CN109536541A - The method of corn pulp preprocess method and production citric acid - Google Patents
The method of corn pulp preprocess method and production citric acid Download PDFInfo
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Abstract
Pretreated method is carried out the present invention relates to a kind of pair of corn pulp and using the method for pretreated corn pulp production citric acid, wherein, by being pre-processed using compound protease to corn pulp, and pretreated corn pulp is used for fermentation production of citric acid, the microorganism that corn pulp can be obviously improved utilizes, and the foam volume of fermentation process is significantly reduced, to improve citric acid yield.
Description
Technical field
The invention belongs to bioengineering and microorganism field, be related to corn pulp in the fermentation manufacturing technique to citric acid into
Method row pretreated method and citric acid is produced using pretreated fermenting corn steep liquor.
Background technique
Citric acid (CA) is a kind of important mesostate of tricarboxylic acid cycle, is widely used in food, medicine, fine
The fields such as chemicals.China's Citric Acid Production is started late, and initial stage mainly carries out shallow tray fermentation research with molasses or granulated sugar；1968
Year, it realizes be made from starch submerged fermentation citric acid for the first time, same period Tianjin industrial microorganism, which is developed, to be done with potato as raw material
The technique of submerged fermentation production citric acid.On the other hand, it is light to be constantly in act foot in citric acid industry for Producing Strain breeding work
The status of weight, research of the China in terms of strain improvement is very active, and by the unremitting effort of several generations, having selected has bottom
Object extensive (without pretreatment), substrate extensive (starchiness, saccharine material), tolerance strong (resistance to high sugar, resistance to homocitric acid and anti-gold
Belong to ion), produce the strain of the high good characteristic of acid, have developed the mainstream production technology of aspergillus niger liquid submerged fermentation.
Currently, domestic citric acid fermentation production technology is mainly to be fermented using the full powder band slag that corn is carried out as raw material.It is non-
The factors such as Fermented raw material solid ingredient and the winding of black-koji mould filament seriously affect the mass transfer of fermentation process, cause starch sharp
With rate is insufficient, ineffective energy consumption is more, impurity content is high, to influence the purification of product separation and Extraction.Therefore, it is badly in need of developing a kind of high
The citric acid fermentation production new technique of effect is greatly lowered Citric Acid Production cost and promotes citric acid product quality.With jade
Rice starch yield is continuously increased, and Starch Production technology is constantly progressive, and starch cost constantly reduces, the yield of corn pulp also phase
It should increase.Corn pulp is used as the culture medium raw material of production fermented product to produce all kinds of high value added products, to extend jade
The purposes of Rice & peanut milk has very important meaning.
Currently, the citric acid all clear liquid zymotechnique being made from starch becomes focal point, mainly by cornstarch
It is prepared into liquid glucose by liquefying, being saccharified, adds cornstarch processing byproduct-corn pulp as nitrogen source, then add a small amount of N, P
Equal nutriments ferment, and the major advantage of the technique is: 1) full liquor fermentation, therefore mass transfer dissolved oxygen level is high, fermentation produces
Sour height, high conversion rate, the period is short, production cost is low；2) black-koji mould filament is free of corn peel and fiber, is conducive to it into one
Walk higher value application.
Corn pulp is cornstarch by-product produced during the preparation process, is the water with sulfurous acid soaking corn by concentration
It is processed into, is filemot thick opaque cotton-shaped suspended matter, wherein solid content is in 50wt% or so, due to corn pulp
The nutriments such as amino acid, reduced sugar, phosphorus, microelement and auxin rich in, and preparation cost is cheap, exists for many years
Fermentation industry has extremely wide application.
But there is also obvious disadvantages for the technique using corn pulp as nitrogen source at present: making when using a large amount of corn pulp
When for nitrogen source culture aspergillus niger, the foam in fermentation process is caused to increase, as corn pulp dosage increases, fermentor utilization efficiency
It decreases.In addition, the microbial spores that insoluble granule and part are not easy high temperature killing in corn pulp also increase therewith, ferment
Microbiological contamination risk increases.Moreover, a large amount of unemployed impurity with fermentation liquid enter extraction link in corn pulp, equally to extracting
Journey adversely affects, such as removal of impurities difficulty increases, major product yield reduces, these impurity finally enter waste liquid, often at
For some factors of liquid waste processing difficulty.
To solve these problems, pretreatment is carried out to the raw material containing abundant organic nitrogen source to have become Recent study more
One field, for example, soybean meal hydrolysate has more research report at present；Compared with non-hydrolysis material, microorganism is to organic nitrogen
Source utilization efficiency is higher, can significantly improve fermentation yield, but these raw materials are mostly expensive, be unfavorable for the control of fermentation costs
System.
In addition, such as patent CN104046674B disclose a kind of producing beta-carotene by fermentation improved corn steep liquor and
Preparation method and application.The improved corn steep liquor is by corn pulp raw material and Na2It is centrifuged after sufficiently being reacted after S mixing, in adjustment
Clear liquid pH value and obtain.The improved corn steep liquor of the method preparation is applied in the fermentation of beta carotene, as main nitrogen source and
Microelement source, beta carotene output increased, is greatly lowered fermentation costs.However, in the method preparation corn pulp
Sulfur content is higher, and the growth metabolism of thallus will be had an impact by being applied in fermentation process, in addition, also can be to a certain extent
Difficulty is brought to the purification and purification of downstream product.
The present invention is pre-processed by the way of enzyme hydrolysis using corn pulp cheap and easy to get as raw material, to obtain more
The pretreated corn pulp easily utilized for microorganism, while being sent out using resulting corn pulp as one of raw material applied to citric acid
Ferment, thus improved citric acid fermentation technique.
Summary of the invention
With using cornstarch as the continuous development of the technique of fermenting raw materials production citric acid, jade in the urgent need to address
Rice & peanut milk largely uses the problems such as the control of bring fermentation process foam is difficult, fermentor utilization rate is low, by fresh corn starch into
The specific pretreatment of row improves microorganism to the utilization rate of nutriment, promotes the resource utilization of corn pulp, so as to further
Promote the accumulation of citric acid.
