CN107312804B - A kind of biofermentation new method of positive long-chain tridecanyldicarboxylic acid - Google Patents

A kind of biofermentation new method of positive long-chain tridecanyldicarboxylic acid Download PDF

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CN107312804B
CN107312804B CN201710601371.9A CN201710601371A CN107312804B CN 107312804 B CN107312804 B CN 107312804B CN 201710601371 A CN201710601371 A CN 201710601371A CN 107312804 B CN107312804 B CN 107312804B
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candida tropicalis
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CN107312804A (en
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史兰东
修德恒
李刚
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Qingdao Think Tank Biotechnology Co., Ltd.
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张艾琳
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    • C12R2001/74—Candida tropicalis

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Abstract

This product is related to the new method that a kind of candida tropicalis biofermentation produces positive tridecanyldicarboxylic acid, the seed growth phase including candida tropicalis C1301 (Candida tropicalis)；The stage is separated and recovered with product tridecanyldicarboxylic acid in the fermentation culture stage and fermentation liquid of candida tropicalis C1301 (Candida tropicalis).The positive tridecanyldicarboxylic acid of the method for the present invention production has satisfactory lamellar morphology, it is easier to meet European dustlessization, the requirement of self raising flour dirt products export；And it is utilized particularly suitable as the Raw material processing of senior lubricant, advanced nylon resin.

Description

A kind of biofermentation new method of positive long-chain tridecanyldicarboxylic acid
Technical field
The invention belongs to biological chemical field, be related to a kind of bioanalysis be synthetically produced the method for long-chain tridecanyldicarboxylic acid with Bacterial strain and positive long-chain tridecanyldicarboxylic acid product.
Background technique
Long-chain biatomic acid refers to that carbon atom number in ten and above binary acid, refers generally to Straight chain diatomic acid.And Moschus Ketone be in natural musk with physiological activity one of principle active component, can replace natural musk be configured to it is a variety of rare Chinese patent drug has the curative effects such as antibacterial anti-inflammatory, clearing and activating the channels and collaterals.Moschus-the M made of mixed dibasic acid and glycol ester, for production Fine perfumery provides the important source material of new raw material and clothes high-grade nylon hot-melt adhesive and high grade paint.With tridecanyldicarboxylic acid (DC13) it is the Moschus-T of Material synthesis, fixastive can be made.
In addition, the important source material of tridecanyldicarboxylic acid or lube oil additive and cold-resistant plasticizer etc.；Esters lubrication The molecular structure of oil base oil is easy to control, corresponding index obvious effect of the different structure to lube base oil.Esters Lube molecular weight is bigger, and flash-point is higher；Branch is more, and pour point is lower；Branch increases, and viscosity index (VI) improves.Therefore from unitary Pure and mild long-chain tridecanyldicarboxylic acid synthesizes excellent lube base oil, for example, with tridecanyldicarboxylic acid with branched aliphatic Monohydric alcohol direct esterification obtains diester-type compound, and condensation point is not higher than 20 DEG C, and flash-point is not less than 210 DEG C, usual 40 DEG C of lucks Kinetic viscosity is 10-100mm2/s, and viscosity index (VI) is not less than 130, and intersolubility is good between different product, as lube base oil energy Enough requirements met under most high temperature or cryogenic conditions.Compared with common mineral oil, lubricating oil in esters has more Add excellent viscosity-temperature characteristics and low temperature properties, volatility is low, stable chemical performance, and degradable, renewable, flame resistance is good, is used extensively In aviation engine lubricating oil, high-temperature chain oil, compressor oil, superior automobile engine oil, precision instrument oil, metal cutting solution Etc. each high-end field.
