CN102533877A - Method for preparing citric acid by fermentation - Google Patents
Method for preparing citric acid by fermentation Download PDFInfo
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- CN102533877A CN102533877A CN2012100092945A CN201210009294A CN102533877A CN 102533877 A CN102533877 A CN 102533877A CN 2012100092945 A CN2012100092945 A CN 2012100092945A CN 201210009294 A CN201210009294 A CN 201210009294A CN 102533877 A CN102533877 A CN 102533877A
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Abstract
The invention discloses a method for preparing citric acid by fermentation, which comprises the following steps: under the conditions of generating citric acid, inoculating citric acid fermentation strain into fermentation media, and carrying out fermentation, thereby obtaining the fermentation liquid. The invention is characterized in that the method also comprises the step of adding saccharifying enzyme into the fermentation media 0-8 hours after inoculating the citric acid fermentation strain. The method disclosed by the invention enhances the saccharifying capacity in the fermentation process, solves the contradiction between the pH value requirements from citric acid synthesis and saccharification in the fermentation process, and avoids the adverse effect of high DE (dextrose equivalent) value on Aspergillus niger, so that the sugar solution in the fermentation media is saccharified more thoroughly, thereby lowering the residual sugar amount in the fermentation liquid, enhancing the end citric acid content and single-tank acid supply amount, increasing the conversion rate of sugar acid and shortening the fermentation period.
Description
Technical field
The present invention relates to a kind of fermentative prepn methods of citric acid.
Background technology
The fermenting process of tradition citric acid fermentation industry comprises: the pre-treatment of starchy material, the starchy material enzymolysis is become the dextrin liquid glucose, and its DE value is prepared fermention medium about 25; Aspergillus niger spore inserted in the seed culture medium to cultivate to be transferred to after the maturation carry out fermentation production of citric acid in the fermention medium.During the fermentation; Through black mold thalline self synthetic enzyme system the liquid glucose enzymatic saccharification is become to be fit to glucose and the fructose that black mold directly utilizes; Generally about 60 hours, fermenting acidity stops fermentation and puts jar about 14% for metabolism synthesizing citric acid then, fermentation period.
Tradition citric acid fermentation method utilizes Aspergillus niger strain self excretory saccharifying enzyme to carry out the fermentation of saccharification limit, limit.The shortcoming of this method is that black mold grows into the secretion saccharifying enzyme and needs a time course, and the pH value of saccharifying enzyme effect is about 4.0-4.6, and along with producing the acid increase; Acidity continues to raise; Saccharifying enzymic activity is suppressed, and the later stage can't play normal saccharification in fermentation, and the later stage black mold thalline rate of producing acid that causes fermenting receives the restriction of saccharification capability; Final Hydrocerol A output is lower, and residual sugar is higher in the fermented liquid, fermentation period is longer.
Some researchs have been carried out to the saccharification in the citric acid fermentation process; CN101942487A discloses a kind of interpolation saccharifying enzyme fermentative prepn methods of citric acid; Point out in the temperature-fall period of fermention medium sterilization back, to add the saccharifying enzyme of 50-100 enzyme activity unit, after cooling to normal inoculation temp, insert seed liquor and carry out citric acid fermentation.This method is owing to be before inoculation, to add saccharifying enzyme; Though envrionment temperature helps saccharification; But the DE value of fermention medium is too high when inserting Aspergillus niger strain, is unfavorable for the synthetic and effect of Aspergillus niger strain self saccharifying enzyme, is unfavorable for the growth of Aspergillus niger strain; The large usage quantity of saccharifying enzyme, cost is higher; Residual sugar is higher in the fermented liquid.
Therefore, await further to study for the saccharification in the citric acid fermentation process.
Summary of the invention
The objective of the invention is for solve Hydrocerol A in the prior art synthetic with saccharification to the contradiction between the requirement of pH value, and avoid the higher disadvantageous effect of fermention medium DE value to black mold self, a kind of new fermentative prepn methods of citric acid is provided.
Contriver of the present invention is unexpected under study for action to be found;, black mold in fermention medium, adds saccharifying enzyme after being seeded to fermention medium in 0-8 hour; Can quicken the saccharification of liquid glucose in the fermention medium; The later stage citric acid content of avoiding fermenting increases the inhibition to saccharification, and can make the DE value of fermention medium maintain lower level, avoids the disadvantageous effect of high DE value to black mold self.