The invention discloses a kind of pair of corn pulps to carry out pretreated method, wherein the described method comprises the following steps:
(1) take corn pulp be added fermentor in, according to 1:(0.5-1.5) volume ratio water is added into the corn pulp,
Obtain mixed liquor；
(2) pH to 6-8 for adjusting the mixed liquor, then raises temperature to 60 DEG C -70 DEG C, is added in Xiang Suoshu mixed liquor compound
Protease simultaneously carries out enzyme digestion reaction 7-9h；
(3) after the enzyme digestion reaction, reaction system is warming up to 85 DEG C of -95 DEG C of progress enzyme deactivations, be subsequently cooled to 20 DEG C -
35 DEG C, obtain pretreated corn pulp.
On the other hand, the present invention relates to a kind of methods for producing citric acid, wherein described method includes following steps:
(i) corn starch solution that powder slurry concentration is 13%-18% (w/v) is prepared with water, by the corn starch solution
40 DEG C -70 DEG C at a temperature of be standing and soaking, then adjust its pH to 5.0-6.0, add alpha-amylase thereto, in enzyme activity temperature
It keeps the temperature and is stirred continuously under degree, obtain cornstarch liquefier；
(ii) it adds in Xiang Suoshu cornstarch liquefier according to pretreated corn pulp obtained by the above method, ammonium hydroxide
And water, obtain seed culture medium；
(iii) it will be inoculated with the aspergillus niger spore suspension of sterile saline or sterile water preparation into the seed culture medium
In, it is cultivated at 30 DEG C -40 DEG C, obtains seed culture fluid；
(iv) pH for the cornstarch liquefier that step (i) obtains is adjusted to 3.8-5.0, carbohydrase is added thereto in enzyme
It is saccharified at temperature living, total reducing sugar and reduced sugar therein is measured during saccharification, when DE value reaches described in 80% or more end
Saccharification obtains cornstarch saccharification liquid；
(v) it adds in Xiang Suoshu cornstarch saccharification liquid according to pretreated corn pulp obtained by the above method and water,
Fermentation medium is obtained, the seed culture fluid is inoculated in Xiang Suoshu fermentation medium, fermentation training is carried out at 30 DEG C -40 DEG C
It supports, obtains citric acid.
The present invention pre-processes corn pulp using enzyme hydrolysis, and protein molecule therein is degraded to polypeptide and is dissociated
Amino acid, other than making microorganism be easier to utilize and improve citric acid yield, and due to reducing in fermentation process
Foam volume, dosage (the case where relative to using unpretreated corn pulp of the defoaming agent in fermentation process can also be saved
For, the amount of defoaming agent reduces 15wt% or more), so as to reduce cost；And dissolved oxygen is also added, is thus promoted thin
Intracellular growth improves acid production rate；Shorten fermentation period；Increase fermentation tank capacity, improves per unit area yield.
Specific embodiment
In order to absolutely prove the present invention, some illustrative embodiments are listed herein, but the present invention is not by these
The limitation of embodiment.In the case where not influencing present subject matter, the present invention can carry out various modifications and deformation.
Herein, unless otherwise indicated, term " room temperature " refers to 20 DEG C -35 DEG C of temperature.
Herein, unless otherwise indicated, term " DE value " refers to glucose equivalent (Dextrose Equivalent)
Value.
The pretreatment of corn pulp
Corn pulp involved in the present invention can be commercially available corn pulp；Or it can be according to routine known in the art
Knowledge is prepared, for example, being recorded in the method in following document: old Jing is compiled, " cornstarch manual industry ", light industry of China
Publishing house, 2009-09-01；Bai Kun is compiled, " cornstarch engineering technology ", China Light Industry Press, and light industry of China is published
Society is incorporated into herein by reference as reference.
In one embodiment, the present invention relates to a kind of pair of corn pulps to carry out pretreated method, wherein the method
The following steps are included:
(1) take corn pulp be added fermentor in, according to 1:(0.5-1.5) volume ratio water is added into the corn pulp,
Obtain mixed liquor；
(2) pH to 6-8 for adjusting the mixed liquor, then raises temperature to 60 DEG C -70 DEG C, is added in Xiang Suoshu mixed liquor compound
Protease simultaneously carries out enzyme digestion reaction 7-9h；
(3) after the enzyme digestion reaction, reaction system is warming up to 85 DEG C of -95 DEG C of progress enzyme deactivations, be subsequently cooled to 20 DEG C -
35 DEG C, obtain pretreated corn pulp.
In a preferred embodiment, it in above-mentioned steps (1), is added according to the volume ratio of 1:1 into the corn pulp
Water.
In a preferred embodiment, in above-mentioned steps (2), it is described mixed to adjust that ammonia is passed through in Xiang Suoshu mixed liquor
Close the pH of liquid, it is preferable that adjust the pH to 7.00 ± 0.05 of the mixed liquor.
In a preferred embodiment, in above-mentioned steps (2), 65 DEG C are warming up to.
In a preferred embodiment, in above-mentioned steps (2), the body for accounting for the mixed liquor is added in Xiang Suoshu mixed liquor
The compound protease of long-pending 1%.
In a preferred embodiment, in above-mentioned steps (2), the compound protease includes acid protease, alkalinity
One or more of protease and neutral proteinase.
In a preferred embodiment, in above-mentioned steps (2), while carrying out the enzyme digestion reaction, with 190-
The speed of 220r/min is stirred the mixed liquor.
In a preferred embodiment, in above-mentioned steps (2), the enzyme digestion reaction carries out 8h.
In a preferred embodiment, in above-mentioned steps (3), the reaction system is warming up to 90 DEG C of progress enzyme deactivations；
Preferably, the enzyme deactivation carries out 10-20min, such as 15min.
In a preferred embodiment, the various amino acid in above-mentioned steps (3), in the pretreated corn pulp
Content be calculated as with g/100g: Asp 0.939；Thr 0.523；Ser 0.579；Glu2.162；Gly 0.799；Ala
1.768；Val 0.883；Ile 0.511；Leu 1.330；Tyr 0.246；Phe 0.525；His 0.490；Lys 0.431；
Arg 0.439。
The fermenting and producing of citric acid
The cornstarch used in the present invention can be commercially available cornstarch；Or it can be according to known in the art
Conventional wisdom be prepared, for example, being recorded in the method in following document: old Jing is compiled, " cornstarch manual industry ", China
Light industry publishing house, 2009-09-01；Bai Kun is compiled, " cornstarch engineering technology ", China Light Industry Press, light industry of China
Publishing house is incorporated into herein by reference as reference.
