of the Hydrocerol A of embodiment 5 and single output are lower slightly; Explain that fructose also can as the carbon source that is converted into Hydrocerol A, and can be accessed higher Hydrocerol A transformation efficiency and single output by the black mold utilization; But in present industrial production, the preparation cost of fructose is higher than glucose, and is farther far above glucose mother liquid; Therefore, using glucose mother liquid is preferred implementation of the present invention.
Embodiment 6 compares with embodiment 1; Do not add urea; The transformation efficiency of the Hydrocerol A of embodiment 6 is compared with embodiment 1 with single output and is differed bigger; Explanation is seeded to behind the fermention medium 32 hours with black mold and accomplishes in the preceding 5 hours time period to fermentation, and not only having added monose but also added nitrogenous source is preferred implementation of the present invention.
Embodiment 7 compares with embodiment 1, and monose and nitrogenous source all are to add several times, compares with embodiment 1, and the single rate ratio embodiment 1 of Hydrocerol A is lower slightly among the embodiment 7, explains that adding monose and nitrogenous source continuously is preferred implementation of the present invention.
Claims (17)
1. the working method of a Hydrocerol A; This method is included under the condition that generates Hydrocerol A; Black mold is seeded in the fermention medium ferments, obtain fermented liquid, it is characterized in that; This method also comprises in fermented liquid adds the monose with 6 carbon atoms, and the time of adding the monose with 6 carbon atoms accomplished in the preceding 5 hours time period to fermentation after black mold is seeded to fermention medium in 24 hours.
2. method according to claim 1, wherein, the weight ratio of the addition of said monose and fermented liquid total amount is 1: 8-100.
3. method according to claim 2, wherein, the weight ratio of the addition of said monose and fermented liquid total amount is 1: 13-28.
4. method according to claim 1, wherein, the time that begins to add the monose with 6 carbon atoms is for being seeded to black mold behind the fermention medium 24-56 hour.
5. method according to claim 4, wherein, the time that begins to add the monose with 6 carbon atoms is for being seeded to black mold behind the fermention medium 40-50 hour.
6. method according to claim 5, wherein, the time of finish adding the monose with 6 carbon atoms is for being seeded to black mold behind the fermention medium 60-70 hour.
7. according to any described method among the claim 1-6, wherein, said monose adds several times, and the timed interval of adjacent twice interpolation is not more than 8 hours, and the amount of each monose that adds is identical.
8. method according to claim 7, wherein, the mode of said interpolation is to add continuously.
9. method according to claim 8, wherein, the amount of the monose of said continuous interpolation makes that the total sugar content of fermented liquid described in the interpolation process is 2-5 weight %.
10. method according to claim 1, wherein, said monose adds with monose solid and/or the form that contains the aqueous solution of monose, and the concentration of monose is more than the 25 weight % in the said aqueous solution that contains monose.
11. according to claim 1 or 10 described methods, wherein, said monose is glucose, the said aqueous solution that contains monose is glucose mother liquid.
12. according to any described method among claim 1-6, the 8-10 and 11; Wherein, This method also comprises adds nitrogenous source in fermented liquid; The time of adding nitrogenous source accomplished in the preceding 5 hours time period to fermentation after black mold is seeded to fermention medium in 24 hours, and in the nitrogen element, and the addition of said nitrogenous source is the 0.033-0.35 weight % of amount of the monose of said interpolation.
13. method according to claim 12, wherein, said nitrogenous source and monose add simultaneously.
14. method according to claim 13, wherein, said nitrogenous source is at least a in urea, ammonium sulfate, an ammonium nitrate, zein and the steeping water.
15. method according to claim 1 wherein, is a benchmark with every gram fermention medium, the inoculum size of black mold is 1.8 * 10
4-5.3 * 10
4Individual colony-forming unit, the condition of said fermentation comprises: temperature is 30-40 ℃, air flow is the 0.1-1 volume: volume minute, the time of fermentation is 75-90 hour.
16. method according to claim 1 wherein, is a benchmark with every gram fermention medium, the inoculum size of black mold is 2.5 * 10
4-4.0 * 10
4Individual colony-forming unit.
17. method according to claim 1; Wherein, said fermention medium contains the starchy material enzymolysis product, and carbon source content is 13-21 weight % in the said starchy material enzymolysis product; Nitrogenous source content is 0.06-0.14 weight %; Phosphorus source content is 0.005-0.07 weight %, and inorganic salt content 0.1-2.6 weight %, water-content are 77-86 weight %.
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| CN103382491A (en) * | 2013-07-03 | 2013-11-06 | 山东寿光巨能金玉米开发有限公司 | Preparation method of citric acid |
| CN103382491B (en) * | 2013-07-03 | 2015-05-20 | 山东寿光巨能金玉米开发有限公司 | Preparation method of citric acid |
| CN103497977A (en) * | 2013-09-30 | 2014-01-08 | 中粮生物化学（安徽）股份有限公司 | Method for preparing citric acid by fermentation |
| CN109536541A (en) * | 2018-12-29 | 2019-03-29 | 吉林中粮生化有限公司 | The method of corn pulp preprocess method and production citric acid |
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