CN102747121B - Starchy material enzymolysis product and preparation method and application thereof and method for preparing citric acid by fermentation - Google Patents
Starchy material enzymolysis product and preparation method and application thereof and method for preparing citric acid by fermentation Download PDFInfo
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Abstract
The invention discloses a preparation method of a starchy material enzymolysis product. The preparation method of the starchy material enzymolysis product includes: (1) hydrolyzing hydrolyzable sugar in residual sugar of distillage filtrate to simple sugar so as to obtain liquid A; (2) crushing starchy materials, and mixing the obtained crushed starchy materials with the liquid A obtained at the step (1) for slurry mixing so as to obtain slurry liquid B; and (3) subjecting the slurry liquid B to enzymolysis under the first enzymolysis condition so as to obtain an enzymolysis product. The invention further discloses a starchy material enzymolysis product comprising the enzymolysis product and/or clear liquid obtained after solid-liquid separation of the enzymolysis product. The invention further discloses application of the starchy material enzymolysis product to preparation of citric acid by fermentation and a method for preparing the citric acid by fermentation. Using the starchy material enzymolysis product for preparing the citric acid by fermentation can shorten the fermentation period of the citric acid and increase sugar-acid conversion ratio. The distillage filtrate is treated in an economical and environment-friendly way by the preparation method of the starchy material enzymolysis product.
Description
Technical field
The present invention relates to a kind of preparation method of starchy material enzymolysis product, and starchy material enzymolysis product obtained by this method, and this starchy material enzymolysis product prepares the application in citric acid in fermentation, and the method for citric acid is prepared in a kind of fermentation.
Background technology
Starchy material enzymolysis product is widely used in the fermentation preparation of the multiple Industrial products such as citric acid, the starchy material enzymolysis product adopting is at present generally that corn and other starches raw material is pulverized, water is sized mixing the product after pulverizing, and then slurries is carried out to enzymolysis and obtains.Starchy material enzymolysis product contains the liquefier that enzymolysis obtains, or also contains the liquefier obtaining is carried out to the liquefaction clear liquid that solid-liquid separation obtains.
But the starchy material enzymolysis product adopting at present, sizes mixing and need to expend a large amount of water, and in the fermentation preparation for citric acid by starchy material enzymolysis product, the fermentation period of citric acid is longer, and glucose acid invert ratio is lower.
Summary of the invention
The object of the invention is to overcome in prior art starchy material enzymolysis product for fermentation, while preparation especially for the fermentation of citric acid, the fermentation period of citric acid is longer, the defect that glucose acid invert ratio is lower, and provide one can shorten the citric acid fermentation cycle, improve the preparation method of the starchy material enzymolysis product of glucose acid invert ratio, and provide a kind of starchy material enzymolysis product obtained by this method, and this starchy material enzymolysis product is prepared the application in citric acid in fermentation, and the method for citric acid is prepared in a kind of fermentation, and solved the problem of distillage filtrate processing simultaneously.
The present inventor surprisingly finds under study for action, after being hydrolyzed to monose, hydrolyzable sugar in the residual sugar of distillage filtrate sizes mixing for starchy material, and enzymolysis obtains enzymolysis product, this enzymolysis product is prepared to citric acid for fermentation, not only can shorten the fermentation period of citric acid, improve glucose acid invert ratio, can also process distillage filtrate in economic environmental protection ground.
Therefore, to achieve these goals, on the one hand, the invention provides a kind of preparation method of starchy material enzymolysis product, it is characterized in that, described method comprises:
(1) the hydrolyzable sugar in the residual sugar of distillage filtrate is hydrolyzed to monose, obtains liquid A;
(2) starchy material is pulverized, and the liquid A that the crushed products obtaining is obtained with step (1) mixes and size mixing, obtain slurries B;
(3), under the first enzymatic hydrolysis condition, by slurries B enzymolysis, obtain enzymolysis product.
Second aspect, the invention provides a kind of starchy material enzymolysis product, this starchy material enzymolysis product comprises liquefier and/or liquefaction clear liquid, it is characterized in that, described liquefier is the enzymolysis product that method as above makes, and described liquefaction clear liquid is the clear liquid that enzymolysis product that method as above makes obtains after solid-liquid separation.
