CN102373242B - Citric acid preparation method - Google Patents
Citric acid preparation method Download PDFInfo
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- CN102373242B CN102373242B CN201010255961.9A CN201010255961A CN102373242B CN 102373242 B CN102373242 B CN 102373242B CN 201010255961 A CN201010255961 A CN 201010255961A CN 102373242 B CN102373242 B CN 102373242B
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Abstract
A citric acid preparation method comprises a step that aspergillus niger is inoculated to fermentation liquor to produce citric acid in a fermentation way, wherein the fermentation temperature of the rise phase of the aspergillus niger respiratory quotient is 35-45 DEG C, the fermentation temperature of the decline phase of the aspergillus niger respiratory quotient is 30-40 DEG C, the fermentation temperature of the low-level maintenance phase of the aspergillus niger respiratory quotient is 30-40 DEG C, and the fermentation temperature of the rebound phase of the aspergillus niger respiratory quotient is 35-45 DEG C; the fermentation temperature of the rise phase of the aspergillus niger respiratory quotient is higher than that of the decline phase of the aspergillus niger respiratory quotient, the fermentation temperature of the decline phase of the aspergillus niger respiratory quotient is higher than that of the low-level maintenance phase of the aspergillus niger quotient, the fermentation temperature of the rebound phase of the aspergillus niger respiratory quotient is higher than those of the decline phase of the aspergillus niger respiratory quotient and the low-level maintenance phase of the aspergillus niger respiratory quotient. With the adoption of the method, the fermentation efficiency can be effectively improved, and the fermentation period is shortened.
Description
Technical field
The present invention relates to a kind of preparation method of citric acid.
Background technology
Citric acid is a kind of organic acid that is widely used in the industries such as beverage, food and medicine, and China is the big producing country of citric acid, and more than 20 factories are arranged, and output is over 800,000 tons.
At present, in the fermenting process of producing citric acid, after the aspergillus niger seed liquor being cultivated to maturation and moving into fermentor tank, usually in the whole process of fermentation, take same control condition, and cause aspergillus strain can not bring into play its optimum effect, and cause fermentation efficiency lower.Therefore, bring into play its maximum effect to improve the method for citric acid fermentation efficiency in the urgent need to developing a kind of aspergillus niger that can make.
Summary of the invention
The object of the invention is to overcome the lower defect of citric acid fermentation efficiency of prior art, a kind of preparation method that can effectively improve the citric acid of citric acid fermentation efficiency is provided.
The present inventor finds, in the fermenting process of producing citric acid, usually need to take corresponding parameter to control to the fermentation of Aspergillus niger process of producing citric acid, so that aspergillus niger is during the fermentation all the time in best state.But, take same control condition in prior art in the whole process of fermentation, the feature that can not reflect truly each different times of aspergillus niger, and the feature of different growing stage during the fermentation is equipped with the environmental parameter adapted with its state according to aspergillus strain.
The present inventor judges the parameter attribute of aspergillus niger different growing stage of living in after research, and during the fermentation, the environmental parameter adapted according to the parameter attribute of aspergillus niger different growing stage pair and its state is adjusted, so that aspergillus niger can and be brought into play its maximum effect and make aspergillus niger have during the fermentation higher fermentation efficiency in best state at its different times.
The invention provides a kind of preparation method of citric acid, the method comprises and aspergillus niger being seeded in fermented liquid and fermentation produces citric acid, wherein, the process of fermentation generation citric acid comprises the ascent stage of aspergillus niger respiratory quotient, the decline stage of aspergillus niger respiratory quotient, the low-level maintenance stage of aspergillus niger respiratory quotient and the rise stage of aspergillus niger respiratory quotient, leavening temperature at the ascent stage of aspergillus niger respiratory quotient is 35-45 ℃, leavening temperature in decline stage of aspergillus niger respiratory quotient is 30-40 ℃, leavening temperature in low-level maintenance stage of aspergillus niger respiratory quotient is 30-40 ℃, leavening temperature in rise stage of aspergillus niger respiratory quotient is 35-45 ℃, and the leavening temperature at the leavening temperature of the ascent stage of aspergillus niger respiratory quotient higher than the decline stage in the aspergillus niger respiratory quotient, leavening temperature at the leavening temperature of decline stage of aspergillus niger respiratory quotient higher than the low-level maintenance stage in the aspergillus niger respiratory quotient, at the leavening temperature in rise stage of aspergillus niger respiratory quotient higher than the leavening temperature of the decline stage in the aspergillus niger respiratory quotient and at the leavening temperature of low-level maintenance stage of aspergillus niger respiratory quotient.
Method of the present invention judges the physiological status of aspergillus niger by the variation tendency of the respiratory quotient of aspergillus niger in the fermenting process according to producing citric acid, under the different physiological statuss of aspergillus niger, leavening temperature is controlled, thereby make growth during the fermentation of aspergillus niger and produce acid phase all in environmental optima, effectively bring into play its optimum utility, and reach the raising fermentation efficiency, shorten the purpose of fermentation period.
