CN104745644B - Antierythrite manufacturing method, clump stalk spore yeast variant strain and application thereof - Google Patents
Antierythrite manufacturing method, clump stalk spore yeast variant strain and application thereof Download PDFInfo
- Publication number
- CN104745644B CN104745644B CN201410831742.9A CN201410831742A CN104745644B CN 104745644 B CN104745644 B CN 104745644B CN 201410831742 A CN201410831742 A CN 201410831742A CN 104745644 B CN104745644 B CN 104745644B
- Authority
- CN
- China
- Prior art keywords
- antierythrite
- culture
- glucose
- bacterial strain
- manufacture
- 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.)
- Active
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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
-
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/32—Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
-
- 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
- C12N2523/00—Culture process characterised by temperature
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
Abstract
The present invention provides antierythrite manufacturing method, clump stalk spore yeast variant strain and application thereof.The present invention provides the culture media composition of the machining object comprising glucose mother liquid and corn impregnation liquid or corn impregnation liquid, and the method for manufacturing antierythrite by antierythrite production bacterial strain using the culture media composition.In addition, the present invention provides the clump stalk spore yeast SYC ERY1 bacterial strains with antierythrite production capacity and the antierythrite manufacture biocatalyst composition comprising it.
Description
Technical field
The present invention relates to comprising glucose mother liquid (hydrol) and corn impregnation liquid (Corn steep liquor, CSL) or
The culture media composition and use the composition of the machining object of corn impregnation liquid manufacture antierythrite by antierythrite production bacterial strain
Method.In addition, the present invention relates to antierythrite production capacity clump stalk spore yeast SYC-ERY1 bacterial strains and comprising its
Antierythrite manufacture biocatalyst composition.
Background technology
Antierythrite is both as tetrose alcohol, with oligosaccharide (oligosaccharide), xylitol (xylitol) one
Representative functional sweetener, and be present in the plants such as the fermented foods such as grape wine or salty sauce, mushroom and fruit,
The natural materials of the body fluid of mammal.The sugariness of antierythrite is the 70~80% of sugar, is less than the 10% of sugar as heat
The low calorie sweetener material of (0.3Kcal/g) will not be used as the energy, and is largely discharged by urine in vivo.When dissolving
It plays heat-absorbing action and there is refrigerant sense, solubility is low and easy tos produce crystallization, and hygroscopicity and parasitics are low and can be applied to
All food.Further, since can not be by Streptococcus mutans (Streptococcus mutants) profit as saprodontia pathogen
With, therefore not will produce acid or glucan, to the feature also with preventing decayed tooth.Moreover, compared with previous glycitols, even if
Huge uptake also seldom induces diarrhea.Due to these properties, antierythrite quilt in requiring refrigerant sense and crystalline candy etc.
As sugar-free raw material.
For the production of antierythrite, there is extraction method, chemical synthesis and fermentation method.Wherein, for extraction method,
Due to only existing the antierythrite of denier under natural conditions in fruit or vegetables etc., do not have industrial economy；With regard to changing
For learning synthetic method, since raw material valence is high, is generated in building-up process by-product is not easy to remove, yield is low, do not have
Economy is not used to mass production.Therefore, most antierythrite is produced by fermentation method.
It is mainly carried out by yeast using the production of the antierythrite of fermentation method, so far, having reported can produce
The yeast of antierythrite.Wherein, although Moniliella tomentosa var.pollinis can be received with so-called 41% height
Industrialization is also not implemented by glucose production antierythrite, but due to generating a large amount of glycerine, ribitol etc. in rate.Though
Right Aureobasidium (Aureobasidium sp.) can also produce antierythrite, but productivity and sugar tolerance are poor.
In addition to this, the manufacturing method as the antierythrite using fermentation method utilizes Pseudozyma although existing
The method (Ebrean Registered Patent 0434518) of tsukubaensis utilizes the method side of Trichosporonoides madida
Method (Ebrean Registered Patent 0210958) utilizes the method for class trichosporon cutaneum (Trichosporonoides megachilensis)
Method (US publication 2001-0008769), but when using Pseudozyma sp. and class trichosporon cutaneum, safety has
It is likely to become problem.It can be used for regard to the class trichosporon cutaneum the case where, but yield and transition rate are low, therefore industrial economy
Property is poor.
On the other hand, in the production using the antierythrite of fermentation, the glucose as main material is in manufacturing cost
Occupy the overwhelming majority.In this case, in order to reduce manufacturing cost, the yield and productivity that improve antierythrite are needed,
And need to reduce the price of main material.This is most basic in the industry of production antierythrite and is asking for the task of top priority
Topic.
In this context, the present invention is confirmed by using the by-product generated during manufacturing glucose i.e.
By-product when dehydration mother liquor and manufacture sorghum starch replaces glucose as matrix, can admirably manufacture antierythrite,
And it confirms and improves the yield of antierythrite and productive mutant strain is suitable for by so that clump is obstructed the mutation of spore yeast strain
Antierythrite is manufactured, antierythrite productivity is improved while manufacturing cost so as to reduce in being produced in antierythrite, by
This completes the present invention.
Invention content
Project to be solved
There is provided a kind of method of manufacture antierythrite comprising including glucose mother liquid and the leaching of corn impregnation liquid or corn
The step of antierythrite production bacterial strain is cultivated in the culture medium of the machining object of stain liquid.
It is another object of the invention to provide the antierythrites for producing antierythrite to produce strain culturing culture medium
Composition, it includes the machining objects of glucose mother liquid and corn impregnation liquid or corn impregnation liquid.
It is another object of the invention to provide the clumps with antierythrite production capacity to obstruct spore yeast SYC-ERY1 bacterial strains
(preserving number KCTC18261P).
It is another object of the invention to provide antierythrite manufacture biocatalyst compositions, and it includes clumps to obstruct spore ferment
Female SYC-ERY1 bacterial strains.
The method to solve the problem
As a mode for achieving the above object, the present invention relates to a kind of method of manufacture antierythrite, packets
It includes and cultivates antierythrite production in the culture medium of the machining object comprising glucose mother liquid and corn impregnation liquid or corn impregnation liquid
The step of bacterial strain.
Preferably, above-mentioned culture medium can include Portugal in such a way that the concentration of glucose in culture medium is 5 to 45 (w/v) %
Grape sugar mother liquor.
Moreover it is preferred that above-mentioned glucose mother liquid can include 700 to 900g/L glucose.
Moreover it is preferred that above-mentioned culture medium can include the corn impregnation liquid or corn impregnation liquid of 0.5 to 5 (w/v) %
Machining object.
Corn impregnation liquid is used moreover it is preferred that directly can use or concentrate, or can be used by being soaked to corn
Supernatant obtained by stain liquid is centrifuged or precipitates is with water with 3:1~1:The corn impregnation liquid that 3 ratio mixes
Machining object.Moreover it is preferred that above-mentioned incubation step can be implemented at a temperature of 30~35 DEG C.
Moreover it is preferred that above-mentioned incubation step can be in the lower implementations of pH 5 to 6.
Moreover it is preferred that above-mentioned incubation step can be implemented 3 to 10 days.
Moreover it is preferred that above-mentioned incubation step can be the culture of batch-type, Fed-batch or continous way form.
Moreover it is preferred that in above-mentioned incubation step, if when culture in fermentation tank concentration of glucose become 50g/L with
Under, then intermittent charging (feeding) glucose mother liquid.
Moreover it is preferred that above-mentioned charging glucose mother liquid can make in above-mentioned fermentation tank concentration of glucose remain 10 to
100g/L。
Moreover it is preferred that above-mentioned culture medium can further include yeast extract (Yeast extract).
