CN102443611B - Production method of citric acid - Google Patents
Production method of citric acid Download PDFInfo
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- CN102443611B CN102443611B CN201010511953.6A CN201010511953A CN102443611B CN 102443611 B CN102443611 B CN 102443611B CN 201010511953 A CN201010511953 A CN 201010511953A CN 102443611 B CN102443611 B CN 102443611B
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Abstract
The invention provides a production method of citric acid, which comprises a step of inoculating aspergillus niger into a fermentation culture medium for fermenting under a condition of generating citric acid to obtain fermentation liquid. The method is characterized by also comprising the following steps: adding monosaccharide with 6 carbon atoms into the fermentation liquid, wherein the time for adding the monosaccharide with 6 carbon atoms is in a time range from 24 hours after the aspergillus niger is inoculated into the fermentation culture liquid to 5 hours before the fermentation is completed. More citric acid can be produced by adding the monosaccharide and sufficiently utilizing the conversion function of the aspergillus niger on the premise of not changing equipment, reaction conditions and other energy consumptions. Compared with the prior art, the utilization rate of whole equipment is improved, and the capacity of the whole process is increased.
Description
Technical field
The present invention relates to a kind of production method of citric acid.
Background technology
Citric acid is first acid in organic acid, due to the excellent properties of the aspects such as physics, chemistry, is widely used in the industrial circles such as medicine, chemistry, electronics, weaving, oil, leather, building, photography, plastics, casting and pottery.
The production method of citric acid mainly contains two kinds: a kind of is to extract containing the fruit juice of citric acid from natural; Another kind is to produce with fermentation method, at present industrially mainly take fermentation of Aspergillus niger method and produces citric acid as main.Concrete way is that aspergillus niger is inoculated in fermention medium, contains starchy material enzymolysis product and nitrogenous source in fermention medium, obtains by fermentation the solution of citric acid with solid-liquid separation.At present, industrial fermentative Production citric acid conventional 3 * 10
5the fermentor tank of L, the starchy material that each fermentation is used is 5.6 * 10
4kilogram, the citric acid 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 method for the number of increase fermentor tank or other change existing installations certainly will cause the very big increase of industrial production cost like this.
Therefore, in the urgent need to developing a kind of existing installation that do not change, just can make aspergillus niger bring into play the method for its maximum utility, raising citric acid production capacity.
Summary of the invention
The object of this invention is to provide a kind of production method that can effectively improve the citric acid of citric acid production capacity.
The present inventor finds through research, the lower reason of fermentation of Aspergillus niger method integrated artistic production capacity is that the utilization ratio of aspergillus niger is not high at present, the fermenting process of aspergillus niger roughly can be divided into three phases, the one, earlier fermentation, it is mainly the growth phase of aspergillus niger, thalline produces saccharifying enzyme simultaneously, saccharifying enzyme is converted into the utilizable glucose of aspergillus niger by the polysaccharide in raw material, glucose is through a series of pathways metabolisms, by conversion of glucose, be citric acid, because sugar has been synthesized organic acid by aspergillus niger utilization, make the decline of pH value enter lord ferment period; The 2nd, lord ferment period, produces acid phase, and in this stage, citric acid accumulates in a large number; The 3rd, the fermentation later stage, i.e. in the self-dissolving stage, along with exhausting of nutrient, tropina enzyme comes to life, and causes pH value in rising trend again, and now thalline is tending towards self-dissolving and Metabolic activity termination.In whole fermenting process, enter after subordinate phase, because pH value declines, the activity of saccharifying enzyme is suppressed, and causes in lord ferment period process, and part aspergillus niger does not have enough glucose to transform, and has caused the significant wastage of bioenergy and equipment.
The invention provides a kind of production method of citric acid, the method is included under the condition that generates citric acid, aspergillus niger is seeded in fermention medium and is fermented, obtain fermented liquid, it is characterized in that, the method also comprises to adding the monose with 6 carbon atoms in fermented liquid, the time of adding the monose with 6 carbon atoms after aspergillus niger is inoculated into fermention medium 24 hours to time period of 5 hours before having fermented.
The present invention is by adding monose, take full advantage of the transformation function of aspergillus niger, do not changing under the prerequisite of the energy consumptions such as equipment and reaction conditions, obtained higher citric acid production capacity, in embodiment 1 and comparative example 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 citric acids, and comparative example 2 only obtains 33.32 kilograms of citric acids.As can be seen here, compared to existing technology, 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 production method of citric acid, the method is included under the condition that generates citric acid, aspergillus niger is seeded in fermention medium and is fermented, obtain fermented liquid, it is characterized in that, the method also comprises to adding the monose with 6 carbon atoms in fermented liquid, the time of adding the monose with 6 carbon atoms after aspergillus niger is seeded to fermention medium 24 hours to time period of 5 hours before having fermented.
Wherein, the concept that described fermention medium is known to the skilled person, refer to the nutriment with the artificial preparation maintaining for microorganism growth that microorganism fermentation is required, generally all contain carbohydrate, nitrogenous substances, inorganic salt (comprising trace element) and VITAMIN and water etc.The concept that described fermented liquid is also known to the skilled person, refers to an access the liquid nutrient medium (this liquid nutrient medium is also alleged fermention medium in the present invention) of microbial strains, products therefrom after cultivation after a while.
