CN102533877B - Method for preparing citric acid by fermentation - Google Patents
Method for preparing citric acid by fermentation Download PDFInfo
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- CN102533877B CN102533877B CN201210009294.5A CN201210009294A CN102533877B CN 102533877 B CN102533877 B CN 102533877B CN 201210009294 A CN201210009294 A CN 201210009294A CN 102533877 B CN102533877 B CN 102533877B
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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 fermentation and prepare the method for citric acid.
Background technology
The fermenting process of tradition citric acid fermentation industry comprises: the pre-treatment of starchy material, starchy material enzymolysis is become to dextrin liquid glucose, and its DE value, in 25 left and right, is prepared fermention medium; Aspergillus niger spore is accessed in seed culture medium, to cultivate to be transferred to after maturation and in fermention medium, carry out fermentation production of citric acid.During the fermentation, the enzyme system synthetic by aspergillus strain self becomes to be applicable to by liquid glucose enzymatic saccharification glucose and the fructose that aspergillus niger directly utilizes, then metabolism synthesizing citric acid, fermentation period is generally about 60 hours, and fermenting acidity stops fermentation and puts tank in 14% left and right.
Tradition citric acid fermentation method utilizes the saccharifying enzyme of Aspergillus niger strain self secretion to carry out the fermentation of saccharification limit, limit.The shortcoming of this method is that Aspergillus Niger Growth arrives a time course of secretion saccharifying enzyme needs, and the pH value of saccharifying enzyme effect is in 4.0-4.6 left and right, and increase along with producing acid, acidity continues to raise, saccharifying enzymic activity is suppressed, and in fermentation, the later stage cannot play normal saccharification, and the later stage aspergillus strain rate of producing acid that causes fermenting is subject to the restriction of saccharification capability, final citric acid output is lower, and in fermented liquid, residual sugar is higher, fermentation period is longer.
For the saccharification in Citric Acid Fermentation, some researchs have been carried out, CN101942487A discloses a kind of method that citric acid is prepared in saccharifying enzyme fermentation of adding, point out in temperature-fall period, to add the saccharifying enzyme of 50-100 enzyme activity unit after fermention medium sterilizing, after cooling to normal inoculation temp, access seed liquor and carry out citric acid fermentation.The method is owing to being to add saccharifying enzyme before inoculation, although envrionment temperature is conducive to saccharification, but the DE value of fermention medium is too high when access Aspergillus niger strain, be unfavorable for the synthetic and effect of Aspergillus niger strain self saccharifying enzyme, be unfavorable for the growth of Aspergillus niger strain; The large usage quantity of saccharifying enzyme, cost is higher; In fermented liquid, residual sugar is higher.
Therefore, for the saccharification in Citric Acid Fermentation, need further to study.
Summary of the invention
The object of the invention is for solve citric acid in prior art synthetic with saccharification to the contradiction between the requirement of pH value, and avoid the higher disadvantageous effect to aspergillus niger self of fermention medium DE value, provide a kind of new fermentation to prepare the method for citric acid.
The present inventor surprisingly finds under study for action, after being seeded to fermention medium, aspergillus niger in fermention medium, adds saccharifying enzyme in 0-8 hour, can accelerate the saccharification of liquid glucose in 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, avoid the disadvantageous effect of high DE value to aspergillus niger self.
Therefore, to achieve these goals, the invention provides a kind of fermentation and prepare the method for citric acid, described method is included under the condition that generates citric acid, citric acid fermentation bacterial classification is seeded in fermention medium and is fermented, obtain fermented liquid, it is characterized in that, described method is also included in the postvaccinal 0-8 of citric acid fermentation bacterial classification hour and adds saccharifying enzyme in introversive described fermention medium.
Preferably, in the postvaccinal 0-6 of citric acid fermentation bacterial classification hour, in described fermention medium, add saccharifying enzyme.
