CN107058209A - A kind of use carrier adsorption carries out aspergillus niger spore propagation and its preparation method of xerospore powder - Google Patents
A kind of use carrier adsorption carries out aspergillus niger spore propagation and its preparation method of xerospore powder Download PDFInfo
- Publication number
- CN107058209A CN107058209A CN201710381711.1A CN201710381711A CN107058209A CN 107058209 A CN107058209 A CN 107058209A CN 201710381711 A CN201710381711 A CN 201710381711A CN 107058209 A CN107058209 A CN 107058209A
- Authority
- CN
- China
- Prior art keywords
- aspergillus niger
- spore
- powder
- preparation
- solid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N3/00—Spore forming or isolating processes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
Abstract
Aspergillus niger spore propagation and its preparation method of xerospore powder are carried out the invention discloses a kind of use carrier adsorption, including：By 7:3 ratio mixing corn bran, is 1 according to solid content gross weight and the volume ratio of water:5, corn bran is mixed with water, fire resistant alpha-diastase is then added, 100 DEG C are boiled 30 minutes, is filtered, the nutrient solution of aspergillus niger spore is obtained.Volume will be ground into for 1cm3Corn shaft core and aspergillus niger nutrient solution press 1:3.5 w/vs are mixed, and load solid-state fermenter, are sterilized 20 minutes through 121 DEG C, are cooled to 37 DEG C of accesses 104~105CFU/mL aspergillus niger spores, are calculated with aspergillus niger nutrient solution volume.37 DEG C of solid-state fermenter temperature is maintained, is cultivated 120 hours, composite protectant is added after elution aspergillus niger spore, spore suspension is 2: 3 with composite protectant volume ratio, equilibration time is 40 min, aspergillus niger xerospore powder is collected in vacuum drying.The preparation method of the present invention improves aspergillus niger spore powder survival rate, extends the storage period of strain.The method consumption energy for adding composite drying agent simultaneously is few, and technique is simple, with low cost, is more suitable for large-scale production.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of method of porous carrier absorption aspergillus niger spore propagation with
And the dry composite protective agent and drying means of a kind of aspergillus niger spore powder.
Background technology
In industrial fermentation field, the expansion culture of strain is the important step of zymotechnique.Aspergillus niger is extremely important
Industrial microorganism, is the production bacterium of many fermented products, particularly the industry such as organic acid, enzyme preparation, antibiotic, for producing lemon
The organic acids such as lemon acid, malic acid, gallic acid, itaconic acid, malic acid, can be also used for production carbohydrase, acid protease and
The enzyme preparation that more than 30 kinds of pectase etc., only as defined in China's standard the food enzyme preparation of Aspergillus niger origin just have 26 kinds it
It is many.Aspergillus niger can also be used to add during the fermentation such as excrement, organic waste, bio-feritlizer, straw decomposing of poultry.
During aspergillus niger production various product, inoculation fermentation is mainly carried out with aspergillus niger spore.Because aspergillus niger
Spore has easily storage, holding cost is low, can be readily transported, be difficult pollution, environmental suitability is strong, direct plunges into fermentation, ferment
The advantage such as power of regeneration is strong.General be inoculated in spore of Aspergillus niger treats fermentation materials in actual production, therefore develops black song
There is mould conidia powder preferable production application to be worth.
The submerged fermentation of aspergillus niger citric acid is industrial general to utilize the three of 2L, it is necessary to use substantial amounts of aspergillus niger spore to be inoculated with
Angle bottle is using the wheat bran spore for standing the solid fermentation preparation of button bottle method.Because the sporogony time is longer, the nutrition of wheat bran skin is not
This process of the sprouting propagation of support spore, causes the degeneration of low quality and easy of aspergillus niger spore produced enough.And
A large amount of oxygen are needed during sporogony, wheat bran bottle quiescent culture causes oxygen supply not enough, and the spore quality of generation is unstable,
And every bottle of at most harvest spore quantity is only 1011CFU/ bottles, cause 50 tons of seeding tanks to need 100-110 bottles of inoculation, be big rule
Mould industrialized production brings heavy burden.So need to be optimized to sporogony mode, to obtain high quantity, high-quality black
Aspergillus spore carries out inoculation fermentation.
Drying process is the key for preparing probiotics, and thalline survival rate and activity are to determine thalline optimum drying technique
With the index of best protection agent prescription.The influence to microbial cell is dried mainly to cell membrane and the damage of sensitive proteins
Wound.Cell membrane penetration can be caused to sexually revise in drying process, the property of film, 26S Proteasome Structure and Function change, protein denaturation inactivation, pH
Dynamic equilibrium is destroyed, DNA damage and Membrane fatty acid composition change.