Aspergillus niger (Aspergillus niger) is Deuteromycotina, and Hyphomycetes, hyphomycetales, Moniliaceae, aspergillus is true
A Common Species in bacterium.Aspergillus niger is important fermentation industry strain, can produce amylase, acid protease, cellulase,
Pectase, glucose oxidase, citric acid, gluconic acid and gallic acid etc..Merely for exemplary purpose, fermentation side of the invention
Commercialized aspergillus niger Co827 (Aspergillus niger Co827) bacterial strain is used in method, and (Chai Min, a kind of aspergillus niger are oblique
The optimization method of face culture medium, " Information ", 2014,12 (24): 231-232).The preparation of fungal spore suspension uses ability
The conventional means in domain carry out, and sterile water can be used or sterile saline prepares spore suspension.
In one embodiment, the present invention relates to a kind of methods for producing citric acid, wherein the method includes as follows
Step:
(i) corn starch solution that powder slurry concentration is 13%-18% (w/v) is prepared with water, by the corn starch solution
40 DEG C -70 DEG C at a temperature of be standing and soaking, then adjust its pH to 5.0-6.0, add alpha-amylase thereto, in enzyme activity temperature
It keeps the temperature and is stirred continuously under degree, obtain cornstarch liquefier；
(ii) it adds in Xiang Suoshu cornstarch liquefier according to pretreated corn pulp obtained by the above method, ammonium hydroxide
And water, obtain seed culture medium；
(iii) it will be inoculated with the aspergillus niger spore suspension of sterile saline or sterile water preparation into the seed culture medium
In, it is cultivated at 30 DEG C -40 DEG C, obtains seed culture fluid；
(iv) pH for the cornstarch liquefier that step (i) obtains is adjusted to 3.8-5.0, Xiang Suoshu cornstarch liquefier
Middle addition carbohydrase is saccharified at a temperature of enzyme activity, and total reducing sugar and reduced sugar therein are measured during saccharification, when DE value reaches
80% or more terminates the saccharification, obtains cornstarch saccharification liquid；
(v) it adds in Xiang Suoshu cornstarch saccharification liquid according to pretreated corn pulp obtained by the above method and water,
Fermentation medium is obtained, the seed culture fluid is inoculated in Xiang Suoshu fermentation medium, fermentation training is carried out at 30 DEG C -40 DEG C
It supports, obtains citric acid.
In a preferred embodiment, in above-mentioned steps (i), the corn starch solution is placed on recirculated water bath
In impregnated.
In a preferred embodiment, in above-mentioned steps (i), the corn starch solution is impregnated into 20-60min, excellent
Select 25-55min.
The arbitrary acid or alkali adjusting corn starch solution to alpha-amylase with compatibility known in the art can be used
PH.In a preferred embodiment, in above-mentioned steps (i), the pH is adjusted with calcium hydroxide.
In a preferred embodiment, in above-mentioned steps (i), according to 20Ug-1The amount of cornstarch adds the α-
Amylase.
In a preferred embodiment, in above-mentioned steps (i), 90 DEG C at a temperature of keep the temperature.
In a preferred embodiment, in above-mentioned steps (i), the stirring is carried out with the speed of 100-400rpm.
In a preferred embodiment, in above-mentioned steps (ii), the total sugar content of the seed culture medium is 10%-
14% (g/mL), total nitrogen content are 0.25% (g/mL).
In a preferred embodiment, in above-mentioned steps (iii), the concentration of the aspergillus niger spore suspension is 106A/
mL。
In a preferred embodiment, in above-mentioned steps (iii), by the aspergillus niger spore suspension with 0.2-0.8%
(v/v) ratio is inoculated in the seed culture medium.
In a preferred embodiment, in above-mentioned steps (iii), the culture is in triangular flask or mechanical stirring stainless steel
It is carried out in fermentor.In further preferred embodiment, in above-mentioned steps (iii), it is described culture mechanical stirring not
It is carried out in rust steel fermentor, also, the ventilatory capacity of the fermentor is 0.6-1.0vvm, and controls dissolved oxygen therein (DO) and exist
30%-60%.
In a preferred embodiment, in above-mentioned steps (iv), with dilute sulfuric acid by the pH of the cornstarch liquefier
It is adjusted to 3.8-5.0.
In a preferred embodiment, in above-mentioned steps (iv), 0.1%- is added in Xiang Suoshu cornstarch liquefier
The carbohydrase of 0.5% (v/v).
In a preferred embodiment, in above-mentioned steps (iv), the saccharification is carried out at 60 DEG C.
In a preferred embodiment, in above-mentioned steps (iv), while carrying out the saccharification, with 100-300rpm
Speed be stirred.
According to method disclosed in relevant national standard, the content of total reducing sugar and reduced sugar is measured.According to following formula meter
Calculate DE value: DE value=reduced sugar/total reducing sugar × 100%.
In a preferred embodiment, in above-mentioned steps (v), the total sugar content of the fermentation medium is 12%-
18% (g/mL), total nitrogen content are 0.09% (g/mL).
In a preferred embodiment, in above-mentioned steps (v), according to 5% (v/v)-in Xiang Suoshu fermentation medium
The ratio of 10% (v/v) is inoculated with the seed culture fluid.
In a preferred embodiment, in above-mentioned steps (v), the culture is sent out in triangular flask or mechanical stirring stainless steel
It is carried out in fermentation tank.In further preferred embodiment, in above-mentioned steps (v), the culture is in mechanical stirring stainless steel
It is carried out in fermentor, also, the ventilatory capacity of the fermentor is 0.6-0.8vvm, and controls dissolved oxygen therein (DO) and exist
40%-70%.Total volume in further preferred embodiment, during the fermented and cultured, relative to fermentation system
For, the defoaming agent of 0.02%-0.034% (v/v) is added.
Some purposes of the invention or advantage can be realized by the illustrative technical solution listed in following paragraph:
1. a kind of pair of corn pulp carries out pretreated method, wherein the described method comprises the following steps:
(1) take corn pulp be added fermentor in, according to 1:(0.5-1.5) volume ratio water is added into the corn pulp,
Obtain mixed liquor；
(2) pH to 6-8 for adjusting the mixed liquor, then raises temperature to 60 DEG C -70 DEG C, is added in Xiang Suoshu mixed liquor compound
Protease simultaneously carries out enzyme digestion reaction 7-9h；
(3) after the enzyme digestion reaction, reaction system is warming up to 85 DEG C of -95 DEG C of progress enzyme deactivations, be subsequently cooled to 20 DEG C -
35 DEG C, obtain pretreated corn pulp.
2. the method as described in paragraph 1 is added according to the volume ratio of 1:1 into the corn pulp in the step (1)
Water.