Tridecanyldicarboxylic acid can pass through two methods of microbial fermentation or the preparation production of chemical synthesis.Traditional production is long The chemical method of chain diacid needs 9 complicated reaction steps, high temperature, high pressure and catalyst；Needed when production prevent fires, it is explosion-proof and Anti-virus device, yield is low, at high cost and environmental pollution is serious.And biological fermentation process synthesis technology is simple, energy under normal temperature and pressure Production, pollution-free, high income have cost advantage.However, in the existing method of bioanalysis synthesis tridecanyldicarboxylic acid, still It is not high that there are fermentation levels, product purity be not achieved in the requirement for preparing long-chain biatomic acid ester, especially dicarboxylic acid product due to Fermentation strain α, the difference in omega oxidation site can generate the monoacid impurity of certain content, furthermore the fermentation process of the prior art Obtaining product is usually powdery product, this carrys out huge inconvenience to the process bands for synthesizing fine perfumery or synthetic nylon below.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of new method of lamellar morphology tridecanyldicarboxylic acid, this method It is based on the discovery that and specific method of mutagenesis is combined with gene high flux screening, filter out one plant of candida tropicalis (deposit number CCTCC NO. M2014546, the deposit date is on November 4th, 2014, protect by (Candida tropicalis) C1301 Hiding place is Wuhan University, China typical culture collection center, classification naming Candida tropicalis C1301), On the one hand it is able to maintain the ability of existing high-level fermenting and producing DC13 or slightly improves, and the α single-minded with height, omega oxidation Ability, so that the content of product monoacid substantially reduces, i.e. candida tropicalis (Candida tropicalis) C1301 is tool There is the superior strain of single product binary acid.In addition, the strain fermentation and obtaining product 13 using refining methd of the invention Carbon dicarboxylic acid, form are lamellar structure, greatly reduce powder degree so that product more easily reach subsequent technique dustlessization and Low dirtization requirement, is more in line with requirement of the European & American Market especially EU market to dustlessization product, is particularly suitable for advanced lubrication The processing and utilization of oily, advanced nylon resin.
The physiological characteristic of candida tropicalis (Candida tropicalis) C1301 is as follows:
One, the fermentation of carbohydrate: glucose+, galactolipin+, sucrose+, maltose+, lactose-.
Two, assimilate: glucose+, galactolipin+, sorbose-, sucrose+, maltose+, cellobiose+, trehalose+, cream Sugar-, melibiose-, gossypose-, turanose+, levulin-, soluble starch+, xylose+, L- arabinose+, D- arabinose-, core Sugar-, rhamnose-, α-methylglucoside+, glycerol+, ethyl alcohol+, red fresh alcohol-, mannitol+, inositol-, core furfuryl alcohol+, galactolipin Alcohol-, glucitol+, sodium citrate-, sodium succinate+, calcium lactate-.
Three, the needs of auxin: biotin ++, vitamin B1 ++, vitamin B2+, vitamin B6+, vitamin B12+, Folic acid+, niacin+, pantothenic acid+, inositol+, p-aminobenzoic acid+.
Four, other: nitrate-, jellyization milk-, ursolic acid decomposition-solidify milk-, grease enzyme-.
Morphological feature: creamy-white, gauffer type, bacterium colony are cake shape and walnut cake shape.
Cultural characteristic: when cultivating in malt juice liquid medium, pseudohypha is more and grows；In alkane seed culture medium culture When, there are a certain number of short pseudohyphas；And when fermenting in the fermentation medium, it is largely single oval cell.
The method of invention microbe conversion n-tridecane (nC13) production tridecanyldicarboxylic acid (DC13) is with the torrid zone Candida (Candida tropicalis) C1301 is fermenting microbe, using n-tridecane as the culture medium mixed liquor of matrix Middle synchronous fermentation produces α, the positive tridecanyldicarboxylic acid of ω-.
The step of method of invention microbe conversion n-tridecane (nC13) production tridecanyldicarboxylic acid (DC13) Are as follows:
First stage is the seed culture for producing candida tropicalis (Candida tropicalis) C1301, i.e.,
Preferably, the kind mother liquor being trained with one plant of candida tropicalis C1301 (Candida tropicalis) is connect Entering pH value is 5.5~9.0 normal alkanes containing 5~40% (v/v) 12 carbon atoms and 95~60% (v/v) fermentation mediums In mixed liquor.