According to the present invention, the addition of described monose can change in a big way, consider the transformation function that should make full use of aspergillus niger, again in conjunction with cost factor, under preferable case, the weight ratio of the addition of described monose and fermented liquid total amount is 1: 8-100, more preferably 1: 10-50, most preferably is 1: 13-28.
Adding monose is in order to make full use of the transformation function of aspergillus niger, the time point adding is very crucial, too early interpolation has caused the reduction of transformation efficiency on the contrary, comparative example 1 of the present invention was just being added monose before aspergillus niger is inoculated into fermention medium in fermented liquid, in comparative example 1, the transformation efficiency of citric acid is only 92.5%, also lower than the transformation efficiency 93.31% that does not add citric acid in the comparative example 2 of monose.Trace it to its cause and may have two aspects, the first, in comparative example 1, carbon source is too much, causes monose to be converted into the speed of citric acid too fast, and the speed that pH value reduces is also too fast, causes the not enough and thalline early ageing of thalli growth; The second, in comparative example 1, added monose, at the high foam of fermentation, during the phase, easily produce flash phenomenon, thereby cause the loss of raw material.And the interpolation in evening excessively, aspergillus niger is to the phase of decaying of growth, and the function that monose is converted into citric acid dies down, even if add monose, is also difficult to obtain good effect.
Therefore, the time that preferably starts to add the monose with 6 carbon atoms is for being seeded to aspergillus niger 24-56 hour after fermention medium, 40-50 hour more preferably, the time that preferably finishes to add the monose with 6 carbon atoms is for being seeded to aspergillus niger 60-70 hour after fermention medium, like this can be in the situation that not changing equipment and operating condition, improve the production capacity of citric acid, thereby improve the utilization ratio of integral device.
According to the present invention, for the addition manner of described monose or the aqueous solution that contains monose, there is no special restriction, can once add, repeatedly add or continuous adding.Repeatedly interpolation and continuous adding can make total reducing sugar in fermented liquid keep within the specific limits, and therefore, addition manner is preferably and repeatedly adds or continuous adding.
When addition manner is 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 adding can be identical or different, and under preferable case, the amount of each monose adding is identical.
In order to obtain higher citric acid production capacity and the stability of fermentation reaction, described addition manner is continuous adding more preferably, and the amount of the monose of described continuous adding makes total reducing sugar in described fermented liquid remain 2-5 % by weight.
According to the present invention, for the form of the monose of described interpolation, there is no strict requirement, can be monose solid and/or the aqueous solution that contains monose.In fermented liquid, total reducing sugar keeps improving within the specific limits final citric acid production capacity, and therefore, in the aqueous solution that contains monose preferably adding, the concentration of monose is more than 25 % by weight.
According to the present invention, described monose can be any monose that contains 6 carbon atoms, as glucose, fructose, semi-lactosis etc., are preferably glucose and/or fructose, because glucose is that fermentation of Aspergillus niger generates the natural matter monose in citric acid process, consider cost factor, described monose is preferably glucose simultaneously.
According to the present invention, described in contain monose the aqueous solution be the various aqueous solution that contain monose, preferably, described in contain monose the aqueous solution be glucose mother liquid.Described glucose mother liquid is the known concepts of those skilled in the art, when referring to industrial crystallization process and producing glucose, and remaining glucose solution after crystallization.Comparing embodiment 1 and embodiment 2 can find out, embodiment 1 utilizes glucose mother liquid as the monose adding, embodiment 2 utilizes pure D/W as the monose adding, embodiment 1 and embodiment 2 can access suitable citric acid production capacity, and utilize glucose mother liquid can more effectively to reduce production costs as the monose adding, so the present invention preferably uses glucose mother liquid as the monose adding.
According to method of the present invention, the method also comprises in fermented liquid adds nitrogenous source, the time of adding nitrogenous source after aspergillus niger is seeded to fermention medium 24 hours to time period of 5 hours before having fermented, the effect of adding nitrogenous source is in order better to maintain the growth of aspergillus niger, therefore under preferable case, in nitrogen element, the addition of described nitrogenous source can be the 0.033-0.35 % by weight of the amount of the monose of described interpolation, more preferably 0.1-0.2 % by weight.
According to the present invention, after aspergillus niger is seeded to fermention medium in 24 hours, to adding the concrete time of described nitrogenous source, be not particularly limited, can be identical or different with the interpolation time of monose, preferably, the interpolation time of described nitrogenous source is close with the interpolation time of described monose, and more preferably, described nitrogenous source and monose add simultaneously.It should be explicitly made clear at this point, the amount of nitrogenous source of simultaneously adding and the amount of monose meet above-mentioned nitrogenous source and add the proportionlity between total amount and monose interpolation total amount.
According to the present invention, described nitrogenous source can, as the material of the required nitrogenous source of Aspergillus Niger Growth, comprise organic nitrogen source and/or inorganic nitrogen-sourced for any.Preferably, described nitrogenous source is at least one in urea, ammonium sulfate, ammonium nitrate, zein and corn steep liquor.The nutritive ingredient that contains other in organic nitrogen source, for fermentation of Aspergillus niger, producing citric acid has better promoter action, and therefore more preferably, described nitrogenous source is organic nitrogen source, as zein and/or corn steep liquor.