3. the method as described in paragraph 1 or 2 is passed through ammonia in the step (2) to adjust in Xiang Suoshu mixed liquor
State the pH of mixed liquor, it is preferable that adjust the pH to 7.00 ± 0.05 of the mixed liquor.
4. the method as described in either segment in paragraph 1-3 is warming up to 65 DEG C in the step (2).
5. the method as described in either segment in paragraph 1-4 is added in Xiang Suoshu mixed liquor in the step (2) and accounts for institute
State the compound protease of the 1% of the volume of mixed liquor.
6. the method as described in either segment in paragraph 1-5, in the step (2), the compound protease includes acidity
One or more of protease, alkali protease and neutral proteinase.
7. the method as described in either segment in paragraph 1-6 is carrying out the same of the enzyme digestion reaction in the step (2)
When, the mixed liquor is stirred with the speed of 190-220r/min.
8. the method as described in either segment in paragraph 1-7, in the step (2), the enzyme digestion reaction carries out 8h.
9. the reaction system is warming up to 90 in the step (3) by the method as described in either segment in paragraph 1-8
DEG C carry out enzyme deactivation；Preferably, the enzyme deactivation carries out 10-20min, such as 15min.
10. the method as described in either segment in paragraph 1-9, in the step (3), in the pretreated corn pulp
The contents of various amino acid be calculated as with g/100g: Asp 0.939；Thr0.523；Ser 0.579；Glu2.162；Gly
0.799；Ala 1.768；Val 0.883；Ile 0.511；Leu 1.330；Tyr 0.246；Phe 0.525；His 0.490；
Lys 0.431；Arg 0.439.
11. a kind of method for producing citric acid, wherein described method includes following steps:
(i) corn starch solution that powder slurry concentration is 13%-18% (w/v) is prepared with water, by the corn starch solution
40 DEG C -70 DEG C at a temperature of be standing and soaking, then adjust its pH to 5.0-6.0, add alpha-amylase thereto, in enzyme activity temperature
It keeps the temperature and is stirred continuously under degree, obtain cornstarch liquefier；
(ii) method according to claim 1 to 10 is added in Xiang Suoshu cornstarch liquefier to obtain
Pretreated corn pulp, ammonium hydroxide and water, obtain seed culture medium；
(iii) it will be inoculated with the aspergillus niger spore suspension of sterile saline or sterile water preparation into the seed culture medium
In, it is cultivated at 30 DEG C -40 DEG C, obtains seed culture fluid；
(iv) pH for the cornstarch liquefier that step (i) obtains is adjusted to 3.8-5.0, Xiang Suoshu cornstarch liquefier
Middle addition carbohydrase is saccharified at a temperature of enzyme activity, and total reducing sugar and reduced sugar therein are measured during saccharification, when DE value reaches
80% or more terminates the saccharification, obtains cornstarch saccharification liquid；
(v) it adds in Xiang Suoshu cornstarch saccharification liquid according to pretreated corn pulp obtained by the above method and water,
Fermentation medium is obtained, the seed culture fluid is inoculated in Xiang Suoshu fermentation medium, fermentation training is carried out at 30 DEG C -40 DEG C
It supports, obtains citric acid.
12. the corn starch solution is placed on recirculated water in the step (i) by the method as described in paragraph 11
It is impregnated in bath.
13. the corn starch solution is placed on and follows in the step (i) by the method as described in paragraph 11 or 12
It is impregnated in ring water bath.
14. the method as described in either segment in paragraph 11-13 soaks the corn starch solution in the step (i)
Steep 20-60min, preferably 25-55min.
15. the method as described in either segment in paragraph 11-14, in the step (i), adjusted with calcium hydroxide described in
pH。
16. the method as described in either segment in paragraph 11-15, in the step (i), according to 20Ug-1Cornstarch
Amount add the alpha-amylase.
17. the method as described in either segment in paragraph 11-16, in the step (i), 90 DEG C at a temperature of keep the temperature.
18. the method as described in either segment in paragraph 11-17, in the step (i), with the speed of 100-400rpm into
The row stirring.
19. the method as described in either segment in paragraph 11-18, in the step (ii), the seed culture medium it is total
Sugared content is 10%-14% (g/mL), and total nitrogen content is 0.25% (g/mL).
20. the method as described in either segment in paragraph 11-19, in the step (iii), the aspergillus niger spore suspension
Concentration be 106A/mL.
21. the method as described in either segment in paragraph 11-20 hangs the aspergillus niger spore in the step (iii)
Liquid is inoculated in the seed culture medium with the ratio of 0.2-0.8% (v/v).
22. the method as described in either segment in paragraph 11-21, in the step (iii), the culture in triangular flask or
It is carried out in mechanical stirring stainless steel fermentation tank.
23. the method as described in paragraph 22, in the step (iii), the culture is fermented in mechanical stirring stainless steel
It is carried out in tank, also, the ventilatory capacity of the fermentor is 0.6-1.0vvm, and controls dissolved oxygen therein (DO) in 30%-
60%.
24. the method as described in either segment in paragraph 11-23, in the step (iv), with dilute sulfuric acid by the corn
The pH of liquefied starch is adjusted to 3.8-5.0.
25. the method as described in either segment in paragraph 11-24, in the step (iv), the liquefaction of Xiang Suoshu cornstarch
The carbohydrase of 0.1%-0.5% (v/v) is added in liquid.
26. the method as described in either segment in paragraph 11-25 carries out the sugar in the step (iv) at 60 DEG C
Change.
27. the method as described in either segment in paragraph 11-26 is carrying out the same of the saccharification in the step (iv)
When, it is stirred with the speed of 100-300rpm.
28. the method as described in either segment in paragraph 11-27, in the step (v), the total reducing sugar of the fermentation medium
Content is 12%-18% (g/mL), and total nitrogen content is 0.09% (g/mL).