Preferably, seed culture medium of the invention:
(1), the brewer's wort of 10 Bahrain's pols adds solid slope made of 2% agar；
(2), the malt juice liquid medium of 10 Bahrain's pols；
(3), alkane seed culture medium includes: KH2PO46~12g/L, 3~8g/L of yeast extract, 3~8g/L of corn pulp, sugarcane Sugar 3~6g/L, weight 40~70g/L of wax, tap water configuration, natural pH.
Preferably, the process of seed is cultivated are as follows: take an oese candida tropicalis (Candida tropicalis) C1301 yeast thallus, is coated on brewer's wort solid slope that (15 × 180 test tube of φ, every fills 6~7mL culture medium, after sterilizing Put into inclined-plane), it is cultivated 40 hours in 29~30 DEG C.1~2 above-mentioned cultured strain is taken all to scrape into equipped with 30mL alkane In the 250mL triangular flask of seed culture medium, in being cultivated 40~48 hours on 29~30 DEG C 220 revs/min of rotary shaker, as Shake flask fermentation seed takes two above-mentioned cultured slant strains all to scrape into the 5000mL triangular flask equipped with 500mL culture medium In, 29~30 DEG C of rotary shaker in 200 revs/min are cultivated 44~48 hours, when strain growth OD value reaches 0.6~0.85, are made For the seed of fermentation liquid.
It will be appreciated that the above seed culture medium and cultural method are only preferred method, this is given in description of the invention Under the premise of the cultural characteristic of bacterial strain, nutritional character, above-mentioned condition can be changed, as long as making the normal energy of strain growth Enough reach OD value and reaches 0.6~0.85, the seed as fermentation liquid.
Preferably, with the second stage of C1301 bacterial strain production long-chain biatomic acid, especially tridecanyldicarboxylic acid of the invention It is:
Preferably, pass through microscopy cultured, the fermentation liquid seed access pH value of miscellaneous bacteria does not contain for 5.5~9.0 In the mixed liquor of the normal alkane of 5~40% (v/v) 12 carbon atoms and 95~60% (v/v) fermentation mediums.
It is highly preferred that passing through microscopy cultured, the fermentation liquid seed of miscellaneous bacteria does not access containing for pH 6.0~6.8 The C of 15~30% (v/v)13Normal alkane and 85~70% (v/v) fermentation mediums mixed liquor in.
Preferably, the composition of fermentation medium are as follows: 4~15g/L of alkali metal phosphate, preferably 6~10g/L, sodium chloride 0.5~2.5g/L, preferably 1.0~2.0g/L, 400~1200ppm of defoaming agent, weight wax or 15~30g/L of sucrose and it is some its Nutrient source well known to him.
It will be appreciated that the above seed culture medium and cultural method are only preferred method, this is given in description of the invention Under the premise of the cultural characteristic of bacterial strain, nutritional character, can according to environment of the thallus in different culture tanks to above-mentioned condition into Row changes.
Preferably, the fermentation culture stage of the candida tropicalis C1301 (Candida tropicalis) are as follows: by institute It states mixed liquor to convert 48~168 hours at 24~34 DEG C, then isolates and purifies the tridecanyldicarboxylic acid of production.
It is highly preferred that the fermentation culture stage of the candida tropicalis C1301 (Candida tropicalis) are as follows: By said mixture at 25~34 DEG C under pH6.0~7.5, most preferably 27~31 DEG C aerobic fementation 72~168 hours.
Preferably, in the first stage of fermentation stage, system pH is controlled 6.0~6.8, based on thalli growth, simultaneously Produce a certain number of binary acid；Second stage, between system pH control 7.0~8.0, based on fermentation and acid, also growing part Divide thallus；Phase III only produces acid, not long thallus.It is highly preferred that adding a certain amount of normal alkane daily since 72 hours, making > 5% (v/v), alkali metal phosphate can be from KH always for normal alkane concentration in fermentation liquid2PO4Or NaH2PO4In select one kind.