Fermenting process is the biochemical reaction process being participated in by microorganism with regard to its essence, so the quantity of microorganism cells, state, metabolism situation have important impact to the biosynthesizing of product.The size of cell concentration has important impact to the productive rate of tunning.Cell concentration is larger in theory, and the output of product is also larger, but cell concentration is too high, can produce other influences, as nutritive substance consumes too fast, nutritive ingredient in fermented liquid occurs significantly to change, the accumulation of toxic substance etc., and these may 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 citric acid production capacity, can affect citric acid production capacity on the contrary.
But the present inventor finds under study for action, in the present invention, inoculates the aspergillus niger strain higher than convention amount and has obtained on the contrary higher citric acid transformation efficiency and single output.In embodiments of the invention 1, inoculated 3.5 * 10
4individual aspergillus niger strain forms unit, and has added monose and nitrogenous source, and in embodiment 1, the transformation efficiency of citric acid and single output are respectively 96.77% and 45.69 kilogram, comparative example 2 is inoculated the aspergillus niger of same amount with embodiment 1, but does not add monose and nitrogenous source, and in comparative example 2, the transformation efficiency of citric acid and single output are respectively 93.31% and 33.32 kilogram, the aspergillus niger strain of embodiment 4 inoculation convention amounts, has added monose and nitrogenous source, and in embodiment 4, the transformation efficiency of citric acid and single output are respectively 95.60% and 42.88 kilogram, the aspergillus niger strain of comparative example 3 inoculation convention amounts, but do not add monose and nitrogenous source, in comparative example 3, the transformation efficiency of citric acid and single output are respectively 94% and 33.57 kilogram, comparing embodiment 1 can be found out with comparative example 3 with comparative example 2 and embodiment 4 respectively, adding under the condition of monose and nitrogenous source, no matter whether improve the amount of the aspergillus niger of inoculating in fermentor tank, can both improve transformation efficiency and the single output of citric acid, comparing embodiment 1 and embodiment 4 can find out, in the situation that adding monose and nitrogenous source, the amount that improves the aspergillus niger of inoculating in fermentor tank can improve transformation efficiency and the single output of citric acid, but relatively comparative example 2 and comparative example 3 can be found out, add in the absence of monose and nitrogenous source, the amount that is the aspergillus niger inoculated in simple raising fermentor tank causes the transformation efficiency of citric acid and the reduction of single output on the contrary.
Therefore, in the present invention, take every gram of fermention medium as benchmark, the inoculum size of described aspergillus niger can be 1.8 * 10
4-5.3 * 10
4individual colony-forming unit, preferably, take every gram of fermention medium as benchmark, and the inoculum size of described aspergillus niger is 2.5 * 10
4-4.0 * 10
4individual colony-forming unit.
In the present invention, the condition of described fermentation has no particular limits, and can be the fermentation condition of this area routine, and for example, the condition of described fermentation can comprise: temperature is 30-40 ℃, is preferably 30-35 ℃; PH value is 1-7, is preferably 2-4, and air flow is 0.1-1 volume: volume minute; Be preferably 0.2-0.8 volume: volume minute; Time is 75-95 hour, is preferably 80-90 hour.
Described colony-forming unit is defined as a certain amount of bacterium liquid after dilution by the method for pouring into a mould or being coated with, and allows unicellular being dispersed in one by one on culture medium flat plate of microorganism in it, and after cultivating, each viable cell just forms a bacterium colony.It is the single celled number containing in every milliliter of bacterium liquid.
Described colony-forming unit can be measured by means commonly known in the art, for example, by blood counting chamber, counts.
According to the present invention, the composition of fermention medium is not had to special requirement, as long as can be for the fermention medium of citric acid fermentation.Preferably, it is 13-21 % by weight that described fermention medium contains starchy material enzymolysis product carbon source content, and nitrogenous source content is 0.06-0.14 % by weight, and phosphorus source content is 0.005-0.07 % by weight, inorganic salt content 0.1-2.6 % by weight, water-content is 77-86 % by weight.Usually, starchy material enzymolysis obtains starchy material enzymolysis residue and starchy material enzymatic liquefaction clear liquid, conventionally can be by starchy material enzymatic liquefaction clear liquid for the preparation of fermention medium, after also starchy material enzymatic liquefaction clear liquid can being mixed with starchy material enzymolysis residue for the preparation of fermention medium.Starchy material enzymolysis product of the present invention is preferably mixed with water or is not mixed to get with water by starchy material enzymolysis residue and starchy material enzymatic liquefaction clear liquid, and further preferably take described fermention medium gross weight as 100 weight parts be benchmark, the consumption of described starchy material enzymatic liquefaction clear liquid is 80-95 weight part, the consumption of described starchy material enzymolysis residue is 0.5-10 weight part, and the consumption of water is 0-15 weight part.