The third aspect, the invention provides a kind of starchy material enzymolysis product as above and prepares the application in citric acid in fermentation.
Fourth aspect, 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, aspergillus niger is seeded in fermention medium and is fermented, obtain fermented liquid, it is characterized in that, described fermention medium contains starchy material enzymolysis product as above.
Starchy material enzymolysis product of the present invention is prepared citric acid for fermentation, can shorten the fermentation period of citric acid, improves glucose acid invert ratio; Process to the economic environmental protection of preparation method of starchy material enzymolysis product of the present invention distillage filtrate.The inventive method can be widely used in industrial production.
Other features and advantages of the present invention are described in detail the embodiment part 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.
On the one hand, the invention provides a kind of preparation method of starchy material enzymolysis product, the method comprises:
(1) the hydrolyzable sugar in the residual sugar of distillage filtrate is hydrolyzed to monose, obtains liquid A;
(2) starchy material is pulverized, and the liquid A that the crushed products obtaining is obtained with step (1) mixes and size mixing, obtain slurries B;
(3), under the first enzymatic hydrolysis condition, by slurries B enzymolysis, obtain enzymolysis product.
Learn according to the present situation of fermentative production alcohol, the method of fermentative production alcohol exists the problems such as the rear distillage filtrate contaminate environment producing of fermentation, China's alcohol industry approximately discharges distillage filtrate more than 5,000 ten thousand t every year, biological oxygen demand (BOD) (BOD) and chemical oxygen demand (COD) reach respectively 1,800,000 t and 3,600,000 t, account for 1/8 of national trade effluent BOD and COD.
Understand according to the present inventor, the treatment process of distillage filtrate mainly contains following two kinds at present: the one, and after the wet grain obtaining after distillage filtrate solid-liquid separation is mixed with the concentrated solution of clear liquid, be dried and produce protein fodder (DDGS), its major advantage is to turn waste into wealth, but facility investment is large, working cost is high, business burden is heavier, and the DDGS meeting of production produces a large amount of secondary steam phlegmas, its COD and BOD reach respectively 3000mg/L-4000mg/L and 2000mg/L-3000mg/L, still need to process; The 2nd, directly distillage filtrate is entered to environmental protection and carry out wastewater treatment, wastewater discharge is large, and processing costs is high, and the cycle is long.
Therefore, the present inventor surprisingly finds under study for action, after being hydrolyzed to monose, hydrolyzable sugar in the residual sugar of distillage filtrate sizes mixing for starchy material, and enzymolysis obtains enzymolysis product, this enzymolysis product is prepared to citric acid for fermentation, not only can shorten the fermentation period of citric acid, improve glucose acid invert ratio, can also process distillage filtrate in economic environmental protection ground.
In the present invention, distillage filtrate refers to the filtrate that obtains through solid-liquid separation of useless mash that zymamsis produces.
In step of the present invention (1), the method that hydrolyzable sugar in the residual sugar of distillage filtrate is hydrolyzed to monose is without particular requirement, can adopt the conventional the whole bag of tricks in this area, but quicker for what hydrolysis was carried out, preferably by distillage filtrate Pullulanase exist under, under the second enzymatic hydrolysis condition, carry out enzymolysis.With respect to 1g distillage filtrate, the consumption of Pullulanase is preferably 0.1-1U, and wherein, the residual sugar content of distillage filtrate is 0.5-1.0g/100mL.Residual sugar in distillage filtrate generally refers to the monose, disaccharides and the polysaccharide that in distillage filtrate, exist.
In the present invention, the enzyme activity unit of Pullulanase is defined as: under 60 DEG C, pH4.5 condition, the required enzyme amount of 1min hydrolysis pulullan polysaccharide (taking glucose meter) is enzyme activity unit, i.e. a 1U.
It will be understood by those skilled in the art that the second enzymatic hydrolysis condition is under Pullulanase exists, the hydrolyzable sugar in the residual sugar of distillage filtrate is hydrolyzed to the condition of monose, preferably include: pH value is 4-5, and temperature is 58-65 DEG C, and the time is 20-40min.