Moreover it is preferred that above-mentioned culture medium can be in culture medium comprising more than 0 and less than or equal to 0.5 (w/v) %'s
Yeast extract.
Moreover it is preferred that above-mentioned antierythrite production bacterial strain can carry out kind of a bacterium culture before above-mentioned incubation step
Obtained by (seed culture).
Moreover it is preferred that above-mentioned kind of bacterium culture can be comprising selected from by containing glucose, potassium nitrate (KNO3), di(2-ethylhexyl)phosphate
Hydrogen potassium (KH2PO4), manganese sulfate (MnSO4) and thiamine (Thiamine) composition one or more of group culture medium in carry out
Culture.
In addition, it is highly preferred that above-mentioned kind of bacterium culture can cultivate 1 to 3 day at pH 5 to 6,28 to 32 DEG C of temperature.
Moreover it is preferred that above-mentioned antierythrite production bacterial strain can be selected from by clump stalk spore yeast (Moniliella
Pollinis), Moniliella tomentosa var.pollinis, Moniliella acetoabutens, Aureobasidium
(Aureobasidium sp.), Trichosporonoides madida, candida magnoliae (Candida magnolia),
In torulopsis (Torula sp.), pichia (Pichia sp.) and the group of Pseudozyma tsukubaensis
More than one.
Moreover it is preferred that above-mentioned antierythrite production bacterial strain can be Monilia (Moniliella sp.).
Moreover it is preferred that above-mentioned antierythrite production bacterial strain can be clump stalk spore yeast.
Moreover it is preferred that above-mentioned antierythrite production bacterial strain can be clump stalk spore yeast SYC-ERY1 bacterial strains.
As another mode, the present invention relates to produce strain culturing culture for producing the antierythrite of antierythrite
Base composition, it includes the machining objects of glucose mother liquid and corn impregnation liquid or corn impregnation liquid.
Preferably, above-mentioned antierythrite production bacterial strain can be clump stalk spore yeast SYC-ERY1 bacterial strains.
As another mode, the present invention relates to the clumps with antierythrite production capacity to obstruct spore yeast SYC-ERY1 bacterium
Strain.
As another mode, the present invention relates to antierythrite manufacture biocatalyst compositions, and it includes clumps to obstruct spore
Yeast SYC-ERY1 bacterial strains.
Hereinafter, the present invention is described in more detail.
In a specific embodiment of the present invention, it cultivates honeycomb and filters out 4 kinds of bacterial strains of production antierythrite, from wherein sieving
Select a kind of most excellent bacterial strain (embodiment 1) of antierythrite yield.Later, to Korea Institute of Bioengineering microbial resources
Center application identification confirms that the bacterial strain of screening is that clump obstructs spore yeast strain (embodiment 2).
So that the clump of above-mentioned screening is obstructed spore yeast strain and ultraviolet mutation occurs, detaches and obstruct spore as the clump of the bacterial strain of the present invention
Yeast SYC-ERY1 bacterial strains confirm antierythrite yield.Its result confirms, compared with the mother strains before mutation, antierythrite
Yield and transition rate admirably increase (experimental example 1).
In addition, setting different yeast extract additive amounts and temperature condition to the bacterial strain of the present invention, antierythrite is understood
Yield and transition rate.Its result confirms, although observing red moss in a variety of yeast extract additive amount ranges and temperature range
Production of sugar polyol and transformation, but especially the case where adding the yeast extract of 0.5% (w/v) and the case where 35 DEG C of temperature
Under, antierythrite yield and transition rate are excellent (experimental example 2 to 4).
In another embodiment of the invention, using the machining object of glucose mother liquid and corn impregnation liquid or corn impregnation liquid
It is made whether that the experiment of antierythrite can be produced.Its result can be confirmed, although in corn impregnation liquid or corn impregnation liquid
Machining object a variety of usage amount ranges observe antierythrite production and transformation, but especially add it is total relative to culture medium
In the case that amount is the corn impregnation liquid of 1 (w/v) % or the machining object of corn impregnation liquid, antierythrite yield and transition rate are excellent
(experimental example 5).
In another embodiment, carry out confirming the experiment of the condition of production of antierythrite in 5L fermentation tanks.Its result can
To confirm, compared with the case where using glucose, antierythrite yield, transition rate and life using hydrol
Production property similar or excellent (experimental example 6).
In addition, in the culture medium of machining object and yeast extract for being mixed with corn impregnation liquid or corn impregnation liquid, it is real
Apply antierythrite production.Its result confirms, especially in the yeast extraction for adding the CSL and 0.25 (w/v) % of 1 (w/v) %
In the case of object, antierythrite yield and productivity are excellent (experimental example 7).
In another embodiment of the invention, implement production of the understanding according to the antierythrite of glucose mother liquid charging process
Experiment.Its result confirms, and glucose mother is added in such a way that concentration of glucose is 50g/L or 100g/L in Initial stage of culture
When liquid, culture if concentration of glucose in fermentation tank becomes 50g/L or less in the case of intermittent charging glucose mother liquid, to the greatest extent
Pipe incubation time is short, and the yield and productivity of glucose mother liquid are also excellent (experimental example 8 and 9).
Further, in another embodiment of the invention, glucose mother liquid charging is persistently carried out to carry out fermentation until nothing
Method further utilizes glucose.Its result confirms that the yield of antierythrite is 250g/L, red compared with interrupting the case where fermenting
Moss sugar alcohol yield increases, and transition rate is similar (experimental example 10).
It thereby confirms that, in the culture medium of the by-product comprising such as glucose mother liquid and CSL etc, is sent out by microorganism
Ferment can admirably manufacture antierythrite.In addition it proves, the antierythrite production capacity that clump obstructs spore yeast SYC-ERY1 bacterial strains is excellent
It is different, it is used as matrix especially by by glucose mother liquid and CSL, can admirably produces antierythrite.
Accordingly, above-mentioned clump stalk spore yeast SYC-ERY1 bacterial strains were deposited in South Korea's life engineering on November 5th, 2013
Research institute's microbial resources center obtains preserving number KCTC18261P, and has been carried out by initial on November 28th, 2014
Preserving number KCTC18261P is converted to the application of the preservation under budapest treaty, to obtain preserving number KCTC12720BP.
In an embodiment of the invention, a kind of method of manufacture antierythrite is provided comprising including grape
The step of antierythrite production bacterial strain is cultivated in the culture medium of the machining object of sugared mother liquor and corn impregnation liquid or corn impregnation liquid.
In this application, so-called term " antierythrite " refer to sugariness be sugar 70 to 80% or so, have refrigerant sweet taste
White crystalline powder sweetening material, chemical formula C4H10O4.Specifically, in the present invention, antierythrite refers to leading to
The antierythrite of everfermentation method production, it is preferable that it can be by glucose mother liquid and corn impregnation liquid or corn impregnation liquid
The antierythrite that machining object is produced as matrix, by microbial fermentation.Can pass through antierythrite as an example
The antierythrite of bacterial strain production is produced, and can be the antierythrite for obstructing the production of spore yeast SYC-ERY1 bacterial strains by clump.On
State antierythrite can using the present invention method or by culture the present invention bacterial strain obtained from cell, culture or its carry
Object is taken to obtain, by bacterial strain fermentation liquor by filtering, refining, crystallize or water-washing process and obtain after drying, but not limited to this.