According to the present invention, described starchy material enzymatic liquefaction clear liquid can prepare by several different methods, for example, can prepare by the following method: starchy material is pulverized, the product after pulverizing is carried out to enzymolysis, obtain enzymolysis product, by enzymolysis product solid-liquid separation, obtain starchy material enzymatic liquefaction clear liquid and starchy material enzymolysis residue, it is 5-60 % by weight that the condition of described solid-liquid separation makes the solid content of starchy material enzymolysis residue, more preferably 30-50 % by weight.
According to the present invention, described starchy material can variously can be prepared the raw material that contains starch of citric acid for enzymolysis, fermentation for ability is known, for example, can be selected from one or more in corn, potato class (as cassava) and wheat, under preferable case, described starchy material is corn.
Described enzymolysis step can complete by the conventional method in this area, such as add microbes producing cellulase and/or enzyme in crushed products, at the growth temperature of microbes producing cellulase and/or the great-hearted temperature of enzyme, has been incubated.Described microbes producing cellulase be can secreting amylase microbes producing cellulase.Described enzyme comprises amylase.
Because microorganism growth can produce by product, therefore preferably directly add enzyme.The consumption of described enzyme is The more the better, and for cost consideration, preferably with the dry weight basis of the crushed products after every gram of pulverizing, described diastatic consumption is 15-50 enzyme activity unit.
The enzyme activity unit of enzyme of the present invention is defined as: in pH value, be 6.0, temperature is that under the condition of 70 ℃, 1 minute is converted into the required enzyme amount of reducing sugar by 1 milligram of starch is an enzyme activity unit.
The temperature of described enzymolysis can in very large range change, and is preferably 70-105 ℃, more preferably 80-95 ℃.On the time theory of described enzymolysis, the longer the better, considers plant factor, and the time of preferred described enzymolysis is 90-150 minute, more preferably 100-120 minute.The pH value of described enzymolysis can in very large range change, and is preferably 5.0-7.0, and more preferably pH value is 5.4-5.7.
Amylase refers to the general name of class of enzymes that can starch-splitting glycosidic link, and described amylase generally comprises α-amylase, beta-amylase, saccharifying enzyme and isoamylase.
According to the present invention, preferably use α-amylase and/or isoamylase.
According to the present invention, the method and apparatus of described solid-liquid separation is conventionally known to one of skill in the art, for example, and pressure filter or whizzer.
Described aspergillus niger can adopt the inoculation of conventional method, for example, in being seeded to fermention medium before, described aspergillus niger is processed through seed culture, afterwards the seed liquor obtaining is joined in fermention medium.The degree of aspergillus niger seed culture can be measured and be observed the growth of aspergillus niger by sampling sediments microscope inspection, acid test and pH, when pH 2.0-2.5, acidity 0.5-2.0%, bacterium ball size evenly, mycelia is sturdy stops cultivating while stretching out.
Under preferable case, the method that described seed culture is processed comprises: aspergillus niger is seeded in aspergillus niger nutrient solution and is cultivated, and the Semen Maydis powder that contains 10-17 % by weight in described aspergillus niger nutrient solution, after inoculation, in aspergillus niger nutrient solution, the concentration of aspergillus niger is 3 * 10
5-4 * 10
5individual/milliliter.
According to the present invention, the preparation method of described aspergillus niger nutrient solution has no particular limits, as long as the nutrient solution obtaining can be applicable to the cultivation of aspergillus niger.
According to the present invention, the culture condition of described aspergillus niger can in very large range change, for example the condition of described cultivation can comprise: the temperature of cultivation can be 25-45 ℃, pH value can be 1-7, air flow can be 0.05-0.5 volume: volume minute, and the time of cultivation can be 45-65 hour; In preferred situation, the condition of described cultivation can comprise: the temperature of cultivation can be 30-40 ℃, and pH value can be 2-4, and air flow can be 0.1-0.3 volume: volume minute, the time of described cultivation can be 50-60 hour.
Term " air flow " generally, with recently expression of ventilation, recently represents (V/Vmin) with the volume of air by unit volume nutrient solution in per minute conventionally, and for example ventilation ratio is 1: 0.1-1, being called for short air flow is 0.01-1 volume: volume minute.
The equipment of described cultivation is conventionally known to one of skill in the art, for example, can use fermentor tank to cultivate.
The tunning citric acid preparing according to method of the present invention can be by conventional method, separated and refining according to the requirement of different Industrial products, such as neutralization, acidolysis, decolouring, concentrated, crystallization, packing.
Below in conjunction with embodiment, the present invention will be described in more detail.
Embodiment 1
The present embodiment is for illustrating the production method of citric acid provided by the invention.
(1) 56 kg corn of results are stewing in hot water tank profit, until the water content of corn is 15 % by weight, then pulverize, obtain average particle diameter and be the pulverizing after product of 400 microns.
(2) product after pulverizing is sized mixing by the concentration of 25 % by weight, with respect to the product after every gram of pulverizing, amylase (the Novozymes Company that adds 20 enzyme activity units, α-amylase, equal amylase for this reason in the embodiment of the present invention), enter injector, under the condition that is 5.5 at 85 ℃, pH, enzymolysis is 100 minutes, obtains enzymolysis product A1.
