CN109852640B - 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 - Google Patents
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 Download PDFInfo
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Abstract
The invention relates to the field of citric acid preparation, and discloses a seed culture medium for fermenting citric acid and a culture medium for fermenting citric acid by using full starch and a method for preparing citric acid. Mixing the whole starch slurry with amylase, contacting with steam at pH5.2-6, and maintaining at 90-105 deg.C for 60-90 min; then adding nitrogen source, adjusting pH to 4.5-6.5 and optionally saccharifying to obtain seed culture medium. Mixing the non-saccharified seed culture medium with saccharifying enzyme, adjusting pH to 3.8-4.5, and saccharifying at 60-63 deg.C for 5-20 hr; or adjusting pH of the saccharified seed culture medium to 3.8-4.5 to obtain the fermentation culture medium. Inoculating the fermentation strain to a seed culture medium for culture; and (4) obtaining seed liquid, and inoculating the seed liquid into a fermentation culture medium for fermentation to obtain the citric acid. The invention improves the nutrient content of the seed culture medium, and the seed grows fast and has stable quality. The fermentation period is shortened, the end point acidity and the citric acid conversion rate are improved, and the end point residual sugar rate is reduced.
Description
Technical Field
The invention relates to the field of citric acid preparation by fermentation, in particular to a seed culture medium for preparing citric acid by fermenting all starch, a culture medium for fermenting citric acid and a method for preparing citric acid by fermenting all starch.
Background
At present, the citric acid industry mainly adopts corn flour as a raw material to produce citric acid by fermentation, or corn flour is combined with starch raw materials such as wheat and sweet potatoes to produce citric acid by fermentation. The current technical scheme mainly comprises the following operation steps. Firstly, liquefying corn flour to prepare a liquefied liquid, and selecting 5-15% of the liquefied liquid to culture aspergillus niger seeds; secondly, 70-90% of the corn liquefied liquid is selected for filter pressing, and filter pressing clear liquid is prepared to be used as a main carbon source for citric acid fermentation; selecting 5-15% of the corn liquefied liquid as a fermentation substrate culture medium again, and supplementing an organic nitrogen source; finally, other nitrogen sources (such as urea) are supplemented in the fermentation to be used as a fermentation medium. The fermentation of other raw materials is only adjusted on the liquefied clear liquid, and the culture medium of the seeding tank and the fermentation bottom material are starchy raw material liquid.
The treatment method for performing citric acid fermentation by using corn as a starchy raw material in many domestic enterprises comprises the following steps: firstly controlling the temperature of the slurry adjusting water to be 55-65 ℃, then adding corn flour, adding calcium hydroxide to adjust the pH value to be 6.0-6.5, adding all required amylase once, spraying 80-85 ℃ once, maintaining for a certain time, spraying 90-95 ℃ twice, maintaining until iodine is qualified, reserving enough liquefied liquid used by a seed tank culture medium and a fermentation culture medium, and then performing filter pressing to prepare a liquid supernatant. And in the citric acid fermentation, the filter-pressed clear sugar is added into the bottom material liquefied liquid, and the sterilized and cooled liquid is inoculated into the liquefied liquid to culture mature aspergillus niger seeds for citric acid fermentation.
However, the currently adopted method for preparing citric acid by fermentation is long in seed culture time and unstable in quality, and is long in fermentation time, low in acidity at the fermentation end point and citric acid conversion rate, high in residual sugar, and difficult to effectively reduce the residual sugar even if the fermentation time is prolonged.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provides a preparation method of a seed culture medium for fermenting citric acid, the seed culture medium for fermenting citric acid prepared by the method can ensure that the citric acid fermentation strain is quickly matured and the seed property is stable; the culture medium for fermenting the citric acid prepared by the method can effectively shorten the fermentation period, improve the acidity at the fermentation end point and the conversion rate of the citric acid, and reduce the residual sugar content at the end point.
In order to accomplish the above objects, according to one aspect of the present invention, there is provided a method for preparing a seed medium for fermenting citric acid using all starch, the method comprising:
(1) mixing starch slurry obtained from whole starch with amylase to obtain a mixture, contacting the mixture with steam at pH5.2-6 under such conditions that the temperature of the mixture after contacting with steam is 90-105 deg.C, and maintaining at the temperature for 60-90 min to obtain starch liquefied liquid;
(2) adding a nitrogen source into the starch liquefied liquid, adjusting the pH value of the starch liquefied liquid to 4.5-6.5, and optionally saccharifying to obtain the seed culture medium for fermenting the citric acid.
Preferably, the nitrogen source is corn steep liquor.
In a second aspect, the present invention provides a method for preparing a medium for fermented citric acid using whole starch, which comprises mixing an unglycated seed medium for fermented citric acid prepared as described above with a saccharifying enzyme, and saccharifying the resulting mixture at 60-63 ℃ and pH3.8-4.5 for 5-20 hours to obtain a medium for fermented citric acid; or adjusting pH of saccharified seed culture medium for fermenting citric acid prepared by the above method to 3.8-4.5 to obtain culture medium for fermenting citric acid.