Antierythrite produces bacterial strain as the bacterial strain with antierythrite production capacity, refers to utilizing Portugal in the present invention
The machining object of grape sugar mother liquor and/or corn impregnation liquid or corn impregnation liquid can produce the antierythrite production bacterium of antierythrite
Strain.For example, when the bacterial strain of antierythrite can be produced compared with 26S rRNA sequences, has preferably 70% or more, is more excellent
The microorganism for being selected as 80% or more, further preferably 90% or more, most preferably 95% or more sequence homology belongs to
The scope of the present invention.As an example, clump stalk spore yeast strain be antierythrite production bacterial strain, when clump stalk spore yeast strain with
When 26S rRNA sequences compare, have preferably 70% or more, more preferably 80% or more, further preferably 90% or more,
The microorganism of most preferably 95% or more sequence homology all belongs to the scope of the present invention.
So-called term " matrix (substrate) " refers to that antierythrite production bacterial strain uses to produce antierythrite
Raw material.It can be glucose itself as an example, can also be the raw material comprising glucose.In the present invention, as
Matrix has used glucose mother liquid, but not limited to this, as long as antierythrite production bacterial strain makes for the production of antierythrite
Raw material just belongs to the scope of the present invention.
In the present specification, so-called " glucose mother liquid " refers to being manufactured by corn and other starch saccharification (hydrolysis)
The dehydration mother liquor that is additionally generated during glucose and utilize the water such as the washing water used when manufacture glucose and ejected wash water
All liq with identical pol after dissolving glucose.Glucose mother liquid usually contains 700 to 900g/L glucose.This hair
Glucose mother liquid used in bright includes the glucose of about 870g/L degree.Therefore, glucose mother liquid can be used as erythrose
Alcohol production bacterial strain is used to produce the matrix of antierythrite.
As an embodiment, the culture medium of culture antierythrite production bacterial strain can be dense with the glucose in culture medium
The mode that degree is 5 to 45 (w/v) % includes glucose mother liquid.In the case where the concentration of glucose is less than 5 (w/v) %, occur
The phenomenon of concentration of glucose deficiency, leads to cellular damage in culture medium, it is possible to so that cell activity is reduced, in the concentration of glucose
In the case of 45 (w/v) %, hyperosmosis caused by the glucose due to high concentration and hinder microorganism growth and development, have
It may make the reduction of antierythrite speed of production.Specifically, can be with concentration of glucose for 10 to 45 (w/v) %, 5 to 10 (w/
V) mode of % includes, as long as belonging to the glucose concentration range of 5 to 45 (w/v) %, there is no limit belong to the present invention's
Range.
So-called term " corn impregnation liquid (corn steep liquor, CSL) " is obtained during manufacturing cornstarch
The by-product arrived or its concentrate refer to the solution comprising a large amount of amino acid, 3 to 10 (w/v) % nitrogen and other substances.In general,
Corn impregnation liquid can be after corn steeping is removed corn after the water containing sulfurous acid in the manufacturing process of cornstarch
Obtained maceration extract.It in the present invention, can be using corn impregnation liquid come when replacing producing antierythrite by normal fermentation method
Used yeast extract.
As an embodiment, the culture medium of culture antierythrite production bacterial strain can include 0.5 to 5 (w/v) %'s
The machining object of corn impregnation liquid or corn impregnation liquid.It is less than 0.5 in the content of the machining object of corn impregnation liquid or corn impregnation liquid
(w/v) in the case of %, compared with the amount of microorganism nitrogen source needed for growth and development, nitrogen source is insufficient in culture medium, microorganism life
Long development is slack-off, it is possible to produce the influence for keeping antierythrite speed of production slack-off, more than 5 (w/v) %, due to
Nitrogen is excessive in culture medium, therefore microbes are hindered and growth and development and activity is made to reduce.Specifically, corn impregnation liquid
Or the machining object of corn impregnation liquid can be 0.5 to 7 (w/v) %, 0.5 to 1 (w/v) %, 1 to 3 (w/v) %, 0.5 to 3 (w/
V) %.As specific example, in the feelings that the machining object of corn impregnation liquid or corn impregnation liquid and yeast extract are used together
Can include the corn impregnation liquid of 1 (w/v) % or the machining object of corn impregnation liquid under condition, in culture medium, at this point, can in culture medium
To include the yeast extract of 0.25 to 0,5 (w/v) %.
In an embodiment of the invention, above-mentioned corn impregnation liquid or its machining object, corn impregnation liquid can be used
Machining object can be not directly using corn impregnation liquid and will by corn impregnation liquid centrifuge or precipitate obtained by
What clear liquid was obtained by mixing with water.At this point, passing through the mixing of supernatant and water obtained by corn impregnation liquid is centrifuged or precipitated
Ratio can be 1:3 to 3:1.It is higher than in the containing ratio by supernatant obtained by corn impregnation liquid is centrifuged or precipitated
In the case of above range, it is possible to make microorganism growth and development hindered because of the suspended matter in corn impregnation liquid, logical
It crosses by corn impregnation liquid centrifugation or in the case that the containing ratio of supernatant obtained by precipitating is less than above range, the ratio of water
Rate increases, and in contrast, the amount of corn impregnation liquid or its machining object is reduced, it is possible to make nitrogen source supply nothing needed for growth and development
Method is smoothly realized.Specifically, can be 1:2、1:1、2:1, but not limited to this.
For the purposes of the present invention, can include glucose mother liquid in culture antierythrite produces the culture medium of bacterial strain
With corn impregnation liquid or its machining object, glucose mother liquid is by-product when producing glucose, corn impregnation liquid or its machining object
It is to generate the residue left after starch, therefore be dirt cheap.As a result, if using glucose mother liquid and corn impregnation liquid or its
Machining object carries come used glucose and yeast as high price matrix when replacing producing antierythrite according to normal fermentation method
Object is taken, then can establish the especially high process of economy in culture.
In the present invention, glucose mother liquid and corn impregnation liquid or its machining object can be used separately as antierythrite production bacterium
Strain is used for producing the carbon source and nitrogen source of antierythrite, and the present invention not only provides carbon source and nitrogen source by by-product, but also establishes and make
Method with by-product production than previous excellent antierythrite.
Alternatively, culture antierythrite production bacterial strain can further include yeast extract.Yeast carries
It takes object as the substance as bacterial strain nitrogen source, can be used for providing for the culture of bacterial strain together with corn impregnation liquid or its machining object
Sufficient nutrient source.Can include to be more than 0 and the above-mentioned yeast extract less than or equal to 0.5 (w/v) % in culture medium.
Yeast extract may have microorganism just less than above-mentioned content range or in the case of being added without yeast extract completely
It is frequently grown the problem of development itself can not be realized well, more than 0.5 (w/v) %, although microorganism growth and development
It is active, but with the tendency for substantially laying particular emphasis on growth and development is metabolized, consequently, it is possible to there are the reductions of target antierythrite productivity
The problem of.Specifically, can include more than 0 and less than or equal to the above-mentioned ferment of 0.25 (w/v) %, 0.25 to 0.5 (w/v) %
Female extract.
As an embodiment of the invention, the step of antierythrite production bacterial strain is cultivated in the culture medium comprising matrix
Suddenly can 30 to 37 DEG C, preferably at 30 to 35 DEG C at a temperature of implement.In the case where temperature is less than 30 DEG C, there are microorganisms
The problem of growth rate is significantly slack-off, and antierythrite productivity is greatly reduced, more than 37 DEG C, due to being
It is unsuitable for the high temperature of the temperature of microorganism growth and development, it is thus possible to be damaged or be killed because of high temperature there are microorganism
Problem.Specifically, can be 35 to 37 DEG C, 30 DEG C to 35 DEG C, it can be admirably but as long as belonging to antierythrite production bacterial strain
The temperature range of antierythrite is produced with regard to not limited.