Embodiment
According to the present invention, the preparation method of described citric acid comprises and aspergillus niger being seeded in fermented liquid and fermentation produces citric acid, described fermented liquid contains the starchy material enzymolysis product, wherein, the process that fermentation produces citric acid comprises the ascent stage of aspergillus niger respiratory quotient, the decline stage of aspergillus niger respiratory quotient, the low-level maintenance stage of aspergillus niger respiratory quotient and the rise stage of aspergillus niger respiratory quotient, leavening temperature at the ascent stage of aspergillus niger respiratory quotient is 35-45 ℃, is preferably 37-41 ℃; Leavening temperature in decline stage of aspergillus niger respiratory quotient is 30-40 ℃, is preferably 35-39 ℃; Leavening temperature in low-level maintenance stage of aspergillus niger respiratory quotient is 30-40 ℃, is preferably 35-39 ℃; Leavening temperature in rise stage of aspergillus niger respiratory quotient is 35-45 ℃, is preferably 37-41 ℃; And the leavening temperature at the leavening temperature of the ascent stage of aspergillus niger respiratory quotient higher than the decline stage in the aspergillus niger respiratory quotient, leavening temperature at the leavening temperature of decline stage of aspergillus niger respiratory quotient higher than the low-level maintenance stage in the aspergillus niger respiratory quotient, at the leavening temperature in rise stage of aspergillus niger respiratory quotient higher than the leavening temperature of the decline stage in the aspergillus niger respiratory quotient and at the leavening temperature of low-level maintenance stage of aspergillus niger respiratory quotient.
Under preferable case, in order further to control the leavening temperature in aspergillus niger residing stage of respiratory quotient, better to reach the raising fermentation efficiency, shorten the purpose of fermentation period, at the leavening temperature of the ascent stage of aspergillus niger respiratory quotient than the high 0.5-5 ℃ of leavening temperature of the decline stage in the aspergillus niger respiratory quotient, more preferably high 1-3 ℃; At the leavening temperature in rise stage of aspergillus niger respiratory quotient than the leavening temperature of the decline stage in the aspergillus niger respiratory quotient and at the high 0.5-5 ℃ of leavening temperature of low-level maintenance stage of aspergillus niger respiratory quotient, more preferably high 1-3 ℃.
The present inventor finds, the process of fermentation generation citric acid comprises the ascent stage of aspergillus niger respiratory quotient, the decline stage of aspergillus niger respiratory quotient, the low-level maintenance stage of aspergillus niger respiratory quotient and the rise stage of aspergillus niger respiratory quotient, and can judge by the variation tendency of the respiratory quotient of aspergillus niger in the fermenting process according to producing citric acid the physiological status of aspergillus niger, , the variation of the stages of its respiratory quotient is the different physiological stages of corresponding aspergillus niger respectively, be the fast growing period of aspergillus niger, growth is to producing the acid transitional period, produce fast acid phase and decline phase.The present inventor adopts different optimum leavening temperatures according to the variation characteristic physiological stage different at it of aspergillus niger respiratory quotient during the fermentation just, thereby make growth during the fermentation of aspergillus niger and produce acid phase all in environmental optima, the Fast Growth that is particularly conducive to thalline is grown, and can effectively bring into play its optimum utility, and shortened fermentation period, improved fermentation efficiency.Therefore, in order to judge the residing stage of aspergillus niger respiratory quotient, the method can also comprise the respiratory quotient of aspergillus niger in the repeated detection fermenting process, according to the value judgement aspergillus niger residing stage of respiratory quotient of adjacent twice aspergillus niger respiratory quotient.Judge the aspergillus niger respiratory quotient after the residing stage, coming corresponding adjustment fermenting process main control parameters, i.e. leavening temperature.Described repeated detection can be step detection, for example, the timed interval of adjacent twice detection is not particularly limited, generally be not more than 20 minutes, under preferable case, in order to judge more accurately the residing stage of aspergillus niger respiratory quotient, the timed interval of adjacent twice detection is 1-10 minute, more preferably, in situation, the timed interval of adjacent twice detection is 2-5 minute; The method that also can adopt on line real-time monitoring is detected in real time to the respiratory quotient of aspergillus niger in fermenting process.
Term " respiratory quotient " refers to the ratio of release of carbonate dioxide speed and oxygen consumption rate.Respiratory quotient is the various carbon sources indicator values of metabolic condition during the fermentation, sign be that matrix is utilized situation and pathways metabolism situation thereof.
The detection method of described respiratory quotient can be the various methods that can detect the microbial respiratory business, for example, utilize mass spectrograph or exhaust analyzer to detect the exhaust gas component in culturing process, calculate release of carbonate dioxide speed and oxygen consumption rate, then calculate respiratory quotient.
According to the present invention, aspergillus niger is wider in the variation range of the respiratory quotient of its different physiological stages, under preferable case, in order to be easier to the control of fermentation condition, after being seeded in fermented liquid by aspergillus niger, the initial value of the respiratory quotient of aspergillus niger is 0.1-0.5, the respiratory quotient of described aspergillus niger is in the scope in 0.8-1.5 after ascent stage, in the scope in 0.1-0.4 after the decline stage, in the scope of low-level maintenance stage in 0.1-0.4, after the rise stage in more than 0.4; More preferably in situation, the respiratory quotient of aspergillus niger is in the scope in 0.85-1.2 after ascent stage, in the scope in 0.1-0.3 after the decline stage, in the scope of low-level maintenance stage in 0.1-0.3, in the scope in 0.4-0.7 after the rise stage.