(3) by enzymolysis product A1 by carrying out press filtration with fluid pressure type sheet frame pressure filter, isolate enzymatic liquefaction clear liquid and enzymolysis filter residue, wherein, the water content of enzymolysis residue is 50%.
(4) preparation fermention medium, to after the water sterilizing of the above-mentioned enzymatic liquefaction clear liquid of 170 kilograms, the enzymolysis residue of 2.0 kilograms and 13 kilograms, join in the fermentor tank of 300L, obtain fermention medium B1, wherein carbon source content is 16 % by weight, nitrogenous source content is 0.095 % by weight, phosphorus source content is 0.06 % by weight, inorganic salt content 0.4 % by weight, and water-content is 83.4%.
(5) by the Partial digestion liquefier in step (2), be diluted with water to 10 % by weight of total reducing sugar, obtain nutrient solution, nutrient solution is dropped into seeding tank, is heated to 121 ℃ of sterilizations, maintain 30 minutes after fast cooling to 36 ℃, access aspergillus niger strain (aspergillus niger T01, Tianjin industrial microorganism institute, equal aspergillus niger strain for this reason in the embodiment of the present invention, inoculum size is: every gram of 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; By sampling sediments microscope inspection, acid test and pH, measure the growth of aspergillus niger observed, when pH 2.0, acidity 1%, bacterium ball size evenly, mycelia is sturdy while stretching out, stop cultivating.
(6) aspergillus niger strain of step (5) being cultivated joins in the fermentor tank of step (4) and starts fermentation, and detect during the fermentation the total reducing sugar in fermented liquid, inoculum size is: 3.5 * 104 colony-forming units of every gram of fermention medium, fermentation condition comprises that temperature is 35 ℃, pH value is 3, air flow is 0.4 volume: volume minute, when proceeding to the 45th hour, fermentation (from aspergillus niger being inoculated into fermentor tank, starts to calculate, fermentation time in embodiments of the invention all so calculates), adopt the mode of continuous adding, the interpolation solution that adds evenly 28 kilograms, described interpolation solution is that urea concentration is the glucose mother liquid of 0.16 % by weight, wherein the concentration of glucose is 50 % by weight, when proceeding to the 65th hour, fermentation stops adding, the total sugar content that adds fermented liquid in the process of adding solution is 3.5 % by weight, when proceeding to the 80th hour, fermentation carries out solid-liquid separation, obtain citric acid solution.
According to the concentration (abbreviation acidity) of gained citric acid solution in GB 1987-2007 standard detection embodiment 1, calculate transformation efficiency and the single output of citric acid, 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 citric acid, result is as shown in table 1.
Comparative example 1
According to the method described in embodiment 1, produce citric acid, different, in step (6), joined in fermentor tank before fermentation with glucose and the urea of embodiment 1 same amount, step (6) is:
The interpolation solution that adds 28 kilograms in fermentor tank, described interpolation solution is that urea concentration is the glucose mother liquid of 0.16 % by weight, wherein the concentration of glucose is 50 % by weight, the aspergillus niger strain that step (5) is cultivated joins in the fermentor tank of step (4) and starts fermentation, and detecting the total reducing sugar in fermented liquid, inoculum size is: every gram of fermention medium 3.5 * 10
4individual colony-forming unit, fermentation condition comprises that temperature is 35 ℃, and pH value is 3, and air flow is 0.4 volume: volume minute, when proceeding to the 80th hour, fermentation carries out solid-liquid separation, obtain citric acid solution.
Comparative example 2
According to the method described in embodiment 1, produce citric acid, different, in step (6), do not add sugar and urea, but add the fermention medium B1 of 28 kilograms, the step of comparative example 2 (6) is:
The aspergillus niger strain that step (5) is cultivated joins in the fermentor tank of step (4) and starts fermentation, and detects the total reducing sugar in fermented liquid, and inoculum size is: every gram of fermention medium 3.5 * 10
4individual colony-forming unit, fermentation condition comprises that temperature is 35 ℃, pH value is 3, air flow is 0.4 volume: volume minute, when fermentation proceeds to the 45th hour, adopt the mode of continuous adding, add evenly the fermention medium B1 of 28 kilograms, when fermentation proceeds to the 80th hour, carry out solid-liquid separation, obtain citric acid solution.
Embodiment 2
The present embodiment is for illustrating the production method of citric acid provided by the invention.
(1) corn of 56 kilograms of results is stewing in hot water tank profit, until the water content of corn is 20 % by weight, then pulverize, obtain average particle diameter and be the pulverizing after product of 800 microns.
(2) product after pulverizing is sized mixing by the concentration of 25 % by weight, with respect to the product after every gram of pulverizing, add the amylase of 45 enzyme activity units, enter injector, under the condition that is 5 at 90 ℃, pH, enzymolysis is 90 minutes, obtains enzymolysis product A2.
(3) by enzymolysis product A2 by carrying out press filtration with fluid pressure type sheet frame pressure filter, isolate enzymatic liquefaction clear liquid and enzymolysis filter residue, wherein, the water content of enzymolysis residue is 30%.