The invention provides a method for preparing citric acid by adopting full starch fermentation, wherein the method comprises the following steps:
(1) inoculating citric acid zymophyte into the seed culture medium for fermenting citric acid prepared by the method for seed culture to obtain a seed culture solution;
(2) inoculating the seed culture solution obtained in the step (1) into the culture medium for fermenting citric acid prepared by the method for fermenting citric acid under the condition of generating citric acid, and fermenting to obtain a citric acid fermentation solution.
By adopting the technical scheme, the nutrient content of the citric acid fermentation seed culture medium is completely improved, the growth speed of the fermentation seeds is very high, and the shaking of the bottle can be realized within 22 hours; and the seed quality is very stable, and the seed effect is very good. In addition, the components of the citric acid fermentation medium are also greatly improved, the fermentation period is obviously shortened and can be as low as 60 hours, the acidity of the fermentation end point and the conversion rate of citric acid are effectively improved, and meanwhile, the residual sugar rate of the fermentation end point is also greatly reduced, and the residual sugar reduction rate can be even as low as 0.
Detailed Description
The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
In the research of improving the terminal acidity and conversion rate of citric acid and reducing the terminal residual sugar content, the applicant of the present application has been going on the liquefaction process of starchy materials, the composition of fermentation culture liquid and the fermentation process, and although some progress has been made, it is still not satisfactory.
Surprisingly, in continuing research and exploration, applicants have discovered major factors that limit fermentation end point acidity and citric acid conversion, as well as end point residual sugars. Firstly, starchy raw material powder adopted in the prior art contains a large amount of starch and protein-coated starch, on one hand, the starch is not beneficial to liquefaction, which is mainly characterized in that in the sugar making process in the prior art, a solid-liquid separation process is adopted, and if the liquefaction DE value is controlled to be too low (generally controlled to be 25-32%), starch sugar lost by filter residues is very much and has high viscosity, thus being not beneficial to improving the utilization efficiency of the sugar and reducing residual sugar; on the other hand, the prepared starchy material liquefied liquid needs to supplement partial bottom materials, so that the fermentation liquid contains the raw material fiber and insoluble protein, and the dissolved oxygen of the fermentation liquid is limited. Secondly, in the prior art, the starchy raw material liquefied liquid is greatly influenced by the fluctuation of crop components, particularly newly harvested crops greatly influence seeds and a fermentation tank, so that the fermentation period and the fermentation end are influenced. Thirdly, in the middle and later stages of fermentation in the prior art, due to the great reduction of the pH value, most of added saccharifying enzymes in the fermentation process or saccharifying enzymes generated by thalli are inactivated, so that the fermentation period is long, the fermentation end point acidity and citric acid conversion rate are low, and the fermentation residual sugar is relatively high; especially, in the process of producing high citric acid by high sugar, about 0.2 percent of reducing sugar left finally cannot be consumed, so that raw materials are wasted, and COD in the wastewater is increased.
With the above limitations in mind, the applicant of the present application prepared a starch liquefaction solution based on starch by replacing starchy materials used for liquefaction with starch, and further obtained a seed culture medium of a citric acid fermentation strain and a citric acid fermentation culture medium. By the adjustment, macromolecule insoluble substances such as fiber and insoluble protein are not contained in the seed culture medium and the fermentation culture medium any more, so that the oxygen supply of the culture medium is improved, and the seed culture time and the fermentation time are greatly shortened. In addition, the step of preparing liquefied clear liquid of starchy raw materials by filter pressing is omitted by liquefying starch, the DE value of starch liquefaction can be reduced to 10-20% (25-30% in the prior art), and the liquefied liquid is very uniform, so that the DX value after saccharification is effectively improved and can reach more than 95%, the utilization rate of fermentation sugar is further improved, and residual sugar is reduced.
Based on the above research results, in one aspect, the present invention provides a method for preparing a seed culture medium for fermenting citric acid using all-starch, wherein the method comprises:
(1) mixing starch slurry obtained from whole starch with amylase to obtain a mixture, contacting the mixture with steam at pH5.2-6 under such conditions that the temperature of the mixture after contacting with steam is 90-105 deg.C, and maintaining at the temperature for 60-90 min to obtain starch liquefied liquid;
(2) adding a nitrogen source into the starch liquefied liquid, adjusting the pH value of the starch liquefied liquid to 4.5-6.5, and optionally saccharifying to obtain the seed culture medium for fermenting the citric acid.
Preferably, the pH of the jet liquefaction is any value between 5.2 and 6 (e.g., 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9) and any two point composition range. Preferably, the pH of the jet liquefaction is 5.2-5.6.