According to the present invention, air flow makes the dissolved oxygen in fermented liquid can meet of the present invention the requirement under prerequisite, and prevent the impact of air flow on fermenting process, further improve fermentation efficiency, under preferable case, the 20-40% that the amount of dissolved oxygen is calibration value in the ascent stage fermented liquid of aspergillus niger respiratory quotient, the more preferably 25-35% of calibration value; The 30-50% that the amount of dissolved oxygen is calibration value in the decline stage of aspergillus niger respiratory quotient fermented liquid, the more preferably 35-45% of calibration value; The 40-70% that the amount of dissolved oxygen is calibration value in the low-level maintenance stage fermented liquid of aspergillus niger respiratory quotient, the more preferably 45-60% of calibration value; The 40-70% that the amount of dissolved oxygen is calibration value in the rise stage fermentation liquid of aspergillus niger respiratory quotient, the more preferably 40-60% of calibration value; And the amount in the amount of the dissolved oxygen in low-level maintenance stage of aspergillus niger respiratory quotient and the stage of ging up higher than the dissolved oxygen of the ascent stage in the aspergillus niger respiratory quotient and decline stage, the amount in the amount of the dissolved oxygen of decline stage of aspergillus niger respiratory quotient higher than the dissolved oxygen of the ascent stage in the aspergillus niger respiratory quotient.
According to the present invention, described calibration value refers to adjusts to 0.05MPa by tank pressure, at mixing speed, is 100rpm, and ventilation is demarcated dissolved oxygen meter in 1: 0.5 o'clock, and it is 100 that dissolved oxygen meter is demarcated displayed value, that is, now the amount of dissolved oxygen is 10 mg/litre.
According to the present invention, the amount of described dissolved oxygen is subject to the impact of the factors such as air flow and tank pressure.In the present invention, described tank pressure generally can homeostasis between 0.03-0.05MPa.Control etc. condition, as long as above-mentioned condition can meet the requirement of the amount of described dissolved oxygen, the adjustable extent of described air flow is wider, in order to be more suitable for meeting the needs of the aspergillus niger in different physiological stages, further improve fermentation efficiency with the needs that coordinate the amount of dissolved oxygen in fermented liquid, the air flow of the ascent stage of aspergillus niger respiratory quotient is 1: (0.3-0.5), the air flow of the decline stage of aspergillus niger respiratory quotient is 1: (0.4-0.7), the air flow of the low-level maintenance stage of aspergillus niger respiratory quotient is 1: (0.6-1), the air flow in the rise stage of aspergillus niger respiratory quotient is 1: (0.4-0.7), more preferably in situation, the air flow of the ascent stage of aspergillus niger respiratory quotient is 1: (0.4-0.5), the air flow of the decline stage of aspergillus niger respiratory quotient is 1: (0.5-0.7), the air flow of the low-level maintenance stage of aspergillus niger respiratory quotient is 1: (0.7-0.9), the air flow in the rise stage of aspergillus niger respiratory quotient is 1: (0.4-0.7).
Term " air flow " generally recently means with ventilation, usually recently mean (V/Vmin) with the volume of air by unit volume nutrient solution (fermented liquid) in per minute, for example ventilation ratio is 1: 0.1-1, being called for short air flow is the 0.01-1 volume: volume minute.
According to the present invention, the transformation period of aspergillus niger respiratory quotient is relevant according to the variation of the physiological stage of aspergillus niger, under condition of the present invention, can guarantee that aspergillus niger is improved at the growth efficiency of each physiological stage, thereby shortened fermentation period, improved fermentation efficiency, therefore, holding time of the ascent stage of aspergillus niger respiratory quotient can be 2-10 hour, holding time of the decline stage of aspergillus niger respiratory quotient can be 2-10 hour, holding time of the low-level maintenance stage of aspergillus niger respiratory quotient can be 30-38 hour, holding time of the rise stage of aspergillus niger respiratory quotient can be 5-12 hour.More preferably in situation, holding time as 4-8 hour of the ascent stage of aspergillus niger respiratory quotient, holding time as 3-7 hour of the decline stage of aspergillus niger respiratory quotient, holding time as 30-36 hour of the low-level maintenance stage of aspergillus niger respiratory quotient, the holding time as 5-10 hour of the rise stage of aspergillus niger respiratory quotient.
According to the present invention, described fermented liquid can be the fermented liquid for the production of citric acid of this area routine, for example, described fermented liquid contains the starchy material enzymolysis product, described starchy material enzymolysis product contains starchy material enzymolysis residue and starchy material enzymolysis clear liquid, and in order to be easier to the carrying out of fermentation, the gross weight of enzymolysis product of take is benchmark, the content of described starchy material enzymolysis clear liquid is the 80-95 % by weight, and the content of described starchy material enzymolysis residue is the 5-20 % by weight.The water content of described starchy material enzymolysis residue can in very large range change, and under preferable case, the solid content of described starchy material enzymolysis residue is the 5-60 % by weight, more preferably the 20-40 % by weight.
According to the present invention, in described fermented liquid, the content of each component can in very large range change, and under preferable case, can also add supplementary nitrogenous source as required in described fermented liquid, and the content of described supplementary nitrogenous source can be the 0.1-2 % by weight of fermented liquid gross weight.According to the present invention, the kind of described supplementary nitrogenous source is conventionally known to one of skill in the art, and for example, described supplementary nitrogenous source can be one or more in urea, ammonium sulfate and ammonium nitrate.In addition, can also supplement appropriate water in fermented liquid according to the requirement of fermented liquid liquid level, the selectable range of the amount of water is wider, can be decided according to the actual requirements.
According to the present invention, described inorganic nitrogen-sourced kind is conventionally known to one of skill in the art, and for example, described nitrogenous source can be one or more in urea, ammonium sulfate and ammonium nitrate.