In the present invention, starchy material can variously can, for the raw material that contains starch of enzymolysis, for example, can be selected from one or more in corn, potato class (as cassava) and wheat for well known in the art, and under preferable case, described starchy material is corn.
In step of the present invention (2), without particular requirement, can adopt the conventional the whole bag of tricks in this area for the method that starchy material is pulverized, for the degree of pulverizing, as long as crushed products is suitable for enzymolysis after sizing mixing, under preferable case, the particle diameter of crushed products is 300-800 micron.The solid content of slurries B is preferably 22-26 % by weight.
In step of the present invention (3), under the first enzymatic hydrolysis condition, by slurries B enzymolysis, obtain enzymolysis product.What those skilled in the art should understand that is, enzymolysis product of the present invention is for citric acid fermentation, therefore, under the first enzymatic hydrolysis condition, by slurries B enzymolysis, be under the existence of microbes producing cellulase and/or enzyme, at the growth temperature of microbes producing cellulase and/or the great-hearted temperature of enzyme, the starchy material enzymolysis in slurries B become to be suitable for the dextrin liquid glucose that aspergillus niger utilizes.Microbes producing cellulase be can secreting amylase microbes producing cellulase.Enzyme comprises amylase.
Because microorganism growth can produce by product, therefore preferably directly add enzyme.Enzyme is preferably amylase, and 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.In the present invention, preferably use α-amylase and/or isoamylase.
In the present invention, for the consumption of enzyme, The more the better, for cost consideration, preferably with respect to 1g starchy material crushed products, diastatic consumption is 15-50U.
In the present invention, diastatic enzyme activity unit is defined as: under 70 DEG C, pH 6.0 conditions, it is enzyme activity unit, i.e. a 1U that 1 milligram of starch is converted into the required enzyme amount of dextrin liquid glucose by 1min.
Without particular requirement, can adopt the normal condition of this area for the first enzymatic hydrolysis condition, the temperature of enzymolysis can in very large range change, and is preferably 70-105 DEG C, more preferably 80-95 DEG C.On the time theory of enzymolysis, the longer the better, considers plant factor, and preferably the time of enzymolysis is 90-150min, more preferably 100-120min.The pH value of enzymolysis can in very large range change, and is preferably 5.0-7.0, more preferably 5.4-5.7.
Second aspect, the invention provides a kind of starchy material enzymolysis product, this starchy material enzymolysis product comprises liquefier and/or liquefaction clear liquid, liquefier is the enzymolysis product that method as above makes, and liquefaction clear liquid is the clear liquid that enzymolysis product that method as above makes obtains after solid-liquid separation.
In the present invention, the method and apparatus of solid-liquid separation is conventionally known to one of skill in the art, for example, adopts pressure filter or whizzer to carry out press filtration or centrifugal.
The third aspect, the invention provides starchy material enzymolysis product as above and prepares the application in citric acid in fermentation.
Fourth aspect, 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, and aspergillus niger is seeded in fermention medium and is fermented, obtain fermented liquid, fermention medium contains starchy material enzymolysis product as above.
In the present invention, the concept that fermention medium is known to the skilled person, refers to the nutriment with the artificial preparation maintaining for microorganism growth that microorganism fermentation is required.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.
Fermention medium contains starchy material enzymolysis product, and the amount of preferred starch raw material enzymolysis product accounts for the 80-100 % by weight of fermention medium total amount.Starchy material enzymolysis product comprise liquefier and/or liquefaction clear liquid, conventionally can by liquefaction clear liquid for the preparation of fermention medium, also can by liquefaction clear liquid mix with liquefier after for the preparation of fermention medium.Fermention medium is preferably mixed with water or is not mixed to get with water by liquefier and liquefaction clear liquid, and further the preferred total amount taking fermention medium is as benchmark, and liquefaction clear liquid is 80-85 % by weight, and liquefier is 15-20 % by weight.
Before it will be understood by those skilled in the art that in aspergillus niger is seeded to fermention medium, need to, by aspergillus niger through seed tank culture, afterwards the mature seed liquid obtaining be joined in fermention medium.In the present invention, the nutrient solution of seeding tank is preferably above-mentioned liquefier is diluted with water to total reducing sugar is 8-12 % by weight, obtains nutrient solution.