29. the method as described in either segment in paragraph 11-28 is pressed in Xiang Suoshu fermentation medium in the step (v)
The seed culture fluid is inoculated with according to 5% (v/v) -10% (v/v) ratio.
30. the method as described in either segment in paragraph 11-29, in the step (v), the culture is in triangular flask or machine
It is carried out in tool stirring stainless steel fermentation tank.
31. the method as described in paragraph 30, in the step (v), the culture is in mechanical stirring stainless steel fermentation tank
Middle progress, also, the ventilatory capacity of the fermentor is 0.6-0.8vvm, and controls dissolved oxygen therein (DO) in 40%-70%.
32. the method as described in paragraph 31, in the step (v), during the fermented and cultured, relative to fermentation body
For the total volume of system, the defoaming agent of 0.02%-0.034% (v/v) is added.
Embodiment
Examples illustrate the present invention by following, it will be appreciated by those skilled in the art that of the invention
Protection scope be not limited only to this.
Unless otherwise indicated, following preparation examples, the Aspergillus niger strain that uses in embodiment and comparative example is aspergillus nigers
Co827.Moreover, unless otherwise indicated, hereafter involved in each culture medium be known in the art conventional medium, it is commercially available
Obtain or can by those skilled in the art according to the ordinary technical knowledge of this field (for example, Chen Tianshou is edited, " microorganism training
Support the manufacture and application of base ", Chinese agriculture publishing house, June nineteen ninety-five the 1st edition；Horse finds pleasure in good equal chief editor, and " microbiological culture media is real
With handbook ", Jilin science tech publishing house, March the 1st edition in 2006, but not limited to this) prepared.
Unless otherwise indicated, the content of the citric acid in following embodiment and comparative examples measures by the following method: Fehling
Reagent method.Conversion ratio by calculating as follows: citric acid concentration (g/L)/initial total sugar concentration (g/L).
Preparation example 1: the preparation of cornstarch liquefier
The corn starch solution that powder slurry concentration is 13% (w/v) is prepared with water, which is impregnated in recirculated water bath
(soaking temperature is 40 DEG C, soaking time 55min)；Then, pH to 5.0 is adjusted with calcium hydroxide, adds alpha-amylase
(20U·g-1Cornstarch believes Chinese biological Technology Co., Ltd. purchased from Novi), it keeps the temperature and is stirred continuously at 90 DEG C
(100rpm) hydrolyzes 60min, compensates the water evaporated in liquefaction process, obtain cornstarch liquefier.
Preparation example 2: the preparation of cornstarch liquefier
The corn starch solution that powder slurry concentration is 18% (w/v) is prepared with water, which is impregnated in recirculated water bath
(soaking temperature is 68 DEG C, soaking time 25min)；Then, pH to 6.0 is adjusted with calcium hydroxide, adds alpha-amylase
(20U·g-1Cornstarch believes Chinese biological Technology Co., Ltd. purchased from Novi), it keeps the temperature and is stirred continuously at 90 DEG C
(400rpm) hydrolyzes 50min, compensates the water evaporated in liquefaction process, obtain cornstarch liquefier.
Preparation example 3: the preparation of cornstarch saccharification liquid
By the pH of the above-mentioned cornstarch liquefier of 1L with dilute sulfuric acid be adjusted to 3.8 thereto be added 5ml carbohydrase (be purchased from
Novi believes Chinese biological Technology Co., Ltd.) it is saccharified at 60 DEG C, 100rpm stirring after 12 hours, takes solution to measure it
In total reducing sugar and content of reducing sugar, can terminate to be saccharified when DE value reaches 80% or more, obtain cornstarch saccharification liquid.
Preparation example 4: the preparation of cornstarch saccharification liquid
The pH of the above-mentioned cornstarch liquefier of 4L is adjusted to 5.0 with dilute sulfuric acid, (the purchase of 5ml carbohydrase is added thereto
Chinese biological Technology Co., Ltd. is believed from Novi) it is saccharified at 60 DEG C, 300rpm stirring after 6 hours, takes solution to measure it
In total reducing sugar and content of reducing sugar, can terminate to be saccharified when DE value reaches 80% or more, obtain cornstarch saccharification liquid.
Preparation example 5: the preparation of aspergillus niger spore suspension
It is taken the aspergillus niger Co827 bacterial strain of preservation at 4 DEG C with aseptic inoculation ring and is inoculated on PDA solid medium and activated,
It is placed in 35 DEG C of constant incubator culture 7d, fine and close spore is covered on the surface of culture medium；Then a ring spore is taken with oese
Son is seeded on the wheat bran culture medium in eggplant bottle, and eggplant bottle is placed in 35 DEG C of constant temperature to media surface is filled by reciprocal scribing line
Incubator culture 7d, eggplant bottle surface are covered with fine and close spore.
The spore of eggplant bottle surface is washed down completely with 50mL sterile saline, pours into the triangular flask containing bead
In, it sufficiently vibrates, disperses spore, obtain spore suspension mother liquor.1mL spore suspension mother liquor is taken to carry out using sterile saline
10 times of dilutions, and counted using blood counting chamber, so that it is about 10 that concentration, which is made,6The aspergillus niger spore suspension of a/mL.
Preparation example 6: the preparation of pretreated corn pulp
1L corn pulp is taken to be added in 5L mechanical agitating fermentation tank, by water: tap water is added in the volume ratio 1: 1 of corn pulp, obtains
To mixed liquor, it is passed through the pH to 7.00 ± 0.05 that ammonia adjusts mixed liquor thereto, is warming up to 65 DEG C, adds in Xiang Suoshu mixed liquor
Enter to account for 1% compound protease (believing purchased from Novi) of the volume of mixed liquor, mixing speed 200r/min after reacting 8h, rises
Temperature maintains 15min enzyme deactivation, then cools to room temperature, obtain pretreated corn pulp to 90 DEG C.
By quality safety inspection center, Cofco Nutrition And Health Research Institute Co., Ltd. by using efficient liquid-phase chromatography method
The amino acid composition in corn pulp is measured, the content of the various amino acid in above-mentioned pretreated corn pulp is (with g/
100g meter): Asp 0.939；Thr 0.523；Ser 0.579；Glu2.162；Gly 0.799；Ala 1.768；Val 0.883；
Ile 0.511；Leu 1.330；Tyr 0.246；Phe 0.525；His 0.490；Lys 0.431；Arg 0.439.