Therefore; To achieve these goals, the invention provides a kind of fermentative prepn methods of citric acid, said method is included under the condition that generates Hydrocerol A; With fermenting in citric acid fermentation bacterial classification inoculation to the fermention medium; Obtain fermented liquid, it is characterized in that, said method also is included in the 0-8 hour introversive said fermention medium behind the citric acid fermentation bacterial classification inoculation and adds saccharifying enzyme.
Preferably, added saccharifying enzyme in the introversive said fermention medium in the 0-6 behind the citric acid fermentation bacterial classification inoculation hour.
Under the preferable case, with respect to the total reducing sugar in the said fermention medium, the add-on of said saccharifying enzyme is 20-50 enzyme activity unit/g total reducing sugar, further is preferably 30-40 enzyme activity unit/g total reducing sugar.
Fermentative prepn methods of citric acid provided by the invention strengthened the saccharification capability in the fermenting process, solved during the fermentation Hydrocerol A synthetic with saccharification to the contradiction between the requirement of pH value; And avoided the disadvantageous effect of high DE value to black mold self; Thereby make more thorough by saccharification of liquid glucose in the fermention medium, reduced the residual sugar amount in the fermented liquid, improved terminal point citric acid content and single jar and supplied the acid amount; Improve glucose acid invert ratio, shortened fermentation period.
Other features and advantages of the present invention will partly specify in embodiment subsequently.
Embodiment
Following specific embodiments of the invention is elaborated.Should be understood that embodiment described herein only is used for explanation and explains the present invention, is not limited to the present invention.
The invention provides a kind of fermentative prepn methods of citric acid; This method is included under the condition that generates Hydrocerol A; With fermenting in citric acid fermentation bacterial classification inoculation to the fermention medium; Obtain fermented liquid, this method also is included in 0-8 hour behind the citric acid fermentation bacterial classification inoculation and in fermention medium, adds saccharifying enzyme.
According to the present invention, can realize the object of the invention although in fermention medium, add saccharifying enzyme in the 0-8 behind the citric acid fermentation bacterial classification inoculation hour, promptly reduce the residual sugar amount in the fermented liquid; Improve glucose acid invert ratio, shorten fermentation period, but under the preferable case; In fermention medium, add saccharifying enzyme in 0-6 behind the citric acid fermentation bacterial classification inoculation hour; Can further reduce the residual sugar amount in the fermented liquid, improve glucose acid invert ratio, shorten fermentation period.
Among the present invention, in fermention medium, add saccharifying enzyme in 0 hour behind the citric acid fermentation bacterial classification inoculation and be meant citric acid fermentation bacterial classification and saccharifying enzyme are added in the fermention medium simultaneously.
Among the present invention; Fermention medium is for well known to a person skilled in the art notion; Refer to the nutriment of required confession microorganism growth of microbial fermentation and the manual work preparation of keeping usefulness, generally all contain glucide, nitrogenous substances, inorganic salt (comprising trace element) and VITAMINs and water etc.Fermented liquid refers to an access the liquid nutrient medium (this liquid nutrient medium also is an alleged fermention medium among the present invention) of microorganism strains, products therefrom after cultivation after a while also for well known to a person skilled in the art notion.
Among the present invention, saccharifying enzyme promptly is glucoamylase (α-1, a 4-glucose hydrolysis enzyme), and with respect to the total reducing sugar in the fermention medium, the add-on of saccharifying enzyme is preferably 20-50 enzyme activity unit/g total reducing sugar, further is preferably 30-40 enzyme activity unit/g total reducing sugar.Total reducing sugar in the fermention medium of the add-on institute reference of calculating saccharifying enzyme refers to the total reducing sugar that rigidly connects in kind of the post-fermentation and culture base.
Among the present invention, being defined as of enzyme activity unit: be 4.6 in the pH value, temperature is that 1 minute is converted into the required enzyme amount of reducing sugar with 1 milligram of starch is an enzyme activity unit under 60 ℃ the condition.
Among the present invention, do not have particular requirement, can be preferably black mold for bacterial classification commonly used in this area for the kind of citric acid fermentation bacterial classification.