(4) preparation fermention medium, joins in fermentor tank after specifically consisting of the water sterilizing of the enzymatic liquefaction clear liquid of 160 kilograms, the enzymolysis residue of 9 kilograms and 16 kilograms, obtains fermention medium B2.Wherein carbon source content is 18.6 % by weight, and nitrogenous source content is 0.1 % by weight, and phosphorus source content is 0.055 % by weight, inorganic salt content 0.17 % by weight, and water-content is 81%
(5) by the Partial digestion liquefier obtaining in step (2), be diluted with water to total reducing sugar 10%, obtain nutrient solution, nutrient solution is dropped into seeding tank, drop into seeding tank, add urea, the add-on of urea is 0.35% of nutrient solution gross weight, be heated to 120 ℃ of sterilizations, maintain 20 minutes after fast cooling to 36 ℃, access aspergillus niger strain (inoculum size is: every gram of enzymatic liquefaction clear liquid 3.5 * 10
5individual colony-forming unit), at 36 ℃, 0.4 volume: carry out spawn culture under the aeration condition of volume minute; By sampling sediments microscope inspection, acid test and pH, measure the growth of aspergillus niger observed, when pH 2.0, acidity 1%, bacterium ball size evenly, mycelia is sturdy while stretching out, stop cultivating.
(6) aspergillus niger strain of step (5) being cultivated joins in the fermentor tank of step (4) and starts fermentation, and detects during the fermentation the total reducing sugar in fermented liquid, and inoculum size is: every gram of fermention medium 2.5 * 10
4individual colony-forming unit, fermentation condition comprises that temperature is 30 ℃, pH value is 2, air flow is 1 volume: volume minute, when fermentation proceeds to the 40th hour, adopt the mode of continuous adding, the interpolation solution that adds evenly 30 kilograms, described interpolation solution is that urea concentration is that the D/W of 0.125 % by weight (adds and in solution, only has urea, glucose and water), wherein the concentration of glucose is 30 % by weight, when proceeding to the 60th hour, fermentation stops adding, the total sugar content that adds fermented liquid in the process of adding solution is 4.5 % by weight, when proceeding to the 83rd hour, fermentation carries out solid-liquid separation, obtain citric acid solution.
Embodiment 3
The present embodiment is for illustrating the production method of citric acid provided by the invention.
(1) 56 kg corn of results are stewing in hot water tank profit, until the water content of corn is 10 % by weight, then pulverize, obtain average particle diameter and be the pulverizing after product of 500 microns.
(2) product after pulverizing is sized mixing by the concentration of 25 % by weight, with respect to the product after every gram of pulverizing, add the amylase of 30 enzyme activity units, enter injector, under the condition that is 6 at 80 ℃, pH, enzymolysis is 120 minutes, obtains enzymolysis product A3.
(3) by enzymolysis product A3 by carrying out press filtration with fluid pressure type sheet frame pressure filter, isolate enzymatic liquefaction clear liquid and enzymolysis filter residue, wherein, the water content of enzymolysis residue is 15%.
(4) preparation fermention medium, specifically consists of the enzymatic liquefaction clear liquid of 155 kilograms, the enzymolysis residue of 12 kilograms and the water of 18 kilograms, joins in fermentor tank after sterilizing, obtains fermention medium B3.Wherein carbon source content is 14.75 % by weight, and nitrogenous source content is 0.12 % by weight, and phosphorus source content is 0.015 % by weight, inorganic salt content 2.4 % by weight, and water-content is 82.7%.
(5) by the Partial digestion liquefaction clear liquid in step (3), be diluted with water to total reducing sugar 10% and drop into seeding tank, obtain nutrient solution, nutrient solution is dropped into seeding tank, add urea, the add-on of urea is 0.35% of seed tank culture liquid gross weight, is heated to 120 ℃ of sterilizations, fast cooling to 36 ℃ after maintaining 20 minutes, (inoculum size is access aspergillus niger strain: every gram of enzymatic liquefaction clear liquid 4 * 10
5individual colony-forming unit), at 36 ℃, 0.4 volume: carry out spawn culture under the aeration condition of volume minute; By sampling sediments microscope inspection, acid test and pH, measure the growth of aspergillus niger observed, when pH 2.0, acidity 1%, bacterium ball size evenly, mycelia is sturdy while stretching out, stop cultivating.
(6) aspergillus niger strain of step (5) being cultivated joins in the fermentor tank of step (4) and starts fermentation, and detects during the fermentation the total reducing sugar in fermented liquid, and inoculum size is: every gram of fermention medium 4 * 10
4individual colony-forming unit, fermentation condition comprises that temperature is 32 ℃, pH value is 4, air flow is 0.1 volume: volume minute, when fermentation proceeds to the 50th hour, adopt the mode of continuous adding, the interpolation solution that adds evenly 18 kilograms, described interpolation solution is that urea concentration is the glucose mother liquid of 0.088 % by weight, wherein the concentration of glucose is 40 % by weight, when proceeding to the 70th hour, fermentation stops adding, the total sugar content that adds fermented liquid in the process of adding solution is 2.5 % by weight, when proceeding to the 88th hour, fermentation carries out solid-liquid separation, obtain citric acid solution.
Embodiment 4
The present embodiment is for illustrating the production method of citric acid provided by the invention.