Preferably, the invention also includes product tridecanyldicarboxylic acid in fermentation liquid is separated and recovered the stage:
I.e. after fermentation, by fermentation liquid heating plus alkali demulsification, it is heated to 80-90 DEG C, adds alkali to pH10 or so, is pressed into quiet Set layering tank layering.Preferably, the invention also includes residual normal alkanes are recycled, i.e., UF membrane removes thallus, Clear liquid is placed, and is cooled to 20 DEG C, collects the crystal of DC13 sodium salt, and the direct acidizing crystal of mother liquor collects DC13 sodium salt and DC13, It is recrystallized with water or organic solvent, obtains DC13 white crystals.
It, can be in 50m with candida tropicalis C1301 bacterial strain of the invention and fermentation process3Fermentor, from nC13Fermentation life Produce DC13 when, ferment 150 hours, produce acid amount reach 265g/L or more, conversion ratio reach 90% or more, DC13 purity reach 99% with On, wherein the content of monoacid is less than 0.01%.In addition, unexpectedly, DC13 product of the invention has satisfactory Sheet form, this is more competitive for European dustlessization, low dusting product import require.
Compared with prior art, the present invention the invention has the following advantages that DC13 product of the invention is with satisfactory Sheet form, this is more competitive for European dustlessization, low dusting product import requirement.
Detailed description of the invention
Fig. 1 is tridecanyldicarboxylic acid sheet product prepared by embodiment of the present invention method 1；
Fig. 2 is the tridecanyldicarboxylic acid powder-product of comparative example 1 of the present invention preparation；
Fig. 3 is tridecanyldicarboxylic acid sheet product prepared by embodiment of the present invention method 2；
Fig. 4 is the tridecanyldicarboxylic acid sheet product of comparative example 2 of the present invention preparation.
Specific embodiment
In one embodiment of the invention, it provides with candida tropicalis (Candida tropicalis) C1301 For fermenting microbe, the synchronous fermentation in the culture medium mixed liquor using n-tridecane as matrix produces α, the positive tridecanyldicarboxylic acid of ω- Method.
Preferably, in one embodiment of the invention, the fermentation process is conversion n-tridecane (nC13) production The step of method of tridecanyldicarboxylic acid (DC13) are as follows:
First stage is the seed culture for producing candida tropicalis (Candida tropicalis) C1301, i.e.,
Preferably, the kind mother liquor being trained with one plant of candida tropicalis C1301 (Candida tropicalis) is connect Entering pH value is 5.5~9.0 normal alkanes containing 5~40% (v/v) 12 carbon atoms and 95~60% (v/v) fermentation mediums In mixed liquor.
Preferably, in one embodiment of the invention, the seed culture medium are as follows:
(1), the brewer's wort of 10 Bahrain's pols adds solid slope made of 2% agar；
(2), the malt juice liquid medium of 10 Bahrain's pols；
(3), alkane seed culture medium includes: KH2PO46~12g/L, 3~8g/L of yeast extract, 3~8g/L of corn pulp, sugarcane Sugar 3~6g/L, weight 40~70g/L of wax, tap water configuration, natural pH.
Preferably, in one embodiment of the invention, the process of seed is cultivated are as follows: take an oese candida tropicalis (Candida tropicalis) C1301 yeast thallus is coated on brewer's wort solid slope (15 × 180 test tube of φ, every dress 6~7mL culture medium, puts into inclined-plane after sterilizing), it is cultivated 40 hours in 29~30 DEG C.Take 1~2 above-mentioned cultured strain complete Portion is scraped in the 250mL triangular flask equipped with 30mL alkane seed culture medium, is trained on 29~30 DEG C 220 revs/min of rotary shaker It supports 40~48 hours, as shake flask fermentation seed or two above-mentioned cultured slant strains is taken all to scrape into equipped with 500mL training In the 5000mL triangular flask for supporting base, 29~30 DEG C of rotary shaker in 200 revs/min are cultivated 44~48 hours, strain growth OD value Seed when reaching 0.6~0.85, as fermentation liquid.