Fermenting process is the biochemical reaction process of being participated in by mikrobe with regard to its essence, so the quantity of microorganism cells, state, metabolism situation are to the biosynthesizing important influence of product.The size of cell concentration is to the productive rate important influence of tunning.Cell concentration is big more in theory, and the output of product is also big more, can produce other influences but cell concentration is too high; Consume too fast like nutritive substance; Nutritive ingredient in the fermented liquid takes place significantly to change, the accumulation of toxic substance etc., and these possibly change the pathways metabolism of thalline.Therefore, among the present invention, be benchmark with every liter of fermention medium, the inoculum size of black mold is preferably 1.0 * 10
7-5 * 10
7Individual spore further is preferably 2.0 * 10
7-3.0 * 10
7Individual spore.
The quantity of spore can be measured by means commonly known in the art, for example, counts through blood counting chamber.
Because when inserting Aspergillus niger strain, the too high synthetic and effect that is unfavorable for Aspergillus niger strain self saccharifying enzyme of the DE value of fermention medium is unfavorable for the growth of Aspergillus niger strain; The DE value of fermention medium is low excessively; Lack again and can directly be utilized the reducing sugar that ferments by black mold, therefore, before the black mold inoculation; The DE value of fermention medium is preferably 15-40%, further is preferably 15-25%.
Among the present invention, the DE value refers to glucose meter, and reducing sugar accounts for the per-cent of total reducing sugar in the fermention medium.The DE value of citric acid fermentation substratum commonly used is general all at 15-40% in this area, but for the DE value that makes fermention medium is controlled in the 15-25% scope, the fermention medium among the present invention preferably contains the starchy material enzymolysis product.
Usually; The starchy material enzymolysis obtains starchy material enzymolysis residue and starchy material enzymatic liquefaction clear liquid; Usually can starchy material enzymatic liquefaction clear liquid be used to prepare fermention medium, also can with starchy material enzymatic liquefaction clear liquid be used to prepare fermention medium after starchy material enzymolysis residue mixes.Among the present invention; The starchy material enzymolysis product is preferably mixed with water or is not mixed with water by starchy material enzymolysis residue and starchy material enzymatic liquefaction clear liquid and obtains; And further preferred gross weight with fermention medium is that 100 weight parts are benchmark; The consumption of starchy material enzymatic liquefaction clear liquid is the 80-95 weight part, and the consumption of starchy material enzymolysis residue is the 5-10 weight part, and the consumption of water is the 0-15 weight part.
According to the present invention, starchy material enzymatic liquefaction clear liquid can prepare through several different methods, for example; Can prepare through following method: starchy material is pulverized, the product after pulverizing is carried out enzymolysis, obtain enzymolysis product; With the enzymolysis product solid-liquid separation; Obtain starchy material enzymatic liquefaction clear liquid and starchy material enzymolysis residue, it is 45-60 weight %, more preferably 55-60 weight % that the condition of solid-liquid separation makes the solid content of starchy material enzymolysis residue.
According to the present invention; Starchy material can be the known various raw materials that contain starch that can be used for enzymolysis, fermentative prepn Hydrocerol A of ability, for example, can be selected from corn, potato class (like cassava) and the wheat one or more; Under the preferable case, said starchy material is a corn.
Said enzymolysis step can be accomplished through this area method commonly used, and such as in crushed products, adding microbes producing cellulase and/or enzyme, insulation is accomplished under the growth temperature of microbes producing cellulase and/or the great-hearted temperature of enzyme.Said microbes producing cellulase be can secreting amylase microbes producing cellulase.Said enzyme comprises glycase.
Because microorganism growth can produce by product, the therefore preferred enzyme that directly adds.The consumption of said enzyme is The more the better, from cost consideration, and the dry weight basis of the crushed products after preferably pulverizing with every gram, diastatic consumption is a 15-50 enzyme activity unit.
The temperature of enzymolysis can in very large range change, and is preferably 70-105 ℃, more preferably 80-95 ℃.The longer the better on the time theory of enzymolysis, considers plant factor, and the time of preferred enzymolysis is 90-150 minute, more preferably 100-120 minute.The pH value of enzymolysis can in very large range change, and is preferably 5.0-7.0, more preferably 5.4-5.7.
Glycase is meant the general name of class of enzymes that can the starch-splitting glycosidic link, and glycase generally comprises AMS, beta-amylase, saccharifying enzyme and isoamylase.
According to the present invention, preferably use AMS and/or isoamylase.