Under preferable case, with respect to the total reducing sugar in described fermention medium, the add-on of described saccharifying enzyme is 20-50 enzyme activity unit/g total reducing sugar, more preferably 30-40 enzyme activity unit/g total reducing sugar.
The method of citric acid is prepared in fermentation provided by the invention, strengthened the saccharification capability in fermenting process, solved during the fermentation citric acid synthetic with saccharification to the contradiction between the requirement of pH value, and avoided the disadvantageous effect of high DE value to aspergillus niger self, thereby make more thorough by saccharification of liquid glucose in fermention medium, reduced the residual sugar amount in fermented liquid, improved terminal citric acid content and single tank for acid amount, improve glucose acid invert ratio, shortened fermentation period.
Other features and advantages of the present invention partly in detail are described the embodiment subsequently.
Embodiment
Below the specific embodiment of the present invention is elaborated.Should be understood that, embodiment described herein only, for description and interpretation the present invention, is not limited to the present invention.
The invention provides a kind of fermentation and prepare the method for citric acid, the method is included under the condition that generates citric acid, citric acid fermentation bacterial classification is seeded in fermention medium and is fermented, obtain fermented liquid, the method is also included in the postvaccinal 0-8 of citric acid fermentation bacterial classification hour and adds saccharifying enzyme in fermention medium.
According to the present invention, although added saccharifying enzyme can realize object of the present invention in fermention medium in the postvaccinal 0-8 of citric acid fermentation bacterial classification hour, reduce the residual sugar amount in fermented liquid, improve glucose acid invert ratio, shorten fermentation period, but under preferable case, in the postvaccinal 0-6 of citric acid fermentation bacterial classification hour, in fermention medium, add saccharifying enzyme, can further reduce the residual sugar amount in fermented liquid, improve glucose acid invert ratio, shorten fermentation period.
In the present invention, in postvaccinal 0 hour of citric acid fermentation bacterial classification, to adding saccharifying enzyme to refer in fermention medium, citric acid fermentation bacterial classification and saccharifying enzyme are added in fermention medium simultaneously.
In the present invention, the concept that fermention medium is known to the skilled person, refer to the nutriment with the artificial preparation maintaining for microorganism growth that microorganism fermentation is required, generally all contain carbohydrate, nitrogenous substances, inorganic salt (comprising trace element) and VITAMIN and water etc.The concept that fermented liquid is also known to the skilled person, refers to an access the liquid nutrient medium (this liquid nutrient medium is also alleged fermention medium in the present invention) of microorganism strains, products therefrom after cultivation after a while.
In the present invention, saccharifying enzyme is glucoamylase (α-Isosorbide-5-Nitrae-glucose hydrolysis enzyme), with respect to the total reducing sugar in fermention medium, the add-on of saccharifying enzyme is preferably 20-50 enzyme activity unit/g total reducing sugar, more 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 rigidly connecting in kind of post-fermentation and culture base.
In the present invention, enzyme activity unit is defined as: in pH value, be 4.6, temperature is that under the condition of 60 ℃, 1 minute is converted into the required enzyme amount of reducing sugar by 1 milligram of starch is an enzyme activity unit.
In the present invention, for the kind of citric acid fermentation bacterial classification, without particular requirement, can, for bacterial classification conventional in this area, be preferably aspergillus niger.
Fermenting process is the biochemical reaction process being participated in by microorganism with regard to its essence, so the quantity of microorganism cells, state, metabolism situation have important impact to the biosynthesizing of product.The size of cell concentration has important impact to the productive rate of tunning.Cell concentration is larger in theory, and the output of product is also larger, but cell concentration is too high, can produce other influences, as nutritive substance consumes too fast, nutritive ingredient in fermented liquid occurs significantly to change, the accumulation of toxic substance etc., and these may change the pathways metabolism of thalline.Therefore, in the present invention, the every liter of fermention medium of take is benchmark, and the inoculum size of aspergillus niger is preferably 1.0 * 10
7-5 * 10
7individual spore, more 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, by blood counting chamber, counts.