In addition, the step of cultivating antierythrite production bacterial strain in the culture medium comprising matrix can be lower real in pH 5 to 6
It applies.If pH in culture medium is less than 3 or is impacted to microorganism growth and development more than 7, pH, to make bacterial strain almost without
Method is grown.Specifically, if the antierythrite production bacterial strain of the present invention does not adjust pH in culture, pH would generally be about to certainly
4 or so are maintained at, so as to growth and development.In other words, when pH is 4 to 7, although antierythrite productivity reduces, not
Microorganism growth and development can be impacted.However, for effective production of antierythrite, preferably in the pH (pH5 of above range
~6) it is cultivated under.For example, it may be pH 5 to 5.5, pH 5.5 to 6 or about pH 5.5, as long as belonging to antierythrite production
Bacterial strain can admirably produce the pH ranges of antierythrite with regard to not limited.
In the present invention, the step of cultivating bacterial strain can implement 3 to 9 days.For example, the step of above-mentioned culture bacterial strain, can be into
Row about 5 to 8 days or about 6 to 7 days, but not limited to this.As an example, in the case where persistently being cultivated, antierythrite
Speed of production or glucose consumption rate may be decreased, but final antierythrite yield can be made to increase.
Alternatively, the step of antierythrite produces bacterial strain being cultivated in the culture medium comprising matrix can be
The culture of batch-type, Fed-batch or continous way form.
So-called batch-type culture refers to cultivating as follows：In the reactive tanks such as fermentation or enzymatic conversion, using microorganism or enzyme into
In the case of row reaction, make its reaction after the liquid containing starting material and microorganism or enzyme are all added, it later, will be anti-
It is recycled obtained by answering, the culture of water-washing process is undergone to prepare lower secondary response again.In the present invention, can be by
Antierythrite production bacterial strain be used for produce antierythrite medium component all make an addition in fermentation tank cultivated after through going through
Recycle the culture of the process of culture solution.
So-called Fed-batch culture is a kind of method of batch-type culture, refers to that organic nutrition is added in incubation
Ingredient improves the productive culture of desired product.In the present invention, it can be the culture that bacterial strain is produced at antierythrite
The culture of glucose mother liquid is added in journey, but not limited to this, can be added in the incubation that antierythrite produces bacterial strain
The machining object of corn impregnation liquid or corn impregnation liquid, glucose, yeast extract etc. be used for increase antierythrite production at
The culture divided.
So-called continous way culture refers to persistently providing new nutrient medium, while lasting removal includes cell and production
The culture of the culture solution of object.For example, it may be in the incubation that antierythrite produces bacterial strain, grape is constantly provided on one side
Sugared mother liquor, on one side removal produce the culture of the culture solution of antierythrite, but not limited to this.
In the present invention, so-called term " charging " refers to that the culture medium that bacterial strain is produced to culture antierythrite injects grape
Sugared mother liquor adjusts the concentration of glucose in culture medium.For example, if the concentration of glucose in culture medium become 50g/L hereinafter,
Glucose mother liquid then is injected to culture medium, but the concentration of glucose benchmark in the culture medium for the glucose mother liquid that feeds can be
100g/L、200g/L、450g/L。
As an embodiment of the invention, charging intermittent can be implemented, and intermittence is implemented charging and meant, can
To implement charging repeatedly at a certain time interval, in the present invention, the certain time interval that intermittence implements charging can be with
Referring to concentration of glucose becomes certain benchmark time below.
By above-mentioned intermittent charging, the concentration of glucose in fermentation tank can be maintained to 10 to 100g/L degree.Tool
It says to body, can be 40 to 80g/L, 45 to 70g/L.In a specific embodiment of the present invention, if when cultivating in fermentation tank
Concentration of glucose become 50g/L hereinafter, then intermittent charging glucose mother liquid and the concentration of glucose in culture solution is remained
50 to 80g/L.Intermittent charging in this way, can make the concentration of glucose in fermentation tank be maintained above range, to
Purpose according to the present invention can admirably manufacture antierythrite.
It in an embodiment of the invention, can be before being fermented to produce antierythrite to erythrose
Alcohol production bacterial strain carries out kind of a bacterium culture.For above-mentioned kind of bacterium is cultivated, before formally starting fermentation, the activity of bacterial strain can be made
Increase, be fully proliferated to implement antierythrite fermentation, so as to admirably produce antierythrite.In the present invention, kind bacterium training
Supporting can implement in the container (for example, flask etc.) different from for producing the culture culture medium of antierythrite, in addition,
It can implement in container (for example, fermentation tank) identical with for producing the culture culture medium of antierythrite.
It is above-mentioned to include glucose (glucose), potassium nitrate (KNO for kind of a culture medium for bacterium culture3), biphosphate
Potassium (KH2PO4), manganese sulfate ((MnSO4), thiamine (Thiamine), but not limited to this, if it is suitable antierythrite produce bacterium
The ingredient of the culture of strain, then belong to the scope of the present invention.Specifically, can be comprising glucose 0.1 to 10 (w/v) %, ferment
Female 0.1 to 0.5 (w/v) % of extract (yeast extract), potassium nitrate 0.01 to 0.35 (w/v) %, potassium dihydrogen phosphate
0.001 to 0.025 (w/v) %, manganese sulfate 1 to 5ppm and thiamine 1 to 3ppm culture medium.
In addition, above-mentioned kind of bacterium culture can cultivate 1 to 3 day at 28 to 32 DEG C of pH 5 to 6 and temperature, but not limited to this,
It is contemplated that nutritional status etc. suitably changes in the activity of bacterial strain, concentration and culture medium.
In one embodiment of the present invention, the present invention relates to the antierythrites for producing antierythrite to produce strain culturing
With culture media composition, it includes the machining objects of glucose mother liquid and corn impregnation liquid or corn impregnation liquid.By-product will be used as
In the case that the glucose mother liquid and corn impregnation liquid of object or the machining object of corn impregnation liquid are contained in culture medium as matrix, energy
Enough be dirt cheap ground production medium.Specifically, above-mentioned culture media composition can be used for Monilia, clump obstructing spore ferment
Female, clump stalk spore yeast SYC-ERY1 bacterial strains are used to produce the fermentation of antierythrite.
In the present invention, antierythrite production bacterial strain refers to the bacterium that antierythrite can be produced as the metabolite of bacterial strain
Strain.Specifically, can be the microorganism of Monilia, you can be clump stalk spore yeast, Moniliella tomentosa
Var.pollinis, Moniliella acetoabutens, Aureobasidium, Trichosporonoides madida, lily magnolia are false
Silk yeast, Tolula sp., pichia (Pichia sp.) or Pseudozyma tsukubaensis, but not limited to this,
When above-mentioned bacterial strains are compared with 26S rRNA sequences, have preferably 70% or more, be more preferably 80% or more, be further preferred
Microorganism for 90% or more, most preferably 95% or more sequence homology all belongs to the scope of the present invention.For example, erythrose
Alcohol production bacterial strain can be that clump obstructs spore yeast, clump stalk spore yeast SYC-ERY1, but not limited to this.
In the present invention, the method for manufacturing antierythrite can obstruct spore yeast SYC-ERY1 bacterial strains to implement using clump.On
The dissociant that bacterial strain obstructs spore yeast as the clump with antierythrite production capacity is stated, obstructs spore yeast strain phase with other clumps
Than not only antierythrite production capacity is excellent, but also using as the by-product of matrix such as glucose mother liquid and/or CSL etc
Object, can be dirt cheap ground and admirably produce antierythrite.For example, for above-mentioned bacterial strains, as clump stalk spore yeast SYC-
When ERY1 bacterial strains are compared with 26S rRNA sequences (sequence number 1), have preferably 70% or more, more preferably 80% or more, into
One step is preferably that the microorganism of 90% or more, most preferably 95% or more sequence homology all belongs to the scope of the present invention.