According to the present invention, the preparation method of described fermented liquid has no particular limits, as long as can be suitable for fermentation production of citric acid.For example, starchy material is pulverized, the crushed products of starchy material is mixed with microbes producing cellulase and/or enzyme, be incubated enzymolysis and obtained enzymolysis product at the growth temperature of microbes producing cellulase and/or the great-hearted temperature of enzyme, and enzymolysis product is carried out to solid-liquid separation obtained starchy material enzymolysis solid phase residue and starchy material enzymolysis clear liquid.And preferably according to aforementioned proportion preparation fermented liquid.
Wherein, 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, for cost consideration, and the dry weight basis of the crushed products after preferably pulverizing with every gram, described diastatic consumption is 15-50 enzyme activity unit.
The enzyme activity unit of enzyme of the present invention is defined as: under the condition that be 6.0 in the pH value, temperature is 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 the pH value is 5.4-5.7.
Amylase refers to the general name of class of enzymes that can the starch-splitting glycosidic link, and described amylase generally comprises α-amylase, beta-amylase, saccharifying enzyme and isoamylase.
α-amylase claims again starch Isosorbide-5-Nitrae-dextrinase, and it can cut the α-Isosorbide-5-Nitrae of starch chain inside-glycosidic link at random, brokenly, by Starch Hydrolysis be maltose, the oligosaccharides that contains 6 glucose units and with the oligosaccharides of side chain.The microorganism that produces this enzyme mainly has Bacillus subtilus, aspergillus niger, aspergillus oryzae and head mold.
Beta-amylase claims again starch Isosorbide-5-Nitrae-maltoside enzyme, can cut Isosorbide-5-Nitrae-glycosidic link from the starch molecule non reducing end, generates maltose.The product that this enzyme acts on starch is maltose and limit dextrin.This enzyme is mainly produced by aspergillus, head mold and endomyces.
Saccharifying enzyme claims again starch α-Isosorbide-5-Nitrae-glucuroide, and this enzyme acts on the non reducing end of starch molecule, take glucose as unit, acts on successively α-Isosorbide-5-Nitrae in starch molecule-glycosidic link, generates glucose.Saccharifying enzyme acts on that product after amylopectin has glucose and with α-1, the oligosaccharides of 6-glycosidic link; The product acted on after amylose starch is all almost glucose.It is mainly aspergillus niger (left U.S. aspergillus, Aspergillus awamori), head mold (snow-white enzyme, De Shi head mold), endomycopsi.sp, monascus that this enzyme produces bacterium.
Isoamylase claims again starch α-1,6-glucuroide, branching enzyme, and this enzyme acts on the α-1 at amylopectin molecule branching-point place, and the 6-glycosidic link cuts the whole side chain of amylopectin to become amylose starch.It is mainly to dislike the bacteriums such as gas bacillus, genus bacillus and some Pseudomonas that this enzyme produces bacterium.
According to the present invention, preferably use α-amylase and/or isoamylase.
According to the present invention, described starchy material can variously can prepare 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.
According to the present invention, the inoculum size of described aspergillus niger can in very large range change, and under preferable case, every gram fermented liquid of take is benchmark, and the inoculum size of aspergillus niger is 5 * 10
4-2.5 * 10
5individual colony-forming unit, more preferably 1 * 10
5-1.5 * 10
5individual colony-forming unit.
Described colony-forming unit is defined as the method by cast or coating by a certain amount of bacterium liquid after dilution, 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 contained 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.
The aspergillus niger that the present invention's fermentation is used can be aspergillus niger solid preparation or the aspergillus niger strain be purchased, for example, and aspergillus niger Co827 (Shanghai Industrial institute of microbiology) and aspergillus niger T01 (Tianjin industrial microorganism institute).
Described aspergillus niger can adopt the inoculation of conventional method, for example, in being seeded to fermented liquid before, described aspergillus niger is processed through seed culture, afterwards the seed liquor obtained is joined in fermented liquid.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 the aspergillus niger nutrient solution and is cultivated, and it is 0.8-1.5 that the condition of described cultivation makes the respiratory quotient of aspergillus strain, and keeps 2-8 hour under this respiratory quotient.
On the one hand, it is 0.8-1.5 that the condition of described cultivation makes the respiratory quotient of aspergillus niger, is preferably 0.85-1.2; Respiratory quotient in this scope can make the aspergillus niger of cultivating be more suitable in follow-up fermentation, makes its fermentation efficiency even more ideal.On the other hand, the time of described maintenance is 2-8 hour, is preferably 3-5 hour, can improve further the efficiency of aspergillus niger in follow-up fermentation, thereby makes it in being more suitable for the state of fermentation.
According to the present invention, the preparation method of described aspergillus niger nutrient solution has no particular limits, as long as the nutrient solution obtained can be applicable to the cultivation of aspergillus niger, for example, the preparation method of described aspergillus niger nutrient solution can comprise: add amylase in farinaceous size, be warming up to 90-100 ℃ and once spray with 20-150 ℃/speed hourly at this temperature, carry out flash distillation after 5-30 minute, when temperature is down to 80-95 ℃ at this temperature enzymolysis starch 90-140 minute, obtaining liquefier and being diluted to total reducing sugar is the 5-20 % by weight, add afterwards nitrogenous source sterilizing.The term total reducing sugar refers to total content sugary in enzymatic liquefaction liquid.
According to the present invention, in described farinaceous size, the content of starch and water and the pH of slurries can in very large range change, and under preferable case, the content of described starch is the 10-25 % by weight, and the content of water is the 75-90 % by weight, and the pH value of described farinaceous size is 5-7.