In the present invention, the citric acid of concrete grammar prepare to(for) fermentation, without particular requirement, can adopt the conventional the whole bag of tricks in this area.For example, can adopt fermentation with the following method and prepare citric acid.
Nutrient solution is dropped into seeding tank, is heated to 115-125 DEG C of sterilization, maintain 25-35 minute after fast cooling to 34-38 DEG C, access Aspergillus niger strain A, in every liter of nutrient solution, the inoculum size of aspergillus niger is 2 × 10
8-3 × 10
8individual spore.Be to carry out spawn culture under 5-6, pressure 0.02-0.04MPa at 34-38 DEG C, Initial pH, wherein, in cultivation 0-14h process, air flow is 0.2-0.4 volume: (volume minute), in cultivation 14-20h process, air flow is 0.7-0.9 volume: (volume minute), to cultivate after 20h, air flow is 0.5-0.7 volume: (volume minute).
Term " air flow " generally with ventilation recently represents, conventionally recently to represent (V/Vmin) by the volume of air of unit volume nutrient solution in per minute, for example ventilation ratio is 1:0.1-1, abbreviation air flow be 0.1-1 volume: (volume minute).Lower same.
Measure the growth of aspergillus niger is observed by sampling sediments microscope inspection, acid test and pH, when pH < 2.0, acidity > 1g/100mL, bacterium ball size evenly, mycelia is sturdy while stretching out, stop cultivating, obtain mature seed liquid.
Mature seed liquid is joined and in the fermention medium of fermentor tank, start fermentation.Taking every liter of fermention medium as benchmark, the inoculum size of aspergillus niger is 2.2 × 10
7-2.6 × 10
7individual spore.Inoculum size recently represents with the percentage that the volume of mature seed liquid of access fermention medium accounts for the volume of access seed liquor post-fermentation and culture base conventionally, in the time that the volume of mature seed liquid of access fermention medium accounts for the 8-11% of volume of access mature seed liquid post-fermentation and culture base, can meet taking every liter of fermention medium as benchmark, the inoculum size of aspergillus niger is 2.2 × 10
7-2.6 × 10
7within the scope of individual spore, therefore, the inoculum size of the aspergillus niger of access fermention medium can be expressed as: inoculum size is 8-11%.Fermentation condition comprises that temperature is 34-38 DEG C, Initial pH is 4-5, pressure 0.03-0.06MPa, in cultivation 0-8h process, air flow is 0.7-0.9 volume: (volume minute), in cultivation 8-24h process, air flow is 0.9-1.1 volume: (volume minute), and in cultivation 24-48h process, air flow is 0.7-0.9 volume: (volume minute), cultivating after 48h, air flow is 0.5-0.7 volume: (volume minute).
In the time that reducing sugar concentration detected value is lower than 0.2g/100ml in fermented liquid, stop fermentation, be defined as fermentation termination.From fermention medium, access aspergillus niger strain start to the time that fermentation termination experiences be fermentation period.Then solid-liquid separation obtains citric acid solution.
The tunning citric acid preparing according to method of the present invention can, by conventional method, separate and refine according to the requirement of different Industrial products, such as neutralization, acidolysis, decolouring, concentrated, crystallization, packaging.
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, also can carry out arbitrary combination between various embodiment of the present invention, 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).
Transformation efficiency (%)=mono-tank, for acid amount/sugared gross weight × 100%, wherein comprises seeding tank sugar weight and fermentor tank sugar weight by sugared gross weight.
In following examples and comparative example, all stop fermentation when 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.
Measure the concentration of reducing sugar in fermention medium according to the method for GB/T5009.7-2008.
Embodiment 1
The present embodiment is prepared the method for citric acid for starchy material enzymolysis product provided by the invention and fermentation are described.
(1) the pH value of distillage filtrate (residual sugar content is 0.8g/100mL) is adjusted to 4.5, with respect to 1g distillage filtrate, add 0.5U Pullulanase (Novozymes Company, Promozyme Novi letter Pullulanase, equal Pullulanase for this reason in the embodiment of the present invention and comparative example), at 60 DEG C, leave standstill 30min, obtain liquid A;
(2) corn is pulverized, obtained average particle diameter and be the crushed products of 500 microns, crushed products is mixed and sized mixing with liquid A, obtain slurries B, the solid content of slurries B is 24 % by weight.