Due to when accessing Aspergillus niger strain, the too high synthetic and effect that is unfavorable for Aspergillus niger strain self saccharifying enzyme of DE value of fermention medium, be unfavorable for the growth of Aspergillus niger strain, the DE value of fermention medium is too low, lack again and can directly be utilized the reducing sugar fermenting by aspergillus niger, therefore, before aspergillus niger inoculation, the DE value of fermention medium is preferably 15-40%, more preferably 15-25%.
In the present invention, DE value refers to glucose meter, and reducing sugar accounts for the per-cent of total reducing sugar in fermention medium.In this area, the DE value of conventional citric acid fermentation substratum is general all at 15-40%, but for the DE value of fermention medium is controlled within the scope of 15-25%, the fermention medium in the present invention preferably contains starchy material enzymolysis product.
Usually, starchy material enzymolysis obtains starchy material enzymolysis residue and starchy material enzymatic liquefaction clear liquid, conventionally can be by starchy material enzymatic liquefaction clear liquid for the preparation of fermention medium, after also starchy material enzymatic liquefaction clear liquid can being mixed with starchy material enzymolysis residue for the preparation of fermention medium.In the present invention, starchy material enzymolysis product is preferably mixed with water or is not mixed to get with water by starchy material enzymolysis residue and starchy material enzymatic liquefaction clear liquid, and further preferably take fermention medium gross weight as 100 weight parts be benchmark, the consumption of starchy material enzymatic liquefaction clear liquid is 80-95 weight part, the consumption of starchy material enzymolysis residue is 5-10 weight part, and the consumption of water is 0-15 weight part.
According to the present invention, starchy material enzymatic liquefaction clear liquid can prepare by several different methods, for example, can prepare by the following method: starchy material is pulverized, the product after pulverizing is carried out to enzymolysis, obtain enzymolysis product, by enzymolysis product solid-liquid separation, obtain starchy material enzymatic liquefaction clear liquid and starchy material enzymolysis residue, it is 45-60 % by weight that the condition of solid-liquid separation makes the solid content of starchy material enzymolysis residue, more preferably 55-60 % by weight.
According to the present invention, starchy material can variously can be prepared the raw material that contains starch of citric acid for enzymolysis, fermentation for ability is known, for example, can be selected from one or more in corn, potato class (as cassava) and wheat, under preferable case, described starchy material is corn.
Described enzymolysis step can complete by the conventional method in this area, such as add microbes producing cellulase and/or enzyme in crushed products, at the growth temperature of microbes producing cellulase and/or the great-hearted temperature of enzyme, has been incubated.Described microbes producing cellulase be can secreting amylase microbes producing cellulase.Described enzyme comprises amylase.
Because microorganism growth can produce by product, therefore preferably directly add enzyme.The consumption of described enzyme is The more the better, and for cost consideration, preferably with the dry weight basis of the crushed products after every gram of pulverizing, diastatic consumption is 15-50 enzyme activity unit.
The temperature of enzymolysis can in very large range change, and is preferably 70-105 ℃, more preferably 80-95 ℃.On the time theory of enzymolysis, the longer the better, considers plant factor, and preferably the time of 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.
Amylase refers to the general name of class of enzymes that can starch-splitting glycosidic link, and amylase generally comprises α-amylase, beta-amylase, saccharifying enzyme and isoamylase.
According to the present invention, preferably use α-amylase and/or isoamylase.
According to the present invention, the method and apparatus of solid-liquid separation is conventionally known to one of skill in the art, for example, and pressure filter or whizzer.
Aspergillus niger can adopt the inoculation of conventional method, for example, in being seeded to fermention medium before, aspergillus niger, through seed tank culture, is joined the mature seed liquid obtaining in fermention medium afterwards.The degree of aspergillus niger seed culture can be measured and be determined by 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 cultivating while stretching out, seed liquor is now called mature seed liquid.