Described diastatic kind is by the agency of hereinbefore, does not repeat them here.
According to the present invention, the kind of described nitrogenous source is conventionally known to one of skill in the art, for example, described nitrogenous source can be one or more in urea, ammonium sulfate and ammonium nitrate, the add-on of described nitrogenous source can in very large range change, under preferable case, the gross weight of described aspergillus niger nutrient solution of take is benchmark, and the add-on of described nitrogenous source is the 0.05-0.5 % by weight.
In the present invention, the inoculum size of described aspergillus niger can in very large range change, and under preferable case, every gram aspergillus niger nutrient solution of take is benchmark, and the inoculum size of aspergillus niger is 1-3 * 10
5individual colony-forming unit.
According to the present invention, the condition of described seed culture can in very large range change, and for example the condition of described cultivation can comprise: the temperature of cultivation can be 25-45 ℃, and the pH value can be 2-7, air flow can be the 0.1-1 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 the pH value can be 2.5-6.5, and air flow can be the 0.2-0.8 volume: volume minute, the time of described cultivation can be 50-60 hour.
The equipment of described cultivation is conventionally known to one of skill in the art, for example, can use fermentor tank to be cultivated.
The tunning citric acid prepared according to method of the present invention can separate and make with extra care according to the requirement of different Industrial products by conventional method, such as neutralization, acidolysis, decolouring, concentrated, crystallization, packing.
Below will to the present invention, be further described in detail by specific embodiment.
In the following embodiments, adopt mass spectrograph MAX300-LG to detect the exhaust gas component in culturing process, calculate release of carbonate dioxide speed and oxygen consumption rate, then calculate respiratory quotient; In fermented liquid, the measuring method of the amount of dissolved oxygen is for adopting online dissolved oxygen electrode (plum Teller) to detect.
Embodiment 1
The present embodiment is for illustrating the preparation method of citric acid provided by the invention.
(1) the 100 weight part corns that will gather in the crops (moisture content is 14 % by weight) are pulverized, and obtain the crushed products that average particle diameter is 400 microns.
(2) by the product after pulverizing by the concentration of 25 % by weight with size mixing after water mixes, dry weight with respect to every gram crushed products, amylase (the Novozymes Company that adds 20 enzyme activity units, a-amylase), enter injector, under the condition that is 5.5 at 85 ℃, pH, enzymolysis is 100 minutes, obtains enzymolysis product A1.Wherein, the A1 enzymolysis product by 80%, by with fluid pressure type sheet frame pressure filter, carrying out press filtration, is isolated enzymolysis clear liquid and enzymolysis solid phase residue.
(3) enzymolysis product that uses step (2) to obtain and the enzymolysis clear liquid configuration fermented liquid of separating, join in fermentor tank after specifically consisting of the enzymolysis clear liquid of 90 weight parts and the enzymolysis solid phase residue of 10 weight parts (solid content is 15 % by weight) sterilizing, obtain fermented liquid B1.
(4) the enzymolysis product A1 that step (2) obtains, be diluted with water to 10 % by weight of total reducing sugar, and input seeding tank, add urea, the add-on of urea is 0.35% of seed tank culture liquid gross weight, is heated to 121 ℃ of sterilizations, fast cooling to 36 ℃ after maintaining 30 minutes, (inoculum size is the access aspergillus niger strain for aspergillus niger T01, Tianjin industrial microorganism institute: every gram enzymolysis clear liquid 10
5individual colony-forming unit), at 36 ℃, 0.4 volume: under the aeration condition of volume minute, carry out spawn culture; Measure the growth of aspergillus niger observed by sampling sediments microscope inspection, acid test and pH, as pH 2.0, acidity 1%, bacterium ball size evenly, mycelia is sturdy while stretching out, stop cultivating.
(5) step (4) cultivation aspergillus niger strain is joined in the fermentor tank in step (1) and fermented, and detect the total reducing sugar in fermented liquid, inoculum size is: every gram fermented liquid 5 * 10
4individual colony-forming unit, fermentation tail gas is accessed to mass spectrograph repeatedly to be measured the respiratory quotient (respiratory quotient means with RQ) of aspergillus niger in fermenting process, according to value (the 0th hour to the 6th hour aspergillus niger residing stage of respiratory quotient of judgement of adjacent twice aspergillus niger respiratory quotient, every 18 minutes, detect once, the 6th hour later to the 10th hour, every 2 minutes, detect once, the 10th hour later to the 15th hour, every 10 minutes, detect once, the 15th hour later to the 18th hour, every 1 minute, detect once, the 18th hour later to the 48th hour, every 15 minutes, detect once, from the 48th hour later to fermentation ends, every 2 minutes, detect once), tank pressure is adjusted to 0.05MPa, at mixing speed, be 100rpm, ventilation is demarcated dissolved oxygen meter in 1: 0.5 o'clock, it is 100 that dissolved oxygen meter is demarcated displayed value, , now the amount of dissolved oxygen is 10 mg/litre.The 0th hour RQ that ferment is 0.31, it is that 39.5 ℃, ventilation are 1: 0.4 that leavening temperature is controlled, to fermentation the 8.7th hour the time RQ reach 1.21 (at the ascent stage of this aspergillus niger respiratory quotient, 30% that in fermented liquid, the amount of dissolved oxygen is calibration value), then RQ starts to descend; Adjusting leavening temperature and be 38 ℃, ventilation is 1: 0.5, during by the 16.9th hour, RQ is 0.3 (in the decline stage of this aspergillus niger respiratory quotient, in fermented liquid, the amount of dissolved oxygen is calibration value 40%), then adjusting leavening temperature and be 37 ℃, ventilation is 1: 0.6, until ferment the 50th hour, RQ maintains between 0.2-0.3 (in the low-level maintenance stage of this aspergillus niger respiratory quotient, 50% that in fermented liquid, the amount of dissolved oxygen is calibration value) substantially; After fermenting the 50th hour, RQ starts from 0.3 rising (rising in 0.65), now improving leavening temperature and be 39.5 ℃, ventilation is to ventilate at 1: 0.4, until fermentation is to the 56th hour fermentation ends (in the rise stage of this aspergillus niger respiratory quotient, 45% that in fermented liquid, the amount of dissolved oxygen is calibration value).