According to the present invention, the method and apparatus of solid-liquid separation is conventionally known to one of skill in the art, for example, and pressure filter or whizzer.
Black mold can adopt conventional method inoculation, for example, in being seeded to fermention medium before, black mold through seed tank culture, is joined the mature seed liquid that obtains in the fermention medium afterwards.The degree of black mold seed culture can be measured to confirm through sampling sediments microscope inspection, acid test and pH; When pH 2.0-2.5, acidity be 0.5-2.0g/100ml, bacterium ball size evenly, mycelia is sturdy stops to cultivate when stretching out, the seed liquor of this moment is called mature seed liquid.
Under the preferable case, the method for seed tank culture comprises: black mold being seeded in the seed tank culture base cultivating, contain the Semen Maydis powder of 10-17 weight % in the seed tank culture base, is benchmark with every liter of nutrient solution, and the inoculum size of black mold is 2 * 10
8-3 * 10
8Individual spore.
According to the present invention, the preparation method of seed tank culture base has no particular limits, as long as the seed tank culture base that obtains can be applicable to the cultivation of black mold.
According to the present invention, black mold can in very large range change at the culture condition of seed tank culture, and for example culture condition can comprise: the temperature of cultivation is 25-45 ℃, and initial pH value is 5-6, and air flow is the 0.1-1 volume: (volume minute); Under the preferred situation, culture condition can comprise: the temperature of cultivation is 30-40 ℃, and initial pH value is 5-6, and air flow is the 0.3-0.8 volume: (volume minute).
Term " air flow " is generally with ventilation expression recently, and usually recently to represent (V/Vmin) through the volume of air of unit volume nutrient solution in the PM, for example ventilation is than being 1: 0.1-1, the abbreviation air flow is the 0.1-1 volume: (volume minute).
Among the present invention, having no particular limits for the condition of fermenting, can be the conventional fermentation condition in this area, and for example, the condition of fermentation can comprise: temperature is 30-40 ℃, is preferably 33-38 ℃; Initial pH value is 4-5; Air flow is the 0.1-1 volume: (volume minute); Be preferably the 0.3-0.8 volume: (volume minute).
The equipment of fermentation is conventionally known to one of skill in the art, for example, can use fermentor tank to ferment.
The tunning Hydrocerol A for preparing according to method of the present invention can be used conventional method, separate and refining according to the requirement of different Industrial products, such as neutralization, acidolysis, decolouring, concentrate, crystallization, packing.
More than describe preferred implementation of the present invention in detail; But the present invention is not limited to the detail in the above-mentioned embodiment, in technical conceive scope of the present invention; Can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
Need to prove in addition; Each concrete technical characterictic described in above-mentioned embodiment under reconcilable situation, can make up through any suitable manner; For fear of unnecessary repetition, the present invention is to the explanation no longer separately of various possible array modes.
In addition, also can carry out arbitrary combination between the various embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be regarded as the disclosed content of the present invention equally.
Embodiment
Following embodiment will be further described the present invention, but therefore not limit the present invention.
In following examples and Comparative Examples:
Aspergillus niger strain is black mold Co827.
Concentration (being terminal point citric acid content or title acidity) according to GB 1987-2007 standard detection gained citric acid solution.
Measure the concentration of reducing sugar in the fermention medium according to the method for GB/T5009.7-2008.
Measure the concentration of total reducing sugar in the fermention medium according to the method for GB/T5009.8-2003.
Total sugar content in reducing sugar content/fermention medium in DE value=fermention medium.
All stop fermentation in following examples and the Comparative Examples so that reducing sugar concentration detected value is lower than 0.2g/100ml in the fermention medium, confirm as fermentation termination.The time that begins to experience to fermentation termination from access aspergillus niger strain fermention medium is fermentation period.
The concentration of total reducing sugar in the fermention medium when residual sugar amount is fermentation termination.
Single jar of volume that supplies the concentration * citric acid solution of acid amount=citric acid solution.
Transformation efficiency (%)=single jar supplies gross weight * 100% of acid amount/usefulness sugar, wherein uses sugared gross weight to comprise that seeding tank uses sugar weight with sugar weight and fermentor tank.
Embodiment 1
Present embodiment is used to explain fermentative prepn methods of citric acid provided by the invention.