It will be appreciated that the above seed culture medium and cultural method are only preferred method, this is given in description of the invention Under the premise of the cultural characteristic of bacterial strain, nutritional character, above-mentioned condition can be changed, as long as making the normal energy of strain growth Enough reach OD value and reaches 0.6~0.85, the seed as fermentation liquid.
Preferably, in one embodiment of the invention, long-chain biatomic acid is produced with C1301 bacterial strain of the invention, especially It is that the second stage of tridecanyldicarboxylic acid is:
Preferably, in one embodiment of the invention, pass through microscopy cultured, not the fermentation liquid seed of miscellaneous bacteria Accessing pH value is 5.5~9.0 normal alkanes and 95~60% (v/v) fermentation mediums for containing 5~40% (v/v) 12 carbon atoms Mixed liquor in.
It is highly preferred that in another embodiment of the present invention, passing through microscopy cultured, not the fermentation liquid of miscellaneous bacteria The C containing 15~30% (v/v) of seed access pH 6.0~6.813Normal alkane and 85~70% (v/v) fermentation mediums In mixed liquor.
Preferably, in one embodiment of the invention, the composition of fermentation medium are as follows: 4~15g/ of alkali metal phosphate L, preferably 6~10g/L, 0.5~2.5g/L of sodium chloride, preferably 1.0~2.0g/L, 400~1200ppm of defoaming agent, weight wax Or 15~30g/L of sucrose.
It will be appreciated that the above seed culture medium and cultural method are only preferred method, this is given in description of the invention Under the premise of the cultural characteristic of bacterial strain, nutritional character, can according to environment of the thallus in different culture tanks to above-mentioned condition into Row changes.
Preferably, in one embodiment of the invention, the candida tropicalis C1301 (Candida Tropicalis fermentation culture stage) are as follows: the mixed liquor is converted 48~168 hours at 24~34 DEG C, then will be given birth to The tridecanyldicarboxylic acid of production is isolated and purified.
Preferably, in one embodiment of the invention, the candida tropicalis C1301 (Candida Tropicalis fermentation culture stage) are as follows: by said mixture at 25~34 DEG C under pH6.0~7.5, most preferably 27 ~31 DEG C aerobic fementation 72~168 hours.
Preferably, in the first stage of fermentation stage, system pH is controlled 6.0~6.8, based on thalli growth, simultaneously Produce a certain number of binary acid；Second stage, between system pH control 7.0~8.0, based on fermentation and acid, also growing part Divide thallus；Phase III only produces acid, not long thallus.It is highly preferred that adding a certain amount of normal alkane daily since 72 hours, making > 5% (v/v), alkali metal phosphate can be from KH always for normal alkane concentration in fermentation liquid2PO4Or NaH2PO4In select one kind.
It preferably, in one embodiment of the invention, further include separating back product tridecanyldicarboxylic acid in fermentation liquid The receipts stage:
I.e. after fermentation, by fermentation liquid heating plus alkali demulsification, it is heated to 80-90 DEG C, adds alkali to pH10 or so, is pressed into quiet Set layering tank layering.Preferably, the invention also includes residual normal alkanes are recycled, i.e., UF membrane removes thallus, Clear liquid is placed, and is cooled to 20 DEG C, collects the crystal of DC13 sodium salt, and the direct acidizing crystal of mother liquor collects DC13 sodium salt and DC13, It is recrystallized with water or organic solvent, obtains DC13 white crystals.
Below in conjunction with the embodiment in the present invention, the technical solution in the present invention is clearly and completely described.It is aobvious So, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to In the scope of protection of the invention.
Embodiment 1
(1), an oese candida tropicalis C1301 thallus is taken, 15 × 180 Boiling tube brewer's wort solid slopes are coated on On, it is cultivated 2 days at 28 DEG C.
(2), above-mentioned strain one is taken, access is equipped in the 250ml triangular flask of 30ml liquid seed culture medium in 28-30 DEG C It is cultivated 40 hours on 220 revs/min of rotary shaker.Contain KH in liquid seed culture medium2PO4 8g/l, yeast extract 5g/l, Corn pulp 3g/l, sucrose 30g/l, urea 3g/l, tap water configuration, pH5.0.
(3), in the 500ml triangular flask equipped with 15ml fermentation medium, the above-mentioned cultured seed liquor of 3.5ml is accessed, In 28-30 DEG C, 220 revs/min rotary shaker top fermentation 4 days, every 24 hours with pH to 7.5-8.0 of 6N NaOH tune.Fermentation training It supports and contains KH in base mixed liquor2PO410g/l, sucrose 20/l, yeast extract 3g/l, corn pulp 3.5g/l, urea 1.5g/l, and NC13 200ml/l, tap water configuration, pH7.2.It sterilizes 30 minutes at 110 DEG C.
After fermentation, pH to 2-3 being adjusted with the HCl of 6N and boiling off ether after 120ml ether extraction, it is white to obtain DC13 Color crystallization is titrated after the dissolution of 15ml neutral alcohol with standard NaOH solution, and calculating DC13 content in fermentation liquid is 130g/ L, purity 98.23%, wherein unitary acid content 0.030%.
Embodiment 2:
(1) seed culture medium and cultural method and fermentation medium are the same as example 1.
(2) 3000mL is cultivated two days, OD (× 30,620nm) is 0.81pH 3.8, healthy and strong, no miscellaneous strain liquid access dress There is 700L seed culture medium, in the first class seed pot to sterilize 40 minutes through 121 DEG C, 29 DEG C 350 revs/min, tank presses 0.8kg/cm2, Ventilatory capacity 1: 0.8 is cultivated 36 hours, the seed as second level kind mother.
(3) the stalwartness cultivated in (2), the 700L kind liquid access of no miscellaneous bacteria is equipped with 6.5m3Seed culture medium, by 121 DEG C 40 minutes 10m of sterilizing3In second level kind mother's tank, 30 DEG C, 200 revs/min, tank presses 1kg/cm2, ventilatory capacity 1: 0.7, culture 40~ 48 hours, the seed as fermentation.
(4) the stalwartness of culture, the kind liquid without miscellaneous bacteria in (3), access is equipped with 33m3Fermentation medium sterilizes through 121 DEG C 40 minutes 50m3In fermentor, 30 DEG C, 200 revs/min, tank presses 1kg/cm2, ventilatory capacity 1: 0.5, when beginning, nC13 4m3, 30 In hour, system pH is controlled 7.0 hereinafter, thallus mushrooms out, while generating 28.5g/L DC13, and then system pH exists 7.0~8.0, continue to ferment, by 67 hours, produces acid amount and reach 120g/L, add a certain amount of nC13 after 70 hours daily, make to ferment Normal alkane concentration > 5% (v/v) always in liquid.Fermentation 139 hours produces acid amount and reaches 235.8g/L, and fermentation terminated by 163 hours, Acid amount is produced up to 265 g/L.
After fermentation, demulsification layering is carried out, upper layer remnants nC13 is recycled, lower layer's thallus layer removes thallus by filters pressing, Merge clear liquid, 90 DEG C of 0.6~0.7% active carbon of addition decolourize 20 minutes, and filters pressing removes active carbon, add after decoloration clear liquid heating Enter dense H2SO4It to pH3, is cooled to room temperature, filters pressing, air blow drying, the drying of solid content drying machine obtains white crystalline DC13 production Product.NC13 conversion ratio is 94.5%, post-processing total recovery reaches 91.5%, DC13 purity and reaches 99.65%, wherein monoacid Content 0.002%.
Comparative example 1:
The comparative example permits patented strain candida tropicalis candida tropicalis using Microbe Inst., Chinese Academy of Sciences (Candidatropicalis) mutant strain P-12-242, deposit number are CGMCC NO.0297 bacterial strain (referring to patent ZL 97103876.7), other methods with embodiment 1 i.e.:
(1), an oese candida tropicalis P-12-242 thallus is taken, 15 × 180 Boiling tube brewer's wort solids are coated on On inclined-plane, cultivated 2 days at 28 DEG C.
(2), above-mentioned strain one is taken, access is equipped in the 250ml triangular flask of 30ml liquid seed culture medium in 28-30 It DEG C is cultivated 40 hours on 220 revs/min of rotary shaker.Contain KH in liquid seed culture medium2PO48g/l, yeast extract 5g/l, Corn pulp 3g/l, sucrose 30g/l, urea 3g/l, tap water configuration, pH5.0.
(3), in the 500ml triangular flask equipped with 15ml fermentation medium, the above-mentioned cultured seed liquor of 3.5ml is accessed, In 28-30 DEG C, 220 revs/min rotary shaker top fermentation 4 days, every 24 hours with pH to 7.5-8.0 of 6N NaOH tune.Fermentation Contain KH in culture medium mixed liquor2PO410g/l, sucrose 20/l, yeast extract 3g/l, corn pulp 3.5g/l, urea 1.5g/l, and NC13 200ml/l, tap water configuration, pH7.2.It sterilizes 30 minutes at 110 DEG C.
After fermentation, pH to 2-3 being adjusted with the HCl of 6N and boiling off ether after 120ml ether extraction, it is white to obtain DC13 Color powder is titrated, calculating DC13 content in fermentation liquid is after the medium-sized ethyl alcohol dissolution of 15ml with standard NaOH solution 81.6g/l, purity 90.23%, wherein the content of monoacid is greater than 3.6%.
In addition, passing through the comparison of attached drawing 1, it is bigger that the embodiment of the present invention 1 obtains product crystal, and comparative example 1 is produced Product are essentially powdered.
Comparative example 2:
The comparative example permits patented strain candida tropicalis candida tropicalis using Microbe Inst., Chinese Academy of Sciences (Candidatropicalis) mutant strain P-12-242, deposit number are CGMCC NO.0297 bacterial strain (referring to patent ZL 97103876.7), other methods with embodiment 2 i.e.:
(1), seed culture medium and cultural method and fermentation medium, fermentation process are the same as example 2.
(2), 3000mL is cultivated two days, OD (× 30,620nm) is 0.81pH 3.8, healthy and strong, candida tropicalis P- 12-242 is equipped with 700L seed culture medium without the access of miscellaneous strain liquid, in the first class seed pot to sterilize 40 minutes through 121 DEG C, 29 DEG C 350 revs/min, tank presses 0.8kg/cm2, ventilatory capacity 1: 0.8, culture 36 hours, the seed as second level kind mother.
(3), the stalwartness cultivated in step (2), the 700L kind liquid access of no miscellaneous bacteria is equipped with 6.5m3Seed culture medium, warp Cross the 10m of 121 DEG C of sterilizings 40 minutes3In second level kind mother's tank, 30 DEG C, 200 revs/min, tank presses 1kg/cm2, ventilatory capacity 1: 0.7, training It supports 40~48 hours, the seed as fermentation.
(4) the stalwartness cultivated in (3), without miscellaneous bacteria without miscellaneous strain liquid kind liquid, access is equipped with 33m3Fermentation medium, warp The 50m of 121 DEG C of sterilizings 40 minutes3In fermentor, 30 DEG C, 200 revs/min, tank presses 1kg/cm2, ventilatory capacity 1: 0.5, when beginning, nC13 4m3, in 30 hours, system pH control hereinafter, thallus mushrooms out, while generating 21.3g/L DC13 7.0, then System pH continues to ferment 7.0~8.0, adds a certain amount of nC13 after 70 hours daily, makes in fermentation liquid normal alkane concentration always > 5% (v/v).Fermentation 139 hours produces acid amount up to 142 g/L, and fermentation terminated by 163 hours, produces acid amount and reaches 190g/L.