According to the present invention, in step (1), the conditions for contacting with steam are selected from a wide range so long as the mixture of the starch slurry and the enzyme after the contacting can be maintained at the appropriate temperatures, respectively, and for example, the conditions for contacting with steam include the contacting temperature, the ratio of the steam to the mixture, the contacting time, and the like. Preferably, the steam temperature may be 180 ℃ and 270 ℃, the steam to mixture ratio may be 0.03-0.08:1, and the contact time may be 1-5 seconds. More preferably, the temperature of the steam is 240 ℃ to 260 ℃, the ratio of the steam to the mixture is 0.04-0.06:1, and the contact time is 1-3 seconds in step (1) considering the energy and cost together. The method of contacting the mixture of starch slurry and enzyme with steam is not particularly limited, but preferably, the mixture is contacted by spraying in a sprayer known to those skilled in the art, that is, the mixture of starch slurry and enzyme and steam are simultaneously and rapidly sprayed into the sprayer to be instantaneously contacted, and the contacted mixture is sent into another storage tank to be subjected to heat preservation treatment at a certain temperature.
According to the invention, the amylase used can be added in the form of an amylase-producing thermophilic microorganism or in the form of a separate amylase, and the enzymatic hydrolysis of the starch is carried out at the growth temperature of the amylase-producing thermophilic microorganism and/or at a temperature at which the enzyme is viable.
Since microbial growth produces by-products, direct addition of amylase is preferred. The more the amylase is used, the better, and for cost reasons, the preferred amount is 8-25 enzyme activity units per gram dry weight of the pulverized product.
The enzyme activity unit of the enzyme of the invention is defined as: the amount of enzyme required to convert 1 mg of starch to reducing sugars in 1 minute at a pH of 6.0 and a temperature of 70 ℃ was one enzyme activity unit.
Amylases are generic terms referring to a class of enzymes capable of breaking down starch glycosidic bonds and generally include alpha-amylases, beta-amylases, and isoamylases.
Alpha-amylase, also known as starch 1, 4-dextrinase, can randomly and irregularly cut alpha-1, 4-glycosidic bonds inside starch chains to hydrolyze starch into maltose, oligosaccharides containing 6 glucose units and oligosaccharides with branched chains. The microorganisms producing this enzyme are mainly Bacillus subtilis, Aspergillus niger, Aspergillus oryzae and Rhizopus.
Beta-amylase, also known as starch 1, 4-maltosidase, cleaves 1, 4-glycosidic bonds from the non-reducing ends of starch molecules to form maltose. The products of the enzyme acting on starch are maltose and limit dextrin. This enzyme is produced mainly by aspergillus, rhizopus and endospore.
Isoamylase is also called starch alpha-1, 6-glucosidase, branching enzyme, and the enzyme acts on alpha-1, 6-glycosidic bond at branch point of branch chain starch molecule to cut off whole side chain of branch chain starch to become amylose. The enzyme producing bacteria are mainly bacteria such as bacillus, bacillus and some pseudomonas.
According to the present invention, conditions for mixing the starch slurry obtained from starch with amylase are widely selected, and usually, in order to more facilitate the enzyme to exert its effect and in view of energy saving, the mixing of the starch slurry obtained from starch with amylase is carried out at 50 to 60 ℃ for 20 to 30 minutes. The above mixing is more preferably carried out under stirring.
The starch slurry may be prepared by various conventional methods known to those skilled in the art, for example, by mixing starch with water to obtain a starch slurry having a starch concentration of 15 to 35% by weight.
According to the invention, the amount of nitrogen source added in step (2) can vary within wide limits, provided that it meets the nitrogen source requirements of the fermenting species. In general, the amount of the nitrogen source added may be 0.01 to 0.2% by weight, preferably 0.05 to 0.1% by weight, based on the total weight of the medium, in terms of nitrogen element. The kind of the supplementary nitrogen source is well known to those skilled in the art, and for example, the supplementary nitrogen source may be one or more of urea, ammonium sulfate, ammonium nitrate, corn steep liquor, and the like.
More preferably, the nitrogen source is corn steep liquor. In the preferable case, the pH of the starch liquefaction liquid after adding the corn steep liquor can be in the range of 3.8-4.5 (for example, can be 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4), no adjustment is needed, the cost is saved, the process control point is saved, the starch sugar and the corn steep liquor can be mixed uniformly, and the growth of citric acid seeds and the acid production are facilitated.
The corn steep liquor is a byproduct of corn starch production, and the raw materials comprise corn grits, water and corn juice. Corn starch silk is produced by soaking corn kernels in sulfurous acid, and concentrating the soaking solution to obtain a yellow brown liquid, called corn steep liquor, which is rich in soluble protein, auxin and some precursor substances and contains about 40-50% of solid substances.
