CN108220349A - For efficient co-production of citric acid and Glucosamine fermentation medium and use its fermentation process - Google Patents
For efficient co-production of citric acid and Glucosamine fermentation medium and use its fermentation process Download PDFInfo
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Abstract
Fermentation medium and the fermentation process using the fermentation medium used by the present invention relates to fermentation of Aspergillus niger co-production of citric acid and Glucosamine.Specifically, the fermentation medium includes the water of the nutritional ingredients such as carbon source, nitrogen source and phosphate and surplus, wherein, the content of the carbon source is calculated as 7wt% 20wt% with total reducing sugar therein, the content of the nitrogen source is calculated as 0.04wt% 0.3wt% with nitrogen therein, and the phosphatic content is calculated as 0.05wt% 0.2wt% with phosphate radical therein.After the aspergillus niger for being in exponential phase is inoculated in the fermentation medium, by fermented and cultured, efficiently coproduction citric acid and Glucosamine can be obtained.
Description
Technical field
The invention belongs to biological fermentation field, specifically, the present invention relates to the use of aspergillus niger carries out fermentation coproduction lemon
Fermentation medium and the fermentation process using the fermentation medium used by acid and Glucosamine.
Background technology
Citric acid, the entitled 2- hydroxy propanes -1,2 of chemistry, 3- tricarboxylic acids, be output maximum in current world wide food
With organic acid and the organic acid of consumption maximum in the world, it is widely used in the industries such as daily-use chemical industry, food, medicine.In
State is the country of current lemon acid yield maximum.The method of majority production citric acid is first by starchy material (such as corn, rice
With beans etc.) fermentation medium is made, carry out submerged fermentation followed by aspergillus niger (Aspergillus Niger).Fermentation knot
Separating thallus and fermented supernatant fluid after beam by fermented supernatant fluid for extracting citric acid, and are used for by the bacteria residue that thalline is formed
Feed, fertilizer material are processed further.
Aspergillus niger is important industrial strain, is widely used in fermentation production of organic acid, enzyme preparation, flavouring and feed etc.
Product.It, can also be to fermenting in addition to fermenting and producing obtains purpose product due to containing chitin in black-koji mould filament cell wall
The aspergillus niger accumulated in journey is used in itself.Chitin is respectively provided with extensively in health care, agricultural breeding, industry manufacture field
Purposes.The chitosan that chitin is formed after chemical method or bioanalysis removing acetyl group, and through further hydrolyzing to form shell widow
Sugar and glucosamine monomer, are beneficial nutraceutical product, and can be used as medical treatment, aquaculture, manufacturing heavy
Want raw material or additive.
Glucosamine currently on the market is mostly what is obtained by being processed to shellfish shell,
There are the problems such as raw material sources are unstable, quality fluctuation is big, easy sensitization for it.And microbe-derived Glucosamine is developed recently
New development trend is become.Particularly, if it is possible to by the by-product aspergillus niger bacteria residue of citric acid fermentation (herein also referred to as
For " citric acid fermentation bacteria residue ", " fermentation bacteria residue ", " citric acid waste residues " or " waste residue " etc.) as raw material extraction acquisition aminoglucose
Sugar, it will while realize significant economic benefit.
That reports at present obtains the technology of Glucosamine by carrying out conventional treatment to citric acid fermentation bacteria residue
Two class of chemical method and enzyme process can be divided into.Wherein, the glycosidic bond in chitin mainly is interrupted to obtain by chemical method by acid or alkali
Chitosan or chitosan oligosaccharide then remove acetyl and further degradation acquisition Glucosamine.Enzyme process is replaced using enzymatic
For all or part of reaction in chemical method, Glucosamine final product is obtained.Since enzyme process technology is still immature, scale and
Cost cannot still meet industrialized requirement, be typically chosen chemical method in industrial processes at present and bacteria residue is handled to obtain
Glucosamine product.
It is refined that the technological design of chemical method mainly includes pre-treatment, degradation, decoloration purifying.Specifically, in reality
Before applying degradation, first pass through the techniques such as desalination, soda boiling, cleaning removal of impurities and carry out pre-treatment.Then raw material is dropped using acid medium
Solution obtains product；Common acid includes hydrochloric acid, sulfuric acid and the lewis acid in polar solvent, and the acid added in is assisted to have ice
Acetic acid, oxalic acid or assisting ultrasonic processing are so that raw material fully degraded.Finally, using concentration, adsorption bleaching, membrane filtration, tie again
The operation such as crystalline substance is purified and is refined to degradation of mixture, obtains product.
For the prior art of citric acid is obtained using fermentation of Aspergillus niger,《Fed-batch fermentation production citric acid is ground
Study carefully》(Wang Youxu, Hebei University of Science and Technology's master thesis, 2013) pass through experimental study aspergillus niger citric acid batch fermentation
Biomass, specific growth rate, zymotic fluid nitrogen content, acidity, dissolved oxygen and total sugar concentration variation tendency in the process, to aspergillus niger lemon
Citric acid synthesis is analyzed with growth coupling relationship during lemon acid fermentation, and combines regulation and control zymotic fluid nitrogen content, DO values
Aspergillus niger citric acid fermentation is optimized with total sugar concentration.Its seed culture medium used for：Liquefied corn with originally
Water is according to volume ratio 1:2.5% (NH is added in 1 weight ratio mixing4)2SO4With 0.1% silicone emulsion；Fermentation medium
For：Initial nitrogen content 120mg/100mL, first total sugar concentration (16.15-20.15) g/100mL.
In addition, patent application CN102864182A discloses the side that citric acid is prepared using fermentation of Aspergillus niger cornstarch
Method, it is determined that optimal culture condition is cornstarch 20%, (NH4)2SO40.2%th, KH2PO40.2%th, MgSO4·7H2O
0.05%th, methanol 4%, initial pH 3.0.Meanwhile patent application CN104087624A discloses a kind of aspergillus niger and continuously ferments life
The method for producing citric acid, includes the following steps：(1) aspergillus niger obtains ripe spore by expanding culture step by step；(2) spore
Liquid is seeded to seed culture medium, and culture is as ripe seed liquor；(3) ripe seed liquor is forwarded to fermentation medium F1；(4)
After fermented and cultured, stuck fermentation liquid is two parts；A portion zymotic fluid continues fermentation and finishes, and obtains citric acid；(5) divide
Another part zymotic fluid gone out is dispersed through device decentralized processing, obtains the zymotic fluid of dispersion mycelia；(6) dispersion hypha fermentation liquid is connect
Enter fermentation medium F2；After fermented and cultured, step (4) is returned to, so repeats to realize and continuously ferment.Wherein, seed culture medium kind
Total reducing sugar control is in 8-12%, total nitrogen 0.15-0.4% afterwards.In 14-16%, total nitrogen is for total reducing sugar control after fermentation medium F1 kinds
0.05-0.2%.Total reducing sugar control is in 14-16%, total nitrogen 0.10-0.25% after fermentation medium F2 kinds.
For producing the prior art of Glucosamine as raw material using citric acid fermentation bacteria residue, patent application
CN102167713A (Nantong Foreign Trade Medical Health Products Co., Ltd.) is disclosed during a kind of production citric acid with fermentation method
The method that the by-product citric acid waste residues of generation produce Glucosamine for raw material, wherein, with 30-35% at 90-105 DEG C
Hydrochloric acid to raw material hydrolyze 2-2.5h, rate of charge be waste residue weight:Hydrochloric acid weight=1:1.6-2.5；Thereafter it is directly that mother liquor is dense
Crystallization is reduced to, generates aminoglucose hydrochloride crude product, and polishing obtains product.Patent application CN103319547A (is raised
Zhou Xing biotech inc) similar method is used, black-koji mould filament is separated from waste residue first,
Then it is aided with microwave treatment 10-30 minutes using the hydrochloric acid solution of 15-20%, individually hydrolysis is then 70-80 DEG C of hydrolysis 1.8-
2.3h obtains the solution containing Glucosamine, then is decolourized with activated carbon after hydrolysis, crystallizing and drying.Patent application
CN101497634A (Lianyungang Titam Bioengineering Co., Ltd.) by acid solution add in fermentation method generate contain chitin
Substance and can promote fungal cell wall destroy and chitosan chain fracture catalyst, from citric-acid fermentation waste residue produce amino
Glucose.Wherein, composition of the catalyst for organic matter and inorganic matter, preferred weight ratio 1:(0.1-10) so that urge
Agent is reacted 0.5-16 hours with mycosin under conditions of 60-110 DEG C, and the proportion that feeds intake is the 0.1-30% of raw material；
After carrying out catalysis reaction, it is filtered, concentrates, crystallisation by cooling；It carries out being recrystallized to give glucosamine product again.
Extracting the scheme of Glucosamine from citric acid fermentation bacteria residue above by chemical method, there are acidic and alkaline waste water rows
The problem of high-volume big, environmentally friendly of high cost.In existing soda acid process for producing scheme, it is also noted that the problem of spent acid post-processes.
By taking chitin acidolysis produces Glucosamine as an example, one ton of product of production probably needs to generate 5-8 tons of spent acid, wherein containing
The hydrochloric acid of 16-25% and the acetic acid of 4-5%, can not directly be recycled, and can give sewage directly as wastewater treatment
Processing system brings huge pressure.Patent CN1019930411B provides a kind of method for recycling hydrochloric acid in waste water system, leads to
Dichloromethane and the extraction that 10-12% volumes are added in acid pickle are crossed, the dichloromethane in 40-50 DEG C of evaporation subnatant obtains
Take acetic acid；And dichloromethane remaining in upper liquid is evaporated and be passed through hydrogen chloride gas so that its acid concentration reach 31% with
On, to recycle.The discharge of sour waste water can be effectively reduced using the technology, however increased processing step can be dramatically increased into
This.Simultaneously as bacteria residue ingredient and content are increasingly complex, directly the technique is diverted to utilize aspergillus niger bacteria residue production amino Portugal
The flow of grape sugar is difficult to obtain satisfactory as a result, being also unfavorable for the recycling to protein ingredient in bacteria residue.
According to existing literature data, aspergillus niger is the most abundant fungi strain of cell wall chitin content, crust therein
Element can account for the 40wt% of cell wall constituent.However, when using not optimized aspergillus niger bacteria residue as raw material to produce amino Portugal
During grape sugar, the problem of chitin content in the feed is relatively low and Glucosamine recovery rate is low is deposited.Therefore, use is not optimized
Bacteria residue raw material goods and materials unit consumption under identical process conditions and yield can be caused to increase and the problem of the increase of unit sewage quantity, it is comprehensive
It is not high to close benefit.
There are no be improved to balance lemon acid yield and black-koji mould for citric acid fermentation link in the prior art
The research of the chitin content of body, however, the inventors discovered that obtaining citric acid and aminoglucose by fermentation of Aspergillus niger
The fermenting step of sugar optimizes, it will help under the premise of the wastewater flow rate and energy expenditure for reducing post processing, while with height
Efficiency co-production of citric acid and Glucosamine.
Invention content
In order to solve existing fermentation of Aspergillus niger production citric acid and the technique for preparing Glucosamine using aspergillus niger bacteria residue
It middle waste water more (environmental protection pressure is big), the shortcomings that Glucosamine yield is low, by-product utilized rate is low and comprehensive benefit is not high, provides
It is a kind of for efficient co-production of citric acid and fermentation medium and the corresponding fermentation process of Glucosamine.
On the one hand, there is provided herein a kind of fermentation trainings for coming co-production of citric acid and Glucosamine for fermentation of Aspergillus niger
Base is supported, the fermentation medium includes carbon source, nitrogen source and phosphate and the water of surplus, wherein, the content of the carbon source is with it
In total reducing sugar be calculated as 7wt%-20wt%, the content of the nitrogen source is calculated as 0.04wt%-0.3wt% with nitrogen therein, institute
It states phosphatic content and 0.05wt%-0.2wt% is calculated as with phosphate radical therein.
On the other hand, come preparation of citric acid by fermentation and Glucosamine using above-mentioned fermentation medium there is provided herein a kind of
Method, described method includes following steps：
(1) aspergillus niger in exponential phase is inoculated in above-mentioned fermentation medium and fermented, so as to be sent out
Zymotic fluid and fermentation bacteria residue, detach the zymotic fluid and the fermentation bacteria residue；
(2) from the broth extraction citric acid；
(3) the fermentation bacteria residue is handled, so as to obtain Glucosamine.
Specifically, the exemplary embodiments of the present invention are realized by the content described in following entry：
1. a kind of fermentation medium for coming co-production of citric acid and Glucosamine for fermentation of Aspergillus niger, the fermented and cultured
Base includes carbon source, nitrogen source and phosphate and the water of surplus, wherein, the content of the carbon source is calculated as 7wt%- with total reducing sugar therein
20wt%, the content of the nitrogen source are calculated as 0.04wt%-0.3wt% with nitrogen therein, and the phosphatic content is with it
In phosphate radical be calculated as 0.05wt%-0.2wt%.
2. the fermentation medium as described in paragraph 1, wherein, the carbon source is selected from dextrin, maltose and/or starchy material
Liquefaction filtrate.
3. the fermentation medium as described in paragraph 1 or 2, wherein, the nitrogen source is inorganic nitrogen-sourced, organic nitrogen source or the two
Mixture.
4. the fermentation medium as described in paragraph 3, wherein, it is described inorganic nitrogen-sourced for inorganic ammonium salt.
5. the fermentation medium as described in paragraph 4, wherein, the inorganic ammonium salt is ammonium sulfate and/or ammonium nitrate.
6. the fermentation medium as described in paragraph 3, wherein, the organic nitrogen source is urea, the liquid of albumen, starchy material
Change filter residue or their mixture.
7. the fermentation medium as described in paragraph 6, wherein, the protein content of the liquefaction filter residue is 20w%-45wt%.
8. the fermentation medium as described in paragraph 6, wherein, the albumen comes from corn pulp.
9. the fermentation medium as described in paragraph 2 or 6, wherein, the starchy material be selected from by corn, rice,
It is one or more in the group that wheat, beans, potato and cassava are formed.
10. the fermentation medium as described in paragraph 9, wherein, the liquefaction filtrate and the liquefaction filter residue by obtaining as follows
It arrives：Liquefaction processing is carried out to the starchy material using amylase and forms liquefied solution, the liquefied solution is filtered and is distinguished
Obtain the liquefaction filtrate and the liquefaction filter residue.
11. the fermentation medium as described in paragraph 10, wherein, the liquefaction filtrate and the liquefaction filter residue are from identical
Or the different starchy material.
12. the fermentation medium as described in paragraph 10 or 11, wherein, the liquefaction before processing is being carried out, to the starch
Matter raw material carries out one or more in pre-processing as follows：Screening, is crushed and is sized mixing removal of impurities.
13. the fermentation medium as described in paragraph 12, wherein, the liquefaction before processing is being carried out, successively to the starch
Matter raw material is sieved, is cleaned, crushed and is sized mixing.
14. the fermentation medium as described in paragraph 12 or 13, wherein, the screening and removal of impurities include making the starchiness former
Material is by receiving sieve and pipe magnetic separator, to remove the impurity in the starchy material.
15. the fermentation medium as described in either segment in paragraph 12-14, wherein, it is described broken including being pressed by machinery mill
It is crushed.
16. the fermentation medium as described in either segment in paragraph 12-15, wherein, it is described to size mixing including by broken shallow lake
The starch slurry that silty starting material with water is deployed into a concentration of -19.0 Baume of 12.0 Baume and pH value is 5.8-6.2.
17. the fermentation medium as described in paragraph 16, wherein, the starch slurry is in contact with the amylase, so that
Starch Conversion in the starch slurry is dextrin and oligosaccharide.
18. the fermentation medium as described in paragraph 16 or 17, wherein, relative to every gram of starch in the starch slurry, institute
The dosage for stating amylase is -60 enzyme-activity unit of 5 enzyme-activity unit, is preferably -50 enzyme-activity unit of 10 enzyme-activity unit.
19. the fermentation medium as described in either segment in paragraph 10-18, wherein, the amylase be selected from alpha-amylase and/
Or beta amylase.
20. the fermentation medium as described in paragraph 19, wherein, using the alpha-amylase at a temperature of 80 DEG C -110 DEG C
Carry out the liquefaction processing.
21. the fermentation medium as described in either segment in paragraph 10-20, wherein, the time of the liquefaction processing is
90min-140min。
22. the fermentation medium as described in either segment in paragraph 2-21, wherein, the total sugar content in the liquefaction filtrate is
16wt%-25wt%.
23. the fermentation medium as described in either segment in paragraph 1-22, wherein, the phosphate is selected from potassium dihydrogen phosphate
And/or dipotassium hydrogen phosphate.
24. the fermentation medium as described in either segment in paragraph 1-23, wherein, the fermentation medium is included in terms of total reducing sugar
It carbon source for 16wt%-20wt%, the nitrogen source that 0.04wt%-0.15wt% is calculated as with nitrogen and is calculated as with phosphate radical
The phosphate of 0.05wt%-0.1wt%.
25. the fermentation medium in a kind of 1-24 using paragraph described in either segment comes preparation of citric acid by fermentation and aminoglucose
The method of sugar, described method includes following steps：
(1) by the aspergillus niger in exponential phase be inoculated in the fermentation medium in paragraph 1-24 described in either segment into
Row fermentation so as to obtain zymotic fluid and fermentation bacteria residue, detaches the zymotic fluid and the fermentation bacteria residue；
(2) from the broth extraction citric acid；
(3) the fermentation bacteria residue is handled, so as to obtain Glucosamine.
26. the fermentation medium as described in paragraph 25, wherein, the aspergillus niger is selected from by aspergillus niger Co827, black song
It is one or more in the group that mould T01 and aspergillus niger HN-2004 are formed.
27. the method as described in paragraph 25 or 26, wherein, in step (1), the aspergillus niger is inoculated with by oese
In the fermentation medium.
28. the method as described in either segment in paragraph 25-27, wherein, in step (1), by adding to the aspergillus niger
Enter sterile water to obtain the suspension of the aspergillus niger and add in the suspension in the fermentation medium followed by liquid-transfering device
To carry out the inoculation.
29. the method as described in either segment in paragraph 25-28, wherein, in step (1), the fermentation is using selected from deep
The fermentation method of layer liquid fermentation or batch fermentation carries out.
30. the method as described in either segment in paragraph 25-29, wherein, in step (1), before use, by the fermentation
Culture is based on the 25min that sterilizes under 121 DEG C and 0.1MPa.
31. the method as described in either segment in paragraph 25-30, wherein, in step (1), relative to fermentation every gram described
Culture medium, inoculation 1 × 104-1.5×105A Colony Forming Unit, preferably 5 × 104-1×105A Colony Forming Unit it is described
Aspergillus niger.
32. the method as described in either segment in paragraph 25-31, wherein, in step (1), the fermentation 30-40 DEG C,
It is preferred that it is carried out at 36-38 DEG C.
33. the method as described in either segment in paragraph 25-32, wherein, in step (1), the progress 40 hours of fermenting-
70 hours, preferably -65 hours 50 hours.
34. the method as described in either segment in paragraph 25-33, wherein, in step (1), by centrifuging or being separated by filtration
The zymotic fluid and the fermentation bacteria residue.
35. the method as described in paragraph 34, wherein, detach the fermentation by centrifuging 15min under the rotating speed of 8000rpm
Liquid and the fermentation bacteria residue.
The co-production of citric acid of the present invention and the fermentation process of Glucosamine by using specific fermentation medium, so as to
It solves the problems, such as to produce acid and production sugar balance during fermentation of Aspergillus niger, not only ensure that has high citric acid in zymocyte liquid
Content, while can improve the chitin content of aspergillus strain, so that when subsequent processing prepares Glucosamine
Quantity of wastewater effluent can be effectively reduced, the yield that the extraction from bacteria residue obtains Glucosamine is improved, improves the comprehensive of industry
Close benefit.Method of the present invention can realize more than 95% citric acid conversion ratio and more than 10% Glucosamine
Yield.In some preferred embodiments, method of the present invention can even realize more than 98% citric acid conversion
Rate and more than 12% Glucosamine yield.
Specific embodiment
The specific embodiment of the present invention is described in detail below.It is it should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
In the present invention, unless otherwise indicated, there are term " exponential phase " those skilled in the art to be generally understood
Meaning, also known as exponential phase of growth referred to microorganism and grown after lag phase with maximum rate and division is so that this is micro-
Biomass is in logarithm increased period.Aspergillus niger of the present invention in exponential phase can for example, by but it is unlimited
It is obtained in following manner：Aspergillus niger strain is seeded in aspergillus culture medium known in the art or the fermentation medium of the present invention
In, it is then cultivated at suitable temperature (such as 30-40 DEG C), and pass through spectrophotometer etc. and measure absorbance (OD) value come really
Determine aspergillus niger and be in exponential phase.
In the present invention, unless otherwise indicated, term " conversion ratio " refers to the lemon in zymotic fluid after fermentation
The amount (volume for being equal to concentration × zymotic fluid of the citric acid in zymotic fluid) of acid is relative to the total reducing sugar in the culture medium before fermentation
Weight for percentage.
In the present invention, unless otherwise indicated, term " yield " refers to extracting the Glucosamine obtained from bacteria residue
Relative to the weight percent of fermentation bacteria residue dry weight.
On the one hand, there is provided herein a kind of fermented and cultureds for coming co-production of citric acid and Glucosamine for fermentation of Aspergillus niger
Base.In the present invention, unless otherwise indicated, term " fermentation medium " refers to be used for coproduction lemon in the zymotechnique of aspergillus niger
The fluid nutrient medium of lemon acid and Glucosamine.The fermentation medium includes the nutritional ingredients such as nitrogen source, carbon source and inorganic salts,
In, the inorganic salts can be phosphate.Since the carbon source in fermentation medium is to provide the nutrient source of carbon, wherein, it is main
Carbon is provided by carbohydrate, therefore, for purpose definitely, be used as carbon source in fermentation medium by calculating
The percent mass content of total reducing sugar included in substance shows the carbon source content in fermentation medium, that is, will be of the present invention
Fermentation medium in carbon source content shown with the percent mass content of total reducing sugar contained in the carbon source.It is preferred that the fermentation training
It supports base and includes carbon source, nitrogen source and phosphate and the water of surplus, wherein, the content of the carbon source is calculated as with total reducing sugar therein
7wt%-20wt%, the content of the nitrogen source are calculated as 0.04wt%-0.3wt% with nitrogen therein, described phosphatic to contain
Amount is calculated as 0.05wt%-0.2wt% with phosphate radical therein.
Aspergillus niger for use in the present invention includes but not limited to aspergillus niger Co827 (purchased from Shanghai Xin Li industrial microorganisms section
Skill Co., Ltd), aspergillus niger T01 (be purchased from Tianjin industrial microorganism research institute) and aspergillus niger HN-2004 be (purchased from the micro- life of the Chinese Academy of Sciences
Object institute).
In some embodiments, the carbon source in fermentation medium can be dextrin, maltose etc..In order to further reduce life
Cost is produced, the carbon source in fermentation medium can be the liquefaction filtrate of starchy material.This field knows, utilizes amylase pair
Starchy material, which carries out liquefaction processing, can form liquefied solution, be filtrate of liquefying to the filtrate that the liquefied solution is obtained by filtration,
And the part in addition to the liquefaction filtrate is liquefaction filter residue.Sugar is mainly contained in liquefaction filtrate and is therefore used as fermentation medium
In carbon source, and mainly contain albumen in the filter residue that liquefies and be therefore used as the nitrogen source in fermentation medium.It is appreciated that
It is the liquefaction filtrate used in fermentation medium and the filter residue that liquefies can derives from identical or different starchy material.
The starchy material can be any convenient source containing much starch, such as corn, rice, wheat, beans,
Potato and cassava etc..In actual production, commercially available starch product also can be used as starchy material.
Depending on specifically used starchy material, starchy material can also be pre-processed as follows in liquefaction before processing
In it is one or more：Screening, is crushed and is sized mixing removal of impurities.For example, it can be sieved, cleaned, crushed successively in liquefaction before processing
With size mixing.Starchy material is sieved, is cleaned, is crushed, is sized mixing and the common process that liquefied technique is this field, for this
Known to field technology personnel.For example, the screening, impurity removal process may include starchy material is made to pass through receiving sieve, pipe magnetic separator
Conventional equipments are waited, to remove the stone in raw material, metal impurities.The broken method may include that grinding pressure by machinery carries out
It is broken.The method sized mixing may include by broken starchy material with water be deployed into for example a concentration of 12.0 Baume-
19.0 Baumes and the starch slurry that pH value is 5.8-6.2.
Above-mentioned liquefaction processing includes contacting the starch slurry of liquid with amylase, makes the Starch Conversion in the starch slurry be
Dextrin and oligosaccharide.In a preferred embodiment, relative to every gram of starch in starch slurry, (starch is former from starchiness
Material), the dosage of amylase can be -60 enzyme-activity unit of 5 enzyme-activity unit, preferably -50 enzyme-activity unit of 10 enzyme-activity unit.In the present invention
In, any amylase for being capable of hydrolysis starch, including but not limited to alpha-amylase, beta amylase etc. can be used.According to being used
Amylase effective temperature scope itself difference (for example, mesophilicα-diastase effective temperature scope be 35 DEG C -90 DEG C, it is high
Warm alpha-amylase effective temperature scope is 80 DEG C -110 DEG C etc.), can be used different liquefaction treatment temperatures to starchy material or
Starch slurry carries out liquefaction processing.For example, it is preferable to liquefaction processing is carried out at a temperature of 80 DEG C -110 DEG C using high-temperatureα-amylase.
In addition, the time of liquefaction processing is preferably 90min-140min.
In a preferred embodiment, as the total reducing sugar in the liquefaction filtrate of the starchy material of the carbon source of fermentation medium
Content is 16wt%-25wt%.
In some embodiments, the nitrogen source in fermentation medium can be the mixing of inorganic nitrogen-sourced or organic nitrogen source or the two
Object.It is described inorganic nitrogen-sourced for inorganic ammonium salt, such as ammonium sulfate and/or ammonium nitrate.The organic nitrogen source can be such as urea, egg
In vain, the liquefaction filter residue or their mixture of above-mentioned starchy material.There is no limit, such as from corn in the source of the albumen
Slurry.Particularly, in order to more effectively be utilized to starchy material, liquefaction filter residue is preferably used as the organic nitrogen source.It is excellent
Selection of land, the protein content of used liquefaction filter residue is 20wt%-45wt%, and the nitrogen element content in fermentation medium is at this time
The percentage composition of the nitrogen in liquefaction filter residue for the total weight of the fermentation medium.Wherein, the liquefaction filter
The content of albumen in slag is then to be multiplied by conversion coefficient 6.25 by the nitrogen content in Kjeldahl nitrogen determination sample to calculate
Crude protein content out.
In some embodiments, the phosphate in the fermentation medium can be selected from potassium dihydrogen phosphate and/or phosphoric acid
Hydrogen dipotassium, it is not limited to this.
In the present invention, in order to further improve the conversion ratio of citric acid, fermentation medium is included and is calculated as with total reducing sugar
It the carbon source of 16wt%-20wt%, the nitrogen source that 0.04wt%-0.15wt% is calculated as with nitrogen and is calculated as with phosphate radical
The phosphate of 0.05wt%-0.1wt%.Wherein, the content of the total reducing sugar refers to by specified in standard GB/T 6194-86
The content for the total Soluble Sugar that method measures, liquefaction filtrate of the total reducing sugar from starchy material.
On the other hand, come preparation of citric acid by fermentation and Glucosamine using above-mentioned fermentation medium there is provided herein a kind of
Method, described method includes following steps：
(1) aspergillus niger in exponential phase is inoculated in above-mentioned fermentation medium and fermented, so as to be sent out
Zymotic fluid and fermentation bacteria residue, detach the zymotic fluid and the fermentation bacteria residue；
(2) from the broth extraction citric acid；
(3) the fermentation bacteria residue is handled, so as to obtain Glucosamine.
In some embodiments, the aspergillus niger inoculation in step (1) and fermentation method do not require particularly, can be this
The inoculation means and fermentation method of field routine.For example, aspergillus niger is directly inoculated in fermentation by classification inoculation apparatus such as oeses
It obtains aspergillus niger suspension and adds in the suspension using liquid-transfering device to send out in culture medium or by adding in sterile water to aspergillus niger
In ferment culture medium.In addition, for fermentation method, the fermentation side of the routine such as deep fermentation or batch fermentation may be used
Formula.
In a preferred embodiment, before use, the fermented and cultured in step (1) is based under 121 DEG C and 0.1MPa
Sterilize 25min.
In a preferred embodiment, the inoculum concentration of the aspergillus niger in exponential phase is：Relative to hair every gram described
For ferment culture medium, inoculation 1 × 104-1.5×105A Colony Forming Unit, more preferable 5 × 104-1×105A bacterium colony forms list
The aspergillus niger of position.Preferably, the fermentation of step (1) 30-40 DEG C, it is 36-38 DEG C more preferable at carry out.The further preferred hair
Ferment carries out -70 hours 40 hours, preferably -65 hours 50 hours.
It can be by conventional the means separation and fermentation bacteria residue and zymotic fluid such as centrifuging, filtering.Preferably, by 8000rpm
Rotating speed under centrifugation 15min separation and fermentations bacteria residue and zymotic fluid.
The present invention is directed to be adjusted to generate the citric acid in fermentation process by the formula to fermentation medium
It is balanced with the chitin accumulation in thalline, thus optimizes the chitin in the citric acid content and thalline in zymotic fluid
Amount without citric acid extraction or Glucosamine are prepared the technique of itself and optimized, therefore, can be used known in the art
Any applicable method handled to obtain amino Portugal from broth extraction citric acid and to the fermentation bacteria residue of aspergillus niger
Grape sugar, cited method such as, but not limited in background of invention.
Other features and advantages of the present invention will be described in detail in subsequent embodiment part.
Embodiment
Below by way of a series of embodiments, the present invention is described in further detail.These embodiments are only illustrative
, and it is limiting the scope of the present invention that should not be construed.Every technical side realized based on the above of the present invention
Case and its deformation are within the scope of the present invention.
Embodiment 1
The aspergillus niger used in following preparation example, experimental example and comparative example is aspergillus niger T01, purchased from the micro- life of Tianjin industry
Object research institute.Corn seed (niblet) is commercially available conventional corn seed, and amylase is the alpha-amylase purchased from Novozymes Company
(effective temperature scope is 80 DEG C -110 DEG C).
Corn seed machinery mill crush it is broken using purchased from Jiangsu MuYang Group, Ltd. 968-3 types grinder into
Row.Filter press is purchased from the prosperous suitable chemical in Wuxi.
The liquefaction filtrate used in following preparation examples, experimental example and comparative example and the preparation method for the filter residue that liquefies are as follows：Profit
Dry corn seed is crushed with above-mentioned grinder and passes through 40 mesh mesh screens, obtains corn flour.It weighs to corn flour.
The corn flour and water are sized mixing to a concentration of 19 Baume, and its pH value is adjusted to 6.0 with 0.5% (w/w) dilute hydrochloric acid, formed sediment
Slurry.Relative to every gram of starch in starch slurry, the alpha-amylase of 25 enzyme-activity units is added in, 95 DEG C is then raised temperature to and liquefies
Processing, processing were filtered obtained solution using filter press after 140 minutes, and filtrate is filtrate of liquefying, and filter residue is filter of liquefying
Slag.
According to method specified in standard GB/T 6194-86, the total sugar content measured in the liquefaction filtrate is
25wt%.According to Kjeldahl's method, the protein content measured in the liquefaction filter residue is 40wt%.
Reference《The exploration of lemon acid-extraction and hydrogen calcium method industrial practice》(Liu Chen etc., finely and specialty chemicals,
The 1st phase of volume 23 in January, 2015) disclosed in technique, citric acid is extracted from zymotic fluid using hydrogen calcium method therein.
It is carried from fermentation bacteria residue using the chemical acid hydrolysis method disclosed in patent application CN1496408A (Cargill Inc)
Take Glucosamine.
According to the citric acid concentration obtained in the following each experimental example of GB 1987-2007 standard tests and comparative example (referred to as
Acidity), and according to the conversion ratio of equation below calculating citric acid：
The concentration × volume of zymotic fluid/of citric acid in conversion ratio (%)=zymotic fluid is in the culture medium before being fermented
Total reducing sugar weight × 100%.
HPLC methods in 2015 editions Chinese Pharmacopoeias (exposure draft) detect to be obtained in each experimental example and comparative example
Glucosamine quality, and according to equation below calculate Glucosamine yield：
Yield (%)=extract Glucosamine quality/fermentation bacteria residue dry weight × 100% for obtaining from fermentation bacteria residue.
Preparation example 1
Liquefy described in 320g filtrate, 0.43g organic nitrogen sources (urea) and 0.60g dipotassium hydrogen phosphates are mixed, then to it
Middle addition water ad pond om is 500g, thus obtains fermentation medium 1, in the fermentation medium 1, total sugar content is
16wt%, nitrogen content be 0.04wt% and the content of phosphate radical is 0.05wt%.
Preparation example 2
By filtrate of liquefying described in 400g, 2.21g mixed nitrogens (etc. weight mix urea and ammonium sulfate) and 0.72g phosphoric acid
Potassium dihydrogen mixes, and it is 500g then to add in water ad pond om thereto, fermentation medium 2 is thus obtained, in the fermentation medium 2
In, total sugar content 20wt%, nitrogen content be 0.15wt% and the content of phosphate radical is 0.1wt%.
Preparation example 3
Liquefy described in 140g filtrate, 3.07g inorganic nitrogen-sourced (ammonium sulfate) and 1.20g dipotassium hydrogen phosphates are mixed, then to
It is 500g wherein to add in water ad pond om, thus obtains fermentation medium 3, in the fermentation medium 3, total sugar content is
7wt%, nitrogen content be 0.13wt% and the content of phosphate radical is 0.1wt%.
Preparation example 4
Liquefy described in 300g filtrate, 3.21g organic nitrogen sources (urea) and 1.43g potassium dihydrogen phosphates are mixed, then to it
Middle addition water ad pond om is 500g, thus obtains fermentation medium 4, in the fermentation medium 4, total sugar content is
15wt%, nitrogen content be 0.3wt% and the content of phosphate radical is 0.2wt%.
Preparation example 5
Liquefaction filter residue and 0.96g dipotassium hydrogen phosphates described in filtrate of liquefying described in 360g, 19.53g are mixed, then thereto
It is 500g to add in water ad pond om, thus obtains fermentation medium 5, in the fermentation medium 5, total sugar content 15wt%,
The content of nitrogen is 0.25wt% and the content of phosphate radical is 0.08wt%.
Preparation example 6
Liquefaction filter residue and 1.07g potassium dihydrogen phosphates described in filtrate of liquefying described in 240g, 17.2g are mixed, then thereto
It is 500g to add in water ad pond om, thus obtains fermentation medium 6, in the fermentation medium 6, total sugar content 15wt%,
The content of nitrogen is 0.22wt% and the content of phosphate radical is 0.15wt%.
Preparation example 7
Liquefy described in 320g filtrate, 0.32g organic nitrogen sources (urea) and 0.60g dipotassium hydrogen phosphates are mixed, then to it
Middle addition water ad pond om is 500g, thus obtains fermentation medium 7, in the fermentation medium 7, total sugar content is
16wt%, nitrogen content be 0.03wt% and the content of phosphate radical is 0.05wt%.
Preparation example 8
By filtrate of liquefying described in 400g, 2.21g mixed nitrogens (etc. weight mix urea and ammonium sulfate) and 0.29g phosphoric acid
Potassium dihydrogen mixes, and it is 500g then to add in water ad pond om thereto, fermentation medium 8 is thus obtained, in the fermentation medium 8
In, total sugar content 20wt%, nitrogen content be 0.15wt% and the content of phosphate radical is 0.04wt%.
Preparation example 9
Inorganic nitrogen-sourced (ammonium sulfate) and 1.20g dipotassium hydrogen phosphates described in filtrate of liquefying described in 120g, 3.07g are mixed, with
It is 500g to add in water ad pond om thereto afterwards, thus obtains fermentation medium 9, in the fermentation medium 9, total sugar content is
6wt%, nitrogen content be 0.13wt% and the content of phosphate radical is 0.1wt%.
Preparation example 10
Organic nitrogen source (urea) and 1.43g potassium dihydrogen phosphates described in filtrate of liquefying described in 300g, 4.28g are mixed, then
It is 500g to add in water ad pond om thereto, thus obtains fermentation medium 10, in the fermentation medium 10, total sugar content is
15wt%, nitrogen content be 0.4wt% and the content of phosphate radical is 0.2wt%.
Preparation example 11
Liquefaction filter residue and 0.96g dipotassium hydrogen phosphates described in filtrate of liquefying described in 420g, 19.53g are mixed, then thereto
It is 500g to add in water ad pond om, thus obtains fermentation medium 11, in the fermentation medium 11, total sugar content is
21wt%, nitrogen content be 0.25wt% and the content of phosphate radical is 0.08wt%.
Preparation example 12
Liquefaction filter residue and 1.79g potassium dihydrogen phosphates described in filtrate of liquefying described in 240g, 17.2g are mixed, then thereto
It is 500g to add in water ad pond om, thus obtains fermentation medium 12, in the fermentation medium 12, total sugar content is
15wt%, nitrogen content be 0.22wt% and the content of phosphate radical is 0.25wt%.
Experimental example 1
The fermentation medium that preparation example 1 obtains under 121 DEG C and 0.1MPa is sterilized 25min, then naturally cools to 36
DEG C, relative to fermentation medium every gram described, by the aspergillus niger in exponential phase with 5 × 104A Colony Forming Unit
Inoculum concentration is inoculated in the fermentation medium, shaking speed 200rpm, is then cultivated 60 hours at 37 DEG C, fermentation ends,
Zymotic fluid and fermentation bacteria residue are separated by filtration by filter press.Zymotic fluid and fermentation bacteria residue is taken to be handled and detected.
Experimental example 2
The fermentation medium that preparation example 2 obtains is subjected to sterilizing 25min under 121 DEG C and 0.1MPa, then natural cooling
To 36 DEG C, relative to fermentation medium every gram described, by the aspergillus niger in exponential phase with 1 × 104A bacterium colony forms list
The inoculum concentration of position is inoculated in the fermentation medium, then shaking speed 200rpm is cultivated 70 hours at 36 DEG C, fermentation knot
Beam, by centrifuging 15min separation and fermentations bacteria residue and zymotic fluid under the rotating speed of 8000rpm.Zymotic fluid and fermentation bacteria residue is taken to carry out
Processing and detection.
Experimental example 3
The fermentation medium that preparation example 3 obtains is subjected to sterilizing 25min under 121 DEG C and 0.1MPa, then natural cooling
To 36 DEG C, relative to fermentation medium every gram described, by the aspergillus niger in exponential phase with 1.5 × 105A bacterium colony is formed
The inoculum concentration of unit is inoculated in the fermentation medium, shaking speed 200rpm, is then cultivated 40 hours at 38 DEG C, fermentation
Terminate, by centrifuging 15min separation and fermentations bacteria residue and zymotic fluid under the rotating speed of 8000rpm.Take zymotic fluid and fermentation bacteria residue into
Row processing and detection.
Experimental example 4
The fermentation medium that preparation example 4 obtains is subjected to sterilizing 25min under 121 DEG C and 0.1MPa, then natural cooling
To 36 DEG C, relative to fermentation medium every gram described, by the aspergillus niger in exponential phase with 1 × 105A bacterium colony forms list
The inoculum concentration of position is inoculated in the fermentation medium, then shaking speed 200rpm is cultivated 50 hours at 37.5 DEG C, fermentation
Terminate, zymotic fluid and fermentation bacteria residue are separated by filtration by vacuum filtration.Zymotic fluid and fermentation bacteria residue is taken to be handled and detected.It is real
Test example 5
The fermentation medium that preparation example 5 obtains is subjected to high pressure sterilization 25min under 121 DEG C and 0.1MPa, it is then natural
36 DEG C are cooled to, relative to fermentation medium every gram described, by the aspergillus niger in exponential phase with 7 × 104A bacterium colony shape
Inoculum concentration into unit is inoculated in the fermentation medium, shaking speed 200rpm, is then cultivated 65 hours at 36.5 DEG C,
Fermentation ends are separated by filtration zymotic fluid and fermentation bacteria residue by filter press.Zymotic fluid and fermentation bacteria residue is taken to be handled and detected.
Experimental example 6
The fermentation medium that preparation example 6 obtains is subjected to sterilizing 25min under 121 DEG C and 0.1MPa, then natural cooling
To 36 DEG C, relative to fermentation medium every gram described, by the aspergillus niger in exponential phase with 1.2 × 105A bacterium colony is formed
The inoculum concentration of unit is inoculated in the fermentation medium, shaking speed 200rpm, is then cultivated 55 hours at 37.8 DEG C, hair
Ferment terminates, by centrifuging 15min separation and fermentations bacteria residue and zymotic fluid under the rotating speed of 8000rpm.Take zymotic fluid and fermentation bacteria residue
It is handled and is detected.
Comparative example 1-6
In addition to the fermentation medium used in experimental example 1-6 is replaced with the fermented and cultured obtained in preparation example 7-12 respectively
Outside base, ferment respectively according to the method for experimental example 1-6.Fermentation ends take zymotic fluid and fermentation bacteria residue to be handled and examined
It surveys.
Each experimental example of 1 part of embodiment and the citric acid conversion ratio of comparative example and Glucosamine yield are as shown in table 1.
Table 1
| Zymotic fluid and bacteria residue | Citric acid conversion ratio (%) | Glucosamine yield (%) |
| Experimental example 1 | 98.2 | 10.7 |
| Experimental example 2 | 98.8 | 12.1 |
| Experimental example 3 | 97.9 | 11.8 |
| Experimental example 4 | 98.1 | 12.9 |
| Experimental example 5 | 98.4 | 12.5 |
| Experimental example 6 | 98.0 | 12.3 |
| Comparative example 1 | 75.3 | 6.5 |
| Comparative example 2 | 72.5 | 5.4 |
| Comparative example 3 | 69.2 | 6.2 |
| Comparative example 4 | 67.1 | 4.6 |
| Comparative example 5 | 71.0 | 6.7 |
| Comparative example 6 | 73.2 | 5.8 |
Embodiment 2
The aspergillus niger used in following preparation example, experimental example and comparative example is aspergillus niger Co827, and work is newly found purchased from Shanghai
Industry microorganism Science and Technology Ltd..Corn seed is the commercially available conventional corn seed from Anhui, and amylase is believes purchased from Novi
The alpha-amylase of company (effective temperature scope is 80 DEG C -110 DEG C).
Corn seed machinery mill crush it is broken using purchased from Jiangsu MuYang Group, Ltd. 968-3 types grinder into
Row.Filter press is purchased from the prosperous suitable chemical in Wuxi.
The liquefaction filtrate used in following preparation examples, experimental example and comparative example and the preparation method for the filter residue that liquefies are as follows：Profit
Dry corn seed is crushed with above-mentioned grinder and passes through 40 mesh mesh screens, obtains corn flour.It weighs to corn flour.
The corn flour and water are sized mixing to a concentration of 12 Baume, and its pH value is adjusted to 6.2 with 0.5% (w/w) dilute hydrochloric acid and is formed sediment
Slurry.Relative to every gram of starch in starch slurry, the alpha-amylase of 50 enzyme-activity units is added in, 80 DEG C is then raised temperature to and liquefies
Processing, processing were filtered obtained solution using filter press after 90 minutes, and filtrate is filtrate of liquefying, and filter residue is filter of liquefying
Slag.
According to method specified in standard GB/T 6194-86, the total sugar content measured in the liquefaction filtrate is
16wt%.According to Kjeldahl's method, the protein content measured in the liquefaction filter residue is 25wt%.
Citric acid and the extraction amino Portugal from fermentation bacteria residue are extracted from zymotic fluid in the same manner as shown in Example 1
Grape sugar.
The calculation formula of the conversion ratio of citric acid and the yield of Glucosamine is the same as embodiment 1.
The group ingredient of the total reducing sugar of the fermentation medium in preparation example 13-24 set by embodiment 2, nitrogen and phosphate radical
It is not identical with the accordingly composition of the fermentation medium in the preparation example 1-12 of 1 part of embodiment (that is, according on 2 part of embodiment
State the protein content in the total sugar content and liquefaction filter residue in the liquefaction filtrate of acquisition, each preparation example of 2 part of adjustment embodiment
In liquefaction filtrate and the filter residue that liquefies additive amount, ensure that the total reducing sugar of the fermentation medium that each preparation example obtains in embodiment 2 contains
Amount, nitrogen element content and phosphate content correspond to containing for the respective substance of the fermentation medium of preparation example acquisition with embodiment 1
Measure identical), meanwhile, the experimental example 7-12 and comparative example 7-12 of 2 part of embodiment and the experimental example 1-6 of 1 part of embodiment and right
Ratio 1-6 is identical.
Each experimental example of 2 part of embodiment and the citric acid conversion ratio of comparative example and Glucosamine yield are as shown in table 2.
Table 2
| Zymotic fluid and bacteria residue | Citric acid conversion ratio (%) | Glucosamine yield (%) |
| Experimental example 7 | 98.1 | 10.2 |
| Experimental example 8 | 98.2 | 12.6 |
| Experimental example 9 | 97.4 | 12.4 |
| Experimental example 10 | 98.1 | 12.2 |
| Experimental example 11 | 98.5 | 12.3 |
| Experimental example 12 | 98.0 | 12.5 |
| Comparative example 7 | 75.5 | 6.5 |
| Comparative example 8 | 72.8 | 5.5 |
| Comparative example 9 | 69.3 | 6.7 |
| Comparative example 10 | 67.0 | 4.9 |
| Comparative example 11 | 71.7 | 6.0 |
| Comparative example 12 | 73.3 | 5.6 |
Embodiment 3
The aspergillus niger used in following preparation example, experimental example and comparative example is aspergillus niger HN-2004, micro- purchased from the Chinese Academy of Sciences
Biological institute.Corn seed is commercially available conventional corn seed, and amylase is alpha-amylase (the effective temperature model purchased from Novozymes Company
Enclose is 80 DEG C -110 DEG C).
Corn seed machinery mill crush it is broken using purchased from Jiangsu MuYang Group, Ltd. 968-3 types grinder into
Row.Filter press is purchased from the prosperous suitable chemical in Wuxi.
The liquefaction filtrate used in following preparation examples, experimental example and comparative example and the preparation method for the filter residue that liquefies are as follows：Profit
Dry corn seed is crushed with above-mentioned grinder and passes through 40 mesh mesh screens, obtains corn flour.It weighs to corn flour.
The corn flour and water are sized mixing to a concentration of 16 Baume, and its pH value is adjusted to 5.8 with 0.5% (w/w) dilute hydrochloric acid and is formed sediment
Slurry.Relative to every gram of starch in starch slurry, the alpha-amylase of 5 enzyme-activity units is added in, 110 DEG C is then raised temperature to and liquefies
Processing, processing were filtered obtained solution using filter press after 120 minutes, and filtrate is filtrate of liquefying, and filter residue is filter of liquefying
Slag.
According to method specified in standard GB/T 6194-86, the total sugar content measured in the liquefaction filtrate is
22wt%.According to Kjeldahl's method, the protein content measured in the liquefaction filter residue is 45wt%.
Citric acid and the extraction amino Portugal from fermentation bacteria residue are extracted from zymotic fluid in the same manner as shown in Example 1
Grape sugar.
The calculation formula of the conversion ratio of citric acid and the yield of Glucosamine while embodiment 1.
The group ingredient of the total reducing sugar of the fermentation medium in preparation example 25-36 set by embodiment 3, nitrogen and phosphate radical
It is not identical with the accordingly composition of the fermentation medium in the preparation example 1-12 of 1 part of embodiment (that is, according on 3 part of embodiment
State the protein content in the total sugar content and liquefaction filter residue in the liquefaction filtrate of acquisition, each preparation example of 3 part of adjustment embodiment
In liquefaction filtrate and the filter residue that liquefies additive amount, ensure that the total reducing sugar of the fermentation medium that each preparation example obtains in embodiment 3 contains
Amount, nitrogen element content and phosphate content correspond to containing for the respective substance of the fermentation medium of preparation example acquisition with embodiment 1
Measure identical), meanwhile, the experimental example 13-18 and comparative example 13-18 of 3 part of embodiment and the experimental example 1-6 of 1 part of embodiment and
Comparative example 1-6 is identical.
Each experimental example of 3 part of embodiment and the citric acid conversion ratio of comparative example and Glucosamine yield are as shown in table 3.
Table 3
| Zymotic fluid and bacteria residue | Citric acid conversion ratio (%) | Glucosamine yield (%) |
| Experimental example 13 | 98.5 | 10.8 |
| Experimental example 14 | 98.6 | 12.3 |
| Experimental example 15 | 97.8 | 12.1 |
| Experimental example 16 | 98.5 | 12.5 |
| Experimental example 17 | 98.2 | 12.2 |
| Experimental example 18 | 98.3 | 12.0 |
| Comparative example 13 | 75.2 | 6.2 |
| Comparative example 14 | 72.9 | 5.9 |
| Comparative example 15 | 69.5 | 6.0 |
| Comparative example 16 | 67.8 | 4.5 |
| Comparative example 17 | 71.8 | 6.2 |
| Comparative example 18 | 73.9 | 5.2 |
It can be seen that from the result in upper table 1- tables 3 compared to using the fermentation medium not fallen in the scope of the invention
When being cultivated, high citric acid conversion ratio and high amino Portugal can be achieved at the same time using fermentation medium of the present invention
Grape sugar yield.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail, within the scope of the technical concept of the present invention, a variety of simple variants can be carried out to technical scheme of the present invention, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (10)
1. a kind of fermentation medium for coming co-production of citric acid and Glucosamine for fermentation of Aspergillus niger, the fermentation medium packet
The water of carbonaceous sources, nitrogen source and phosphate and surplus, wherein, the content of the carbon source is calculated as 7wt%- with total reducing sugar therein
20wt%, the content of the nitrogen source are calculated as 0.04wt%-0.3wt% with nitrogen therein, and the phosphatic content is with it
In phosphate radical be calculated as 0.05wt%-0.2wt%.
2. fermentation medium as described in claim 1, wherein, the carbon source is selected from dextrin, maltose and/or starchy material
Liquefaction filtrate.
3. fermentation medium as claimed in claim 1 or 2, wherein, the nitrogen source is inorganic nitrogen-sourced, organic nitrogen source or the two
Mixture.
4. fermentation medium as claimed in claim 3, wherein, it is described it is inorganic nitrogen-sourced for inorganic ammonium salt, preferably ammonium sulfate and/
Or ammonium nitrate；Or the organic nitrogen source is urea, the liquefaction filter residue or their mixture of albumen, starchy material.
5. the fermentation medium as described in claim 2 or 4, wherein, the starchy material be selected from by corn, rice,
It is one or more in the group that wheat, beans, potato and cassava are formed.
6. the fermentation medium as described in any one of claim 1-5, wherein, the phosphate be selected from potassium dihydrogen phosphate and/
Or dipotassium hydrogen phosphate.
7. a kind of fermentation medium using described in any one of claim 1-6 comes preparation of citric acid by fermentation and Glucosamine
Method, described method includes following steps：
(1) by the aspergillus niger in exponential phase be inoculated in the fermentation medium described in any one of claim 1-6 into
Row fermentation so as to obtain zymotic fluid and fermentation bacteria residue, detaches the zymotic fluid and the fermentation bacteria residue；
(2) from the broth extraction citric acid；
(3) the fermentation bacteria residue is handled, so as to obtain Glucosamine.
8. the method as described in paragraph 4, wherein, the aspergillus niger is selected from by aspergillus niger Co827, aspergillus niger T01 and black song
It is one or more in the group that mould HN-2004 is formed.
9. method as claimed in claim 7 or 8, wherein, in step (1), relative to fermentation medium every gram described, inoculation
1×104-1.5×105A Colony Forming Unit, preferably 5 × 104-1×105The aspergillus niger of a Colony Forming Unit.
10. method as claimed in any one of claims 7-9, wherein, in step (1), the fermentation 30-40 DEG C, it is excellent
It selects and is carried out at 36-38 DEG C.
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