Embodiment 2
The present embodiment is for illustrating the preparation method of citric acid provided by the invention.
Method according to embodiment 1 prepares citric acid, different: as to use the enzymolysis product A1 configuration fermented liquid obtained in embodiment 1, join in fermentor tank after specifically consisting of the enzymatic liquefaction clear liquid of 85 weight parts, the enzymolysis solid phase residue of 14.8 weight parts (solid content is 30 % by weight) and 0.2 weight part urea sterilizing, obtain fermented liquid B2.
By the enzymatic liquefaction clear liquid obtained in embodiment 1, be diluted with water to total reducing sugar 10 % by weight and drop into seeding tank, add urea, the add-on of urea is 0.35% of seed tank culture liquid gross weight, be heated to 120 ℃ of sterilizations, maintain 20 minutes after fast cooling to 36 ℃, access aspergillus niger strain (aspergillus niger T01, Tianjin industrial microorganism institute, inoculum size is: every gram enzymatic liquefaction clear liquid 10
5individual colony-forming unit), at 36 ℃, 0.4 volume: under the aeration condition of volume minute, carry out spawn culture; Measure the growth of aspergillus niger observed by sampling sediments microscope inspection, acid test and pH, as pH 2.0, acidity 1%, bacterium ball size evenly, mycelia is sturdy while stretching out, stop cultivating.
Above-mentioned cultivation aspergillus niger strain is joined in fermentor tank and fermented, and detect the total reducing sugar in fermented liquid, inoculum size is: every gram fermented liquid 10
5individual colony-forming unit, fermentation tail gas is accessed to mass spectrograph repeatedly to be measured the respiratory quotient (respiratory quotient means with RQ) of aspergillus niger in fermenting process, according to value (the 0th hour to the 6th hour aspergillus niger residing stage of respiratory quotient of judgement of adjacent twice aspergillus niger respiratory quotient, every 16 minutes, detect once, the 6th hour later to the 10th hour, every 1.5 minutes, detect once, the 10th hour later to the 14th hour, every 5 minutes, detect once, the 14th hour later to the 17th hour, every 2 minutes, detect once, the 17th hour later to the 46th hour, every 18 minutes, detect once, from the 46th hour later to fermentation ends, every 1 minute, detect once), the 0th hour RQ that ferment is 0.36, it is that 39.5 ℃, ventilation are 1: 0.4 that leavening temperature is controlled, to fermentation the 8th hour the time RQ reach 1.21 (at the ascent stage of this aspergillus niger respiratory quotient, 28% that in fermented liquid, the amount of dissolved oxygen is calibration value), then RQ starts to descend, adjusting leavening temperature and be 38 ℃, ventilation is 1: 0.6, to fermentation the 15.7th hour the time RQ be 0.3 (in the decline stage of this aspergillus niger respiratory quotient, in fermented liquid, the amount of dissolved oxygen is calibration value 36%), then adjusting leavening temperature and be 37 ℃, ventilation is 1: 0.8, until fermentation is after the 49th hour, RQ maintains between 0.2-0.3 (in the low-level maintenance stage of this aspergillus niger respiratory quotient, 46% that in fermented liquid, the amount of dissolved oxygen is calibration value) substantially, after fermenting the 49th hour, RQ starts from 0.3 rising (rising in 0.7), now improving leavening temperature and be 39 ℃, ventilation is to ventilate at 1: 0.4, until fermentation is to the 55th hour fermentation ends (in the rise stage of this aspergillus niger respiratory quotient, 44% that in fermented liquid, the amount of dissolved oxygen is calibration value).
Embodiment 3
The present embodiment is for illustrating the preparation method of citric acid provided by the invention.
Method according to embodiment 1 prepares citric acid, different: as to use the enzymolysis product A1 configuration fermented liquid obtained in embodiment 1, join in fermentor tank after specifically consisting of the water sterilizing of the enzymolysis solid phase residue of the enzymatic liquefaction clear liquid of 90 weight parts and 5 weight parts (solid content is 30 % by weight) and 0.1 weight part urea, 4.9 weight parts, obtain fermented liquid B3.
By the enzymatic liquefaction clear liquid obtained in embodiment 1, be diluted with water to total reducing sugar 10 % by weight and drop into seeding tank, add urea, the add-on of urea is 0.35% of seed tank culture liquid gross weight, be heated to 120 ℃ of sterilizations, maintain 20 minutes after fast cooling to 36 ℃, access aspergillus niger strain (aspergillus niger T01, Tianjin industrial microorganism institute, inoculum size is: every gram enzymatic liquefaction clear liquid 2 * 10
5individual colony-forming unit), at 36 ℃, 0.4 volume: under the aeration condition of volume minute, carry out spawn culture; Measure the growth of aspergillus niger observed by sampling sediments microscope inspection, acid test and pH, as pH 2.0, acidity 1%, bacterium ball size evenly, mycelia is sturdy while stretching out, stop cultivating.