As an embodiment of the invention, bacterial strain of the invention can be urged with being contained in antierythrite manufacture with biology
Form in agent composition provides.In above-mentioned biocatalyst composition, the production for making antierythrite can be further included
Amount or the increased ingredient of transition rate.
Invention effect
The present invention is provided is used as micro- life by the by-product of such as glucose mother liquid and/or corn impregnation liquid or its machining object etc
Object fermentation substrate manufactures the method for antierythrite and comprising such as glucose mother liquid and/or corn impregnation liquid or its machining object
Etc by-product culture media composition, and provide be suitable for the present invention antierythrite manufacturing method and antierythrite give birth to
The excellent clump of production capacity power obstructs spore yeast SYC-ERY1 bacterial strains.As a result, by by glucose mother liquid and corn impregnation liquid or its processing
Object is used as matrix, produces antierythrite with capable of being dirt cheap, if using above-mentioned bacterial strains, can make the yield of antierythrite
And productivity increases, and cultivated days can be shortened.
Description of the drawings
Fig. 1 shows the variations according to the OD values of charging (feeding) method of experimental example 8." Conventional feed introduction
(conventional feeding) " indicates culture and the feed conditions 1 of embodiment 8；" continuous charging (continuous
Feeding culture and the feed conditions 2 of embodiment 8) " are indicated；" interval charging (intermittent feeding) " indicates real
Apply culture and the feed conditions 3 of example 8.
Specific implementation mode
Hereinafter, embodiment according to the present invention is described in detail.But following embodiments are only used for illustrating the present invention, this
Invention is not limited to following embodiments.
The separation of 1. clumps of stalk spore yeast SYC-ERY1 mother strains of embodiment
Microorganism used in the present invention detaches according to following methods.Honeycomb is being included into 35 (w/v) % glucose and 1.5
(w/v) with 30 degree, the CMC model of 250rpm 6 days in the fluid nutrient medium of % yeast extracts.The culture solution of culture is smeared
In the solid medium of same composition, about 5000 bacterium colonies (colony) are obtained.
By the colony inoculation obtained by strain isolation in include 45 (w/v) % glucose, 0.5 (w/v) % yeast extraction
Object, 0.35 (w/v) %KNO3, 0.025 (w/v) %KH2PO4、5ppm MnSO4, 3ppm thiamines (Thiamine) 4 ml (examination
Pipe) culture medium (pH 5.5), and cultivated 6 days with 30 DEG C, 200rpm.
6 days culture solutions of culture are carried out centrifuging for 10 minutes at 13,000rpm, cell is removed and is only trained
Supernatant is supported, analyzes supernatant using thin-layer chromatography (TLC), screening antierythrite produces bacterial strain.
TLC analysis conditions are as follows.TLC plates (plate) use 60 F254 of TLC Silica gel, and developing solvent is second
Nitrile：Ethyl acetate：1- propyl alcohol：Water (water)=85:20:20:15 (v/v/v/v), by plate at alkaline (Alkaline) after expansion
AgNO3It is impregnated 5 minutes in solvent.Later, after being impregnated 30 minutes in methanol (MeOH) solution, rapid soaking is in 1.5M
Na2S2O3Solution simultaneously takes out, and color dot (spot) is confirmed after being completely dried.
By TLC, only the bacterium colony of screening production antierythrite passes through the sample of color dot distinctness in 5000 bacterium colonies
The amount of RI the analyses quantitative remaining glucose and the antierythrite produced of high performance liquid chromatography (HPLC).HPLC analysis conditions are such as
Shown in table 1, whole embodiments later are analyzed under the same conditions.HPLC analyses use Aminex HPC-87H columns,
Mobile phase solvent uses 0.0025N H2SO4, 65 DEG C of speed with 0.5ml/min on one side trickle solvent while use RI detectors
Analysis.
[table 1]
| Column | Rezex ROA- organic acid H+ columns (Phenomenex companies) |
| Solvent | 0.0025N H2SO4 |
| Flow velocity | 0.5ml/min |
| Temperature | 65℃ |
| Detector | RI detectors |
| Sample injected slurry volume | 10ul |
By HPLC, the bacterial strain of 4 production antierythrites has been screened in total, by 4 strain culturings in 250ml flasks
(flask), yield is reaffirmed.When 250ml flask cultures, using with carried out bacterium colony culture culture medium (antierythrite give birth to
Produce culture medium) identical culture medium, after, in all embodiments, only the amount of glucose and yeast extract changes, remaining
Medium component be carried out similarly.It is cultivated to 250ml flasks addition culture medium 25ml, is trained at 30 DEG C, 250rpm
It supports 9 days.After culture, compare antierythrite yield, a most excellent bacterial strain is filtered out from 4 bacterial strains.
Microorganism growth and development, which confirms, utilizes UV- spectrophotometry (UV-spectrophotometry), and culture solution is adopted
Sample and after collecting corresponding thalline, absorbance (optical density, OD) is measured at 600nm, culture solution is suitably diluted
It is measured after (10~100 times), so that absorbance is fallen into the range of 0.1~1.0.
The characteristic of the bacterial strain screened in 2. embodiment 1 of embodiment
In order to understand the characteristic of the bacterial strain screened in above-described embodiment 1 and identify bacterial strain, to Korean Collection
for Type cultures(KCTC)of Korea Research institute of Bioscience and
(South Korea's Culture Collection of South Korea's bioscience and biotechnology research institute is located at Taejon, Korea to Biotechnology
The scholar city section ways for education 125) apply and is identified.
The physio-biochemical characteristics of this bacterial strain are as follows：
1) sugared, organic acid anabolic
Soluble starch (Soluble starch) (+)
Xylose (D-xylose) (+)
L-arabinose (L-arabinose) (-)
Glycerine (Glycerol) (+)
2) areas the D1/D2 and ITS base sequence analysis of 26S rRNA genes
It investigates, since the sequence similarity of the CSB 461.67T bacterial strains with the reference culture for obstructing spore yeast as clump is
100%, therefore be identical bacterial strain.
3) the Physiology and biochemistry detection of investigation utilization of carbon source sex differernce
Show the utilization of carbon source characteristic of clump stalk spore yeast strain.
In summary result considers, the bacterial strain screened in above-described embodiment 1 can be accredited as clump stalk spore yeast strain.
*Experimental example 1. screens dissociant and confirms antierythrite yield
Ultraviolet mutation (UV mutation), which is carried out, by the bacterial strain (mother strains) that will be screened in embodiment 1 detaches mutation
Bacterial strain.Specifically, by the bacterial strain (wild type) screened in embodiment 1 in 2 (w/v) % glucose, 2 (w/v) % malt extracts, 0.1
(w/v) after being cultivated 2 days in % peptone culture mediums, ultraviolet mutation is carried out using culture solution.In ultraviolet mutation, bacterium is trained
Nutrient solution dilutes and is applied to ultraviolet mutation culture medium (45 (w/v) % glucose, yeast nitrogen).By the plate of smearing in carcinogenic item
After irradiating 5 minutes ultraviolet lights under part, is wrapped up with foil paper and cultivated in 30 DEG C of incubators (incubator), obtain bacterium colony.It obtains
Bacterium colony when strain isolation in the same manner as cultivate and carry out TLC analyses and HPLC RI analysis, in 250ml flask (culture mediums
Culture 9 days in 50ml), confirm yield (table 2).