Research shows, appropriate protective agent is added before it is dried can change physics when thalline is dried, chemical environment, subtract
It is light or prevent from drying or infringement of the rehydration to cell, original various physiological and biochemical properties and bioactivity are kept as far as possible, from
And the survival rate in microorganism drying process is improved, extend strain storage period.Protective agent widely used at present has trehalose, taken off
Fat milk powder, sucrose, glycerine, tween, sodium glutamate etc..And in fact, protectant effect meeting is with the difference of bacterial strain species, protection
The species of agent and certain difference of proportioning performance.Therefore, optimal drying means need to be found and protection agent prescription is dry to reduce
Influence of the dry process to microbial cell.Influence of the protective agent to microorganism drying effect is very big, and protectant selection is the most
A crucial step.
Freeze-drying is the main method that current most of microbe is dried.But numerous studies are shown, in extracellular microbial
Most enzymes can be a greater impact in freeze-drying process, but not yet have preferable method at present to prevent these enzymes when lyophilized
Occurs the change of activity.On the other hand, freeze-drying has many deficiencies simultaneously:Freeze-drying process complex operation, instrument cost be high,
Drying time length, process conditions are more just carved, and have impact on probiotics industrialization, commercialization process.Comparatively speaking, room temperature
The seasoning consumption energy is few, technique is simple, with low cost, is more suitable for large-scale production.
The content of the invention
The present invention changes the industrial triangular flask quiescent culture using 2L, the method that solid fermentation prepares wheat bran spore.
By being optimized to sporogony mode, high quantity, high-quality aspergillus niger xerospore powder are obtained.
To achieve the above object, the invention discloses following technology contents：
Aspergillus niger used is that CMCC2450 is known bacterial strain in research process of the present invention.
A kind of use carrier adsorption carries out the preparation method of aspergillus niger spore propagation, it is characterised in that by a certain proportion of jade
Rice is mixed with wheat bran, and then alpha-amylase liquefies, filtering, adds the corncob particle mixing crushed, sterilizing, inoculated aspergillus niger
Composite protectant is added after spore, solid-state fermenter culture, elution spore suspension, spore suspension is with composite protectant volume ratio
2: 3, equilibration time is 40 min, is finally dried in vacuo, and collects aspergillus niger xerospore powder；And carried out by the steps；
37 DEG C of culture 120h → elution spore → addition protective agent → balances 40 minutes of solid-state fermenter → 35 DEG C of vacuum drying 16h
→ collect xerospore.
（1）The preparation of aspergillus niger spore Multiplying culture liquid：
It is 7 by weight by corn and wheat bran:3 ratio mixing, is 1 according to solid content gross weight and the volume ratio of water:5, will
Corn bran is mixed with water, and alpha-amylase is added in said mixture by every gram of 12 units of corn weight and liquefied, and 100
DEG C boil 30 minutes, filter, the nutrient solution of filtrate fixed molten added water to original volume, i.e. acquisition aspergillus niger spore；
（2）The preparation of solid-state fermenter vector propagation aspergillus niger spore culture：
Volume will be ground into for 1cm3Corn shaft core and above-mentioned aspergillus niger nutrient solution volume ratio 1 by weight:3.5 mixing, load
Solid-state fermenter, sterilizes 20 minutes through 121 DEG C, is cooled to 37 DEG C of access aspergillus niger spores, inoculum concentration is with aspergillus niger culture liquid
Product calculates 104~105CFU/mL, maintains 37 DEG C of solid-state fermenter temperature, cultivates 120 hours, spore suspension is obtained after elution；
（3）The composite protectant of aspergillus niger spore powder：
Each component protective agent precise is dissolved completely in certain distilled water, 115 DEG C, 20min sterilizings are cooled to room
Temperature, is added in spore suspension：Described composite protectant is：
The g/100mL of the g/100mL of trehalose 5 ~ 10
The g/100mL of the g/100mL of peptone 5 ~ 10
Glycerine 2g/100mL ~ 4g/100mL；
（4）The drying of aspergillus niger spore powder：Aspergillus niger spore dried bean noodles drying process be 35 DEG C, 0.4 × 105Pa vacuums dry 16h,
Collection obtains aspergillus niger xerospore powder.
Preparation method of the present invention, wherein corn shaft core and above-mentioned aspergillus niger nutrient solution volume ratio 1 by weight:3.5
It is most suitable for aspergillus niger spore propagation.Described aspergillus niger spore be through culture, washing obtained by concentration be 5 × 109CFU/mL ~6
×109CFU/mL spore suspension.
The present invention further discloses the composite protectant in preparation method.Its composite protectant feature group turns into：Sea
Algae sugar 7.5 g/100 mL, peptone 10g/100 mL and glycerine 3g/100 mL.