Above-mentioned cultivation aspergillus niger strain is joined in fermentor tank and fermented, and detect the total reducing sugar in fermented liquid, inoculum size is: every gram fermented liquid 1.5 * 10
5individual colony-forming unit, access mass spectrograph by fermentation tail gas the respiratory quotient (respiratory quotient means with RQ) of aspergillus niger in fermenting process carried out to on-line real time monitoring; The 0th hour RQ that ferment is 0.27, it is that 37.5 ℃, ventilation are 1: 0.5 that leavening temperature is controlled, to fermentation the 7.1st hour the time RQ reach 1.14 (at the ascent stage of this aspergillus niger respiratory quotient, 35% that in fermented liquid, the amount of dissolved oxygen is calibration value), then RQ starts to descend; Adjusting leavening temperature and be 35.5 ℃, ventilation is 1: 0.6, to fermentation the 14.3rd hour the time RQ be 0.25 (in the decline stage of this aspergillus niger respiratory quotient, in fermented liquid, the amount of dissolved oxygen is calibration value 45%), then adjusting leavening temperature and be 35 ℃, ventilation is 1: 0.8, until fermentation is after the 48.2nd hour, RQ maintains between 0.15-0.25 (in the low-level maintenance stage of this aspergillus niger respiratory quotient, 55% that in fermented liquid, the amount of dissolved oxygen is calibration value) substantially; After fermenting the 48.2nd hour, RQ starts from 0.25 rising (rising in 0.58), now improving leavening temperature and be 38 ℃, ventilation is to ventilate at 1: 0.4, until fermentation is to the 54th hour fermentation ends (in the rise stage of this aspergillus niger respiratory quotient, 49% that in fermented liquid, the amount of dissolved oxygen is calibration value).
Embodiment 4
The present embodiment is for illustrating the preparation method of citric acid provided by the invention.
Method according to embodiment 1 prepares citric acid, different, in step (5), step (4) cultivation aspergillus niger strain is joined in the fermentor tank in step (1) and fermented, and the total reducing sugar in the detection fermented liquid, inoculum size is: every gram fermented liquid 5 * 10
4individual colony-forming unit, access mass spectrograph by fermentation tail gas the respiratory quotient (respiratory quotient means with RQ) of aspergillus niger in fermenting process carried out to on-line real time monitoring; The 0th hour RQ that ferment is 0.22, it is that 41 ℃, ventilation are 1: 0.3 that leavening temperature is controlled, to fermentation the 6.6th hour the time RQ reach 0.95 (at the ascent stage of this aspergillus niger respiratory quotient, 30% that in fermented liquid, the amount of dissolved oxygen is calibration value), then RQ starts to descend; Adjusting leavening temperature and be 39 ℃, ventilation is 1: 0.65, to the 14th hour RQ, be 0.2 (in the decline stage of this aspergillus niger respiratory quotient, in fermented liquid, the amount of dissolved oxygen is calibration value 37%), then adjusting leavening temperature and be 38 ℃, ventilation is 1: 0.9, until ferment the 49th hour, RQ maintains between 0.2-0.3 (in the low-level maintenance stage of this aspergillus niger respiratory quotient, 48% that in fermented liquid, the amount of dissolved oxygen is calibration value) substantially; After fermenting the 49th hour, RQ starts from 0.2 rising (rising in 0.62), now improving leavening temperature and be 40.5 ℃, ventilation is to ventilate at 1: 0.6, until fermentation is to the 56th hour fermentation ends (in the rise stage of this aspergillus niger respiratory quotient, 43% that in fermented liquid, the amount of dissolved oxygen is calibration value).
Embodiment 5
The present embodiment is for illustrating the preparation method of citric acid provided by the invention.
According to the method identical with embodiment 2, fermented, different is, control by the following method the cultivation of aspergillus niger: will cultivate tail gas access mass spectrograph in culturing process, online record release of carbonate dioxide speed and oxygen consumption rate, and, in the line computation respiratory quotient, in respiratory quotient, be to maintain 7 hours 0.75 time.
Embodiment 6
The present embodiment is for illustrating the preparation method of citric acid provided by the invention.
According to the method identical with embodiment 2, fermented, different is, control by the following method the cultivation of aspergillus niger: will cultivate tail gas access mass spectrograph in culturing process, online record release of carbonate dioxide speed and oxygen consumption rate, and, in the line computation respiratory quotient, in respiratory quotient, be to maintain 3 hours 1.2 times.
Comparative Examples 1
This Comparative Examples is for illustrating the preparation method of citric acid of prior art.
Method according to embodiment 1 prepares citric acid, different, and the condition of fermentation remains: at 30 ℃, ventilation, be 0.8 volume: the condition bottom fermentation of volume minute finishes fermentation after 68 hours.
Embodiment 7-12
Concentration (abbreviation acidity) according to fermentation secondary fermentation liquid in GB 1987-2007 standard detection embodiment 1-6, and the transformation efficiency of calculating citric acid, the weight of the volume/total reducing sugar of the concentration of transformation efficiency (%)=fermented liquid (abbreviation acidity) * fermented liquid * 100%, result is as shown in table 1.