According to the invention, in the step (2), the nitrogen source is added into the starch liquefied liquid when the starch liquefied liquid is cooled to below 65 ℃, various methods known in the art can be adopted as the cooling method, preferably, in order to better achieve the purpose of leading the expansion of starch molecules to be more sufficient due to rapid temperature change so as to enable large-particle starch to be expanded and broken into small-particle starch, and leading the contact effect of amylase and starch particles to be better so as to achieve better enzymolysis (liquefaction) effect, preferably, the mixture obtained after the step (1) is contacted with steam is cooled to below 65 ℃ in a flash evaporation mode, and the steam obtained after the flash evaporation can be returned to the step (1) in the continuous production process for contacting with the mixture. Furthermore, the condensed water obtained after flashing can also be returned to step (1) for the preparation of a starch slurry, i.e. for mixing with starch to prepare a starch slurry. Wherein flashing means that saturated water at high pressure enters a vessel at a relatively low pressure and then becomes a portion of the saturated vapor and water at the vessel pressure due to a sudden drop in pressure. According to the invention, the flash evaporation conditions can be selected from a wide range as long as the purpose of reducing the temperature after the flash evaporation is achieved, and preferably, the flash evaporation conditions comprise that the vacuum degree of the flash evaporation can be 0.05-0.09MPa, and the flash evaporation time can be 5-20 seconds. The value read from the vacuum gauge is called the vacuum degree. The vacuum value is a value indicating that the actual value of the system pressure is lower than the atmospheric pressure, that is: the vacuum degree is equal to atmospheric pressure to absolute pressure (absolute value of atmospheric pressure and absolute pressure). "vacuum" is the degree of vacuum. By "vacuum" is meant a gaseous state at a pressure below 101325 pascals (i.e., about 101KPa above one standard atmospheric pressure) within a given space.
According to the present invention, the whole starch may be any kind of whole starch known in the art, and may be selected from one or more of corn whole starch, potato (e.g., tapioca) whole starch, wheat whole starch, and sorghum whole starch, for example. It should be noted here that "whole starch" is relatively pure white starch powder, which is prepared from corn by soaking in sulfurous acid, crushing, sieving, fine grinding, precipitating, and drying, and has a dry starch content of 98.5% or more and a small amount of fat and protein. The potato, wheat and sorghum starch is prepared by similar process.
According to the present invention, the starch liquefaction liquid after adjusting the pH value to 4.5-6.5 can be saccharified, and the type and amount of saccharifying enzyme used in the saccharification and the saccharification conditions are described below and will not be repeated.
In a second aspect, the present invention provides a method for preparing a medium for fermented citric acid using whole starch, wherein the method comprises mixing a seed medium for fermented citric acid prepared as described above to be saccharified with a saccharifying enzyme, and saccharifying the resulting mixture at 60 to 63 ℃ and ph3.8 to 4.5 for 5 to 20 hours to obtain a medium for fermented citric acid; or adjusting pH of saccharified fermented citric acid seed culture medium prepared by the above method to 3.8-4.5 to obtain fermented citric acid culture medium.
According to a preferred embodiment of the present invention, the liquefied liquid after contacting with steam is cooled to 60 to 63 ℃ by the above flash evaporation method, and saccharified after adding components such as saccharifying enzyme and optionally nitrogen source and adjusting the pH. Wherein, the selection of the nitrogen source has been described in detail above, and is not repeated herein.
Wherein the time for saccharification is preferably 5 to 10 hours.
According to the invention, the saccharifying enzyme to be used can be added in the form of a saccharifying enzyme-producing thermophilic microorganism or in the form of a separate saccharifying enzyme, and the saccharification of the starch liquefied liquid is completed at the growth temperature of the saccharifying enzyme-producing thermophilic microorganism and/or at the temperature at which the enzyme is viable.
Since microbial growth produces by-products, direct addition of saccharifying enzymes is preferred. The more the saccharifying enzyme is used, the better, and for cost reasons, it is preferable that the saccharifying enzyme is used in an amount of 50 to 100 enzyme activity units per gram of dry weight of the pulverized product.
Saccharifying enzyme is also called starch alpha-1, 4-glucosidase, and the saccharifying enzyme acts on the non-reducing end of starch molecule, takes glucose as unit, and acts on alpha-1, 4-glycosidic bond in starch molecule in sequence to generate glucose. The product of the glucoamylase after acting on the amylopectin contains glucose and oligosaccharide with alpha-1, 6-glycosidic bond; the product after amylose is almost exclusively glucose. The enzyme producing strain is mainly Aspergillus niger (left Aspergillus oryzae, Aspergillus awamori), Rhizopus (Rhizopus niveus, Rhizopus delemar), Neurospora sp, and Monascus ruber.
In a third aspect, the present invention provides a method for preparing citric acid by fermenting whole starch, comprising:
(1) inoculating citric acid zymophyte into the seed culture medium for fermenting citric acid prepared by the method for seed culture to obtain a seed culture solution;
(2) inoculating the seed culture solution obtained in the step (1) into the culture medium for preparing fermented citric acid by the method and fermenting under the condition of generating citric acid to obtain a citric acid fermentation liquid.
According to the present invention, there is no particular limitation on the fermentation species, and microorganisms capable of fermenting monosaccharides such as glucose and/or fructose, oligosaccharides such as sucrose and/or galactose can be used for the fermentation process of the present invention, preferably the fermentation species uses Aspergillus niger.
The Aspergillus niger used in the fermentation of the present invention may be a commercially available Aspergillus niger solid preparation or an Aspergillus niger species, for example, Aspergillus niger Co827 (New Industrial microbiology, Inc. of Shanghai), Aspergillus niger T01 (Tianjin Industrial Microbiol., Ltd.), and Aspergillus niger strain (Zhongkojie Microbiol., Ltd.).