(3) with respect to 1 gram of crushed products, to the amylase (Novozymes Company that adds 30U in slurries B, Aquazym Ultra alpha-amylase, equal amylase for this reason in the embodiment of the present invention and comparative example), under the condition that is 5.9 at 93 DEG C, pH, slurries B enzymolysis is obtained to enzymolysis product, i.e. liquefier for 100 minutes.
(4) Partial digestion product is carried out to press filtration by fluid pressure type sheet frame pressure filter, separate obtain liquefying clear liquid and enzymolysis residue, wherein, the solid content of enzymolysis residue is 51 % by weight.
(5) preparation fermention medium, by joining in the fermentor tank of 300L after the above-mentioned liquefaction clear liquid of 180.5 kilograms, the liquefier sterilizing of 35.5 kilograms, obtains fermention medium.
(6) partial liquefaction liquid being diluted with water to total reducing sugar is 10 % by weight, obtains nutrient solution, and nutrient solution is dropped into seeding tank, be heated to 121 DEG C of sterilizations, fast cooling to 36 DEG C after maintaining 30 minutes, access aspergillus niger strain, in every liter of nutrient solution, the inoculum size of aspergillus niger is 2 × 10
8individual spore.Be 5 at 36 DEG C, Initial pH, carry out spawn culture under pressure 0.03MPa, wherein, in cultivation 0-14h process, air flow is 0.3 volume: (volume minute), in cultivation 14-20h process, air flow is 0.8 volume: (volume minute), to cultivate after 20h, air flow is 0.6 volume: (volume minute).
Measure the growth of aspergillus niger is observed by sampling sediments microscope inspection, acid test and pH, when pH < 2.0, acidity > 1g/100mL, bacterium ball size evenly, mycelia is sturdy while stretching out, stop cultivating, obtain mature seed liquid.
(7) the mature seed liquid obtaining is joined and in the fermention medium of fermentor tank, start fermentation, inoculum size is 10%, fermentation condition comprises that temperature is 35 DEG C, Initial pH is 5, pressure is 0.04MPa, in cultivation 0-8h process, air flow is 0.8 volume: (volume minute), in cultivation 8-24h process, air flow is 1.0 volumes: (volume minute), and in cultivation 24-48h process, air flow is 0.8 volume: (volume minute), cultivating after 48h, air flow is 0.6 volume: (volume minute).
In the time that reducing sugar concentration detected value is lower than 0.2g/100ml in fermented liquid, stop fermentation, statistics fermentation period.Then carry out solid-liquid separation, obtain citric acid solution.Measure the concentration (being terminal citric acid content, lower same) of citric acid solution, calculate transformation efficiency in table 1.
Embodiment 2
The present embodiment is prepared the method for citric acid for starchy material enzymolysis product provided by the invention and fermentation are described.
(1) the pH value of distillage filtrate (residual sugar content is 0.5g/100mL) is adjusted to 4, with respect to 1g distillage filtrate, adds 0.1U Pullulanase, at 58 DEG C, leave standstill 40min, obtain liquid A;
(2) corn is pulverized, obtained average particle diameter and be the crushed products of 800 microns, crushed products is mixed and sized mixing with liquid A, obtain slurries B, the solid content of slurries B is 26 % by weight.
(3), with respect to 1 gram of crushed products, to the amylase that adds 50U in slurries B, under the condition that is 7 at 105 DEG C, pH, slurries B enzymolysis is obtained to enzymolysis product, i.e. liquefier for 90 minutes.
(4) Partial digestion product is carried out to press filtration by fluid pressure type sheet frame pressure filter, separate obtain liquefying clear liquid and enzymolysis residue, wherein, the solid content of enzymolysis residue is 50 % by weight.
(5) preparation fermention medium, by joining in the fermentor tank of 300L after the above-mentioned liquefaction clear liquid of 177.1 kilograms, the liquefier sterilizing of 38.9 kilograms, obtains fermention medium.