Under preferable case, the method for seed tank culture comprises: aspergillus niger is seeded in seed tank culture base and is cultivated, contain the Semen Maydis powder of 10-17 % by weight in seed tank culture base, the every liter of nutrient solution of take is benchmark, and the inoculum size of aspergillus niger 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 obtaining can be applicable to the cultivation of aspergillus niger.
According to the present invention, aspergillus niger can in very large range change at the culture condition of seed tank culture, and the condition of for example cultivating can comprise: the temperature of cultivation is 25-45 ℃, and Initial pH is 5-6, and air flow is 0.1-1 volume: (volume minute); In preferred situation, the condition of cultivation can comprise: the temperature of cultivation is 30-40 ℃, and Initial pH is 5-6, and air flow is 0.3-0.8 volume: (volume minute).
Term " air flow " generally, with recently expression of ventilation, recently represents (V/Vmin) with the volume of air by unit volume nutrient solution in per minute conventionally, and for example ventilation ratio is 1: 0.1-1, being called for short air flow is 0.1-1 volume: (volume minute).
In the present invention, for the condition of fermenting, having no particular limits, can be the fermentation condition of this area routine, and for example, the condition of fermentation can comprise: temperature is 30-40 ℃, is preferably 33-38 ℃; Initial pH is 4-5; Air flow is 0.1-1 volume: (volume minute); Be preferably 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 citric acid preparing according to method of the present invention can be by conventional method, separated and refining according to the requirement of different Industrial products, such as neutralization, acidolysis, decolouring, concentrated, crystallization, packing.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive 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.
It should be noted that in addition, each concrete technical characterictic described in above-mentioned embodiment, in reconcilable situation, can combine by any suitable mode, for fear of unnecessary repetition, the present invention is to the explanation no longer separately of various possible array modes.
In addition, between various embodiment of the present invention, also can carry out arbitrary combination, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Embodiment
The present invention is further illustrated for following embodiment, but therefore do not limit the present invention.
In the following Examples and Comparative Examples:
Aspergillus niger strain is aspergillus niger Co827.
According to the concentration of GB 1987-2007 standard detection gained citric acid solution (being terminal citric acid content or title acidity).
According to the method for GB/T5009.7-2008, measure the concentration of reducing sugar in fermention medium.
According to the method for GB/T5009.8-2003, measure the concentration of total reducing sugar in fermention medium.
Total sugar content in reducing sugar content/fermention medium in DE value=fermention medium.
In following examples and comparative example, all stop fermentation so that reducing sugar concentration detected value is lower than 0.2g/100ml in fermention medium, be defined as fermentation termination.From fermention medium, access aspergillus niger strain start to the time that fermentation termination experiences be fermentation period.
The concentration of total reducing sugar in fermention medium when residual sugar amount is fermentation termination.
Single tank is for the volume of the concentration * citric acid solution of acid amount=citric acid solution.
Transformation efficiency (%)=mono-tank, for acid amount/sugared gross weight * 100%, wherein comprises sugar weight and fermentor tank sugar weight for seeding tank by sugared gross weight.
Embodiment 1
The present embodiment is prepared the method for citric acid for fermentation provided by the invention is described.
(1) 56 kg corn of results are stewing in hot water tank profit, until the water content of corn is 15 % by weight, then pulverize, obtain average particle diameter and be the pulverizing after product of 400 microns.
(2) product after pulverizing is sized mixing by the concentration of 25 % by weight, with respect to the product after every gram of pulverizing, amylase (the Novozymes Company that adds 20 enzyme activity units, α-amylase, equal amylase for this reason in the embodiment of the present invention and comparative example), enter injector, under the condition that is 5.5 at 85 ℃, pH, enzymolysis is 100 minutes, obtains enzymolysis product.
(3) by enzymolysis product by carrying out press filtration with fluid pressure type sheet frame pressure filter, isolate enzymatic liquefaction clear liquid and enzymolysis filter residue, wherein, the water content of enzymolysis residue is 45 % by weight.