[table 2]
* mother strains：The bacterial strain screened in embodiment 1
* control groups：Clump stalk spore yeast KCTC 6813
**SYC-ERY1：By being mutated the bacterial strain obtained in experimental example 1
As a result, in the bacterial strain of the bacterium colony obtained by ultraviolet mutation process, the yield of antierythrite dramatically increases,
To confirm mutant strain that transition rate also greatly improves.Above-mentioned mutant strain is named as clump stalk spore yeast SYC-ERY1, in
It is preserved in Korean Collection for Type cultures (KCTC) of Korea Research on November 5th, 2013
The institute of Bioscience and Biotechnology (South Korea of South Korea's bioscience and biotechnology research institute
Culture Collection is located at the Taejon, Korea scholar city section ways for education 125), obtain preserving number：KCTC18261P, and in
On November 28th, 2014 be converted to by initial preserving number KCTC18261P the application of the preservation under budapest treaty,
To obtain preserving number KCTC12720BP.The genetic analysis result of above-mentioned preservation strain clump stalk spore yeast SYC-ERY1：It analyzes
26s rRNA have the base sequence of sequence number 1.
The productivity of antierythrite of 2. dissociant of experimental example under different yeast extract additive amounts
Antierythrite production medium (0.35 (w/v) % added with 45% glucose is added in SYC-ERY1 bacterial strains
KNO3, 0.025 (w/v) %KH2PO4、5ppm MnSO4, 3ppm thiamines, pH5.5), and the amount for changing yeast extract come it is true
Recognize antierythrite yield.For condition of culture, cultivated 9 days at 30 DEG C, 250rpm.Table 3 is indicated in different yeast extracts
Additive amount under antierythrite yield.
[table 3]
It can be confirmed, wherein the yield of antierythrite and transformation in the case where adding 0.5% (w/v) yeast extract
Rate is excellent.
Experimental example 3. confirms again in the antierythrite production medium added with 0.5 (w/v) % yeast extracts
In order to know whether the result confirmed in above-mentioned experimental example 2 is reproducible, cultivated again with same procedure and true
Recognize antierythrite yield.Its cultivation results is as shown in table 4.
[table 4]
| Experiment number | Glucose (g/L) | Antierythrite (g/L) | Transition rate ((g/g) %) |
| 1 | 24 | 234 | 55 |
| 2 | 23 | 232 | 54 |
| 3 | 25 | 234 | 55 |
As a result, showing similar with the result of experimental example 2 as a result, reproducible to confirm.
Experimental example 4. is according to the production of the antierythrite of temperature
Implement to cultivate with condition identical with above-mentioned experimental example 3.But in order to know the influence of temperature, change culture temperature respectively
Degree is cultivated.All conditions (condition of culture, cultivated days, rpm) all same, and at 3 kinds of temperature conditions 30,35,37 DEG C
Implement.The results are shown in Table 5 for it.
[table 5]
| Temperature (DEG C) | Glucose (g/L) | Antierythrite (g/L) | Transition rate ((g/g) %) |
| 30 | 0 | 194 | 43 |
| 35 | 0 | 233 | 52 |
| 37 | 98 | 145 | 41 |
Its result confirms, and at 35 DEG C, the yield of antierythrite and transition rate are excellent.
Experimental example 5. utilizes the production of glucose mother liquid and the antierythrite of CSL
In order to reduce culture medium expense, using glucose mother liquid (Samyang Genex Co., Ltd.) and CSL, (lucky Buddhist nun is supported in (strain) three
Ke Si) carry out antierythrite production whether possible experiment.For CSL, since suspended matter is more, use will pass through centrifugation
The supernatant being isolated is with water with 1:1 ratio mixing is to dilute 2 times of CSL.Including control group (previous antierythrite life
Produce culture medium) inside altogether implement 6 kinds of conditions experimental group.In experimental group except for a control group, add relative to culture medium
Total amount replaces 0.5 (w/v) % yeast extracts to be tested by the CSL of 1,3,5,7,9 (w/v) %.Except yeast extract with
Outside, other medium components are similarly added.When analyzing experimental result, the glucose amount that is converted into glucose mother liquid.Culture
Implement at 30 DEG C, 250rpm.Experimental result is shown in the following table 6.
[table 6]
| Experiment condition | Glucose (g/L) | Antierythrite (g/L) | Transition rate ((g/g) %) |
| Control group | 2 | 220 | 49 |
| CSL 1 (w/v) % | 54 | 201 | 51 |
| CSL 3 (w/v) % | 155 | 88 | 30 |
| CSL 5 (w/v) % | 256 | 42 | 22 |
| CSL 7 (w/v) % | 297 | 33 | 22 |
| CSL 9 (w/v) % | 315 | 31 | 23 |
Its result confirms, in the case where addition is relative to the CSL that culture medium total amount is 1 (w/v) %, antierythrite production
Amount and transition rate are excellent.
Experimental example 6. confirms the antierythrite production in 5L fermentation tanks
Based on the result carried out in flask, carry out that the pattern experiment how in 5L fermentation tanks confirmed.In flask
With the CMC model kind bacterium (Seed) of experimental example 3, when the OD values when cell growth the become 10, (culture medium into 5L fermentation tanks
Volume 3L) 10 (v/v) % of inoculation.Change culture medium with following condition and implements culture.
<Culture medium condition 1>
Glucose mother liquid is added in such a way that concentration of glucose in culture medium is 450g/L, with a concentration of 1 (w/ in culture medium
V) mode of % adds CSL, and is cultivated.
<Culture medium condition 2>
Glucose mother liquid is added in such a way that concentration of glucose in culture medium is 450g/L, in culture medium a concentration of 0.5
(w/v) mode of % adds yeast extract, and is cultivated.
<Culture medium condition 3>
Glucose is added in such a way that concentration of glucose in culture medium is 450g/L, with a concentration of 0.5 (w/ in culture medium
V) mode of % adds yeast extract, and the condition cultivated.
For fermentation tank condition, it is kept stirring speed 600rpm, 35 DEG C of temperature, pH 5.5, ventilatory capacity 0.5vvm and carries out
Culture.Concentration of glucose confirms while sampling analysis, if glucose is down to 50g/L, female using glucose
In the case of the culture medium of the culture medium condition 1 and 2 of liquid by the glucose mother liquid containing 870g/L glucose, using glucose
Culture medium condition 3 culture medium in the case of by the Glucose Liquid of 800g/L charging (feeding) to culture solution so that training
Concentration of glucose is maintained near 50g/L in nutrient solution.Cultivation results are shown in table 7.
[table 7]
| Condition of culture | Antierythrite (g/L) | Transition rate (%) | Incubation time (h) | Productivity (antierythrite) |
| 1 | 220 | 48 | 146 | 1.5 |
| 2 | 243 | 49 | 126 | 1.9 |
| 3 | 230 | 45 | 146 | 1.6 |
Its result confirms, compared with the case where using glucose, the antierythrite using glucose mother liquid
Yield, transition rate and productivity are similar or excellent.
Experimental example 7. cultivates antierythrite in being mixed with the culture medium of CSL and yeast extract
Based on the result of experimental example 6, mixes CSL and yeast extract and cultivated.Since compound barm extracts
Object and the additive amount for reducing yeast extract.Half 0.25 (w/v) % of (w/v) % 0.5 before only using, CSL are using therewith
Preceding identical 1 (w/v) %.Glucose is not used, is all replaced by glucose mother liquid.All experiments later are similarly only
Glucose mother liquid is used.In addition, fermentation tank condition is all carried out similarly operation.As a result it is shown in table 8.
[table 8]
Its result confirms, in the case where adding (w/v) % of CSL 1 and yeast extract 0.25 (w/v) %, erythrose
Alcohol yield and productivity are excellent.