The present invention further discloses the method that corncob carries out sporogony as carrier adsorption, it is characterised in that beautiful
Meter Xin be it is size-reduced into volume be 1cm3Corncob, add solid-state fermenter, add inoculum concentration be 104~105The black songs of CFU/mL
The aspergillus niger nutrient solution of mould spore, w/v is 1:3.5, calculated with aspergillus niger nutrient solution volume.Its drying means, it is special
Levy and be：35℃、0.4×10516h is dried under Pa vacuums.
Aspergillus niger spore powder survival rate, extension are being improved the present invention further discloses prepared aspergillus niger xerospore powder
Application in terms of strain storage period.The composite protectant of the present invention is dry for aspergillus niger spore dried bean noodles, and survival rate is up to 95.81%,
And be not used it is protectant in the case of, Conidia persistence only 15.23%.Composite protectant in particularly described preparation method
Improving the application during aspergillus spore is dried.For example in increase sporogony spatial table area and dissolved oxygen, sporogony side is improved
The application in face.
The more detailed preparation method of the present invention and it is discussed below：
The corncob particle that corn bran → amylase liquefaction → filtering that the present invention mixes certain proportion → addition is crushed is mixed
Conjunction → sterilizing → 37 DEG C of inoculation → solid-state fermenter culture 120h → elution spore → addition protective agent → balance 40 minutes → 35
DEG C vacuum drying 16h → collection xerospore.
Its detailed method is as follows：
First, the method for aspergillus niger spore of the invention propagation is comprised the following steps that：
1st, the preparation of aspergillus niger spore Multiplying culture liquid
It is 7 by weight by corn and wheat bran:3 ratio mixing, is 1 according to solid content gross weight and the volume ratio of water:5, will
Corn bran is mixed with water, by every gram of 12 unit high temperature amylase of corn weight, is added in said mixture, 100 DEG C are boiled
Boiling 30 minutes, filtering, the nutrient solution of filtrate fixed molten added water to original volume, i.e. acquisition aspergillus niger spore.
（1）Corn and wheat bran weight ratio are determined
Influence of the corn bran different weight mixing ratio of table 1 to sporogony
The result difference for the nutrient solution propagation aspergillus niger spore that the corn of different proportion, wheat bran ratio are obtained is larger, final to obtain
Best ratio is：Corn and wheat bran are 7 by weight:3.
（2）The ratio-dependent of corn and wheat bran and water：
Influence of the ratio of the corn bran of table 2 and water to sporogony
Corn and wheat bran ratio are 7:3, sporogony effect is best when addition water volume is 5 times of both weight sums.
The innovative point of the present invention：Aspergillus niger spore Multiplying culture liquid.It instead of the solid wheat bran that traditional industry is used, black song
Mould sporogony quantity is more, does not degenerate, and growth is fast.
2nd, solid-state fermenter vector propagation aspergillus niger spore culture
The granular size of corncob is 1cm3, corncob particle and the ratio of aspergillus niger spore nutrient solution added be, corncob
Particle fully absorbs aspergillus niger spore nutrient solution, substantially without free water.Spore is so conducive to be adsorbed onto the table of corncob particle
Sufficient sporogony is carried out on face.Corncob particle fully increases the surface of spore absorption propagation as the carrier of sporogony
Product, improves ventilation efficiency in solid-state fermenter, reaches spatial table area, spatial volume and the dissolved oxygen of increase single spore growth,
And then the growth of spore has been greatly accelerated, finally improve the quantity and quality of spore.Solid-state fermenter control parameter：Tank pressure
0.05-0.1Mpa, ventilate 0.5-1m3/m3.h, 37 DEG C of temperature, rotating speed 30-50/min, incubation time 120h.
（1）Corncob granular absorption aspergillus niger spore culture liquid proportional
The different proportion corncob particle of table 3 and influence of the aspergillus niger spore nutrient solution to sporogony
As seen from Table 3, when corncob particle and aspergillus niger spore culture liquid proportional（w/v）For 1:When 3.5, it is most suitable for aspergillus niger
Sporogony.
（2）The influence that inoculum concentration is bred to aspergillus niger spore
Influence of the inoculum concentration of table 4 to sporogony
As seen from Table 4, when corncob particle and aspergillus niger spore culture liquid proportional（CFU/L）For 104When be most suitable for aspergillus niger
Sporogony.
2nd, the dry composite protective agent and drying means of aspergillus niger spore powder：
One of the problem of present invention is solved is to provide a kind of dry composite protective agent of aspergillus niger spore powder, reduces and dry to bacterium
The loss of body, improves the survival rate and bin stability of aspergillus niger spore powder.The two of the problem of present invention is solved are to determine most
Good is used for the drying means that a kind of aspergillus niger spore is dried.The few technique of this method consumption energy is simple, with low cost, is more suitable for big
Type is produced.