(4) preparation fermention medium, by being cooled to 35 ℃ after the water sterilizing of the above-mentioned enzymatic liquefaction clear liquid of 180 kilograms, the enzymolysis residue of 10 kilograms and 13 kilograms, joins in the fermentor tank of 300L, obtains fermention medium.Measure the concentration of reducing sugar and the concentration of total reducing sugar in fermention medium, calculating DE value is 20%.
(5) by the Partial digestion product in step (2), being diluted with water to total reducing sugar is 10 % by weight, obtain seed tank culture base, seed tank culture base is dropped into seeding tank, be heated to 121 ℃ of sterilizations, fast cooling to 36 ℃ after maintaining 30 minutes, access aspergillus niger strain, in every liter of seed tank culture base, the inoculum size of aspergillus niger is 3 * 10
8individual spore.At 36 ℃, Initial pH, be 5,0.3 volume: under the aeration condition of (volume minute), carry out spawn culture; By sampling sediments microscope inspection, acid test and pH, measure the growth of aspergillus niger observed, when pH 2.0, acidity 1g/100ml, bacterium ball size evenly, mycelia is sturdy while stretching out, and stops cultivating, and obtains mature seed liquid.
(6) mature seed liquid step (5) being obtained joins in the fermentor tank of step (4) and starts fermentation, and inoculum size is every liter of fermention medium 2.4 * 10
7individual spore, fermentation condition comprises that temperature is 35 ℃, Initial pH is 5, air flow is 0.3 volume: (volume minute), when fermentation culture added saccharifying enzyme in the time of 3 hours in substratum, with respect to the total reducing sugar rigidly connecting in kind of post-fermentation and culture base, the add-on of saccharifying enzyme is 35 enzyme activity units/g total reducing sugar, when reducing sugar detected value is lower than 0.2g/100ml in fermention medium, stop fermentation, statistics fermentation period, the total sugar concentration of measuring in fermention medium (is residual sugar amount, lower same), then carry out solid-liquid separation, obtain citric acid solution, the concentration of measuring citric acid solution (is terminal citric acid content, or title acidity, lower same), calculating single tank supplies acid amount and transformation efficiency in Table 1.
Embodiment 2
The present embodiment is prepared the method for citric acid for fermentation provided by the invention is described.
(1) 56 kg corn of results are stewing in hot water tank profit, until the water content of corn is 20 % by weight, then pulverize, obtain average particle diameter and be the pulverizing after product of 800 microns.
(2) product after pulverizing is sized mixing by the concentration of 25 % by weight, with respect to the product after every gram of pulverizing, add the amylase of 50 enzyme activity units, enter injector, under the condition that is 5.7 at 95 ℃, pH, enzymolysis is 110 minutes, obtains enzymolysis product.
(3) by enzymolysis product by carrying out press filtration with fluid pressure type sheet frame pressure filter, isolate enzymatic liquefaction clear liquid and enzymolysis filter residue, wherein, the water content of enzymolysis residue is 40 % by weight.
(4) preparation fermention medium, by being cooled to 38 ℃ after the above-mentioned enzymatic liquefaction clear liquid of 190 kilograms, the enzymolysis residue sterilizing of 10 kilograms, joins in the fermentor tank of 300L, obtains fermention medium.Measure the concentration of reducing sugar and the concentration of total reducing sugar in fermention medium, calculating DE value is 25%.
(5) by the Partial digestion liquefaction clear liquid in step (2), being diluted with water to total reducing sugar is 10 % by weight, obtain seed tank culture base, seed tank culture base is dropped into seeding tank, be heated to 121 ℃ of sterilizations, fast cooling to 33 ℃ after maintaining 30 minutes, access aspergillus niger strain, in every liter of seed tank culture base, the inoculum size of aspergillus niger is 2 * 10
8individual spore.At 33 ℃, Initial pH, be 5.5,0.6 volume: under the aeration condition of (volume minute), carry out spawn culture; By sampling sediments microscope inspection, acid test and pH, measure the growth of aspergillus niger observed, when pH 2.5, acidity 0.5g/100ml, bacterium ball size evenly, mycelia is sturdy while stretching out, and stops cultivating, and obtains mature seed liquid.