According to the present invention, the fermentative species seed solution may be prepared according to various methods known in the art, for example, the fermentative species seed solution may be prepared by: putting the seed culture medium for fermenting citric acid prepared by the method into a seed tank, inoculating Aspergillus niger strain, culturing at 30-45 deg.C, pH 1-7, and ventilation amount of 0.03-0.4V/V.min; observing the growth of Aspergillus niger by microscopic examination, acidity measurement and pH measurement of a sampling microscope, and stopping culturing when the pH is 2.0-2.5, the acidity is 5-20g/L, the size of a fungus ball is uniform, and hyphae grow robustly. The amount of Aspergillus niger inoculum can vary within a wide range, and preferably the concentration of Aspergillus niger in the seed medium after inoculation is 2X 10/ml Aspergillus niger culture medium5-5×105And (4) spores. The Aspergillus niger seed liquid prepared by the method can effectively shorten the culture period, and the obtained blackThe aspergillus seeds have stable quality and good effect.
The number of spores can be determined by methods known in the art, for example, by counting on a hemocytometer. Specifically, the measurement can be carried out by the following method: taking a bottle of mouldy bran for culturing Aspergillus niger, pouring 0.5% of Tween-80 into the bottle to reach a constant volume of V1-500 mL, and simultaneously placing the bottle into a rotor to stir until suspension is uniform. Diluting the uniform spore liquid with V2-5 mL to a constant volume of V3-100 mL, ultrasonically dispersing for 10 minutes, pouring into a beaker, homogenizing by using a magnetic stirrer, sampling, counting by using a blood counting plate, and counting to obtain the spore number N of the diluted spore suspension per milliliter, wherein the bran spore number per bottle is V1 x (N multiplied by V3/V2).
According to the invention, the conditions of the fermentation in said fermentation step may vary widely, as long as citric acid is obtained by fermentation, for example, the conditions of the fermentation include: fermenting at 25-45 deg.C, pH 1-7, tank pressure 0.04-0.1Mpa, and ventilation amount 0.1-1V/V.min; more preferably, the fermentation conditions include: the fermentation temperature is 30-40 deg.C, pH value is 1-4, tank pressure is 0.05-0.09Mpa, and ventilation volume is 0.2-0.3V/V.min.
The term "aeration" is generally expressed in terms of aeration ratio, usually in terms of the volume of air per minute per unit volume of culture broth (V/V.min), for example an aeration ratio of 1:0.05 to 0.5, abbreviated to an aeration of 0.05 to 0.5 by volume: volume. min.
In the present invention, the amount of the Aspergillus niger inoculum can be widely varied, and is preferably 5 × 10/g Aspergillus niger culture solution4-1.5×105Individual colony forming units.
The colony forming unit is defined as that a certain amount of diluted bacterial liquid is poured or coated to disperse microbial single cells in the bacterial liquid on a culture medium flat plate one by one, and each living cell forms a colony after culture. I.e.the number of single cells contained per ml of bacterial suspension.
The fermentation equipment is well known to those skilled in the art, and for example, a fermenter can be used for the cultivation. The fermenter is not particularly limited, and various fermenters commonly used in the art may be used, and preferably, the fermenter has a volume of 300 cubic meters.
The citric acid as fermentation product can be separated and refined by conventional method according to different industrial product requirements, such as neutralization, acidolysis, decolorization, concentration, crystallization and packaging.
Examples
The invention is further illustrated in more detail by the following examples, which are intended to illustrate, but not to limit the invention. It will be understood by those of ordinary skill in the art that these examples are not intended to limit the present invention in any way and that modifications may be made without departing from the spirit and scope of the present invention as set forth in the following claims.
The fermentation method of citric acid according to the present invention will be described in detail below with reference to examples and comparative examples.
Determining the total sugar content in the enzymolysis solid phase residue according to the total sugar direct titration method of GBT 15038-2006;
residual reducing sugars were determined according to GBT 50099-2008;
the concentration (acidity for short) of the fermentation broth after fermentation is detected according to the GB 1987-2007 standard, and the conversion rate of the citric acid is calculated, wherein the conversion rate (%) is the concentration (acidity for short) of the fermentation broth x the volume of the fermentation broth/the weight of the total sugar (total sugar: total sugar in a seed tank + total sugar in a fermentation tank x 100%).
Example 1
This example illustrates the preparation of citric acid according to the invention
1) Size mixing: and (3) mixing the corn starch with water to obtain starch slurry, wherein the adding amount of the water is such that the mixing concentration of the starch slurry is 20 wt%.
2) Enzymolysis: mixing the starch slurry obtained in step (1) with amylase (Novitin, alpha-amylase, which is used in the examples of the present invention) at 50 ℃ to obtain a mixture (the amount of amylase is 15 enzyme activity units per gram of dry starch), subjecting the mixture to jet contact with steam at 240 ℃ in a jet injector (the weight ratio of steam to mixture is 0.04:1), wherein the contact time is 3 seconds, so that the temperature of the mixture after the contact with steam is 100 ℃, adjusting the pH value to 5.3, and keeping the temperature for 70 minutes.