(1) 56 kg corn that will gather in the crops are stewing in hot water tank profit, are 15 weight % until the water cut of corn, pulverize then, obtain average particle diameter and be 400 microns pulverizing after product.
(2) product after will pulverizing is sized mixing by the concentration of 25 weight %; With respect to the product after every gram pulverizing, add glycase (Novozymes Company, the AMS of 20 enzyme activity units; Equal glycase for this reason in the embodiment of the invention and the Comparative Examples); Getting into injector, is enzymolysis 100 minutes under 5.5 the condition at 85 ℃, pH, obtains enzymolysis product.
(3) with enzymolysis product through carrying out press filtration with the fluid pressure type plate-and-frame filter press, isolate enzymatic liquefaction clear liquid and enzymolysis filter residue, wherein, the water cut of enzymolysis residue is 45 weight %.
(4) preparation fermention medium with being cooled to 35 ℃ after 180 kilograms above-mentioned enzymatic liquefaction clear liquids, 10 kilograms the water sterilization of enzymolysis residue and 13 kilograms, joins in the fermentor tank of 300L, obtains fermention medium.Measure the concentration of the reducing sugar in the fermention medium and the concentration of total reducing sugar, calculating the DE value is 20%.
(5) with the part enzymolysis product in the step (2); Thin up to total reducing sugar is 10 weight %, obtains the seed tank culture base, and the seed tank culture base is dropped into seeding tank; Be heated to 121 ℃ of sterilizations; Keep after 30 minutes fast cooling to 36 ℃, insert aspergillus niger strain, the inoculum size of black mold is 3 * 10 in every liter of seed tank culture base
8Individual spore.At 36 ℃, initial pH value is 5,0.3 volume: carry out spawn culture under the aeration condition of (volume minute); Measure through sampling sediments microscope inspection, acid test and pH the growth of black mold observed, when pH 2.0, acidity 1g/100ml, bacterium ball size evenly, mycelia is sturdy when stretching out, and stops to cultivate, and obtains mature seed liquid.
(6) the mature seed liquid that step (5) is obtained joins and begins fermentation in the fermentor tank of step (4), and inoculum size is every liter of fermention medium 2.4 * 10
7Individual spore, fermentation condition comprise that temperature is 35 ℃, and initial pH value is 5; Air flow is 0.3 volume: (volume minute), and when fermentation culture adds saccharifying enzyme in substratum in the time of 3 hours, with respect to the total reducing sugar that rigidly connects in kind of the post-fermentation and culture base; The add-on of saccharifying enzyme is 35 enzyme activity units/g total reducing sugar, when the reducing sugar detected value is lower than 0.2g/100ml in the fermention medium, stops fermentation, the statistics fermentation period; Measure the total sugar concentration (being the residual sugar amount, down together) in the fermention medium, carry out solid-liquid separation then; Obtain citric acid solution, the concentration of measuring citric acid solution (is the terminal point citric acid content, or claims acidity; Down together), calculating single jar supplies acid amount and transformation efficiency to see table 1.
Embodiment 2
Present embodiment is used to explain fermentative prepn methods of citric acid provided by the invention.
(1) 56 kg corn that will gather in the crops are stewing in hot water tank profit, are 20 weight % until the water cut of corn, pulverize then, obtain average particle diameter and be 800 microns pulverizing after product.
(2) product after will pulverizing is sized mixing by the concentration of 25 weight %, and the product after pulverizing with respect to every gram adds the glycase of 50 enzyme activity units, gets into injector, is enzymolysis 110 minutes under 5.7 the condition at 95 ℃, pH, obtains enzymolysis product.
(3) with enzymolysis product through carrying out press filtration with the fluid pressure type plate-and-frame filter press, isolate enzymatic liquefaction clear liquid and enzymolysis filter residue, wherein, the water cut of enzymolysis residue is 40 weight %.
(4) preparation fermention medium with being cooled to 38 ℃ after 190 kilograms above-mentioned enzymatic liquefaction clear liquids, 10 kilograms the enzymolysis residue sterilization, joins in the fermentor tank of 300L, obtains fermention medium.Measure the concentration of the reducing sugar in the fermention medium and the concentration of total reducing sugar, calculating the DE value is 25%.