(6) mature seed liquid step (5) being obtained joins in the fermentor tank of step (4) and starts fermentation, and inoculum size is every liter of fermention medium 2.2 * 10
7individual spore, fermentation condition comprises that temperature is 38 ℃, Initial pH is 4.5, air flow is 0.6 volume: (volume minute), when fermentation culture added saccharifying enzyme in the time of 6 hours in substratum, with respect to the total reducing sugar rigidly connecting in kind of post-fermentation and culture base, the add-on of saccharifying enzyme is 30 enzyme activity units/g total reducing sugar, when reducing sugar detected value is lower than 0.2g/100ml in fermention medium, stop fermentation, statistics fermentation period, measure the total sugar concentration in fermention medium, then carry out solid-liquid separation, obtain citric acid solution, measure the concentration of citric acid solution, calculating single tank supplies acid amount and transformation efficiency in Table 1.
Embodiment 3
The present embodiment is prepared the method for citric acid for fermentation provided by the invention is described.
(1) 56 kg corn of results are stewing in hot water tank profit, until the water content of corn is 10 % by weight, then pulverize, obtain average particle diameter and be the pulverizing after product of 500 microns.
(2) product after pulverizing is sized mixing by the concentration of 25 % by weight, with respect to the product after every gram of pulverizing, add the amylase of 15 enzyme activity units, enter injector, under the condition that is 5.4 at 80 ℃, pH, enzymolysis is 120 minutes, obtains enzymolysis product.
(3) by enzymolysis product by carrying out press filtration with fluid pressure type sheet frame pressure filter, isolate enzymatic liquefaction clear liquid and enzymolysis filter residue, wherein, the water content of enzymolysis residue is 70 % by weight.
(4) preparation fermention medium, by being cooled to 40 ℃ after the water sterilizing of the enzymolysis residue of the above-mentioned enzymatic liquefaction clear liquid of 160 kilograms and 20 kilograms and 20 kilograms, joins in the fermentor tank of 300L, obtains fermention medium.Measure the concentration of reducing sugar and the concentration of total reducing sugar in fermention medium, calculating DE value is 15%.
(5) by the Partial digestion liquefaction clear liquid in step (2), being diluted with water to total reducing sugar is 10 % by weight, obtain seed tank culture base, seed tank culture base is dropped into seeding tank, be heated to 121 ℃ of sterilizations, fast cooling to 38 ℃ after maintaining 30 minutes, access aspergillus niger strain, in every liter of seed tank culture base, the inoculum size of aspergillus niger is 2.5 * 10
8individual spore.At 38 ℃, Initial pH, be 6,0.8 volume: under the aeration condition of (volume minute), carry out spawn culture; By sampling sediments microscope inspection, acid test and pH, measure the growth of aspergillus niger observed, when pH 2.5, acidity 2g/100ml, bacterium ball size evenly, mycelia is sturdy while stretching out, and stops cultivating, and obtains mature seed liquid.
(6) mature seed liquid step (5) being obtained joins in the fermentor tank of step (4) and starts fermentation, and inoculum size is every liter of fermention medium 2.6 * 10
7individual spore, fermentation condition comprises that temperature is 33 ℃, Initial pH is 4, air flow is 0.8 volume: (volume minute), after adding mature seed liquid, in substratum, add saccharifying enzyme immediately, with respect to the total reducing sugar rigidly connecting in kind of post-fermentation and culture base, the add-on of saccharifying enzyme is 40 enzyme activity units/g total reducing sugar, when reducing sugar detected value is lower than 0.2g/100ml in fermention medium, stop fermentation, statistics fermentation period, measure the total sugar concentration in fermention medium, then carry out solid-liquid separation, obtain citric acid solution, measure the concentration of citric acid solution, calculating single tank supplies acid amount and transformation efficiency in Table 1.