(6) partial liquefaction liquid being diluted with water to total reducing sugar is 8 % by weight, obtains nutrient solution, and nutrient solution is dropped into seeding tank, be heated to 115 DEG C of sterilizations, fast cooling to 34 DEG C after maintaining 35 minutes, access aspergillus niger strain, in every liter of nutrient solution, the inoculum size of aspergillus niger is 2.5 × 10
8individual spore.Be 5.5 at 34 DEG C, Initial pH, carry out spawn culture under pressure 0.02MPa, wherein, in cultivation 0-14h process, air flow is 0.2 volume: (volume minute), in cultivation 14-20h process, air flow is 0.7 volume: (volume minute), to cultivate after 20h, air flow is 0.5 volume: (volume minute).
Measure the growth of aspergillus niger is observed by sampling sediments microscope inspection, acid test and pH, when pH < 2.0, acidity > 1g/100mL, bacterium ball size evenly, mycelia is sturdy while stretching out, stop cultivating, obtain mature seed liquid.
(7) the mature seed liquid obtaining is joined and in the fermention medium of fermentor tank, start fermentation, inoculum size is 8%, fermentation condition comprises that temperature is 34 DEG C, Initial pH is 4.5, pressure is 0.03MPa, in cultivation 0-8h process, air flow is 0.7 volume: (volume minute), in cultivation 8-24h process, air flow is 0.9 volume: (volume minute), and in cultivation 24-48h process, air flow is 0.7 volume: (volume minute), cultivating after 48h, air flow is 0.5 volume: (volume minute).
In the time that reducing sugar concentration detected value is lower than 0.2g/100ml in fermented liquid, stop fermentation, statistics fermentation period.Then carry out solid-liquid separation, obtain citric acid solution.Measure the concentration (being terminal citric acid content, lower same) of citric acid solution, calculate transformation efficiency in table 1.
Embodiment 3
The present embodiment is prepared the method for citric acid for starchy material enzymolysis product provided by the invention and fermentation are described.
(1) the pH value of distillage filtrate (residual sugar content is 1.0g/100mL) is adjusted to 5, with respect to 1g distillage filtrate, adds 1U Pullulanase, at 65 DEG C, leave standstill 20min, obtain liquid A;
(2) corn is pulverized, obtained average particle diameter and be the crushed products of 300 microns, crushed products is mixed and sized mixing with liquid A, obtain slurries B, the solid content of slurries B is 22 % by weight.
(3), with respect to 1 gram of crushed products, to the amylase that adds 15U in slurries B, under the condition that is 5 at 70 DEG C, pH, slurries B enzymolysis is obtained to enzymolysis product, i.e. liquefier for 150 minutes.
(4) Partial digestion product is carried out to press filtration by fluid pressure type sheet frame pressure filter, separate obtain liquefying clear liquid and enzymolysis residue, wherein, the solid content of enzymolysis residue is 49 % by weight.
(5) preparation fermention medium, by joining in the fermentor tank of 300L after the above-mentioned liquefaction clear liquid of 169 kilograms, the liquefier sterilizing of 42.2 kilograms, obtains fermention medium.
(6) partial liquefaction liquid being diluted with water to total reducing sugar is 12 % by weight, obtains nutrient solution, and nutrient solution is dropped into seeding tank, be heated to 125 DEG C of sterilizations, fast cooling to 38 DEG C after maintaining 25 minutes, access aspergillus niger strain, in every liter of nutrient solution, the inoculum size of aspergillus niger is 3 × 10
8individual spore.Be 6 at 38 DEG C, Initial pH, carry out spawn culture under pressure 0.04MPa, wherein, in cultivation 0-14h process, air flow is 0.4 volume: (volume minute), in cultivation 14-20h process, air flow is 0.9 volume: (volume minute), to cultivate after 20h, air flow is 0.7 volume: (volume minute).
Measure the growth of aspergillus niger is observed by sampling sediments microscope inspection, acid test and pH, when pH < 2.0, acidity > 1g/100mL, bacterium ball size evenly, mycelia is sturdy while stretching out, stop cultivating, obtain mature seed liquid.