（1）The preparation process of the aspergillus niger spore powder of the present invention is as follows：
It is prepared by A protection liquid：Each component protective agent precise is dissolved completely in certain distilled water, 115 DEG C, 20min goes out
Bacterium, is cooled to room temperature.
The outstanding configuration of B spores：The aspergillus niger spore provided in the present invention be through culture, washing obtained by concentration be 5 × 109CFU/
mL~ 6×109CFU/mL spore suspension.
C is mixed：The drying protectant that the present invention is provided, is sufficiently mixed with aspergillus niger spore suspension before it is dried, and spore hangs
Liquid is 2: 3 with protective agent volume ratio, and equilibration time is 40 min
D is dried：35 DEG C and it is adjusted in the drying box of certain vacuum degree, dries and obtain conidia powder after certain time.
（2）The protectant screening of dry composite of aspergillus niger spore powder is determined
The present invention is to 14 kinds of materials such as peptone, trehalose, glucose, trehalose, glycerine, sorbierite, peptone, polyethylene glycol
Dry-run protection effect to aspergillus niger spore compares research, is shown in Table 5, preliminary screening goes out the preferably single guarantor of protecting effect
Protect agent：Trehalose, peptone, sorbierite, fructose, glycerine.
The single protective agent of table 5 dries the influence of survival rate to aspergillus niger spore
On the basis of more than, the influence that different composite protective agent is dried to a kind of aspergillus niger spore is have studied, 6 are shown in Table.
As a result show, trehalose, peptone, glycerine are best complex protective agent.
The composite protectant of table 6 dries the influence of survival rate to aspergillus niger spore
According to result above, using L16（43）Orthogonal test determines that trehalose, peptone, 3 kinds of protective agents of glycerine are protected as compound
The optimum addition of agent is protected, is as a result shown, best complex protection agent prescription is：The g/ of 7.5 g/100mL peptones of trehalose 10
100mL, glycerine 3g/100mL.Complex protection agent prescription is determined：
The orthogonal test factor level table of table 7
The Orthogonal experiment results of table 8 and its range analysis
Verify that best complex protection agent prescription is：The g/100mL of 7.5 g/100mL peptones of trehalose 10, glycerine 3g/100mL
Drying protectant, determine dried Conidia persistence, up to 87.83%, obtained preferable effect.Therefore utilize the present invention's
It is beneficial that composite protectant, which prepares aspergillus niger spore,.
（3）The determination of drying condition
Drying means
The survival rate of spore is dried under the different vacuum different times of table 9
Temperature：A kind of drying temperature of aspergillus niger spore powder provided by the present invention is set as 35 DEG C, optimal because of the aspergillus niger
Growth temperature is 35 DEG C.
Vacuum：Set different vacuum vacuums（×105Pa）：0、0.2、0.4、06、0.8、1.0（Normal pressure, control）,
The dry-run protection effect of aspergillus niger spore to being dried under different vacuums compares research.
Determine：The quality for drying Conidia persistence and conidia powder after different vacuums and different time is determined, to determine
Optimal vacuum and optimal drying time, so that it is determined that optimum drying technique.
Present invention determine that drying time be 16h, conidia powder quality is no longer after being shown in dry 16h because of determination data
Change, illustrates that 16h is dried complete, therefore think spore powder drying to 16h just with enough.
The vacuum provided by the present invention for ensureing 90% survival rate（×105Pa）There are 0.4,0.6,0.8, Conidia persistence
Respectively 92.30%, 95.81%, 93.14%.In order to which cost-effective vacuum of choosing is 0.4 × 105Pa in industrial production.
Integrated comparative, the optimum drying technique of aspergillus niger spore powder provided by the present invention is 35 DEG C, 0.4 × 105Pa is true
Reciprocal of duty cycle dries 16h.
Use carrier adsorption disclosed by the invention carries out the positive effect that the method and purposes of aspergillus niger spore propagation have
Fruit is：
（1）The quantity increase of aspergillus niger spore propagation, production spore cycle time, spore quality increase are difficult to degenerate；
（2）Aspergillus niger spore has easily storage, holding cost is low, can be readily transported, be difficult pollution, environmental suitability by force, directly
The advantage such as input fermentation, fermentation regenerated ability be strong
（3）Solve because spore quality is unstable, manual labor is more caused by lazy weight, the problem of low production efficiency.
Reduce the production cost of factory.
Brief description of the drawings
Fig. 1 is the yield of different time citric acid；
Fig. 2 is different vaccination amount to production acid and with the influence of solid content.