Embodiment 4
Citric acid is prepared in method fermentation according to embodiment 1, different, when fermentation culture added saccharifying enzyme in the time of 8 hours in substratum.Statistics fermentation period, measures the total sugar concentration in fermention medium, measures the concentration of citric acid solution, calculates single tank and supplies acid amount and transformation efficiency in Table 1.
Embodiment 5
Citric acid is prepared in method fermentation according to embodiment 1, and different, with respect to the total reducing sugar rigidly connecting in kind of post-fermentation and culture base, the add-on of saccharifying enzyme is 50 enzyme activity units/g total reducing sugar.Statistics fermentation period, measures the total sugar concentration in fermention medium, measures the concentration of citric acid solution, calculates single tank and supplies acid amount and transformation efficiency in Table 1.
Embodiment 6
Citric acid is prepared in method fermentation according to embodiment 1, and different, with respect to the total reducing sugar rigidly connecting in kind of post-fermentation and culture base, the add-on of saccharifying enzyme is 20 enzyme activity units/g total reducing sugar.Statistics fermentation period, measures the total sugar concentration in fermention medium, measures the concentration of citric acid solution, calculates single tank and supplies acid amount and transformation efficiency in Table 1.
Comparative example 1
Citric acid is prepared in method fermentation according to embodiment 1, different, when fermentation culture added saccharifying enzyme in the time of 10 hours in substratum.Statistics fermentation period, measures the total sugar concentration in fermention medium, measures the concentration of citric acid solution, calculates single tank and supplies acid amount and transformation efficiency in Table 1.
Comparative example 2
Citric acid is prepared in method fermentation according to embodiment 1, different is, during step (4) preparation fermention medium, while being cooled to 60 ℃ after sterilizing, in substratum, add saccharifying enzyme, with respect to the starch in 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 no longer add saccharifying enzyme after in the fermentor tank that the aspergillus niger strain of step (5) cultivation is joined to step (4).Statistics fermentation period, measures the total sugar concentration in fermention medium, measures the concentration of citric acid solution, calculates single tank and supplies acid amount and transformation efficiency in Table 1.
Comparative example 3
Citric acid is prepared in method fermentation according to embodiment 1, different, does not add saccharifying enzyme.Statistics fermentation period, measures the total sugar concentration in fermention medium, measures the concentration of citric acid solution, calculates single tank and supplies acid amount and transformation efficiency in Table 1.
Table 1
As can be seen from Table 1, adopt the inventive method, fermentation period is short, and residual sugar amount is low, and acidity and single tank are high for acid amount, and transformation efficiency is high.Embodiment 1-6 and comparative example 1 are compared and can be found out, in fermentation culture, after 8 hours, add saccharifying enzyme, be unfavorable for the fermentation of citric acid; Embodiment 1-6 and comparative example 2 are compared and can be found out, after fermention medium sterilizing, in temperature-fall period, add saccharifying enzyme, not as added the effective of saccharifying enzyme in fermentation culture 0-8 hour; Embodiment 1-6 and comparative example 3 are compared and can be found out, in fermentation culture 0-8 hour, add saccharifying enzyme than not adding saccharifying enzyme, be conducive to the fermentation of citric acid.
Embodiment 1 and embodiment 4 are compared and can be found out, in fermentation culture 0-6 hour, add saccharifying enzyme, be more conducive to the fermentation of citric acid; Embodiment 1 is compared and can be found out with embodiment 5 and embodiment 6 respectively, and with respect to the total reducing sugar rigidly connecting in kind of post-fermentation and culture base, the add-on of saccharifying enzyme is 30-40 enzyme activity unit/g total reducing sugar, is more conducive to the fermentation of citric acid.