3) Flash evaporation: flashing the mixture with steam (vacuum degree of 0.07Mpa, time of 10 s) to 60 deg.C to obtain liquefied corn liquid with DE value of 15%.
4) Seed culture medium: adding water into part of the corn liquefaction liquid obtained in the step 3) to dilute the corn liquefaction liquid until the total sugar concentration is 12%, and simultaneously adding 4.5 wt% of corn steep liquor (calculated by 30 wt% of the dry basis of the corn steep liquor) to obtain a seed culture medium.
5) Fermentation medium: adding water into the remaining part of the corn liquefied liquid obtained in the step 3) to dilute until the total sugar concentration is 18%, adding 2 wt% of corn steep liquor (the pH value is 4.2 after the corn steep liquor is added according to 30 wt% of a corn steep liquor dry basis), simultaneously adding saccharifying enzyme (Novitin company, the using amount of the saccharifying enzyme is 70 enzyme activity units relative to per gram of dry starch), and maintaining for 10 hours at 60 ℃ to obtain a fermentation medium, wherein the DX value is 97%.
6) Seed culture: putting the seed culture medium prepared in the step 4) into a seed tank, heating to 121 ℃ for disinfection, maintaining for 30 minutes, quickly cooling to 36 ℃, inoculating Aspergillus niger (Aspergillus niger T01, Tianjin Industrial Microbiol, all of which are in the embodiment of the invention, and the inoculation amount is as follows: inoculating 2X 10 of the culture medium per liter5Spores) are cultured under the condition of aeration with pH value of 3, temperature of 36 ℃ and 0.4V/V.min; the growth of Aspergillus niger was observed by microscopic examination, acidity measurement and pH measurement of a sampling microscope, and when the pH was 2.0, the acidity was 10g/L, the pellet size was uniform, and the hyphae were thick and protruded, the culture was stopped, and the time taken for the culture was recorded as shown in Table 1.
7) And (3) fermenting to prepare citric acid: adding the cultured aspergillus niger strains into a sterilized fermentation tank containing the prepared fermentation liquid for fermentation, and detecting the total sugar in the fermentation liquid, wherein the inoculation amount is as follows: 5X 10 per gram of fermentation liquor4Culturing the colony forming unit under conditions of stirring at 37 deg.C and 120 rpm and ventilating at a ratio of 1:0.4 for 60 hr, detecting acidity at the end of fermentation, calculating conversion rate of citric acid, and detecting residual reducing sugar at the end of fermentationAnd the amount of residual total sugars, the results are shown in Table 1.
Example 2
This example illustrates the preparation of citric acid according to the invention
1) Size mixing: and (3) mixing the corn starch with water to obtain starch slurry, wherein the adding amount of the water is such that the mixing concentration of the starch slurry is 15 wt%.
2) Enzymolysis: mixing the starch slurry obtained in the step (1) with amylase (Novitin, alpha-amylase, which is used in the examples of the present invention) at 50 ℃ to obtain a mixture (the amount of the amylase is 10 enzyme activity units per gram of the dry starch), and subjecting the mixture to jet contact with steam at 230 ℃ in a jet ejector (the weight ratio of the steam to the mixture is 0.04:1), wherein the contact time is 3 seconds, so that the temperature of the mixture after the contact with the steam is 91 ℃, adjusting the pH value to 5.4, and keeping the temperature for 60 minutes.
3) Flash evaporation: flashing the mixture with steam (vacuum degree of 0.07Mpa, time of 10 s) to 62 deg.C to obtain liquefied corn liquid with DE value of 16%.
4) Seed culture medium: adding water into part of the corn liquefaction liquid obtained in the step 3) to dilute until the total sugar concentration is 10%, and simultaneously adding 4 wt% of corn steep liquor (calculated by 30 wt% of the dry corn steep liquor basis) to obtain a seed culture medium.
5) Fermentation medium: adding water into the remaining part of the corn liquefied liquid obtained in the step 3) to dilute until the total sugar concentration is 16%, adding 2 wt% of corn steep liquor (the pH value is 4.0 after adding the corn steep liquor calculated by 30 wt% of a corn steep liquor dry base), simultaneously adding saccharifying enzyme (Novitin company, the using amount of the saccharifying enzyme is 60 enzyme activity units relative to each gram of dry starch), and maintaining for 8 hours at 62 ℃ to obtain a fermentation culture medium, wherein the DX value is 95%.
6) Seed culture: putting the seed culture medium prepared in the step 4) into a seed tank, heating to 121 ℃ for disinfection, maintaining for 30 minutes, quickly cooling to 36 ℃, inoculating Aspergillus niger (Aspergillus niger T01, Tianjin Industrial Microbiol, all of which are in the embodiment of the invention, and the inoculation amount is as follows: inoculating the culture medium per liter2×105Spores) are cultured under the condition of aeration with pH value of 3, temperature of 36 ℃ and 0.4V/V.min; the growth of Aspergillus niger was observed by microscopic examination, acidity measurement and pH measurement of a sampling microscope, and when the pH was 2.0, the acidity was 10g/L, the pellet size was uniform, and the hyphae were thick and protruded, the culture was stopped, and the time taken for the culture was recorded as shown in Table 1.