(5) with the part enzymatic liquefaction clear liquid in the step (2); Thin up to total reducing sugar is 10 weight %, obtains the seed tank culture base, and the seed tank culture base is dropped into seeding tank; Be heated to 121 ℃ of sterilizations; Keep after 30 minutes fast cooling to 33 ℃, insert aspergillus niger strain, the inoculum size of black mold is 2 * 10 in every liter of seed tank culture base
8Individual spore.At 33 ℃, initial pH value is 5.5,0.6 volume: carry out spawn culture under the aeration condition of (volume minute); Measure through sampling sediments microscope inspection, acid test and pH the growth of black mold observed, when pH 2.5, acidity 0.5g/100ml, bacterium ball size evenly, mycelia is sturdy when stretching out, and stops to cultivate, and obtains mature seed liquid.
(6) the mature seed liquid that step (5) is obtained joins and begins fermentation in the fermentor tank of step (4), and inoculum size is every liter of fermention medium 2.2 * 10
7Individual spore, fermentation condition comprise that temperature is 38 ℃, and initial pH value is 4.5; Air flow is 0.6 volume: (volume minute), and when fermentation culture adds saccharifying enzyme in substratum in the time of 6 hours, with respect to the total reducing sugar that rigidly connects in kind of the post-fermentation and culture base; The add-on of saccharifying enzyme is 30 enzyme activity units/g total reducing sugar, when the reducing sugar detected value is lower than 0.2g/100ml in the fermention medium, stops fermentation, the statistics fermentation period; Measure the total sugar concentration in the fermention medium, carry out solid-liquid separation then, obtain citric acid solution; Measure the concentration of citric acid solution, calculate single jar and supply acid amount and transformation efficiency to see table 1.
Embodiment 3
Present embodiment is used to explain fermentative prepn methods of citric acid provided by the invention.
(1) 56 kg corn that will gather in the crops are stewing in hot water tank profit, are 10 weight % until the water cut of corn, pulverize then, obtain average particle diameter and be 500 microns pulverizing after product.
(2) product after will pulverizing is sized mixing by the concentration of 25 weight %, and the product after pulverizing with respect to every gram adds the glycase of 15 enzyme activity units, gets into injector, is enzymolysis 120 minutes under 5.4 the condition at 80 ℃, pH, obtains enzymolysis product.
(3) with enzymolysis product through carrying out press filtration with the fluid pressure type plate-and-frame filter press, isolate enzymatic liquefaction clear liquid and enzymolysis filter residue, wherein, the water cut of enzymolysis residue is 70 weight %.
(4) preparation fermention medium is cooled to 40 ℃ after the water sterilization with the enzymolysis residue of 160 kilograms above-mentioned enzymatic liquefaction clear liquids and 20 kilograms and 20 kilograms, joins in the fermentor tank of 300L, obtains fermention medium.Measure the concentration of the reducing sugar in the fermention medium and the concentration of total reducing sugar, calculating the DE value is 15%.
(5) with the part enzymatic liquefaction clear liquid in the step (2); Thin up to total reducing sugar is 10 weight %, obtains the seed tank culture base, and the seed tank culture base is dropped into seeding tank; Be heated to 121 ℃ of sterilizations; Keep after 30 minutes fast cooling to 38 ℃, insert aspergillus niger strain, the inoculum size of black mold is 2.5 * 10 in every liter of seed tank culture base
8Individual spore.At 38 ℃, initial pH value is 6,0.8 volume: carry out spawn culture under the aeration condition of (volume minute); Measure through sampling sediments microscope inspection, acid test and pH the growth of black mold observed, when pH 2.5, acidity 2g/100ml, bacterium ball size evenly, mycelia is sturdy when stretching out, and stops to cultivate, and obtains mature seed liquid.
(6) the mature seed liquid that step (5) is obtained joins and begins fermentation in the fermentor tank of step (4), and inoculum size is every liter of fermention medium 2.6 * 10
7Individual spore, fermentation condition comprise that temperature is 33 ℃, and initial pH value is 4; Air flow is 0.8 volume: (volume minute) adds saccharifying enzyme after adding mature seed liquid, immediately with respect to the total reducing sugar that rigidly connects in kind of the post-fermentation and culture base in substratum; The add-on of saccharifying enzyme is 40 enzyme activity units/g total reducing sugar, when the reducing sugar detected value is lower than 0.2g/100ml in the fermention medium, stops fermentation, the statistics fermentation period; Measure the total sugar concentration in the fermention medium, carry out solid-liquid separation then, obtain citric acid solution; Measure the concentration of citric acid solution, calculate single jar and supply acid amount and transformation efficiency to see table 1.