The method of citric acid is prepared in fermentation provided by the invention, strengthened the saccharification capability in fermenting process, solved during the fermentation citric acid synthetic with saccharification to the contradiction between the requirement of pH value, and avoided the disadvantageous effect of high DE value to aspergillus niger self, thereby make more thorough by saccharification of liquid glucose in fermention medium, reduced the residual sugar amount in fermented liquid, improved terminal citric acid content and single tank for acid amount, improve glucose acid invert ratio, shortened fermentation period.
Claims (8)
1. the method for citric acid is prepared in a fermentation, described method is included under the condition that generates citric acid, citric acid fermentation bacterial classification is seeded in fermention medium and is fermented, obtain fermented liquid, it is characterized in that, described method is also included in the postvaccinal 0-8 of citric acid fermentation bacterial classification hour and adds saccharifying enzyme in introversive described fermention medium, wherein, before the inoculation of citric acid fermentation bacterial classification, the DE value of fermention medium is 15-25%, DE value refers to glucose meter, and reducing sugar accounts for the per-cent of total reducing sugar in fermention medium; Citric acid fermentation bacterial classification in being seeded to fermention medium before, through seed tank culture, afterwards the mature seed liquid obtaining is joined in fermention medium, described citric acid fermentation bacterial classification is aspergillus niger.
2. method according to claim 1 wherein, added saccharifying enzyme in the postvaccinal 0-6 of citric acid fermentation bacterial classification hour in described fermention medium.
3. method according to claim 1 and 2, wherein, with respect to the total reducing sugar in described fermention medium, the add-on of described 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 described fermention medium, the add-on of described saccharifying enzyme is 30-40 enzyme activity unit/g total reducing sugar.
5. method according to claim 1 and 2, wherein, take every liter of fermention medium as benchmark, and the inoculum size of aspergillus niger is 1 * 10
7-5 * 10
7individual spore.
6. method according to claim 5, wherein, take every liter of fermention medium as benchmark, and the inoculum size of aspergillus niger is 2 * 10
7-3 * 10
7individual spore.
7. method according to claim 1 and 2, wherein, described fermention medium contains starchy material enzymolysis product.
8. method according to claim 1 and 2, wherein, the condition of described fermentation comprises: temperature is 30-40 ℃, and Initial pH is 4-5, and air flow is 0.1-1 volume: (volume minute).
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| 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 |
| 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 |
| 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 |
| CN111172204B (en) * | 2020-03-13 | 2023-01-24 | 合肥五粮泰生物科技有限公司 | Preparation method for improving citric acid fermentation efficiency |
| CN116179617B (en) * | 2023-04-23 | 2023-07-18 | 莱芜泰禾生化有限公司 | Method for preparing citric acid by fermentation |
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| GB878151A (en) * | 1957-12-06 | 1961-09-27 | Kyowa Hakko Kogyo Kk | Process for the production of citric acid by fermentation |
| CN101942487A (en) * | 2010-10-21 | 2011-01-12 | 江南大学 | Method for fermenting and preparing citric acid by adding saccharifying enzyme |
| CN102234672A (en) * | 2011-05-09 | 2011-11-09 | 安徽丰原生物化学股份有限公司 | Enzymolysis method for starchy material and method for preparing citric acid |
| CN102260717A (en) * | 2011-06-16 | 2011-11-30 | 山东柠檬生化有限公司 | Method for producing citric acid by fermentation |
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| GB878151A (en) * | 1957-12-06 | 1961-09-27 | Kyowa Hakko Kogyo Kk | Process for the production of citric acid by fermentation |
| CN101942487A (en) * | 2010-10-21 | 2011-01-12 | 江南大学 | Method for fermenting and preparing citric acid by adding saccharifying enzyme |
| CN102234672A (en) * | 2011-05-09 | 2011-11-09 | 安徽丰原生物化学股份有限公司 | Enzymolysis method for starchy material and method for preparing citric acid |
| CN102260717A (en) * | 2011-06-16 | 2011-11-30 | 山东柠檬生化有限公司 | Method for producing citric acid by fermentation |
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