Comparative Examples 2
Detect acidity and the transformation efficiency of Comparative Examples 1 fermentation secondary fermentation liquid according to the method identical with embodiment 5-8, result is as shown in table 1.
Table 1
From the data of table 1 can find out, the acidity of the citric acid that adopts method fermentation of the present invention to obtain and transformation efficiency are all higher than the citric acid that adopts existing method to obtain, and fermentation period significantly shortens.Explanation thus, method of the present invention judges the physiological status of aspergillus niger by the variation tendency of the respiratory quotient of aspergillus niger in the fermenting process according to producing citric acid, under the different physiological statuss of aspergillus niger, fermentation condition is controlled and adjusted, thereby make growth during the fermentation of aspergillus niger and produce acid phase all in environmental optima, and reach its optimum utility of effective performance, and improved fermentation efficiency and shortened fermentation period.
In addition, with the data of embodiment 7-10, compare, the fermentation efficiency of embodiment 11 and embodiment 12 (acidity and transformation efficiency) is better, this shows, judge the cultivation conditions of aspergillus niger seed according to respiratory quotient, can make aspergillus niger there is higher fermentation efficiency (acidity and transformation efficiency) in follow-up fermenting process, and this method is subject to the impact of human factor very little, the stability that between can keeping batch, aspergillus niger is cultivated.
Claims (6)
1. the preparation method of a citric acid, the method comprises and aspergillus niger being seeded in fermented liquid and fermentation produces citric acid, it is characterized in that, the process of fermentation generation citric acid comprises the ascent stage of aspergillus niger respiratory quotient, the decline stage of aspergillus niger respiratory quotient, the low-level maintenance stage of aspergillus niger respiratory quotient and the rise stage of aspergillus niger respiratory quotient, leavening temperature at the ascent stage of aspergillus niger respiratory quotient is 37-41 ℃, leavening temperature in decline stage of aspergillus niger respiratory quotient is 35-39 ℃, leavening temperature in low-level maintenance stage of aspergillus niger respiratory quotient is 35-39 ℃, leavening temperature in rise stage of aspergillus niger respiratory quotient is 37-41 ℃, and the leavening temperature at the leavening temperature of the ascent stage of aspergillus niger respiratory quotient higher than the decline stage in the aspergillus niger respiratory quotient, leavening temperature at the leavening temperature of decline stage of aspergillus niger respiratory quotient higher than the low-level maintenance stage in the aspergillus niger respiratory quotient, at the leavening temperature in rise stage of aspergillus niger respiratory quotient higher than the leavening temperature of the decline stage in the aspergillus niger respiratory quotient and at the leavening temperature of low-level maintenance stage of aspergillus niger respiratory quotient,
Wherein, after being seeded in fermented liquid by aspergillus niger, the initial value of the respiratory quotient of aspergillus niger is 0.1-0.5, the respiratory quotient of aspergillus niger is in the scope in 0.8-1.5 after ascent stage, in the scope in 0.1-0.4 after the decline stage, in the scope of low-level maintenance stage in 0.1-0.4, after the rise stage in more than 0.4;
At the leavening temperature of the ascent stage of aspergillus niger respiratory quotient than the high 1-3 ℃ of leavening temperature of the decline stage in the aspergillus niger respiratory quotient, at the leavening temperature in rise stage of aspergillus niger respiratory quotient than the leavening temperature of the decline stage in the aspergillus niger respiratory quotient and at the high 1-3 ℃ of leavening temperature of low-level maintenance stage of aspergillus niger respiratory quotient;
The 20-40% that the amount of dissolved oxygen is calibration value in the ascent stage fermented liquid of aspergillus niger respiratory quotient, the 30-50% that the amount of dissolved oxygen is calibration value in the decline stage of aspergillus niger respiratory quotient fermented liquid, the 40-70% that the amount of dissolved oxygen is calibration value in the low-level maintenance stage fermented liquid of aspergillus niger respiratory quotient, the 40-70% that the amount of dissolved oxygen is calibration value in the rise stage fermentation liquid of aspergillus niger respiratory quotient; And the amount in the amount of the dissolved oxygen in low-level maintenance stage of aspergillus niger respiratory quotient and the stage of ging up higher than the dissolved oxygen of the ascent stage in the aspergillus niger respiratory quotient and decline stage, the amount in the amount of the dissolved oxygen of decline stage of aspergillus niger respiratory quotient higher than the dissolved oxygen of the ascent stage in the aspergillus niger respiratory quotient.
2. method according to claim 1, wherein, the method comprises the respiratory quotient of aspergillus niger in the repeated detection fermenting process, according to the value judgement aspergillus niger residing stage of respiratory quotient of adjacent twice aspergillus niger respiratory quotient.
3. method according to claim 2, wherein, the timed interval of adjacent twice detection is not more than 20 minutes.
4. method according to claim 3, wherein, the timed interval of adjacent twice detection is 2-5 minute.
5. method according to claim 1, wherein, every gram fermented liquid of take is benchmark, the inoculum size of aspergillus niger is 5 * 10
4-2.5 * 10
5individual colony-forming unit.
6. method according to claim 1, wherein, before in being seeded to fermented liquid, described aspergillus niger is also processed through seed culture, the method that this seed culture is processed comprises: aspergillus niger is seeded in the aspergillus niger nutrient solution and is cultivated, it is 0.8-1.5 that the condition of described cultivation makes the respiratory quotient of aspergillus niger, and keeps 2-8 hour under this respiratory quotient.
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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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