Experimental example 8. is produced according to the antierythrite of glucose mother liquid charging process
During being tested, the inventors of the present invention recognize that this bacterial strain can be influenced by concentration of glucose, thus into
Whether the following operation of confirmation of having gone correctly tests：Glucose mother is injected in such a way that glucose is 450g/L to culture medium
Liquid；And the concentration of above-mentioned concentration is kept below on one side, continue in the training period to keep corresponding concentration of glucose on one side
It feeds.Other experiment conditions with it is following identical, tested in 5L fermentation tanks.
<Culture and feed conditions 1>
Initial stage of culture (when beginning) adds glucose mother liquid in such a way that concentration of glucose is 450g/L, if sent out when culture
Concentration of glucose becomes 50g/L hereinafter, then intermittent charging glucose mother liquid is so that concentration of glucose is protected in culture solution in ferment slot
Hold the condition of culture near 50g/L.
<Culture and charging (feeding) condition 2>
Initial stage of culture (when beginning) adds glucose mother liquid in such a way that concentration of glucose is 50g/L, if sent out when culture
Concentration of glucose becomes 50g/L hereinafter, then intermittent charging glucose mother liquid is so that concentration of glucose is protected in culture solution in ferment slot
Hold the condition of culture near 50g/L.
<Culture and feed conditions 3>
Initial stage of culture (when beginning) adds glucose mother liquid in such a way that concentration of glucose is 100g/L, if sent out when culture
Concentration of glucose becomes 50g/L hereinafter, then intermittent charging glucose mother liquid is so that concentration of glucose is protected in culture solution in ferment slot
Hold the condition of culture near 50g/L.
As described above, being tested with 3 kinds of experiment conditions.Only, in this experimental example, the culture kind bacterium not in flask
After be transferred to fermentation tank, but implement kind of a bacterium culture in fermentation tank.20% is planted in flask the culture solution inoculation of bacterium culture
In including 100g/L glucose, 0.5% yeast extract, 0.35%KNO3, 0.025%KH2PO4、5ppm MnSO4, 3ppm sulphur
The culture medium of amine element is cultivated 21 hours.After culture 21 hours, carry out being added when this culture 1% is added to fermentation tank
CSL, 0.25% yeast extract, 0.35%KNO3, 0.025%KH2PO4、5ppm MnSO4, 3ppm thiamines, and with corresponding
Concentration injects glucose mother liquid.Due to carrying out kind of a bacterium culture in this way, although three kinds of condition kind bacterium similarly grow, due to first
The amount for the glucose mother liquid that phase is added is different, therefore compared with other conditions, in the case of condition 1 because initial stage start OD it is low due to
Have the tendency that lag time (lag time) is elongated (Fig. 1).In fermentation tank, kind bacterium is cultivated when cultivating with 32 DEG C, red moss
Sugar alcohol is cultivated when fermenting with 35 DEG C.Other fermentation tank conditions are identical as examples detailed above.Experimental result is shown in the following table 9.
[table 9]
| Condition of culture | Antierythrite (g/L) | Transition rate (%) | Incubation time (h) | Productivity (g/L-h) |
| 1 | 249 | 53 | 134 | 1.86 |
| 2 | 254 | 52 | 111 | 2.29 |
| 3 | 251 | 50 | 86 | 2.92 |
Its result confirms, and glucose mother is added in such a way that concentration of glucose is 50g/L or 100g/L in Initial stage of culture
Liquid, when culture if concentration of glucose in fermentation tank become 50g/L hereinafter, if in the case of intermittent charging glucose mother liquid,
Although incubation time is short, the yield and productivity of glucose mother liquid are also excellent.
The production that experimental example 9. passes through the antierythrite of intermittence charging
Whether the inventors of the present invention show reproducibility to confirm when being carried out with intermittent charging process, with experimental example 8
's<Culture and feed conditions 3>Condition carried out repeat test.It carries out in the same way and shows the result in table 10.
[table 10]
| Experiment | Antierythrite (g/L) | Transition rate (%) | Incubation time (h) | Productivity (g/L-h) |
| 1 | 251 | 62 | 79.5 | 3.16 |
| 2 | 252 | 58 | 84 | 3 |
| 3 | 250 | 52 | 84 | 2.98 |
Its result confirms, and shows similar with the result of experimental example 8 as a result, reproducible.
Experimental example 10. is produced by the high of antierythrite of intermittence charging
In the case of above-mentioned experimental example 9, if it is confirmed that the yield to antierythrite becomes 250g/L, then fermentation is interrupted.
But cell is not inactivated state when interruption, only the depletion rate of the speed of production of antierythrite and glucose reduces, red moss
Production of sugar polyol is still in progress.Therefore, the inventors of the present invention continue glucose mother liquid charging to carry out fermentation until again
Also glucose can not be utilized.The results are shown in tables 11.
[table 11]
| Experiment | Antierythrite (g/L) | Transition rate (%) | Incubation time (h) | Productivity (g/L-h) |
| 1 | 329 | 57 | 192 | 1.71 |
| 2 | 270 | 62 | 133.5 | 2.02 |
| 3 | 327 | 58 | 167.5 | 1.95 |
Its result confirms, compared with interrupting the case where fermenting as long as becoming 250g/L as long as the yield of antierythrite, red moss
Sugar alcohol yield increases, and transition rate is similar.
Claims (11)
1. a kind of method of manufacture antierythrite comprising：
Supernatant obtained by being centrifuged or precipitate by the corn impregnation liquid to the nitrogen comprising 3 to 10 (w/v) % with
Water is with 3:1 to 1:3 ratio mixing, the step of preparing the machining object of corn impregnation liquid；
In the machining object of the corn impregnation liquid, added in such a way that the concentration of glucose in culture medium is 5 to 45 (w/v) %
The step of glucose mother liquid adds the yeast extract of 0.25 to 0.5 (w/v) %, manufactures strain culturing culture medium, the training
The machining object that base includes the corn impregnation liquid of 1 to 3 (w/v) % is supported, the glucose mother liquid includes 700 to 900g/L grape
Sugar；And
The step of antierythrite production bacterial strain is cultivated in strain culturing culture medium,
The antierythrite production bacterial strain is that the clump that preserving number is KCTC12720BP obstructs spore yeast SYC-ERY1 (Moniliella
Pollinis SYC-ERY1) bacterial strain.
2. the step of method of manufacture antierythrite as described in claim 1, the culture is at a temperature of 30 to 35 DEG C
Implement.
3. the step of method of manufacture antierythrite as described in claim 1, the culture is in the lower implementations of pH 5 to 6.
4. the step of method of manufacture antierythrite as described in claim 1, the culture, implements 3 to 10 days.
5. the method for manufacture antierythrite as described in claim 1, the step of culture be batch-type, Fed-batch or
The culture of continous way form.
6. the method for manufacture antierythrite as described in claim 1, the culture the step of in, if fermentation tank when culture
Interior concentration of glucose becomes 50g/L hereinafter, then intermittent charging glucose mother liquid.
7. the method for manufacture antierythrite, the charging glucose mother liquid make Portugal in the fermentation tank as claimed in claim 6
Grape sugar concentration remains 10 to 100g/L.
8. the method for manufacture antierythrite as described in claim 1, the antierythrite production bacterial strain is in the culture
It is carried out before step obtained by kind of bacterium culture.
9. the method for manufacture antierythrite as claimed in claim 8, described kind of bacterium culture is comprising selected from by glucose, nitric acid
Potassium KNO3, potassium dihydrogen phosphate KH2PO4, manganese sulfate MnSO4Implement in the culture medium of one or more of the group of thiamine composition.
10. the method for manufacture antierythrite as claimed in claim 8, the culture of described kind of bacterium pH 5 to 6 and temperature 28 to
Implement 1 to 3 day at 32 DEG C.