7) And (3) fermenting to prepare citric acid: adding the cultured aspergillus niger strains into a sterilized fermentation tank containing the prepared fermentation liquid for fermentation, and detecting the total sugar in the fermentation liquid, wherein the inoculation amount is as follows: 5X 10 per gram of fermentation liquor4Each colony forming unit was cultured under conditions of stirring at 37 ℃ and 120 rpm and aeration at a ratio of 1:0.4 for 60 hours, and after completion of fermentation, the acidity at the end of fermentation was measured, and the conversion rate of citric acid was calculated, and the amount of residual reducing sugars and the amount of residual total sugars at the end of fermentation were measured, and the results are shown in Table 1.
Example 3
This example illustrates the preparation of citric acid according to the invention
1) Size mixing: and (3) mixing the corn starch with water to obtain starch slurry, wherein the adding amount of the water is such that the mixing concentration of the starch slurry is 25 wt%.
2) Enzymolysis: mixing the starch slurry obtained in step (1) with amylase (Novitin, alpha-amylase, which is used in the examples of the present invention) at 50 ℃ to obtain a mixture (the amount of amylase is 25 enzyme activity units per gram of dry starch), and subjecting the mixture to jet contact with steam at 240 ℃ in a jet injector (the weight ratio of steam to mixture is 0.04:1), wherein the contact time is 4 seconds, so that the temperature of the mixture after the contact with steam is 105 ℃, adjusting the pH value to 5.6, and keeping the temperature for 90 minutes.
3) Flash evaporation: flashing the mixture with steam (vacuum degree of 0.07Mpa, time of 10 s) to 62 deg.C to obtain liquefied corn liquid with DE value of 10%.
4) Seed culture medium: adding water into part of the corn liquefaction liquid obtained in the step 3) to dilute until the total sugar concentration is 15%, and simultaneously adding 4 wt% of corn steep liquor (calculated by 30 wt% of the dry corn steep liquor basis) to obtain a seed culture medium.
5) Fermentation medium: diluting the rest part of the corn liquefied liquid obtained in the step 3) with water until the total sugar concentration is 20%, adding 2.5 wt% of corn steep liquor (pH value is 4.4 after adding the corn steep liquor calculated by 30 wt% of the dry basis of the corn steep liquor), simultaneously adding saccharifying enzyme (Novit company, the dosage of the saccharifying enzyme is 70 enzyme activity units relative to per gram of dry starch), and maintaining at 62 ℃ for 7 hours to obtain a fermentation medium with a DX value of 98%.
6) Seed culture: putting the seed culture medium prepared in the step 4) into a seed tank, heating to 121 ℃ for disinfection, maintaining for 30 minutes, quickly cooling to 36 ℃, inoculating Aspergillus niger (Aspergillus niger T01, Tianjin Industrial Microbiol, all of which are in the embodiment of the invention, and the inoculation amount is as follows: inoculating 2X 10 of the culture medium per liter5Spores) are cultured under the condition of aeration with pH value of 3, temperature of 36 ℃ and 0.4V/V.min; the growth of Aspergillus niger was observed by microscopic examination, acidity measurement and pH measurement of a sampling microscope, and when the pH was 2.0, the acidity was 10g/L, the pellet size was uniform, and the hyphae were thick and protruded, the culture was stopped, and the time taken for the culture was recorded as shown in Table 1.
7) And (3) fermenting to prepare citric acid: adding the cultured aspergillus niger strains into a sterilized fermentation tank containing the prepared fermentation liquid for fermentation, and detecting the total sugar in the fermentation liquid, wherein the inoculation amount is as follows: 5X 10 per gram of fermentation liquor4Each colony forming unit was cultured under conditions of stirring at 37 ℃ and 120 rpm and aeration at a ratio of 1:0.4 for 60 hours, and after completion of fermentation, the acidity at the end of fermentation was measured, and the conversion rate of citric acid was calculated, and the amount of residual reducing sugars and the amount of residual total sugars at the end of fermentation were measured, and the results are shown in Table 1.
Example 4
This example illustrates the preparation of citric acid according to the invention
The procedure of example 1 was followed to conduct the production of citric acid, except that the nitrogen source added to the seed medium and the citric acid fermentation medium was not corn steep liquor, but urea, and dilute sulfuric acid was added to adjust the pH to the same value as in example 1. The results are shown in Table 1.
Comparative example 1
This comparative example illustrates the preparation of a reference citric acid
1) The procedure is as in example 1
2) Enzymolysis: mixing the starch slurry obtained in step (1) with amylase (Novitin, alpha-amylase, which is used in the examples of the present invention) at 50 ℃ to obtain a mixture (the amount of amylase is 25 enzyme activity units per gram of dry starch), and subjecting the mixture to jet contact with steam at 240 ℃ in a jet injector (the weight ratio of steam to mixture is 0.04:1), wherein the contact time is 4 seconds, so that the temperature of the mixture after the contact with steam is 110 ℃, adjusting the pH value to 6.2, and keeping the temperature for 100 minutes.