After fermentation, demulsification layering is carried out, upper layer remnants nC13 is recycled, lower layer's thallus layer removes thallus by filters pressing, Merge clear liquid, 90 DEG C of 0.6~0.7% active carbon of addition decolourize 20 minutes, and filters pressing removes active carbon, add after decoloration clear liquid heating Enter dense H2SO4It to pH 3, is cooled to room temperature, filters pressing, air blow drying, the drying of solid content drying machine obtains white powder DC13 Product.NC13 conversion ratio is that 93.1%, DC13 purity reaches 96.54%, and wherein the content of monoacid is greater than 3.8%.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Claims (11)

1. a kind of method for being synthetically produced long-chain tridecanyldicarboxylic acid using bioanalysis, comprising the following steps:
The seed growth phase of (1) one plant of candida tropicalis (Candida tropicalis) C1301；
The fermentation culture stage of (2) one plants of candida tropicalis (Candida tropicalis) C1301；
(3) product tridecanyldicarboxylic acid separates and recovers the stage in fermentation liquid；
Wherein, the deposit number CCTCC NO.M2014546 of candida tropicalis (Candida tropicalis) C1301.
2. the method according to claim 1, wherein the candida tropicalis (Candida tropicalis) The seed growth phase of C1301 are as follows:
It is 5.5 that the kind mother liquor being trained with one plant of candida tropicalis (Candida tropicalis) C1301, which is accessed pH value, In the mixed liquor of~9.0 normal alkanes containing 5~40% (v/v) 13 carbon atoms and 95~60% (v/v) fermentation mediums.
3. according to the method described in claim 2, it is characterized in that, the fermentation medium forms are as follows: alkali metal phosphate 4~ 15 grams per liters, 0.5~2.5 grams per liter of sodium chloride, 10~30 grams per liter of sucrose, 1~10 grams per liter of nitrate, 1~5 grams per liter of urea are spat Warm 0.2~1.5 grams per liter and other nutrient sources.
4. according to the method described in claim 2, it is characterized in that, the candida tropicalis (Candida tropicalis) The fermentation culture stage of C1301 are as follows:
The mixed liquor is converted 48~168 hours at 24~34 DEG C, then separates the tridecanyldicarboxylic acid of production Purifying.
5. according to the method described in claim 2, it is characterized in that, the content of normal alkane is 15~40% in the mixed liquor (v/v), fermentation medium content 85~60% (v/v).
6. according to the method described in claim 3, it is characterized in that in the composition of fermentation medium: alkali metal phosphate 6~10 Grams per liter, 2~8 grams per liter of nitrate, 1.5~2.5 grams per liter of urea, 0.4~0.8 grams per liter of tween.
7. according to the method described in claim 2, it is characterized in that, the normal alkane contains 13 carbon atoms.
8. according to the method described in claim 3, it is characterized in that alkali metal phosphate is sodium salt or sylvite；Nitrate is KNO3Or NaNO3, tween is polysorbate60 or Tween 80.
9. according to the method described in claim 2, pH value is it is characterized in that the temperature control of fermentation mixed liquor is at 26~32 DEG C 6.5~8.5.
Candida tropicalis 10. (Candida tropicalis) C1301 bacterial strain, wherein the candida tropicalis The deposit number CCTCC NO.M2014546 of (Candida tropicalis) C1301.
11. the tridecanyldicarboxylic acid product prepared such as claim 1~9 any one method；Wherein, the false silk in the torrid zone The deposit number CCTCC NO.M2014546 of yeast (Candida tropicalis) C1301.
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