Embodiment 4
According to the method fermentative prepn Hydrocerol A of embodiment 1, different is, when fermentation culture adds saccharifying enzyme in substratum in the time of 8 hours.The statistics fermentation period is measured the total sugar concentration in the fermention medium, measures the concentration of citric acid solution, calculates single jar and supplies acid amount and transformation efficiency to see table 1.
Embodiment 5
According to the method fermentative prepn Hydrocerol A of embodiment 1, different is that with respect to the total reducing sugar that rigidly connects in kind of the post-fermentation and culture base, the add-on of saccharifying enzyme is 50 enzyme activity units/g total reducing sugar.The statistics fermentation period is measured the total sugar concentration in the fermention medium, measures the concentration of citric acid solution, calculates single jar and supplies acid amount and transformation efficiency to see table 1.
Embodiment 6
According to the method fermentative prepn Hydrocerol A of embodiment 1, different is that with respect to the total reducing sugar that rigidly connects in kind of the post-fermentation and culture base, the add-on of saccharifying enzyme is 20 enzyme activity units/g total reducing sugar.The statistics fermentation period is measured the total sugar concentration in the fermention medium, measures the concentration of citric acid solution, calculates single jar and supplies acid amount and transformation efficiency to see table 1.
Comparative Examples 1
According to the method fermentative prepn Hydrocerol A of embodiment 1, different is, when fermentation culture adds saccharifying enzyme in substratum in the time of 10 hours.The statistics fermentation period is measured the total sugar concentration in the fermention medium, measures the concentration of citric acid solution, calculates single jar and supplies acid amount and transformation efficiency to see table 1.
Comparative Examples 2
According to the method fermentative prepn Hydrocerol A of embodiment 1, different is, during step (4) preparation fermention medium; In substratum, add saccharifying enzyme when being cooled to 60 ℃ after the sterilization, with respect to the starch in the fermention medium, the add-on of saccharifying enzyme is 80 enzyme activity units/g starch; And then be cooled to 35 ℃; Join in the fermentor tank of 300L, obtain fermention medium, and after the aspergillus niger strain that step (5) is cultivated joins in the fermentor tank of step (4), no longer add saccharifying enzyme.The statistics fermentation period is measured the total sugar concentration in the fermention medium, measures the concentration of citric acid solution, calculates single jar and supplies acid amount and transformation efficiency to see table 1.
Comparative Examples 3
According to the method fermentative prepn Hydrocerol A of embodiment 1, different is not add saccharifying enzyme.The statistics fermentation period is measured the total sugar concentration in the fermention medium, measures the concentration of citric acid solution, calculates single jar and supplies acid amount and transformation efficiency to see table 1.
Table 1
Can find out that from table 1 adopt the inventive method, fermentation period is short, the residual sugar amount is low, and acidity and single jar of confession acid amount are high, and transformation efficiency is high.Embodiment 1-6 and Comparative Examples 1 are compared and can find out, add saccharifying enzyme after 8 hours, be unfavorable for the fermentation of Hydrocerol A in fermentation culture; Embodiment 1-6 and Comparative Examples 2 are compared and can find out, in the temperature-fall period of fermention medium sterilization back, add saccharifying enzyme, not as in fermentation culture 0-8 hour, adding the effective of saccharifying enzyme; Embodiment 1-6 and Comparative Examples 3 are compared and can find out, in fermentation culture 0-8 hour, add saccharifying enzyme, help the fermentation of Hydrocerol A than not adding saccharifying enzyme.
Embodiment 1 and embodiment 4 are compared and can find out, in fermentation culture 0-6 hour, add saccharifying enzyme, more help the fermentation of Hydrocerol A; Embodiment 1 is compared and can find out with embodiment 5 and embodiment 6 respectively, and with respect to the total reducing sugar that rigidly connects in kind of the post-fermentation and culture base, the add-on of saccharifying enzyme is 30-40 enzyme activity unit/g total reducing sugar, more helps the fermentation of Hydrocerol A.
Fermentative prepn methods of citric acid provided by the invention strengthened the saccharification capability in the fermenting process, solved during the fermentation Hydrocerol A synthetic with saccharification to the contradiction between the requirement of pH value; And avoided the disadvantageous effect of high DE value to black mold self; Thereby make more thorough by saccharification of liquid glucose in the fermention medium, reduced the residual sugar amount in the fermented liquid, improved terminal point citric acid content and single jar and supplied the acid amount; Improve glucose acid invert ratio, shortened fermentation period.
Claims (10)
1. fermentative prepn methods of citric acid; Said method is included under the condition that generates Hydrocerol A; With fermenting in citric acid fermentation bacterial classification inoculation to the fermention medium; Obtain fermented liquid, it is characterized in that, said method also is included in the 0-8 hour introversive said fermention medium behind the citric acid fermentation bacterial classification inoculation and adds saccharifying enzyme.
2. method according to claim 1 wherein, adds saccharifying enzyme in the 0-6 behind the citric acid fermentation bacterial classification inoculation hour introversive said fermention medium.
3. method according to claim 1 and 2, wherein, with respect to the total reducing sugar in the said fermention medium, the add-on of said saccharifying enzyme is 20-50 enzyme activity unit/g total reducing sugar.
4. method according to claim 3, wherein, with respect to the total reducing sugar in the said fermention medium, the add-on of said saccharifying enzyme is 30-40 enzyme activity unit/g total reducing sugar.
5. according to any described method among the claim 1-4, wherein, said citric acid fermentation bacterial classification is a black mold.
6. method according to claim 5 wherein, is a benchmark with every liter of fermention medium, and the inoculum size of black mold is 1 * 10
7-5 * 10
7Individual spore.
7. method according to claim 6 wherein, is a benchmark with every liter of fermention medium, and the inoculum size of black mold is 2 * 10
7-3 * 10
7Individual spore.
8. according to any described method among the claim 5-7, wherein, before the black mold inoculation, the DE value of fermention medium is 15-40%.
9. according to any described method among the claim 1-8, wherein, said fermention medium contains the starchy material enzymolysis product.
10. according to any described method among the claim 1-9, wherein, the condition of said fermentation comprises: temperature is 30-40 ℃, and initial pH value is 4-5, and air flow is the 0.1-1 volume: (volume minute).
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| CN104561140A (en) * | 2013-10-23 | 2015-04-29 | 中粮营养健康研究院有限公司 | Method for preparing citric acid by fermentation |
| CN105586367A (en) * | 2016-03-10 | 2016-05-18 | 江南大学 | Method for conducting fermentative production of citric acid by adding saccharifying enzyme stage by stage based on pH responses |
| CN109266695A (en) * | 2018-10-31 | 2019-01-25 | 西北农林科技大学 | A method of utilizing persimmon fermented production citric acid |
| CN109852640A (en) * | 2017-11-30 | 2019-06-07 | 中粮生物化学（安徽）股份有限公司 | Full starch prepares citric acid fermented with seed culture medium and the citric acid fermented method with culture medium and citric acid |
| CN111172204A (en) * | 2020-03-13 | 2020-05-19 | 合肥五粮泰生物科技有限公司 | Preparation method for improving citric acid fermentation efficiency |
| CN116179617A (en) * | 2023-04-23 | 2023-05-30 | 莱芜泰禾生化有限公司 | Method for preparing citric acid by fermentation |
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| CN104561140B (en) * | 2013-10-23 | 2018-03-06 | 中粮营养健康研究院有限公司 | A kind of method of preparation of citric acid by fermentation |
| CN105586367A (en) * | 2016-03-10 | 2016-05-18 | 江南大学 | Method for conducting fermentative production of citric acid by adding saccharifying enzyme stage by stage based on pH responses |
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| CN109852640B (en) * | 2017-11-30 | 2020-12-15 | 中粮生物科技股份有限公司 | Seed culture medium for preparing fermented citric acid from full starch, culture medium for fermenting citric acid and method for preparing citric acid from full starch |
| CN109266695A (en) * | 2018-10-31 | 2019-01-25 | 西北农林科技大学 | A method of utilizing persimmon fermented production citric acid |
| CN109266695B (en) * | 2018-10-31 | 2021-09-03 | 西北农林科技大学 | Method for producing citric acid by fermentation of persimmons |
| CN111172204A (en) * | 2020-03-13 | 2020-05-19 | 合肥五粮泰生物科技有限公司 | Preparation method for improving citric acid fermentation efficiency |
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