3) Flash evaporation: flashing the mixture with steam (vacuum degree of 0.07Mpa for 10 s) to 55 deg.C to obtain liquefied corn liquid with DE value of 25%.
4) Seed culture medium: adding water into part of the corn liquefaction liquid obtained in the step 3) to dilute until the total sugar concentration is 15%, and simultaneously adding 4 wt% of corn steep liquor (calculated by 30 wt% of the dry corn steep liquor basis) to obtain a seed culture medium.
5) Fermentation medium: diluting the rest part of the corn liquefied liquid obtained in the step 3) with water until the total sugar concentration is 20%, adding 2.5 wt% of corn steep liquor (pH value is 5.0 after adding the corn steep liquor calculated by 30 wt% of the dry basis of the corn steep liquor), simultaneously adding saccharifying enzyme (Novit company, the dosage of the saccharifying enzyme is 100 enzyme activity units relative to per gram of dry starch), and maintaining at 55 ℃ for 7 hours to obtain a fermentation medium with a DX value of 80%.
The procedure was as in example 1, and the results are shown in Table 1.
Comparative example 2
This comparative example illustrates the preparation of a reference citric acid
The method of example 1 was followed to prepare citric acid, except that the seed culture medium was prepared according to the prior art using corn liquefied solution, and the fermentation medium was prepared using corn liquefied solution and corn liquefied clear solution (see CN 103146769B for details), and the fermentation time was 75 h. The results are shown in Table 1.
Comparative example 3
This comparative example illustrates the preparation of a reference citric acid
The preparation of citric acid was carried out according to the method of example 1, except that the saccharifying enzyme was added during the fermentation. The results are shown in Table 1.
TABLE 1
According to the results, the technical scheme of the application effectively improves the acidity of the fermentation end point and the citric acid conversion rate, and reduces the residual quantity of total sugar and reducing sugar. Meanwhile, the time of seed culture and the fermentation period can be effectively shortened.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, numerous simple modifications can be made to the technical solution of the invention, including combinations of the individual specific technical features in any suitable way. The invention is not described in detail in order to avoid unnecessary repetition. Such simple modifications and combinations should be considered within the scope of the present disclosure as well.
Claims (7)
1. A method for preparing a seed culture medium for fermenting citric acid by using full starch is characterized by comprising the following steps:
(1) mixing starch slurry obtained from whole starch with amylase to obtain a mixture, contacting the mixture with steam under the condition of pH5.2-6, wherein the contacting condition is that the temperature of the mixture after contacting with the steam is 90-105 ℃, keeping the temperature for 60-90 minutes, and then reducing the temperature to 60-63 ℃ by a flash evaporation method to obtain starch liquefied liquid;
(2) adding a nitrogen source into the starch liquefied liquid to obtain a seed culture medium for fermenting citric acid;
the nitrogen source is corn steep liquor; the addition amount of the nitrogen source is 0.01-0.2 parts by weight calculated by nitrogen element based on 100 parts by weight of the starch liquefied liquid;
wherein the DE value of the starch liquefied liquid is 10-20%.
2. The process of claim 1, wherein in step (1), the concentration of the starch slurry is 15-35 wt%; the dosage of the amylase is 8-25 enzyme activity units relative to the weight of each gram of dry starch.
3. The method of claim 1, wherein the whole starch is from one or more of corn whole starch, potato whole starch, wheat whole starch, and sorghum whole starch.
4. A method for preparing a medium for fermenting citric acid by using whole starch, which comprises mixing a seed medium for fermenting citric acid prepared by the method of any one of claims 1 to 3 with a saccharifying enzyme, and saccharifying the resulting mixture at 60 to 63 ℃ and pH of 3.8 to 4.5 for 5 to 20 hours to obtain a medium for fermenting citric acid;
wherein the DX value of the culture medium for fermenting the citric acid is more than 95 percent.
5. The process according to claim 4, wherein the saccharifying enzyme is used in an amount of 50 to 100 enzyme activity units per gram of dry starch.
6. A method for preparing citric acid by adopting full starch fermentation is characterized by comprising the following steps:
(1) inoculating citric acid zymophyte into a seed culture medium for fermenting citric acid prepared by the method of any one of claims 1-3 for seed culture to obtain a seed culture solution;
(2) inoculating the seed culture solution obtained in the step (1) into the culture medium for fermenting citric acid prepared by the method of claim 4 or 5 under the condition of generating citric acid, and fermenting to obtain a citric acid fermentation solution.
7. The method according to claim 6, wherein the citric acid fermentation medium contains 5 x 10 of citric acid zymophyte per gram of the medium4-1.5×105Individual colony forming units, the conditions of the fermentation comprising: temperature is 25-45 deg.C, pH value is 1-7, tank pressure is 0.04-0.1Mpa, and ventilation volume is 0.1-1 volume: volume, minute.
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