According to the method described in embodiment 1, produce citric acid, different, in step (6), the inoculum size of aspergillus niger is: every gram of fermention medium 2.4 * 10
4individual colony-forming unit.
Comparative example 3
According to the method described in embodiment 4, produce citric acid, different, step is not added sugar and urea in (6), and the step of comparative example 3 (6) is:
The aspergillus niger strain that step (5) is cultivated joins in the fermentor tank of step (4) and starts fermentation, and detects the total reducing sugar in fermented liquid, and inoculum size is: every gram of fermention medium 2.4 * 10
4individual colony-forming unit, fermentation condition comprises that temperature is 35 ℃, and pH value is 3, and air flow is 0.4 volume: volume minute, after fermenting 88 hours, carry out solid-liquid separation, obtain citric acid solution.
Embodiment 5
The present embodiment is for illustrating the production method of citric acid provided by the invention.
According to method described in embodiment 2, produce citric acid, different, in step (6), the concentration of adding 30 kilograms is that to replace the concentration of 30 kilograms be the D/W of 30 % by weight to the fructose water solution of 30 % by weight.
Embodiment 6
The present embodiment is for illustrating the production method of citric acid provided by the invention.
According to the method described in embodiment 1, produce citric acid, different, step is not added urea in (6), and 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 starts fermentation, and detects the total reducing sugar in fermented liquid, and inoculum size is: every gram of fermention medium 3.5 * 10
4individual colony-forming unit, fermentation condition comprises that temperature is 35 ℃, pH value is 3, and air flow is 0.4 volume: volume minute, when fermentation proceeds to the 45th hour, adopt the mode of continuous adding, the interpolation solution that adds evenly 28 kilograms, described interpolation solution is that glucose concn is the glucose mother liquid of 50 % by weight, when fermentation proceeds to the 65th hour, stops adding, when fermentation proceeds to the 80th hour, carry out solid-liquid separation, obtain citric acid solution.
Embodiment 7
The present embodiment is for illustrating the production method of citric acid provided by the invention.
According to the method described in embodiment 1, produce citric acid, different, monose and nitrogenous source in step (6) add several times, and 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 starts fermentation, and detects during the fermentation the total reducing sugar in fermented liquid, and inoculum size is: every gram of fermention medium 3.5 * 10
4individual colony-forming unit, fermentation condition comprises that temperature is 35 ℃, pH value is 3, air flow is 0.4 volume: volume minute, when fermentation proceeds to the 45th hour, divide and add 28 kilograms of glucose mother liquids for 6 times, wherein the concentration of glucose is 50 % by weight, within average every 4 hours, add once, in each 10 minutes of adding after glucose solids, the zein that 0.15 kilogram of concentration of disposable interpolation is 20%, when fermentation proceeds to the 65th hour, stop adding, 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 citric acid |
| Embodiment 1 | 18.65 | 96.77 | 45.69 |
| Comparative example 1 | 15.60 | 92.50 | 38.22 |
| Comparative example 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 example 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 upper table 1 can be found out, the method of production citric acid provided by the invention, do not changing under the prerequisite of equipment and reaction conditions, higher product acidacidity, transformation efficiency and citric acid single output have been obtained, in the situation that each production cycle of producing is constant, the raising of citric acid single output has improved the utilization ratio of integral device undoubtedly, has improved the single output of integrated artistic.
Comparative example 1 starts just to add glucose and urea in fermentation, than embodiment 1, product acidacidity and the transformation efficiency of comparative example 1 differ greatly, and even lower than the comparative example 2 of not adding glucose and urea, illustrate and select suitable time point to add glucose and urea can realize the present invention.
In embodiments of the invention 1, inoculated 3 * 10
4individual aspergillus niger strain forms unit, and has added monose and nitrogenous source, and in embodiment 1, the transformation efficiency of citric acid and single output are respectively 96.77% and 45.69 kilogram, the aspergillus niger of comparative example 2 embodiment 1 inoculation same amounts, but monose and nitrogenous source do not added, and in comparative example 2, the transformation efficiency of citric acid and single output are respectively 93.31% and 33.32 kilogram, the aspergillus niger strain of embodiment 4 inoculation convention amounts, and added monose and nitrogenous source, in embodiment 4, the transformation efficiency of citric acid and single output are respectively 95.60% and 42.88 kilogram, the aspergillus niger strain of comparative example 3 inoculation convention amounts, but do not add monose and nitrogenous source, in comparative example 3, the transformation efficiency of citric acid and single output are respectively 94% and 33.57 kilogram, comparing embodiment 1 can be found out with comparative example 3 with comparative example 2 and embodiment 4 respectively, adding under the condition of monose and nitrogenous source, no matter whether improve the amount of the aspergillus niger of inoculating in fermentor tank, can both improve transformation efficiency and the single output of citric acid, comparing embodiment 1 and embodiment 4 can find out, in the situation that adding monose and nitrogenous source, the amount that improves the aspergillus niger of inoculating in fermentor tank can improve transformation efficiency and the single output of citric acid, but relatively comparative example 2 and comparative example 3 can be found out, add in the absence of monose and nitrogenous source, the amount that is the aspergillus niger inoculated in simple raising fermentor tank causes the transformation efficiency of citric acid and the reduction of single output on the contrary.This shows, in the situation that adding monose and nitrogenous source, it is the preferred embodiment of the present invention that suitable raising is inoculated into the amount of aspergillus niger in fermentor tank.
The sugar adding in embodiment 5 is fructose water solution, compare with embodiment 1, the transformation efficiency of the citric acid of embodiment 5 and single output are lower slightly, illustrate that fructose also can be utilized by aspergillus niger, as the carbon source that is converted into citric acid, and can access higher citric acid transformation efficiency and single output, but in current industrial production, the preparation cost of fructose is higher than glucose, farther far above glucose mother liquid, therefore, using glucose mother liquid is the preferred embodiment of the present invention.
Embodiment 6 compares with embodiment 1, do not add urea, the transformation efficiency of the citric acid of embodiment 6 is compared and is differed larger with embodiment 1 with single output, explanation is seeded to after fermention medium 32 hours to the time period of first 5 hours of having fermented by aspergillus niger, and not only having added monose but also added nitrogenous source is the preferred embodiment of the present invention.
Embodiment 7 compares with embodiment 1, and monose and nitrogenous source are all to add several times, compares with embodiment 1, and in embodiment 7, the single output of citric acid is more lower slightly than embodiment 1, illustrates that adding continuously monose and nitrogenous source is the preferred embodiment of the present invention.
Claims (16)
1. the production method of a citric acid, the method is included under the condition that generates citric acid, aspergillus niger is seeded in fermention medium and is fermented, obtain fermented liquid, it is characterized in that, the method also comprises in fermented liquid adds the monose with 6 carbon atoms, the time of adding the monose with 6 carbon atoms after aspergillus niger is seeded to fermention medium 24 hours to time period of 5 hours before having fermented, the time of fermentation is 75-95 hour, the weight ratio of the addition of described monose and fermented liquid total amount is 1:8-100, described monose is glucose and/or fructose.
2. method according to claim 1, wherein, the weight ratio of the addition of described monose and fermented liquid total amount is 1:13-28.
3. method according to claim 1, wherein, the time that starts to add the monose with 6 carbon atoms is for being seeded to aspergillus niger 24-56 hour after fermention medium.
4. method according to claim 3, wherein, the time that starts to add the monose with 6 carbon atoms is for being seeded to aspergillus niger 40-50 hour after fermention medium.
5. method according to claim 4, wherein, the time that finishes to add the monose with 6 carbon atoms is for being seeded to aspergillus niger 60-70 hour after fermention medium.
6. according to the method described in any one in claim 1-5, wherein, described monose adds several times, and the timed interval of adjacent twice interpolation is not more than 8 hours, and the amount of each monose adding is identical.
7. method according to claim 6, wherein, the mode of described interpolation is continuous adding.
8. method according to claim 7, wherein, it is 2-5 % by weight that the amount of the monose of described continuous adding makes the total sugar content of fermented liquid described in adding procedure.
9. method according to claim 1, wherein, described monose adds with the form of monose solid and/or the aqueous solution that contains monose, described in contain monose the aqueous solution in the concentration of monose be more than 25 % by weight.
10. method according to claim 9, wherein, described monose is glucose, described in contain monose the aqueous solution be glucose mother liquid.
11. according to the method described in any one in claim 1-5 and 7-10, wherein, the method also comprises in fermented liquid adds nitrogenous source, the time of adding nitrogenous source after aspergillus niger is seeded to fermention medium 24 hours to time period of 5 hours before having fermented, and in nitrogen element, the 0.033-0.35 % by weight of the amount of the monose that the addition of described nitrogenous source is described interpolation.
12. methods according to claim 11, wherein, described nitrogenous source and monose add simultaneously.
13. methods according to claim 12, wherein, described nitrogenous source is at least one in urea, ammonium sulfate, ammonium nitrate, zein and corn steep liquor.
14. methods according to claim 1, wherein, take every gram of fermention medium as benchmark, and the inoculum size of aspergillus niger is 1.8 * 10
4-5.3 * 10
4individual colony-forming unit, the condition of described fermentation comprises: temperature is 30-40 ℃, air flow is 0.1-1 volume: (volume minute), the time of fermentation is 80-90 hour.
15. methods according to claim 1, wherein, take every gram of fermention medium as benchmark, and the inoculum size of aspergillus niger is 2.5 * 10
4-4.0 * 10
4individual colony-forming unit.
16. methods according to claim 1, wherein, described fermention medium contains starchy material enzymolysis product, in described starchy material enzymolysis product, carbon source content is 13-21 % by weight, nitrogenous source content is 0.06-0.14 % by weight, phosphorus source content is 0.005-0.07 % by weight, inorganic salt content 0.1-2.6 % by weight, and water-content is 77-86 % by weight.
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| CN101638674A (en) * | 2009-08-26 | 2010-02-03 | 安徽丰原发酵技术工程研究有限公司 | Method for manufacturing citric acid by utilizing cane sugar hydrolysate fermentation method |
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| CN101555497A (en) * | 2009-05-20 | 2009-10-14 | 安徽丰原生物化学股份有限公司 | Preparation method of citric acid fermentation solution |
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