Embodiment
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention
Scope, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
The various changes carried out on the premise of invention spirit and scope to the material component in these embodiments and consumption or change
Belong to protection scope of the present invention.Raw materials used and reagent of the invention is commercially available.Strains tested used in the present invention：It is black
Aspergillus（Aspergillus niger）It is commercially available, the bacterial strain disclosed in CGMCC2450 can also be selected to be tested, can be with
Reach the effect of aspergillus niger spore propagation.
Embodiment 1
A kind of use carrier adsorption carries out the preparation method of aspergillus niger spore propagation；
（1）The preparation of aspergillus niger spore Multiplying culture liquid：
It is 7 by weight by corn and wheat bran:3 ratio mixing, is 1 according to solid content gross weight and the volume ratio of water:5, will
Corn bran is mixed with water, and amylase is added in said mixture by every gram of 12 units of corn weight and liquefied, 100 DEG C are boiled
Boiling 30 minutes, filtering, the nutrient solution of filtrate fixed molten added water to original volume, i.e. acquisition aspergillus niger spore；
（2）The preparation of solid-state fermenter vector propagation aspergillus niger spore culture：
Volume will be ground into for 1cm3Corn shaft core and above-mentioned aspergillus niger nutrient solution volume ratio 1 by weight:3.5 mixing, load
Solid-state fermenter, sterilizes 20 minutes through 121 DEG C, is cooled to 37 DEG C of access aspergillus niger spores, inoculum concentration is with aspergillus niger culture liquid
Product calculates 104CFU/mL, maintains 37 DEG C of solid-state fermenter temperature, cultivates 120 hours, spore suspension is obtained after elution；Described
Aspergillus niger spore be through culture, washing obtained by concentration be 5 × 109CFU/mL ~6×109CFU/mL spore suspension
（3）The composite protectant of aspergillus niger spore powder：
Each component protective agent is accurately weighed into 7.5 g trehaloses, 10 g peptones, 3g glycerine and is dissolved completely in 100mL distilled water
In, 115 DEG C, 20min sterilizings are cooled to room temperature, are added to the volume ratio that protective agent hangs with spore in spore suspension and are：3:2：It is described
Composite protectant be：The g/100mL of trehalose 7.5, the g/100mL of peptone 10, glycerine 3g/100mL.
（4）The drying of aspergillus niger spore powder：
Aspergillus niger spore dried bean noodles drying process be 35 DEG C, 0.4 × 105Pa vacuums dry 16h, and collection obtains aspergillus niger xerospore
Powder, Conidia persistence is 87.83%.
Embodiment 2
A kind of use carrier adsorption carries out the preparation method of aspergillus niger spore propagation；
（1）The preparation of aspergillus niger spore Multiplying culture liquid：
It is 7 by weight by corn and wheat bran:3 ratio mixing, is 1 according to solid content gross weight and the volume ratio of water:5, will
Corn bran is mixed with water, and amylase is added in said mixture by every gram of 12 units of corn weight and liquefied, 100 DEG C are boiled
Boiling 30 minutes, filtering, the nutrient solution of filtrate fixed molten added water to original volume, i.e. acquisition aspergillus niger spore；
（2）The preparation of solid-state fermenter vector propagation aspergillus niger spore culture：
Volume will be ground into for 1cm3Corn shaft core and above-mentioned aspergillus niger nutrient solution volume ratio 1 by weight:3.5 mixing, load
Solid-state fermenter, sterilizes 20 minutes through 121 DEG C, is cooled to 37 DEG C of access aspergillus niger spores, inoculum concentration is with aspergillus niger culture liquid
Product calculates 105CFU/mL, maintains 37 DEG C of solid-state fermenter temperature, cultivates 120 hours, spore suspension is obtained after elution；Described
Aspergillus niger spore be through culture, washing obtained by concentration be 5 × 109CFU/mL ~6×109CFU/mL spore suspension
（3）The composite protectant of aspergillus niger spore powder：
Each component protective agent is accurately weighed into 7.5 g trehaloses, 10 g peptones, 3g glycerine and is dissolved completely in 100mL distilled water
In, 115 DEG C, 20min sterilizings are cooled to room temperature, are added to the volume ratio that protective agent hangs with spore in spore suspension and are：3:2：It is described
Composite protectant be：
The g/100mL of trehalose 10, peptone 5 g/100mL 100mL, glycerine 2g/100mL.
（4）The drying of aspergillus niger spore powder：
Aspergillus niger spore dried bean noodles drying process be 35 DEG C, 0.4 × 105Pa vacuums dry 16h, and collection obtains aspergillus niger xerospore
Powder, Conidia persistence is 87.83%.