Embodiment 1: citric acid is produced using pretreated corn pulp in shaking flask
Seed culture medium (initial total sugar concentration is controlled in 120g/L, total nitrogen content 2.5g/L) is prepared using following: beautiful
Rice starch liquefier (total sugar concentration of 200g/L), the pretreated corn pulp of 30g/L, 10g/L ammonium hydroxide, surplus water match the production of hybrid seeds
Sub- culture medium.
Fermentation medium (initial total sugar concentration is controlled in 160g/L, total nitrogen content 0.9g/L) is prepared using following: beautiful
The water of rice starch saccharified liquid (total sugar concentration of 200g/L), the pretreated corn pulp of 20g/L and surplus prepares fermented and cultured
Base.
50mL seed culture medium is added into the triangular flask of 250mL, the above-mentioned aspergillus niger of 0.2mL is then added thereto
Spore suspension, cultivation temperature are 36 DEG C, revolving speed 400rpm, and culture obtains seed culture fluid for 24 hours.
The above-mentioned fermentation medium of 50mL is added into 250mL triangular flask, is then up to the seed culture of logarithmic growth phase
Liquid is inoculated in above-mentioned fermentation medium by the inoculum concentration of 10% (v/v), is fermented at 37 DEG C of temperature and the revolving speed of 300rpm
84h。
Fermentation liquid is taken to be measured, as a result, it has been found that, lemon acid yield is 124g/L, and corn pulp conversion ratio is 95.38%.
Comparative example 1: citric acid is produced using unpretreated corn pulp in shaking flask
Seed culture medium (initial total sugar concentration is controlled in 120g/L, total nitrogen content 2.5g/L) is prepared using following): it will
The water preparation of cornstarch liquefier (total sugar concentration of 200g/L), the unpretreated corn pulp of 30g/L, 10g/L ammonium hydroxide, surplus
Seed culture medium.
Fermentation medium (initial total sugar concentration is controlled in 160g/L, total nitrogen content 0.9g/L) is prepared using following: will
The water of cornstarch saccharification liquid (total sugar concentration of 200g/L), the unpretreated corn pulp of 20g/L and surplus is sent out according to preparation
Ferment culture medium.
50mL seed culture medium is added into the triangular flask of 250mL, the above-mentioned aspergillus niger of 0.2mL is then added thereto
Spore suspension, cultivation temperature are 36 DEG C, revolving speed 400rpm, and culture obtains seed culture fluid for 24 hours.
The above-mentioned fermentation medium of 50mL is added into 250mL triangular flask, is then up to the seed culture of logarithmic growth phase
Liquid is inoculated in above-mentioned fermentation medium by the inoculum concentration of 10% (v/v), is fermented at 37 DEG C of temperature and the revolving speed of 300rpm
84h。
Fermentation liquid is taken to be measured, as a result, it has been found that, lemon acid yield is 105g/L, and corn pulp conversion ratio is 85.46%.
Citric acid is produced using pretreated corn pulp in embodiment 2:50L mechanical agitating fermentation tank
Seed culture medium (initial total sugar concentration is controlled in 120g/L, total nitrogen content 2.5g/L) is prepared using following: will
Cornstarch liquefier (200g/L total sugar concentration), the pretreated corn pulp of 30g/L, 10g/L ammonium hydroxide, surplus water match the production of hybrid seeds
Sub- culture medium.
Fermentation medium (initial total sugar concentration is controlled in 180g/L, total nitrogen content 0.9g/L) is prepared using following: will
The water of cornstarch saccharification liquid (200g/L total sugar concentration), the pretreated corn pulp of 20g/L and surplus prepares fermented and cultured
Base.
Above-mentioned aspergillus niger spore suspension is inoculated into seed culture medium with the ratio of 0.8% (v/v), in 10L machinery
It is cultivated in stirring stainless steel fermentation tank (protecting emerging 10JS), wherein the liquid amount of seed culture medium is 7L, cultivation temperature 36
DEG C, revolving speed 450rpm, ventilatory capacity 1.0vvm, tank pressure are 0.06Mpa, and revolving speed and dissolved oxygen (DO) value are coupled, DO controlled
40%.Culture obtains seed culture fluid for 24 hours.
The seed culture fluid is inoculated with according to the inoculum concentration of 10% (v/v) and (is protected into 50L mechanical stirring stainless steel fermentation tank
Emerging 50JS) in, the amount of the fermentation medium in the fermentor is 35L, and defoaming agent (900 polyethers GPE) additive amount is 0.034%,
Revolving speed is 350rpm, and ventilatory capacity 0.6vvm, tank pressure is 0.06Mpa, and temperature is 37 DEG C, incubation time 68h, revolving speed and dissolution
The coupling of oxygen (DO) value, DO is controlled 50%.
Fermentation liquid is taken to be measured, as a result, it has been found that, lemon acid yield 170g/L, corn pulp conversion ratio is 94.4%, defoaming
Agent dosage is 0.034%.
Citric acid is produced using unpretreated corn pulp in comparative example 2:50L mechanical agitating fermentation tank
Seed culture medium (initial total sugar concentration is controlled in 120g/L, total nitrogen content 2.5g/L) is prepared using following: beautiful
Rice starch liquefier (200g/L total sugar concentration), the unpretreated corn pulp of 30g/L, 10g/L ammonium hydroxide.
Fermentation medium (initial total sugar concentration is controlled in 180g/L, total nitrogen content 0.9g/L) is prepared using following: beautiful
The water of rice starch saccharified liquid (200g/L total sugar concentration), the untreated corn pulp of 20%g/L and surplus prepares fermentation medium.
Above-mentioned aspergillus niger spore suspension is inoculated into seed culture medium with the ratio of 0.8% (v/v), in 10L machinery
It is cultivated in stirring stainless steel fermentation tank (protecting emerging 10JS), wherein the liquid amount of seed culture medium is 7L, cultivation temperature 36
DEG C, revolving speed 450rpm, ventilatory capacity 1.0vvm, tank pressure are 0.06Mpa, and revolving speed and dissolved oxygen (DO) value are coupled, DO controlled
40%.Culture obtains seed culture fluid for 24 hours.
The seed culture fluid is inoculated with according to the inoculum concentration of 10% (v/v) and (is protected into 50L mechanical stirring stainless steel fermentation tank
Emerging 50JS) in, the liquid amount of the fermentation medium in the fermentor is 35L, and defoaming agent (900 polyethers GPE) additive amount is
0.04%, revolving speed 350rpm, ventilatory capacity 0.6vvm, temperature are 37 DEG C, incubation time 68h, revolving speed and dissolved oxygen (DO)
Value coupling, DO is controlled 50%.
Fermentation liquid is taken to be measured, as a result, it has been found that, lemon acid yield is 158g/L, and corn pulp conversion ratio is 87.77%, is disappeared
Infusion dosage is 0.04%.
Citric acid is produced using pretreated corn pulp in embodiment 3:50L mechanical agitating fermentation tank
Seed culture medium (initial total sugar concentration is controlled in 120g/L, total nitrogen content 2.5g/L) is prepared using following: will
Cornstarch liquefier (200g/L total sugar concentration), the pretreated corn pulp of 30g/L, 10g/L ammonium hydroxide, surplus water match the production of hybrid seeds
Sub- culture medium.
Fermentation medium (initial total sugar concentration is controlled in 180g/L, total nitrogen content 0.9g/L) is prepared using following: will
The water of cornstarch saccharification liquid (200g/L total sugar concentration), the pretreated corn pulp of 20g/L and surplus prepares fermented and cultured
Base.
Above-mentioned aspergillus niger spore is hanged and is inoculated into seed culture medium with the ratio of 0.2% (v/v), is stirred in 10L machinery
It mixes and is cultivated in stainless steel fermentation tank (protecting emerging 10JS), wherein the liquid amount of seed culture medium is 7L, cultivation temperature 36
DEG C, revolving speed 450rpm, ventilatory capacity 0.6vvm, tank pressure are 0.08Mpa, and revolving speed and dissolved oxygen value are coupled, and DO control is existed
30%, culture obtains seed culture fluid for 24 hours.
The seed culture fluid is inoculated with according to the inoculum concentration of 5% (v/v) and (is protected into 50L mechanical stirring stainless steel fermentation tank
Emerging 50JS) in, the liquid amount of the fermentation medium in the fermentor is 40L, and defoaming agent (900 polyethers GPE) additive amount is
0.03% (v/v), revolving speed 350rpm, ventilatory capacity 0.8vvm, tank pressure are 0.08Mpa, 37 DEG C of temperature, revolving speed and dissolved oxygen value
Coupling, by DO control 40%, incubation time 68h.
Fermentation liquid is taken to be measured, as a result, it has been found that, lemon acid yield is 173g/L, and corn pulp conversion ratio is 96.11%, is disappeared
Infusion dosage is 0.03%.
Citric acid is produced using pretreated corn pulp in embodiment 4:500L mechanical agitating fermentation tank
Seed culture medium (initial total sugar concentration is controlled in 120g/L, total nitrogen content 2.5g/L) is prepared using following: will
Cornstarch liquefier (200g/L total sugar concentration), the pretreated corn pulp of 30g/L, 10g/L ammonium hydroxide, surplus water match the production of hybrid seeds
Sub- culture medium
Fermentation medium (initial total sugar concentration is controlled in 180g/L, total nitrogen content 0.9g/L) is prepared using following: will
The water of cornstarch saccharification liquid (200g/L total sugar concentration), the pretreated corn pulp of 20g/L and surplus prepares fermented and cultured
Base.
Above-mentioned aspergillus niger spore suspension is inoculated into seed culture medium with the ratio of 0.6% (v/v), in 50L machinery
It is cultivated in stirring stainless steel fermentation tank (protecting emerging 50JS), wherein the liquid amount of seed culture medium is 35L, and cultivation temperature is
36 DEG C, revolving speed 350rpm, ventilatory capacity 0.8vvm, tank pressure is 0.08Mpa, and revolving speed and dissolved oxygen value are coupled, and DO control is existed
60%, culture obtains seed culture fluid for 24 hours.
The seed culture fluid is inoculated in 500L mechanical agitating fermentation tank according to the inoculum concentration of 8% (v/v), the fermentation
The liquid amount of fermentation medium in tank is 400L, and defoaming agent (900 polyethers GPE) additive amount is 0.02% (v/v), and revolving speed is
200rpm, ventilatory capacity 0.8vvm, 37 DEG C of temperature, tank pressure is 0.08Mpa, and revolving speed and dissolved oxygen value are coupled, and DO control is existed
70%, cultivate 68h.
Fermentation liquid is taken to be measured, as a result, it has been found that, lemon acid yield is 176g/L, and corn pulp conversion ratio is 97.77%, is disappeared
Infusion dosage is 0.02%.
Claims (10)
1. a kind of pair of corn pulp carries out pretreated method, wherein the described method comprises the following steps:
(1) take corn pulp be added fermentor in, according to 1:(0.5-1.5) volume ratio water is added into the corn pulp, obtain
Mixed liquor；
(2) pH to 6-8 for adjusting the mixed liquor, then raises temperature to 60 DEG C -70 DEG C, compound protein is added in Xiang Suoshu mixed liquor
Enzyme simultaneously carries out enzyme digestion reaction 7-9h；
(3) after the enzyme digestion reaction, reaction system is warming up to 85 DEG C of -95 DEG C of progress enzyme deactivations, is subsequently cooled to 20 DEG C -35
DEG C, obtain pretreated corn pulp.
2. the method as described in claim 1 is added according to the volume ratio of 1:1 into the corn pulp in the step (1)
Water.
3. it is method according to claim 1 or 2, in the step (2), ammonia is passed through in Xiang Suoshu mixed liquor to adjust
State the pH of mixed liquor, it is preferable that adjust the pH to 7.00 ± 0.05 of the mixed liquor；
Preferably, in the step (2), 65 DEG C are warming up to；Preferably, in the step (2), add in Xiang Suoshu mixed liquor
Enter the compound protease of the 1% of the volume for accounting for the mixed liquor；
Preferably, in the step (2), the compound protease includes acid protease, alkali protease and neutral protein
One or more of enzyme；
Preferably, in the step (2), while carrying out the enzyme digestion reaction, with the speed of 190-220r/min to institute
Mixed liquor is stated to be stirred；
Preferably, in the step (2), the enzyme digestion reaction carries out 8h.
4. the reaction system is warming up to 90 in the step (3) by method as claimed in any one of claims 1-3
DEG C carry out enzyme deactivation；Preferably, the enzyme deactivation carries out 10-20min, such as 15min；
Preferably, in the step (3), the content of the various amino acid in the pretreated corn pulp is in terms of g/100g
Are as follows: Asp 0.939；Thr 0.523；Ser 0.579；Glu2.162；Gly 0.799；Ala 1.768；Val 0.883；Ile
0.511；Leu 1.330；Tyr 0.246；Phe 0.525；His 0.490；Lys 0.431；Arg 0.439.
5. a kind of method for producing citric acid, wherein described method includes following steps:
(i) corn starch solution that powder slurry concentration is 13%-18% (w/v) is prepared with water, by the corn starch solution 40
It is standing and soaking at a temperature of DEG C -70 DEG C, then adjusts its pH to 5.0-6.0, add alpha-amylase thereto, at a temperature of enzyme activity
It keeps the temperature and is stirred continuously, obtain cornstarch liquefier；
(ii) it is pre- that the warp that method according to any of claims 1-4 obtains is added in Xiang Suoshu cornstarch liquefier
Corn pulp, ammonium hydroxide and the water of processing, obtain seed culture medium；
(iii) it will be inoculated in the seed culture medium with the aspergillus niger spore suspension of sterile saline or sterile water preparation,
It is cultivated at 30 DEG C -40 DEG C, obtains seed culture fluid；
(iv) pH for the cornstarch liquefier that step (i) obtains is adjusted to 3.8-5.0, added in Xiang Suoshu cornstarch liquefier
Enter carbohydrase to be saccharified at a temperature of enzyme activity, total reducing sugar and reduced sugar therein are measured during saccharification, when DE value reaches 80%
Terminate the saccharification above, obtains cornstarch saccharification liquid；
(v) addition is obtained according to pretreated corn pulp obtained by the above method and water in Xiang Suoshu cornstarch saccharification liquid
Fermentation medium is inoculated with the seed culture fluid in Xiang Suoshu fermentation medium, fermented and cultured is carried out at 30 DEG C -40 DEG C, is obtained
To citric acid.
6. the corn starch solution is placed on circulator bath in the step (i) by method as claimed in claim 5
It is impregnated in slot；
Preferably, in the step (i), the corn starch solution is placed in recirculated water bath and is impregnated；
Preferably, in the step (i), the corn starch solution is impregnated into 20-60min, preferably 25-55min；
Preferably, in the step (i), the pH is adjusted with calcium hydroxide；
Preferably, in the step (i), according to 20Ug-1The amount of cornstarch adds the alpha-amylase；
Preferably, in the step (i), 90 DEG C at a temperature of keep the temperature；
Preferably, in the step (i), the stirring is carried out with the speed of 100-400rpm.
7. in the step (ii), the total sugar content of the seed culture medium is such as method described in claim 5 or 6
10%-14% (g/mL), total nitrogen content are 0.25% (g/mL).
8. the method as described in any one of claim 5-7, in the step (iii), the aspergillus niger spore suspension
Concentration is 106A/mL；
Preferably, in the step (iii), by the aspergillus niger spore suspension with the ratio of 0.2-0.8% (v/v) be inoculated with into
In the seed culture medium；
Preferably, in the step (iii), the culture carries out in triangular flask or mechanical stirring stainless steel fermentation tank；
Preferably, in the step (iii), the culture carries out in mechanical stirring stainless steel fermentation tank, also, the hair
The ventilatory capacity of fermentation tank is 0.6-1.0vvm, and controls dissolved oxygen therein (DO) in 30%-60%.
9. the method as described in any one of claim 5-8, in the step (iv), with dilute sulfuric acid by the cornstarch
The pH of liquefier is adjusted to 3.8-5.0；
Preferably, in the step (iv), the saccharification of 0.1%-0.5% (v/v) is added in Xiang Suoshu cornstarch liquefier
Enzyme；
Preferably, in the step (iv), the saccharification is carried out at 60 DEG C；
Preferably, it in the step (iv), while carrying out the saccharification, is stirred with the speed of 100-300rpm.
10. the method as described in any one of claim 5-9, in the step (v), the total reducing sugar of the fermentation medium contains
Amount is 12%-18% (g/mL), and total nitrogen content is 0.09% (g/mL)；
Preferably, it in the step (v), is inoculated in Xiang Suoshu fermentation medium according to 5% (v/v) -10% (v/v) ratio
The seed culture fluid；
Preferably, in the step (v), the culture carries out in triangular flask or mechanical stirring stainless steel fermentation tank；
Preferably, in the step (v), the culture carries out in mechanical stirring stainless steel fermentation tank, also, the fermentation
The ventilatory capacity of tank is 0.6-0.8vvm, and controls dissolved oxygen therein (DO) in 40%-70%；
Preferably, it in the step (v), during the fermented and cultured, for the total volume of fermentation system, is added
The defoaming agent of 0.02%-0.034% (v/v).
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102443611A (en) * | 2010-10-13 | 2012-05-09 | 中粮生物化学（安徽）股份有限公司 | Production method of citric acid |
| CN102517345A (en) * | 2011-12-15 | 2012-06-27 | 中粮生物化学（安徽）股份有限公司 | Citric acid fermented stock solution and preparation method for citric acid and starch sugar |
| CN103642857A (en) * | 2013-09-02 | 2014-03-19 | 福建贝辰生物科技有限公司 | Production method for microbial lipid fermentation by utilization of corn steep liquor enzymatic hydrolysate |
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2018
- 2018-12-29 CN CN201811634360.1A patent/CN109536541A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102443611A (en) * | 2010-10-13 | 2012-05-09 | 中粮生物化学（安徽）股份有限公司 | Production method of citric acid |
| CN102517345A (en) * | 2011-12-15 | 2012-06-27 | 中粮生物化学（安徽）股份有限公司 | Citric acid fermented stock solution and preparation method for citric acid and starch sugar |
| CN103642857A (en) * | 2013-09-02 | 2014-03-19 | 福建贝辰生物科技有限公司 | Production method for microbial lipid fermentation by utilization of corn steep liquor enzymatic hydrolysate |
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| 李黔蜀等: "玉米浆作为氮源对透明质酸发酵的影响", 《杨凌职业技术学院学报》 * |
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