(7) the mature seed liquid obtaining is joined and in the fermention medium of fermentor tank, start fermentation, inoculum size is 11%, fermentation condition comprises that temperature is 38 DEG C, Initial pH is 4, pressure is 0.06MPa, in cultivation 0-8h process, air flow is 0.9 volume: (volume minute), in cultivation 8-24h process, air flow is 1.1 volumes: (volume minute), and in cultivation 24-48h process, air flow is 0.9 volume: (volume minute), cultivating after 48h, air flow is 0.7 volume: (volume minute).
In the time that reducing sugar concentration detected value is lower than 0.2g/100ml in fermented liquid, stop fermentation, statistics fermentation period.Then carry out solid-liquid separation, obtain citric acid solution.Measure the concentration (being terminal citric acid content, lower same) of citric acid solution, calculate transformation efficiency in table 1.
Comparative example 1
Citric acid is prepared in method fermentation according to embodiment 1, different, and corn is pulverized, and crushed products is mixed and sized mixing with water, obtains slurries B, and the solid content of slurries B is 24 % by weight.In the time that reducing sugar concentration detected value is lower than 0.2g/100ml in fermented liquid, stop fermentation, statistics fermentation period.Then carry out solid-liquid separation, obtain citric acid solution.Measure the concentration (being terminal citric acid content, lower same) of citric acid solution, calculate transformation efficiency in table 1.
Table 1
As can be seen from Table 1, adopt the starchy material enzymolysis product that the inventive method makes to prepare citric acid as fermention medium for fermentation, can shorten the fermentation period of citric acid, although the variation of terminal citric acid content is little, but can improve transformation efficiency.
Starchy material enzymolysis product prepared by the inventive method is prepared citric acid for fermentation, not only can shorten the fermentation period of citric acid, improves glucose acid invert ratio, has also processed to economic environmental protection distillage filtrate.The inventive method can be widely used in industrial production.
Claims (10)
1. a preparation method for starchy material enzymolysis product, is characterized in that, described method comprises:
(1) by distillage filtrate Pullulanase exist under, under the second enzymatic hydrolysis condition, carry out enzymolysis, obtain liquid A;
(2) starchy material is pulverized, and the liquid A that the crushed products obtaining is obtained with step (1) mixes and size mixing, obtain slurries B;
(3), under the first enzymatic hydrolysis condition, by slurries B enzymolysis, obtain enzymolysis product;
Wherein, described the first enzymatic hydrolysis condition also comprises use amylase, and with respect to 1g starchy material crushed products, diastatic consumption is 15-50U.
2. method according to claim 1, wherein, in step (1), described distillage filtrate is the filtrate that the useless mash of zymamsis generation obtains through solid-liquid separation.
3. method according to claim 1, wherein, with respect to 1g distillage filtrate, the consumption of Pullulanase is 0.1-1U; The residual sugar content of distillage filtrate is 0.5-1.0g/100mL.
4. method according to claim 1, wherein, described the second enzymatic hydrolysis condition comprises: pH value is 4-5, and temperature is 58-65 DEG C, and the time is 20-40min.
5. method according to claim 1, wherein, in step (2), the particle diameter of described crushed products is 300-800 micron; The solid content of described slurries B is 22-26 % by weight.
6. method according to claim 1 or 5, wherein, described starchy material is selected from one or more in corn, cassava and wheat.
7. method according to claim 1, wherein, in step (3), described the first enzymatic hydrolysis condition comprises: pH value is 5.0-7.0, and temperature is 70-105 DEG C, and the time is 90-150min.
8. the method as described in any one in claim 1-7 is prepared the application in citric acid in fermentation.
9. the method for citric acid is prepared in a fermentation, the method is included under the condition that generates citric acid, aspergillus niger is seeded in fermention medium and is fermented, obtain fermented liquid, it is characterized in that, right to use requires the method described in any one in 1-7 to prepare starchy material enzymolysis product, and uses described starchy material enzymolysis product to prepare described fermention medium.
10. method according to claim 9, wherein, described fermention medium contains liquefier and liquefaction clear liquid, and taking the total amount of fermention medium as benchmark, the content of liquefaction clear liquid is 80-85 % by weight, and the content of liquefier is 15-20 % by weight; Described liquefier is the enzymolysis product that in claim 1-7, the method described in any one makes; Described liquefaction clear liquid is the clear liquid that enzymolysis product that in claim 1-7, the method described in any one makes obtains after solid-liquid separation.
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