11. a kind of purposes of the culture media composition in culture antierythrite production bacterial strain for producing antierythrite, described
Culture media composition includes the corn impregnation liquid of the glucose mother liquid containing 700 to 900g/L glucose, 1 to 3 (w/v) %
The yeast extract of machining object and 0.25 to 0.5 (w/v) %, the machining object of the corn impregnation liquid be will by comprising 3 to
Supernatant obtained by the corn impregnation liquid of the nitrogen of 10 (w/v) % is centrifuged or precipitates is with water with 3:1 to 1:3 ratio is mixed
It closing, the glucose mother liquid adds in such a way that the concentration of glucose in culture medium is 5 to 45 (w/v) %,
The antierythrite production bacterial strain is that the clump that preserving number is KCTC12720BP obstructs spore yeast SYC-ERY1 bacterial strains.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020130164668A KR101576183B1 (en) | 2013-12-26 | 2013-12-26 | Method of producing erythritol using by-products, Moniliella pollinis mutant with high productivity of erythritol and the use thereof |
| KR10-2013-0164668 | 2013-12-26 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104745644A CN104745644A (en) | 2015-07-01 |
| CN104745644B true CN104745644B (en) | 2018-08-17 |
Family
ID=53585899
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410831742.9A Active CN104745644B (en) | 2013-12-26 | 2014-12-26 | Antierythrite manufacturing method, clump stalk spore yeast variant strain and application thereof |
Country Status (2)
| Country | Link |
|---|---|
| KR (1) | KR101576183B1 (en) |
| CN (1) | CN104745644B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102250428B1 (en) | 2017-11-23 | 2021-05-11 | 경북대학교 산학협력단 | New Yeast Strain with high productivity of erythritol production method thereof |
| CN112175847B (en) * | 2020-11-11 | 2022-04-01 | 宜宾五粮液股份有限公司 | New strain of Plectosporium yeast and its use |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1932002A (en) * | 2005-09-14 | 2007-03-21 | 中国食品发酵工业研究院 | Candida lipolytica and the erythritol producing process therewith |
| CN101979616A (en) * | 2010-11-12 | 2011-02-23 | 合肥工业大学 | Method for producing erythritol by using broken rice |
| CN102154383A (en) * | 2010-12-29 | 2011-08-17 | 保龄宝生物股份有限公司 | Method for producing phycite by using corn meal |
| US8187847B2 (en) * | 2006-10-03 | 2012-05-29 | Jungbunzlauer Austria Ag | Process for producing erythritol using Moniliella tomentosa strains in the presence of neutral inorganic nitrates, such as potassium nitrate, ammonium nitrate or sodium nitrate, as nitrogen source |
| CN103374534A (en) * | 2013-07-05 | 2013-10-30 | 上海交通大学 | Yarrowia lipolytica strain and method thereof for synthesizing erythritol |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100271137B1 (en) | 1998-06-24 | 2000-11-01 | 유병택 | Trichosporonoides media ds911 and method for producing erythritol using the same |
| KR100406918B1 (en) * | 2001-04-23 | 2003-11-21 | 조흥화학공업주식회사 | Composition of a low oxygen demand medium for culturing yeast |
-
2013
- 2013-12-26 KR KR1020130164668A patent/KR101576183B1/en active IP Right Grant
-
2014
- 2014-12-26 CN CN201410831742.9A patent/CN104745644B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1932002A (en) * | 2005-09-14 | 2007-03-21 | 中国食品发酵工业研究院 | Candida lipolytica and the erythritol producing process therewith |
| US8187847B2 (en) * | 2006-10-03 | 2012-05-29 | Jungbunzlauer Austria Ag | Process for producing erythritol using Moniliella tomentosa strains in the presence of neutral inorganic nitrates, such as potassium nitrate, ammonium nitrate or sodium nitrate, as nitrogen source |
| CN101979616A (en) * | 2010-11-12 | 2011-02-23 | 合肥工业大学 | Method for producing erythritol by using broken rice |
| CN102154383A (en) * | 2010-12-29 | 2011-08-17 | 保龄宝生物股份有限公司 | Method for producing phycite by using corn meal |
| CN103374534A (en) * | 2013-07-05 | 2013-10-30 | 上海交通大学 | Yarrowia lipolytica strain and method thereof for synthesizing erythritol |
Non-Patent Citations (2)
| Title |
|---|
| 葡萄糖母液的工业利用;章朝晖;《化工生产与技术》;20001225;第7卷(第6期);第32-35页 * |
| 赤藓糖醇研究进展及在食品中的应用;徐莹等;《中国食品添加剂》;20050615(第3期);第92-95页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| KR101576183B1 (en) | 2015-12-09 |
| KR20150076048A (en) | 2015-07-06 |
| CN104745644A (en) | 2015-07-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6329940B2 (en) | Euglena microalgae, polysaccharide manufacturing method, and organic compound manufacturing method | |
| Kumar et al. | Gibberellic acid by solid state fermentation: consistent and improved yields | |
| CN101445786A (en) | Bacillus subtilis highly producing tetramethylpyrazine and method thereof for fermentation producing tetramethylpyrazine | |
| Diaz De Villegas et al. | Conversion of furfural into furfuryl alcohol by Saccharomyces cervisiae 354 | |
| CN100338221C (en) | Preparation of lactic acid from a pentose-containing substrate | |
| JP2011050359A (en) | New microorganism, enzyme derived from the microorganism and method for producing saccharified solution by using the same | |
| CN104745644B (en) | Antierythrite manufacturing method, clump stalk spore yeast variant strain and application thereof | |
| CN1233836C (en) | Method for producing lactic acid from paper-making pulp | |
| CN100569946C (en) | The separation of candida tropicalis bacterial strain and be used for the method that Xylitol is produced | |
| CN101855973A (en) | Fungus strain irpex iacteus for producing laccase, and culturing method and application thereof | |
| CN103725719A (en) | Methods of producing carboxylic acids and/or alcohols | |
| CN1063483C (en) | High-butanol ratio clostridium acetobutylicium and culturing method and use thereof | |
| CN101597627B (en) | Production method of high molecular poly (gamma-glutamic acid) | |
| CN107604036B (en) | Method for preparing beta-carotene and beta-carotene product | |
| Rashid et al. | Mannanase production by Aspergillus niger USM F4 via solid substrate fermentation in a shallow tray using palm kernel cake as a substrate | |
| Maheva et al. | Sporulation of Penicillium roqueforti in solid substrate fermentation | |
| CN102212484B (en) | Method for controlling growthform of filamentous fungi during fermentation process | |
| WO2008062558A1 (en) | Thermotolerant ethanol-producing yeast and ethanol production method utilizing the same | |
| CN103952334B (en) | A kind of strain HD 385 and method of microorganism fermenting and producing L-erythrulose | |
| CN102952761A (en) | Nocardia sp. capable of converting quininone into (R)-3-quinuclidinol and conversion method | |
| CN107475306B (en) | Method for preparing lycopene and lycopene product | |
| RU2186846C2 (en) | Yeast strain saccharomyces cerevisiae used for fermentation of molasses must in production of ethyl alcohol and baking yeast | |
| RU2560585C1 (en) | STRAIN OF BACTERIA Bacillus stratosphericus INTENDED TO PRODUCE ETHANOL FROM LIGNOCELLULOSIC BIOMASS | |
| CN116254189B (en) | Method for producing butyric acid by semi-solid fermentation of rape straw by clostridium beijerinckii | |
| CN117417869B (en) | Flavobacterium johnsonii W24H and application thereof in production of 2, 3-butanediol |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20160411 Address after: Seoul special city Applicant after: SAMYANG Corporation Address before: Seoul special city Applicant before: Samyang Genex Corp. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |