CN108277240A - A kind of technique of preparation of citric acid by fermentation - Google Patents
A kind of technique of preparation of citric acid by fermentation Download PDFInfo
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- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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Abstract
The invention belongs to technical field of microbial fermentation, disclose a kind of technique of preparation of citric acid by fermentation comprising following steps：Step 1）Prepare fermentation medium, step 2）Activate aspergillus niger seed liquor, step 3）Fermentation and acid.Zymotechnique of the present invention is of low cost, and lemon acid yield is high.
Description
Technical field
The invention belongs to technical field of microbial fermentation, and in particular to a kind of technique of preparation of citric acid by fermentation.
Background technology
Citric acid also known as citric acid, scientific name 2- hydroxyls-l, 2,3- tricarballylic acids（2-hydroxy-1,2,3-propane
tricanboxylic acid）, it is white crystalline powder, it is tasteless, often contain a molecular crystalline water or nodeless mesh water, it is soluble easily in water.
It is three one of the main intermediate product for connecing acid cycle in organism, so there is important generation in almost all of organism
Gratitude justice.Citric acid has a wide range of applications in fields such as food, medicine, cosmetics, chemical industry.Since the mouthfeel of citric acid is refreshing
Fast tart flavour, so, it is commonly used as in the antioxidant of various beverages, the acidic flavouring agent of dessert or edible oil.In addition,
Citric acid has the function of preventing or eliminating cutaneous pigmentation, therefore citric acid can be used for pharmacy and cosmetic industry.Lemon
Lemon acid solution and its salting liquid are also used as the builder of textile, can precipitate metal ion on textile, meanwhile, as
Pure cotton textile fabric without formic acid crease proofing agent.About 70% citric acid is used for food service industry in world market, and 10% is used for
Medicine and cosmetics, chemical industry account for 15%, other industry 5%.Currently, citric acid whole world consumption has reached 120
It ten thousand tons, produces per year up to 1,600,000 tons, market trade amount is more than 90 ten thousand tons, and is risen year by year with annual 7% growth rate.In
As maximum citric acid exported country in the world, production technology is at the forefront in the world for state, and annual export volume is more than quantum of world trade
Half mainly sells to the ground such as Europe, Japan.Therefore the production of citric acid is to having great importance.
The production method of citric acid mainly has natural extraction method, chemical synthesis and biological fermentation process.Biological fermentation process is
Most common method.Citric acid fermentation mode is divided into：It is fermented using the solid state surface of Penicillium and Aspergillus fungal；It is true using aspergillus
Deep-fermentation；Solid state rheology, continuous culture and multistage fermentation.Currently, commercial mainly pass through aspergillus niger by substrate of starch
Fermentation production of citric acid.The process of fermentation of Aspergillus niger citric acid includes mainly：Utilize the carbon source in culture medium；After EMP Embden Meyerbof Parnas pathway,
Accumulate citric acid；Citric acid is secreted by mitochondria to cytoplasm；Citric acid is by emiocytosis to culture medium.
Citric acid fermentation industry in industrial production urgent problem to be solved mainly include strain, fermented and cultured based raw material,
Three aspects of extraction.In terms of fermenting microbe, multiple-microorganism can accumulate citric acid, such as aspergillus niger, mould, aspergillus nidulans
Deng in addition, some yeast can also manufacture citric acid using acetic acid, alkane etc..The strain used in actual industrial production
All it is the novel bacterial by screening selection and breeding.The development of molecular bioengineering, for excellent species selection and breeding provide it is more wide
Space replaces the report of original Citric Acid Production bacterium to have much with genetic recombination strain, and the 70-90 ages in 20th century are
The high term of China's citric acid novel bacterial.Aspergillus niger is the filamentous fungi of grade-safe, and aspergillus niger is abundant with enzyme system, fermentation
The advantages such as efficient, by-product is few can be good at the flux for regulating and controlling glycolysis and citric acid from mitochondria and cytoplasmic
Secretion is still the important directions of citric acid fermentation development, and improves the important channel of Citric Acid Production rate.To being at present
Only, aspergillus niger is still the Citric Acid Production bacterium being industrially most widely used.It is carried out for having the production acid mechanism of superior strain
Research, analysis bacterial strain is entirely grown and the physiology course of metabolism, provides foundation for the explaination and strain improvement of the sour mechanism of production, anticipates
Justice is great.It is black that " the strain improvement commentary of citric acid fermentation bacterium aspergillus niger, biotechnology 2005 " discloses citric acid fermentation bacterium
The mode of ameliorating of aspergillus, including the modes such as mutagenesis and transgenic technology are carried out to bacterial strain, bacterium is improved to a certain extent
The production acid amount of strain；It is original that " the citric acid fermentation Primary Study of corn powder raw material, industrial microorganism 2000 ", which has studied corn flour,
Material carries out the technological parameter of citric acid fermentation, optimizes production acid system.How microbial fermentation production acid system is advanced optimized,
To put forward high acid amount, while fermentation costs are reduced, is the fermentation industry technical issues that need to address.
Invention content
Present invention aim to address prior art fermentation medium is of high cost, the defects such as citric acid yield is low provide one kind
The technique of preparation of citric acid by fermentation.
The present invention is achieved by the following technical solution：
A kind of technique of preparation of citric acid by fermentation comprising following steps：Step 1）Prepare fermentation medium, step 2）It activates black
Aspergillus seed liquor, step 3）Fermentation and acid.
Specifically, the technique includes the following steps：
Step 1）Prepare fermentation medium：Maize straw is crushed, then with maize peel according to 2:1 mass ratio mixing, crushes
It is more than 50 mesh to grain size, is then added in the water of 5-7 times of weight, warming while stirring keeps the temperature 30min, then add to 80 DEG C
Add potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L, epsom salt 0.2g/L, ferrous sulfate heptahydrate 0.01g/L, sulfuric acid monohydrate
Manganese 0.01g/L, 121 DEG C of moist heat sterilization 30min, it is cooling, obtain trichoderma culture solution；Trichoderma reesei is connect according to 10% inoculum concentration
Enter to trichoderma culture solution, cultivate 72-96h, control mixing speed is 100rpm when culture, and cultivation temperature is 30-32 DEG C, passes through stream
The pH for ammoniating water management culture ferment process is 4-5；After the completion of culture, it is filtered removing thalline, first is added into gained liquid
Alcohol and calcium hydroxide, stir evenly to get fermentation medium；
Step 2）Activate aspergillus niger seed liquor：Aspergillus niger is obtained into aspergillus niger seed liquor according to routine culture for 24 hours, then by seed
Liquid is ultrasonically treated；
Step 3）Fermentation and acid：By step 2）Gained aspergillus niger seed liquor is linked into according to the inoculum concentration of 8-10% contains step 1）
Fermented and cultured is carried out in the fermentation tank of gained fermentation medium, is controlled in pH in 4-4.5, hair by Feeding ammonia water in fermentation process
The ferment time is 60h.
Preferably,
The step 1）In, the additive amount of methanol is 0.2%, volume ratio, and the additive amount of calcium hydroxide is 10wt%.
Preferably,
The supersound process parameter is：Supersonic frequency 30KHz, power 6W, ultrasound interval 15s, ultrasonic time 1s, total ultrasonic time
For 160s.
Preferably,
The step 3）In, fermentation parameter is：Fermentation temperature is 34-36 DEG C, and tank pressure is 0.03-0.04MPa, air quantity 500-
600L/h。
Compared with prior art, the advantageous effect that the present invention obtains includes but is not limited to mainly the following aspects：
Corn is the primary raw material of fermentation enterprise, and corn flour is for fermenting and preparing carbohydrate, and maize straw is discarded mostly, corn
Skin is used as feed mostly, and added value is relatively low, causes certain waste；Can maize straw and maize peel be subjected to General Office
Reason, realizing turns waste into wealth, and improves added value；
It is directly fermented to agricultural wastes such as straw powder and maize peels using aspergillus niger, lemon acid yield is only 10-20g/
L, sugared conversion ratio are less than 60%, can not industrially be applied.Trichoderma reesei degrading maize straws and maize peel, are contained
The solution of reduced sugar carries out fermentation use suitable for aspergillus niger；However experiment is found, trichoderma reesei and aspergillus niger are sent out jointly
Ferment, although two kinds of bacterial strains can preferable symbiosis, the yield of citric acid reduces, and possible cause is that trichoderma reesei is utilized
More nitrogen source so that the nutriment relative deficiency of aspergillus niger, also or trichoderma reesei produces mucilage secretion to black song
Mould citric acid approach produces inhibiting effect, to reduce the yield of citric acid；Therefore, the present invention is in trichoderma reesei culture
After the completion, degerming is carried out, mycoprotein by-product can also be generated；
Aspergillus niger can utilize the reduced sugar or non-reducing sugar that trichoderma reesei decomposition of cellulose generates, but in its fermentation process,
Along with lemon acid accumulation, fermentation pH drastically declines, and especially when pH is down to 2.00 or less, diastatic activity is broken
It is bad, cause glucose supplies reduced rate, Product formation rate to reduce, the residual total sugar content that ferments is higher；It is needed in fermentation process
It is 4 or so that Feeding ammonia water, which controls pH, and otherwise the conversion ratio of citric acid can reduce；Higher residual sugar problem significantly reduce substrate with
Ratio is converted between product, while can further aggravate the difficulty of product isolation and purification step；
The present invention can increase the ability that citric acid passes through cell membrane using the methanol of addition low concentration in the medium, improve lemon
The secretion level of lemon acid；Low intensive ultrasonic pretreatment seed liquor, promotes bacterial strain moisture penetration, can activate bacterial strain, contracting
The fermented incubation time of short bacterial strain improves the yield of citric acid, compared with be not compared using ultrasonication, lemon acid yield
5% or so can be improved.
Specific implementation mode
Those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.In particular, it should be pointed out that
All similar substitutions and modifications are apparent to those skilled in the art, they are considered as being included in this hair
It is bright.The product and method of the present invention is described by preferred embodiment, and related personnel can obviously not depart from this hair
Product as described herein and method are modified or are suitably changed and combined in bright content, spirit and scope, to realize and answer
Use the technology of the present invention.For a further understanding of the present invention, the following describes the present invention in detail with reference to examples.
Embodiment 1
A kind of technique of preparation of citric acid by fermentation comprising following steps：
Prepare fermentation medium：Maize straw is crushed, then with maize peel according to 2:1 mass ratio mixing, is crushed to grain size
50 mesh are then added in the water of 5 times of weight, and warming while stirring keeps the temperature 30min, then add potassium dihydrogen phosphate to 80 DEG C
1g/L, dipotassium hydrogen phosphate 1g/L, epsom salt 0.2g/L, ferrous sulfate heptahydrate 0.01g/L, manganese sulfate monohydrate 0.01g/L,
121 DEG C of moist heat sterilization 30min, it is cooling, obtain trichoderma culture solution；By trichoderma reesei ATCC 13631 according to 10% inoculum concentration（It connects
Kind density is 5 × 106cfu/mL）It is linked into trichoderma culture solution, cultivates 72h, control mixing speed is 100rpm, culture when culture
Temperature is 30-32 DEG C, and the pH that culture ferment process is controlled by Feeding ammonia water is 4-5；After the completion of culture, it is filtered and removes bacterium
Body adds 0.2% into gained liquid（Volume ratio）Methanol and 10wt% calcium hydroxide, stir evenly to get fermentation train
Support base（Content of reducing sugar is 68g/L, and albumen and polypeptide total content are 8.1g/L）；Thalline can be used for preparing mycoprotein；
Activate aspergillus niger seed liquor：Aspergillus niger Co827 is obtained into aspergillus niger seed liquor according to routine culture for 24 hours, then by seed
Liquid is ultrasonically treated, and supersonic frequency 30KHz, power 6W, ultrasound interval 15s, ultrasonic time 1s are controlled, and total ultrasonic time is
160s；
Fermentation and acid：By step 2）Gained aspergillus niger seed liquor according to 8% inoculum concentration（Inoculum density is 1 × 107cfu/mL）It connects
Enter to containing 30L steps 1）Fermented and cultured is carried out in the 50L fermentation tanks of gained fermentation medium, fermentation temperature is 34 DEG C, tank pressure
For 0.03MPa, air quantity 600L/h, in fermentation process by Feeding ammonia water control in pH 4, fermentation time 60h；Producing acid is
151g/L, average conversion 96%, residual sugar amount 0.18%.
Embodiment 2
A kind of technique of preparation of citric acid by fermentation comprising following steps：
Prepare fermentation medium：Maize straw is crushed, then with maize peel according to 2:1 mass ratio mixing, is crushed to grain size
100 mesh are then added in the water of 6 times of weight, and warming while stirring keeps the temperature 30min, then add potassium dihydrogen phosphate to 80 DEG C
1g/L, dipotassium hydrogen phosphate 1g/L, epsom salt 0.2g/L, ferrous sulfate heptahydrate 0.01g/L, manganese sulfate monohydrate 0.01g/L,
121 DEG C of moist heat sterilization 30min, it is cooling, obtain trichoderma culture solution；By trichoderma reesei according to 10% inoculum concentration（Inoculum density is 5
×106cfu/mL）It is linked into trichoderma culture solution, cultivates 96h, control mixing speed is 100rpm, cultivation temperature 30- when culture
32 DEG C, the pH that culture ferment process is controlled by Feeding ammonia water is 4-5；After the completion of culture, it is filtered removing thalline, toward gained liquid
0.2% is added in body（Volume ratio）Methanol and 10wt% calcium hydroxide, stir evenly to get fermentation medium（Reduced sugar
Content is 73g/L, and albumen and polypeptide total content are 8.7g/L）；Thalline can be used for preparing mycoprotein；
Activate aspergillus niger seed liquor：By aspergillus niger（ATCC1015）It obtains aspergillus niger seed liquor for 24 hours according to routine culture, then will
Seed liquor is ultrasonically treated, and supersonic frequency 30KHz, power 6W, ultrasound interval 15s, ultrasonic time 1s, total ultrasonic time are controlled
For 160s；
Fermentation and acid：By step 2）Gained aspergillus niger seed liquor according to 10% inoculum concentration（Inoculum density is 1 × 107cfu/mL）
It is linked into containing 30L steps 1）Fermented and cultured is carried out in the 50L fermentation tanks of gained fermentation medium, fermentation temperature is 35 DEG C, tank
Pressure is 0.04MPa, air quantity 500L/h, in fermentation process by Feeding ammonia water control in pH 4.3, fermentation time 60h；Production
Acid amount is 143g/L, average conversion 94%, residual sugar amount 0.27%.
Comparative example 1
Fermentation of Aspergillus niger tank culture medium：Corn flour 3kg, alpha-amylase 2000U/g, CaCl2 30g, remaining be water.Preparation method
For：Corn flour is added in water, adjusts pH to 6.0-6.5, and Thermostable α-Amylase is added, and is heated to 70 DEG C and keeps the temperature 1h, fast
Speed is heated to 90 DEG C, heat preservation 45-60 min；Reheating boils 5-10min, adds 5% ammonium sulfate, 10% calcium hydroxide,
10% antifoaming agent.Zymotechnique is the same as embodiment 1；Production acid production citric acid amount is 137g/L, fermentation period 68h.
Embodiment 3
By taking embodiment 1 as an example, trichoderma reesei and the sour situation of aspergillus niger mixed fermentation production are compared, specific comparing result is shown in Table
1：
Table 1
| Fermentation method | Production acid amount g/L, | Residual sugar amount % | Fermentation period h |
| Mixed fermentation | 129 | 0.21 | 60 |
| Embodiment 1 | 151 | 0.18 | 60 |
As shown in Table 1, by trichoderma reesei and aspergillus niger co-fermentation, although two kinds of bacterial strains can preferable symbiosis, lemon
The yield of lemon acid reduces 16%, and possible cause is that more nitrogen source is utilized in trichoderma reesei so that the nutriment phase of aspergillus niger
To deficiency, it is also possible to trichoderma reesei produces mucilage secretion and produces inhibiting effect to the citric acid approach of aspergillus niger, from
And reduce the yield of citric acid；Therefore, the present invention needs to remove thalline after the completion of trichoderma reesei culture.
Embodiment 4
Influence of the methanol additive amount to fermentation and acid effect in fermentation medium, be arranged methanol additive amount be 0,0.1%, 0.2%,
0.4%, 0.8%, remaining fermentation system is specifically shown in Table 2 with embodiment 1：
Table 2
| Additive amount v/v% | Production acid amount g/L, | Fermentation period h |
| 0 | 140 | 60 |
| 0.1 | 147 | 60 |
| 0.2 | 151 | 60 |
| 0.4 | 145 | 60 |
| 0.8 | 137 | 60 |
As shown in table 2, it with the increase of methanol additive amount, produces acid amount and increases, when additive amount is 0.2%, reach maximum value,
With the increase of methanol additive amount, produces acid amount and decrease, therefore, select 0.2% additive amount the most suitable.
The explanation of above example is only intended to facilitate the understanding of the method and its core concept of the invention.It should be pointed out that for this
For the those of ordinary skill of technical field, without departing from the principle of the present invention, the present invention can also be carried out several
Improvement and modification, these improvement and modification are also fallen within the protection scope of the claims of the present invention.
Claims (5)
1. a kind of technique of preparation of citric acid by fermentation comprising following steps：Step 1）Prepare fermentation medium, step 2）Activation
Aspergillus niger seed liquor, step 3）Fermentation and acid.
2. technique according to claim 1, which is characterized in that the technique includes the following steps：
Step 1）Prepare fermentation medium：Maize straw is crushed, then with maize peel according to 2:1 mass ratio mixing, crushes
It is more than 50 mesh to grain size, is then added in the water of 5-7 times of weight, warming while stirring keeps the temperature 30min, then add to 80 DEG C
Add potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L, epsom salt 0.2g/L, ferrous sulfate heptahydrate 0.01g/L, sulfuric acid monohydrate
Manganese 0.01g/L, 121 DEG C of moist heat sterilization 30min, it is cooling, obtain trichoderma culture solution；Trichoderma reesei is connect according to 10% inoculum concentration
Enter to trichoderma culture solution, cultivate 72-96h, control mixing speed is 100rpm when culture, and cultivation temperature is 30-32 DEG C, passes through stream
The pH for ammoniating water management culture ferment process is 4-5；After the completion of culture, it is filtered removing thalline, first is added into gained liquid
Alcohol and calcium hydroxide, stir evenly to get fermentation medium；
Step 2）Activate aspergillus niger seed liquor：Aspergillus niger is obtained into aspergillus niger seed liquor according to routine culture for 24 hours, then by seed
Liquid is ultrasonically treated；
Step 3）Fermentation and acid：By step 2）Gained aspergillus niger seed liquor is linked into according to the inoculum concentration of 8-10% contains step 1）
Fermented and cultured is carried out in the fermentation tank of gained fermentation medium, is controlled in pH in 4-4.5, hair by Feeding ammonia water in fermentation process
The ferment time is 60h.
3. technique according to claim 2, which is characterized in that the step 1）In, the additive amount of methanol is 0.2%, volume
Than the additive amount of calcium hydroxide is 10wt%.
4. technique according to claim 2, which is characterized in that the supersound process parameter is：Supersonic frequency 30KHz, work(
Rate 6W, ultrasound interval 15s, ultrasonic time 1s, total ultrasonic time is 160s.
5. technique according to claim 2, which is characterized in that the step 3）In, fermentation parameter is：Fermentation temperature is
34-36 DEG C, tank pressure is 0.03-0.04MPa, air quantity 500-600L/h.
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| CN108588134A (en) * | 2018-05-03 | 2018-09-28 | 齐齐哈尔龙江阜丰生物科技有限公司 | The extraction and preparation technique of citric acid |
| CN113604365A (en) * | 2021-09-02 | 2021-11-05 | 江南大学 | Method for controlling particle size of mycelium pellet by ultrasonic waves and method for producing citric acid by using mycelium pellet |
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| CN108588134A (en) * | 2018-05-03 | 2018-09-28 | 齐齐哈尔龙江阜丰生物科技有限公司 | The extraction and preparation technique of citric acid |
| CN113604365A (en) * | 2021-09-02 | 2021-11-05 | 江南大学 | Method for controlling particle size of mycelium pellet by ultrasonic waves and method for producing citric acid by using mycelium pellet |
| CN113604365B (en) * | 2021-09-02 | 2023-09-15 | 江南大学 | Ultrasonic wave control mycelium pellet size and method for producing citric acid by using same |
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| CN108277240B (en) | 2020-11-10 |
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