Embodiment 3
Aspergillus niger spore fermentation production of citric acid prepared by the present invention：
Citric acid is a kind of important organic acid, and it is now with biology to be widely used in the industry citric acid such as food, medicine, chemical industry
The maximum organic acid of chemical method yield, while being also most widely used organic acid in the world, is referred to as first in the industry
Using sour agent, more than the 60% of acid condiment consumption is accounted for.Drying conidia powder provided by the present invention connects for citric acid factory
Plant fermentation production of citric acid.And determine its optimum inoculation amount.
（1）It is prepared by fermentation medium：
The band slag corn liquid that the citric acid company of cooperation is provided.It will be added with slag corn liquid in fermentation tank, liquid amount is 8%, warp
121 DEG C sterilize 20 minutes.
（2）Citric Acid Production：
By in dry conidia powder access fermentation tank provided by the present invention, inoculum concentration is respectively 6%, 8%, 10%, 12%, 14%, inoculation
Amount is calculated with fermentation of Aspergillus niger culture volume, controls 36.5 DEG C of fermentation temperature, the r/min of speed of agitator 400,400 L/ of ventilation
Min, tank pressure 0.1 Mpa, incubation time 72h, carries out citric acid fermentation.Fermentation starts to sample from fermentation tank every 12 h, surveys
Determine citric acid content.
（3）Conclusion：
In Citric Acid Fermentation, the size of inoculum concentration directly affects the yield of citric acid, therefore we need to find conjunction
Suitable inoculum concentration carries out citric acid fermentation.It follows that when inoculum concentration is 6% ~ 8%, because inoculum concentration is very few, causing to ferment
Fermentation is short of power in journey, influences lemon acid yield, continues to increase inoculation volume production acid increase, inoculation is worked as in solid content increase
Measure for 10% when, the sour highest of production continues to increase inoculation volume production acid and starts reduction, now because inoculum concentration is excessive, causes mycelium pellet to be given birth to
Length is bad, and solid content, which continues increase, causes fermentation broth viscosity to increase, and is unfavorable for dissolved oxygen, and increase carrying for downstream citric acid
Take difficulty.Consider the influence that lemon acid yield and solid content are extracted to citric acid, experiment control inoculum concentration is 10%.
Embodiment 4
Black mold solid state fermentation production tannase prepared by the present invention：
Tannic acid is widely used in the fields such as process hides, wine brewing, medicine, beverage.It is the single-minded enzyme that enzymolysis turns molten " tea cream ", closely
In depth studied along with the fast development of tea beverage industry over year.
（1）Process for solid state fermentation
The mixture for weighing 8g tannic acid and wheat bran is placed in 500mL triangular flasks, PDA liquid medium 20mL, 121 DEG C respectively
Sterilized 20min, and aspergillus niger spore powder provided by the present invention is accessed in PDA liquid medium, and inoculum concentration is 6 × 109CFU/
ML, vaccinated PDA culture medium is added in the 500mL triangular flasks containing solid content, be placed in after fully shaking up 30 in incubator
DEG C, quiescent culture 120h.
（2）Enzyme activity assay
The enzyme activity of zymotic fluid was determined every 24 hours, 10 are shown in Table
The enzyme activity of Aspergillus Niger tannase under the different time of table 10
As shown in Table 10, the U/ g of highest enzyme activity 56.3 are reached in 96h.
Claims (9)
1. a kind of use carrier adsorption carries out the preparation method of aspergillus niger spore propagation, it is characterised in that by a certain proportion of corn
Mixed with wheat bran, then alpha-amylase liquefies, filtering, add the corncob particle mixing crushed, sterilizing, inoculated aspergillus niger spore
Composite protectant is added after son, solid-state fermenter culture, elution spore suspension, spore suspension is 2 with composite protectant volume ratio:
3, equilibration time is 40 min, is finally dried in vacuo, and collects aspergillus niger xerospore powder；And carried out by the steps；
37 DEG C of culture 120h → elution spore → addition protective agent → balances 40 minutes of solid-state fermenter → 35 DEG C of vacuum drying 16h
→ collect xerospore；
（1）The preparation of aspergillus niger spore Multiplying culture liquid：
It is 7 by weight by corn and wheat bran:3 ratio mixing, is 1 according to solid content gross weight and the volume ratio of water:5, will
Corn bran is mixed with water, and alpha-amylase is added in said mixture by every gram of 12 units of corn weight and liquefied, and 100
DEG C boil 30 minutes, filter, the nutrient solution of filtrate fixed molten added water to original volume, i.e. acquisition aspergillus niger spore；
（2）The preparation of solid-state fermenter vector propagation aspergillus niger spore culture：
Volume will be ground into for 1cm3Corn shaft core and above-mentioned aspergillus niger nutrient solution volume ratio 1 by weight:3.5 mixing, load solid
State fermentation tank, sterilizes 20 minutes through 121 DEG C, is cooled to 37 DEG C of access aspergillus niger spores, inoculum concentration is with aspergillus niger nutrient solution volume
Calculate 104~105CFU/mL, maintains 37 DEG C of solid-state fermenter temperature, cultivates 120 hours, spore suspension is obtained after elution；
（3）The composite protectant of aspergillus niger spore powder：
Each component protective agent precise is dissolved completely in certain distilled water, 115 DEG C, 20min sterilizings are cooled to room
Temperature, is added in spore suspension：Described composite protectant is：
The g/100mL of the g/100mL of trehalose 5 ~ 10
The g/100mL of the g/100mL of peptone 5 ~ 10
Glycerine 2g/100mL ~ 4g/100mL；
（4）The drying of aspergillus niger spore powder：Aspergillus niger spore dried bean noodles drying process be 35 DEG C, 0.4 × 105Pa vacuums dry 16h,
Collection obtains aspergillus niger xerospore powder.
2. the preparation method described in claim 1, wherein corncob particle are with aspergillus niger spore nutrient solution volume ratio by weight
1:It is most suitable for aspergillus niger spore propagation when 3.5.
3. the preparation method described in claim 1, wherein it through the concentration obtained by culture, washing is 5 that described aspergillus niger spore, which is,
×109CFU/mL ~6×109CFU/mL spore suspension.
4. using application of the aspergillus niger xerospore powder prepared by claim 1 in terms of aspergillus niger spore powder survival rate is improved；
Its survival rate is up to 95.81%, and be not used it is protectant in the case of, Conidia persistence only 15.23%.
5. using application of the aspergillus niger xerospore powder prepared by claim 1 in terms of extension strain storage period；Prepared
Aspergillus niger xerospore powder stored 1 year at 4 DEG C after work spore rate still up to 90.2%.
6. the composite protectant in preparation method described in claim 1；Its composite protectant feature group turns into：The g/ of trehalose 5
The g/100mL and glycerine 2g/100mL ~ 4g/100mL of the g/100mL of 100mL ~ 10, the g/100mL of peptone 5 ~ 10.
7. the method that corncob described in claim 1 carries out sporogony as carrier adsorption, it is characterised in that corncob is warp
Volume is ground into for 1cm3Corncob, add solid-state fermenter, add inoculum concentration be 104~105CFU/mL aspergillus niger spores
Aspergillus niger nutrient solution, w/v is 1:3.5, calculated with aspergillus niger nutrient solution volume.
8. a kind of drying means of aspergillus niger spore powder described in claim 1, it is characterised in that：35℃、0.4×105Pa vacuums
Lower dry 16h.
9. the aspergillus niger xerospore powder prepared using the method described in claim 1 is in increase sporogony spatial table area and molten
Oxygen, improves the application in terms of sporogony.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710381711.1A CN107058209A (en) | 2017-05-26 | 2017-05-26 | A kind of use carrier adsorption carries out aspergillus niger spore propagation and its preparation method of xerospore powder |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710381711.1A CN107058209A (en) | 2017-05-26 | 2017-05-26 | A kind of use carrier adsorption carries out aspergillus niger spore propagation and its preparation method of xerospore powder |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107058209A true CN107058209A (en) | 2017-08-18 |
Family
ID=59611171
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710381711.1A Pending CN107058209A (en) | 2017-05-26 | 2017-05-26 | A kind of use carrier adsorption carries out aspergillus niger spore propagation and its preparation method of xerospore powder |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107058209A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107365735A (en) * | 2017-08-30 | 2017-11-21 | 武汉工控工业技术研究院有限公司 | A kind of preparation method of mycotic spore product |
| CN107815421A (en) * | 2017-12-08 | 2018-03-20 | 江苏国信协联能源有限公司 | A kind of aspergillus niger seed culture and its method for preparing citric acid |
| CN108410792A (en) * | 2018-03-01 | 2018-08-17 | 江苏国信协联能源有限公司 | The preparation method of introduces a collection spore microbial inoculum in a kind of aspergillus niger yeast making process |
| CN108485993A (en) * | 2018-07-11 | 2018-09-04 | 佛山皖阳生物科技有限公司 | A kind of preparation method of biological adsorption ocean black-koji mould pompon |
| CN110982712A (en) * | 2020-01-04 | 2020-04-10 | 广东环凯生物科技有限公司 | Stabilizing agent for aspergillus niger spores and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103194398A (en) * | 2013-02-27 | 2013-07-10 | 日照鲁信金禾生化有限公司 | Citric-acid high-yield strain and screening method thereof |
| CN105695341A (en) * | 2016-03-16 | 2016-06-22 | 江苏国信协联能源有限公司 | Mouldy bran culture medium for enlarged culture of aspergillus niger and culture method |
-
2017
- 2017-05-26 CN CN201710381711.1A patent/CN107058209A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103194398A (en) * | 2013-02-27 | 2013-07-10 | 日照鲁信金禾生化有限公司 | Citric-acid high-yield strain and screening method thereof |
| CN105695341A (en) * | 2016-03-16 | 2016-06-22 | 江苏国信协联能源有限公司 | Mouldy bran culture medium for enlarged culture of aspergillus niger and culture method |
Non-Patent Citations (1)
| Title |
|---|
| SHULIN CHEN,ET AL: "High Production of β-Glucosidase by Aspergillus niger on Corncob", 《APPPLIED BIOCHEMISTRY AND BIOTECHNOLOGY》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107365735A (en) * | 2017-08-30 | 2017-11-21 | 武汉工控工业技术研究院有限公司 | A kind of preparation method of mycotic spore product |
| CN107815421A (en) * | 2017-12-08 | 2018-03-20 | 江苏国信协联能源有限公司 | A kind of aspergillus niger seed culture and its method for preparing citric acid |
| CN107815421B (en) * | 2017-12-08 | 2020-06-05 | 江苏国信协联能源有限公司 | Aspergillus niger seed culture and citric acid preparation method |
| CN108410792A (en) * | 2018-03-01 | 2018-08-17 | 江苏国信协联能源有限公司 | The preparation method of introduces a collection spore microbial inoculum in a kind of aspergillus niger yeast making process |
| CN108485993A (en) * | 2018-07-11 | 2018-09-04 | 佛山皖阳生物科技有限公司 | A kind of preparation method of biological adsorption ocean black-koji mould pompon |
| CN110982712A (en) * | 2020-01-04 | 2020-04-10 | 广东环凯生物科技有限公司 | Stabilizing agent for aspergillus niger spores and application thereof |
| CN110982712B (en) * | 2020-01-04 | 2023-04-11 | 广东环凯生物科技有限公司 | Stabilizing agent for aspergillus niger spores and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107058209A (en) | A kind of use carrier adsorption carries out aspergillus niger spore propagation and its preparation method of xerospore powder | |
| CN106635820B (en) | A kind of Aspergillus niger strain of high yield theabrownin and its application | |
| CN104388514A (en) | Method for preparing gamma-aminobutyric acid by virtue of fermentation of composite bacteria | |
| CN103695341B (en) | A kind of algin catenase secreted by marine bacteria and preparation method thereof | |
| CN102965418A (en) | Extraction method for biological oat polypeptide and applications thereof | |
| CN109825444B (en) | Strain for improving food safety of red yeast rice yellow wine and brewing method thereof | |
| CN105766382A (en) | Antrodia cultivating method capable of improving content of triterpene | |
| CN102212479B (en) | Method for pretreating beer waste for biochemical utilization | |
| CN104403953B (en) | A kind of feed S. cervisiae high density fermentation culture medium formula and its application | |
| CN103602590A (en) | Method of preparing functional monascus mycelia and fermentation liquor by liquid fermentation, and products of the monascus mycelia and the fermentation liquor | |
| CN103689550A (en) | Ganoderma lucidum functional food and preparation method thereof | |
| CN102845225A (en) | Hypsizygus marmoreus liquid strain fermenting technique | |
| CN104046569B (en) | Aspergillus tubingensis for high-yield production of glucoamylase, alpha-amylase and acidic protease and application thereof | |
| CN103289925B (en) | Lactic acid bacteria strain for high yield of amylase and application thereof | |
| CN103045487B (en) | Bacterial strain for producing citric acid and method for fermenting and producing critic acid through fermentation of bacterial strain | |
| CN105255745A (en) | Rhizopus for producing beta-glucosidase and method of producing enzyme through rhizopus fermentation | |
| CN107418909A (en) | One plant of marine source bacillus licheniformis is used for the biological reinforced method of vinegar | |
| CN108203729B (en) | Preparation method of kelp antioxidant peptide | |
| CN109576182A (en) | A kind of strong resistance Lactobacillus rhamnosus A-4 and application thereof | |
| CN102986536B (en) | A kind of flammulina velutipes strain and preparation method | |
| CN106035985A (en) | Method for producing single cell proteins by using processed waste from mixed bacteria liquid fermentation of yellow wine | |
| KR20070116202A (en) | Wine preparation using extract of sea tangle(laminaria japonica) and it's powder | |
| CN103497896B (en) | A kind of dehydration pit mud functional bacteria protective material and application thereof | |
| CN112522114B (en) | Cordyceps militaris fungus chaff extract, ganoderma lucidum fermentation product, and preparation methods and applications thereof | |
| CN105647837A (en) | Complex microbial inoculant for pickled vegetable fermentation and application of complex microbial inoculant |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |