WO2021083079A1 - 一种长链组合物、长链组合物套组、其制造方法及其应用 - Google Patents

一种长链组合物、长链组合物套组、其制造方法及其应用 Download PDF

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WO2021083079A1
WO2021083079A1 PCT/CN2020/123700 CN2020123700W WO2021083079A1 WO 2021083079 A1 WO2021083079 A1 WO 2021083079A1 CN 2020123700 W CN2020123700 W CN 2020123700W WO 2021083079 A1 WO2021083079 A1 WO 2021083079A1
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Prior art keywords
long
chain
fermentation
acid
hours
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English (en)
French (fr)
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张霖
樊亚超
师文静
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China Petroleum and Chemical Corp
Sinopec Dalian Research Institute of Petroleum and Petrochemicals
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China Petroleum and Chemical Corp
Sinopec Dalian Research Institute of Petroleum and Petrochemicals
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Priority to KR1020227018170A priority Critical patent/KR102909670B1/ko
Priority to JP2022525052A priority patent/JP7662629B2/ja
Priority to US17/755,315 priority patent/US12540342B2/en
Priority to EP20882024.1A priority patent/EP4036218A4/en
Publication of WO2021083079A1 publication Critical patent/WO2021083079A1/zh
Anticipated expiration legal-status Critical
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    • 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
    • C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
    • 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
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C53/00—Saturated compounds having only one carboxyl group bound to an acyclic carbon atom or hydrogen
    • C07C53/126—Acids containing more than four carbon atoms
    • 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; 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—Fungi 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; 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
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • 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
    • 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
    • C12R2001/72—Candida
    • 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
    • C12R2001/72—Candida
    • C12R2001/74—Candida tropicalis

Definitions

  • the invention belongs to the technical field of biochemical industry, and specifically relates to a long-chain composition or a long-chain composition kit.
  • the invention also relates to a method for manufacturing a long-chain composition or a long-chain composition kit, and its application in the manufacture of long-chain dibasic acids.
  • Dodecanedioic acid refers to aliphatic dicarboxylic acid containing 12 carbon atoms in the carbon chain. It is a class of fine chemical products with important and wide-ranging industrial uses. It is also a synthetic high-grade fragrance in the chemical industry. Important raw materials for high-performance nylon engineering plastics, high-grade nylon hot melt adhesives, high-temperature dielectrics, high-grade paints and coatings, high-grade lubricants, cold-resistant plasticizers, resins, medicines and pesticides.
  • long-chain dibasic acids by fermentation uses the unique oxidizing ability of microorganisms and the action of microbial intracellular enzymes. Under normal temperature and pressure, the methyl groups at both ends of the long-chain n-alkanes are oxidized into carboxyl groups through ⁇ and ⁇ -oxidation, respectively.
  • Various long-chain dibasic acids with corresponding chain lengths.
  • bacteria, molds and actinomycetes that can produce long-chain dibasic acids through fermentation.
  • yeasts of the genus Candida are high-yielding microorganisms that can ferment n-alkanes to produce dibasic acids.
  • yeasts are generally used as fermentation strains, and n-alkanes are used as substrates to produce long-chain dibasic acids with corresponding carbon numbers, such as CN102115767A, CN102115768A, etc., which form undecanedioic acid and hexadecanoic acid, respectively.
  • the production plan of carbodiacid discloses a method for producing long-chain dibasic acids, which is to expand the culture of dibasic acid bacteria to obtain seed liquid, which is subjected to expanded fermentation, emulsified alkanes are added during the fermentation process, and the bacterial protein is removed after the reaction is completed. Crystallization obtains long-chain dibasic acid products.
  • the inventors of the present invention found that the prior art still has problems such as low fermentation level or low substrate utilization when using n-alkanes as substrates to produce long-chain dibasic acids.
  • the present invention has been completed based on this finding.
  • the present invention relates to the following aspects.
  • a long-chain composition (preferably for fermentation), comprising at least one long-chain alkane selected from C9-18 linear or branched (preferably linear) alkanes (preferably selected from n-dodecane, n-decane) At least one long-chain alkane in tetraalkane and n-hexadecane, especially n-dodecane) and aliphatic monobasic selected from C9-18 straight or branched chain (preferably straight chain), saturated or unsaturated (preferably saturated) At least one long-chain carboxylic acid in the carboxylic acid (preferably at least one long-chain carboxylic acid selected from the group consisting of lauric acid, myristic acid and palmitic acid, especially lauric acid), wherein the long-chain alkane and the long-chain carboxylic acid
  • the mass ratio of chain carboxylic acid is 1:1 to 40:1 (preferably 2:1 to 20:1 or 5:1 to 10:1).
  • the long-chain composition according to any one of the preceding or following aspects, further comprising water, wherein the mass of the water is 0.5-10 times the sum of the masses of the long-chain alkane and the long-chain carboxylic acid (Preferably 1-5 times or 1-3 times).
  • the long-chain composition according to any one of the foregoing or the following aspects has a pH value of 5-12 (preferably 7-10, 7.5-9 or 7.5-8.0), and/or, at 32° C.
  • the form of a liquid or a solid-liquid mixture (preferably a liquid form, especially an aqueous liquid form), and/or the long-chain alkane and the long-chain carboxylic acid have the same number of carbon atoms.
  • a long-chain composition kit comprising n long-chain compositions according to any one of the foregoing or following aspects that exist independently of each other (such as independently packaged or separated from each other), wherein n is 2-40 (Preferably 4-20 or 5-10) positive integer, where set in the i-th (i is any positive integer from 2 to n) long-chain composition, the long-chain alkane and the long-chain carboxylic acid
  • the mass ratio of is R i
  • the mass ratio of the long-chain alkane to the long-chain carboxylic acid is R 1
  • the long-chain composition kit according to any one of the foregoing or the following, wherein the n long-chain compositions are manufactured separately, and/or, any two of the n long-chain compositions
  • the weight ratio between each other is the same or different (preferably the same or substantially the same), and/or the weight between any two of the n long-chain compositions
  • the ratio is the same or substantially the same.
  • a method for producing a long-chain composition wherein the long-chain composition (preferably for fermentation) contains at least one long-chain alkane selected from C9-18 linear or branched (preferably linear) alkanes ( Preferably at least one long-chain alkane selected from n-dodecane, n-tetradecane and n-hexadecane, especially n-dodecane) and selected from C9-18 linear or branched (preferably linear), saturated or At least one long-chain carboxylic acid in the unsaturated (preferably saturated) aliphatic monocarboxylic acid (preferably at least one long-chain carboxylic acid selected from the group consisting of lauric acid, myristic acid and palmitic acid, especially lauric acid), Wherein the mass ratio of the long-chain alkane to the long-chain carboxylic acid is 1:1 to 40:1 (preferably 2:1 to 20:1 or 5:1 to 10:1), and the manufacturing method at least includes mixing Step: The long-chain alkane and the long-
  • the manufacturing method further includes optionally heating (preferably at a heating temperature of 45 to 70°C or 50 to 60°C) and stirring (preferably at a stirring speed of 50 to 250 rpm or 150 to 250 rpm).
  • a pH adjusting agent such as an alkali, especially at least one selected from sodium hydroxide and potassium hydroxide
  • the step of adding water and a pH regulator such as an alkali, especially at least one selected from sodium hydroxide and potassium hydroxide
  • a pH regulator such as an alkali, especially at least one selected from sodium hydroxide and potassium hydroxide
  • the quality of the water is the long-chain 0.5-10 times (preferably 1-5 times or 1-3 times) of the sum of the masses of the paraffin and the long-chain carboxylic acid
  • the amount of the pH adjuster is such that the pH of the long-chain composition Reach 5-12 (preferably 7-10, 7.5-9 or 7.5-8.0).
  • a method for producing a long-chain dibasic acid wherein the long-chain dibasic acid is selected from C9-18 linear or branched (preferably linear), saturated or unsaturated (preferably saturated) aliphatic two At least one long-chain dibasic acid in the carboxylic acid (preferably at least one long-chain dibasic acid selected from dodecane dibasic acid, tetradecane dibasic acid and hexadecane dibasic acid, especially Dodecanedioic acid),
  • the manufacturing method includes the following steps:
  • the long-chain composition is produced according to the production method according to any one of the foregoing or the following aspects, the long-chain composition is provided according to any of the foregoing or the following aspects, or the long-chain composition is provided according to any one of the foregoing or the following
  • the long-chain composition kits described in this aspect are collectively referred to as long-chain compositions;
  • the long-chain composition is subjected to a fermentation reaction in the presence of fermentation bacteria and a fermentation medium to convert the long-chain dibasic acid.
  • the fermenting bacteria is a yeast having a complete ⁇ , ⁇ -oxidation pathway (preferably selected from the group consisting of Candida, Cryptococcus, and Endospora
  • a yeast having a complete ⁇ , ⁇ -oxidation pathway preferably selected from the group consisting of Candida, Cryptococcus, and Endospora
  • At least one yeast selected from the genus, Hansenula, Pichia, Rhodotorula, Sphaeropsis, and Trichosporon more preferably at least one yeast selected from the genus Candida , Especially Candida tropicalis, more especially Candida tropicalis mutant strain PF-UV-56 (preserved by the General Microbiology Center of China Microorganism Collection Management Committee, deposit number is CGMCC No.0356)
  • the fermentation medium includes: sucrose 20-28g/L, corn steep liquor 0.8-1.5g/L, yeast extract 2.0-4.0g/L, sodium chloride 0.8-1.2g/L, potassium dihydrogen phosphate 3.0-3.5 g/L, magnesium
  • Preliminary step mixing the fermentation bacteria and the fermentation medium, and performing the fermentation reaction for 5-60 hours (preferably 10-40 hours, more preferably 20-30 hours or 24 hours) to obtain a fermentation base liquid,
  • Transformation step adding the long-chain composition to the fermentation base liquid, and performing the fermentation reaction for 60-400 hours (preferably 100-300 hours, more preferably 100-160 hours or 100-140 hours) to remove The long-chain composition is converted into the long-chain dibasic acid.
  • the addition amount (by volume) of the fermented bacteria seed liquid is 2-30% (preferably 5 to 20% or 10 to 15%) of the control volume of the total liquid ), and/or, the reaction temperature of the fermentation reaction is 25 to 37°C, preferably 28 to 32°C, and/or, the stirring speed of the fermentation reaction is 100 to 1000 rpm (preferably 120 to 500 rpm or 150 to 300 rpm) , And/or, the aeration of the fermentation reaction is 0.2 to 10.0 VVM (preferably 0.2 to 2.0 VVM or 0.5 to 1.0 VVM), and/or, the reaction time of the fermentation reaction is 65 hours or more (preferably more than 120 hours) , More preferably 130 to 400 hours, 130 to 300 hours, 130 to 200 hours or 138 to 160 hours), and/or, the added amount of the long-chain composition is 100 to 1000 g/L total liquid control volume (preferably 100 To 500 g/L total assembly liquid control volume or 150 to 250 g/L total assembly liquid control
  • the long-chain composition is added in n batches, and the time interval between two adjacent batches may be the same Or different (preferably the time interval is 5-60 hours, 10-40 hours, 20-30 hours or 24 hours), where n is a positive integer of 2-40 (preferably 4-20 or 5-10), or according to the reaction time
  • the long chain composition is continuously added.
  • the present invention also relates to the following aspects.
  • a method for preparing dodecanedioic acid characterized in that it comprises the following steps:
  • Fermentation bacteria and fermentation medium are added to the fermentor for fermentation, and the mixed substrate prepared in step (1) is added during the fermentation process until the fermentation is completed;
  • the long-chain dibasic acid product is obtained after demulsification, membrane filtration, acid precipitation, filtration, and drying treatments.
  • the mass ratio of dodecane to lauric acid in step (1) is 2:1-10:1, preferably 5:1- 10:1.
  • step (1) the temperature is raised to 45-70°C, preferably 50-60°C; the stirring speed is 50-250rpm, preferably 150-250rpm.
  • step (1) water of 1-3 times the mass of the mixture is added.
  • step (1) after mixing, add an alkali to adjust the pH to 7-9, preferably 7.5-8.0; the alkali is sodium hydroxide , At least one of potassium hydroxide.
  • the twelve-carbon fermentation bacteria described in step (2) are yeasts with complete ⁇ and ⁇ -oxidation pathways, preferably Candida, At least one of Cryptococcus, Endosporium, Hansenula, Pichia, Rhodotorula, Sphaeropsis, or Trichosporon.
  • the fermentation medium formula of step (2) is: sucrose 20-28g/L, corn steep liquor 0.8-1.5g/L, yeast extract 2.0-4.0g/L, sodium chloride 0.8-1.2g/L, potassium dihydrogen phosphate 3.0-3.5g/L, magnesium sulfate 1.2-1.8g/L, urea 1.2-4.8g/L, ammonium sulfate 1.5-2g /L, sodium acetate 1.5-1.8g/L.
  • the added amount of the fermentation bacteria seed liquid in step (2) is 5%-20% of the volume of the fermentation liquid, preferably 10%-15% .
  • the fermentation temperature in step (2) is 25-37°C, preferably 28-32°C; the stirring speed is 120-500rpm, preferably 150-300rpm; The aeration volume is 0.2-1.0 VVM, preferably 0.5-1.0 VVM; the fermentation time is 138-144 hours.
  • step (2) pH control method is specifically: no pH control is performed within 0-24 hours of fermentation; pH is adjusted to 6.8- after 24 hours Neutral range of 7.0; then increase the pH control value by 0.1-0.3 every 24 hours until the end of the reaction.
  • step (2) fermentation process adopts batch feeding or flow feeding method to add the mixed substrate of step (1).
  • step (2) adopts batch feeding, which is added in 5-10 batches; adopts flow feeding, and calculates according to the fermentation time. Flow acceleration.
  • step (3) is to adjust the pH of the fermentation broth to 8.5-10, preferably 9-9.5, and raise the temperature to 75-90°C , Maintain 20-40min.
  • the membrane filtration in step (3) is to filter the broken emulsion through a membrane for solid-liquid separation to obtain a filtered clear liquid;
  • the filtration temperature is 30-50 °C, preferably 40-50 °C, the diameter of the membrane pores is 10-50nm, preferably 20-25nm.
  • step (3) is to adjust the acidic pH of the filtered supernatant to control the pH to 3-5, preferably 3-4, Long-chain dibasic acid precipitates out.
  • step (3) adopts plate and frame filtration
  • the filtration pressure is 0.5-1.0 MPa
  • the filtration temperature is 20-30°C
  • the drying temperature is 80-105°C
  • the drying time is 5-20h.
  • the present invention can achieve at least one of the following technical effects, preferably all of them.
  • the mass ratio of long-chain alkanes to long-chain carboxylic acid meets the requirements of the present invention.
  • it can not only improve the fermentation level, but also solve the problem of long-chain carboxylic acid. Poor water solubility and difficult to use in fermentation systems.
  • the long-chain composition of the present invention in a preferred case, two steps of physical dissolution and chemical dissociation are adopted, which not only increases the dissolution effect of long-chain carboxylic acid, but also promotes long-chain alkane and long-chain carboxylic acid.
  • the formation of polar soluble micelles further improves the fermentation level.
  • the alkaline adjustment scheme is adopted to reduce the pH fluctuation problem of the fermentation process due to the feeding, and the feeding method is further adopted to feed the material, which improves The substrate utilization rate has improved the fermentation level.
  • the long-chain carboxylic acid can also be used as a carbon source, and part of the long-chain carboxylic acid can be directly metabolized by fatty acid to obtain the important intermediate metabolite acetyl-CoA, which facilitates the rapid accumulation of biomass. Shorten the fermentation time and increase the production intensity of long-chain dicarboxylic acids.
  • the fermentation level and the production intensity of the long-chain dicarboxylic acid can be further improved.
  • the so-called “substantially” refers to allowing deviations that are acceptable or reasonable to those skilled in the art, such as deviations within ⁇ 10%, within ⁇ 5%, within ⁇ 1%, Within ⁇ 0.5% or within ⁇ 0.1%.
  • total liquid control volume means the total volume of the liquid phase of the fermentor, which is usually 70%-80% of the volume of the fermentor.
  • any two or more embodiments of the present invention can be combined arbitrarily, and the technical solutions formed thereby belong to a part of the original disclosure of this specification and also fall into the protection scope of the present invention.
  • the long-chain composition is preferably a long-chain composition for fermentation, and is particularly suitable for use as a raw material to produce a long-chain dibasic acid by a fermentation method.
  • the long-chain composition comprises at least one long-chain alkane selected from C9-18 linear or branched alkanes and C9-18 linear or branched, saturated or unsaturated At least one long-chain carboxylic acid in the aliphatic monocarboxylic acid.
  • the long-chain alkane at least one long-chain alkane selected from C9-18 straight-chain alkanes is preferred, and at least one selected from n-dodecane, n-tetradecane, and n-hexadecane is more preferred Long chain alkanes, especially n-dodecane.
  • the long-chain carboxylic acid at least one long-chain carboxylic acid selected from C9-18 linear and saturated aliphatic monocarboxylic acids is preferred, and particularly preferably selected from lauric acid, myristic acid and palmitic acid At least one long-chain carboxylic acid, especially lauric acid.
  • the mass ratio of the long-chain alkane to the long-chain carboxylic acid is generally 1:1 to 40:1, preferably 2:1 to 20:1 or 5:1 to 10:1.
  • the long-chain composition further comprises water.
  • the mass of the water is generally 0.5-10 times, preferably 1-5 times or 1-3 times the sum of the masses of the long-chain alkane and the long-chain carboxylic acid.
  • the pH value of the long-chain composition is generally 5-12, preferably 7-10, 7.5-9 or 7.5-8.0.
  • the method for measuring the pH value of the long-chain composition may adopt a pH test paper method or a glass electrode method.
  • the long-chain composition is in the form of a liquid or a solid-liquid mixture at 32°C, preferably a liquid form, especially an aqueous liquid form.
  • liquids include homogeneous liquids and heterogeneous liquids, the latter such as a multiphase liquid mixture formed by two or more liquids.
  • the long-chain alkane and the long-chain carboxylic acid have the same number of carbon atoms, such as a combination of n-dodecane and lauric acid.
  • the present invention also relates to a long-chain composition kit, which includes a plurality of long-chain compositions described in any one of the embodiments of this specification.
  • the long-chain composition kit includes n long-chain compositions according to any one of the embodiments of the present specification that exist independently of each other.
  • n is generally a positive integer of 2-40, preferably a positive integer of 4-20 or 5-10.
  • the so-called being independent of each other in particular, can include separate packages from each other, or separate from each other, that is, multiple compartments in the same package.
  • i is any positive integer from 2 to n.
  • the n long-chain compositions are separately manufactured.
  • the manufacturing method of these long-chain compositions can be referred to the following description of this specification.
  • the weight ratio between any two of the n long-chain compositions is the same or Different, preferably the same or substantially the same.
  • the weight ratio between any two of the n long-chain compositions is the same or Basically the same.
  • the long-chain composition can be manufactured by a manufacturing method.
  • the manufacturing method at least includes the following mixing steps.
  • Mixing step mixing the long-chain alkane and the long-chain carboxylic acid according to the mass ratio to obtain the long-chain composition.
  • the mixing is optionally carried out under heating and stirring.
  • a heating temperature of 45 to 70°C or 50 to 60°C can be mentioned.
  • a stirring rotation speed of 50 to 250 rpm or 150 to 250 rpm can be mentioned.
  • water and a pH adjusting agent are further added in the mixing step.
  • a pH adjuster for example, an alkali may be mentioned, particularly at least one selected from sodium hydroxide and potassium hydroxide.
  • the manufacturing method further includes the step of adding water and a pH adjuster to the long-chain composition.
  • the step is optionally carried out under heating and stirring.
  • a heating temperature of 45 to 70°C or 50 to 60°C can be mentioned.
  • stirring particularly, a stirring rotation speed of 50 to 250 rpm or 150 to 250 rpm can be mentioned.
  • the pH adjuster for example, an alkali may be mentioned, particularly at least one selected from sodium hydroxide and potassium hydroxide.
  • the mass of the water when added, is generally 0.5-10 times the sum of the masses of the long-chain alkane and the long-chain carboxylic acid, preferably 1-5 times or 1-3 times. Times.
  • the amount of the pH adjuster when added, generally makes the pH of the long-chain composition reach 5-12, preferably 7-10, 7.5-9 or 7.5-8.0.
  • the long-chain dibasic acid is at least one long-chain dibasic acid selected from C9-18 linear or branched, saturated or unsaturated aliphatic dicarboxylic acids, preferably selected from C9-18 At least one long-chain dibasic acid in straight-chain, saturated aliphatic dicarboxylic acid, preferably at least one selected from dodecanedioic acid, tetradecanedioic acid and hexadecanedioic acid Long-chain dibasic acid, especially dodecane dibasic acid.
  • the manufacturing method includes the following steps:
  • long-chain composition or long-chain composition kit (collectively referred to as the long-chain composition) according to any one of the embodiments of this specification;
  • the long-chain composition is subjected to a fermentation reaction in the presence of fermentation bacteria and a fermentation medium to convert the long-chain dibasic acid.
  • the fermenting bacteria are yeasts with a complete ⁇ , ⁇ -oxidation pathway, preferably selected from the genus Candida, Cryptococcus, Endosporium, At least one yeast of the genus Hansenula, Pichia, Rhodotorula, Sphaeropsis and Trichosporon, more preferably at least one yeast selected from the genus Candida, particularly It is Candida tropicalis, more particularly the mutant Candida tropicalis PF-UV-56 (preserved by the General Microbiology Center of China Microbial Culture Collection Management Committee, and the preservation number is CGMCC No.0356, and the preservation date is August 1998. 31st).
  • the fermentation medium may be a fermentation medium conventionally used in the art when producing long-chain dibasic acids by fermentation, and generally includes a carbon source of 10-60 g/ L, nitrogen source 1-10g/L, phosphorus source 1-10g/L, trace metal element source 0.1-50ppm, etc.
  • a carbon source for example, one or more selected from glucose, sucrose, maltose, fructose, molasses, glycerol, sorbitol, arabinose, rhamnose, methanol, and ethanol can be cited.
  • nitrogen source for example, one or more selected from yeast extract, peptone, corn steep liquor, urea, ammonium salt, and nitrate can be cited.
  • phosphorus source for example, one or more selected from the group consisting of normal phosphate, monohydrogen phosphate and dihydrogen phosphate, preferably selected from potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and ammonium dihydrogen phosphate , One or more of diammonium hydrogen phosphate, sodium dihydrogen phosphate and disodium hydrogen phosphate.
  • the source of the trace metal elements for example, one or more selected from the group consisting of sodium, potassium, calcium, magnesium, iron, copper, zinc and manganese sulfates, hydrochlorides, acetates and nitrates , Preferably selected from one or more of sodium chloride, sodium acetate, potassium chloride, magnesium sulfate, calcium chloride, iron chloride and copper sulfate.
  • composition of the fermentation medium preferably includes: 20 to 28 g/L of sucrose, 0.8 to 1.5 g/L of corn steep liquor, 2.0 to 4.0 g/L of yeast extract, 0.8 to 1.2 g/L of sodium chloride, Potassium dihydrogen phosphate 3.0 to 3.5g/L, magnesium sulfate 1.2 to 1.8g/L, urea 1.2 to 4.8g/L, ammonium sulfate 1.5 to 2g/L, sodium acetate 1.5 to 1.8g/L.
  • the fermentation reaction includes at least the following two steps.
  • Preliminary step mixing the fermentation bacteria and the fermentation medium, and performing the fermentation reaction for 5-60 hours to obtain a fermentation base liquid.
  • the reaction time of the preliminary step is preferably 10-40 hours, more preferably 20-30 hours or 24 hours
  • Conversion step adding the long-chain composition to the fermentation base liquid, and performing the fermentation reaction for 60-400 hours to convert the long-chain composition into the long-chain dibasic acid.
  • reaction time of the conversion step it is preferably 100-300 hours, more preferably 100-160 hours or 100-140 hours.
  • the added amount (by volume) of the fermented bacteria seed liquid is generally 2-30% of the control volume of the total liquid, preferably 5 to 20% or 10 to 15%.
  • the reaction temperature of the fermentation reaction is generally 25 to 37°C, preferably 28 to 32°C.
  • the stirring speed of the fermentation reaction is generally 100 to 1000 rpm, preferably 120 to 500 rpm or 150 to 300 rpm.
  • the aeration of the fermentation reaction is generally 0.2 to 10.0 VVM, preferably 0.2 to 2.0 VVM or 0.5 to 1.0 VVM.
  • the reaction time of the fermentation reaction is generally 65 hours or more, preferably more than 120 hours, more preferably 130 to 400 hours, 130 to 300 hours, 130 to 200 hours or 138 to 160 hours.
  • the reaction time includes the reaction time of the preliminary step and the reaction time of the conversion step.
  • the added amount of the long-chain composition is generally 100 to 1000 g/L total liquid control volume, preferably 100 to 500 g/L total liquid control volume or 150 to 250 g /L Total liquid control volume.
  • the added amount of the fermentation medium is generally 12 to 80 g/L total liquid control volume, preferably 32 to 50 g/L total liquid control volume, 35 to 45 g/L L total liquid control volume, or 37 to 42g/L total liquid control volume.
  • the long-chain composition is not added, and the fermentation reaction
  • the pH value is an autogenous state, that is, no pH value control is performed during this step or within this time period.
  • the pH value of the fermentation reaction is adjusted to between 6.0 and 7.5, preferably Between 6.8 and 7.0. More preferably, then every 5-60 hours, preferably every 10-40 hours or every 20-30 hours or every 24 hours, the pH value of the fermentation reaction is increased by 0.05 to 0.4, preferably by 0.1 to 0.3, until the end of the fermentation reaction.
  • the long-chain composition (also referred to as batch feeding) is added in n batches.
  • the time interval between two adjacent batches may be the same or different, preferably the same.
  • the time interval for example, 5-60 hours, 10-40 hours, 20-30 hours, or 24 hours can be cited.
  • n is a positive integer of 2-40, preferably a positive integer of 4-20 or 5-10.
  • the long-chain composition set according to any one of the embodiments of this specification is used, and the n batches are added to each of the n batches.
  • Long-chain compositions are preferably added to the n long-chain compositions in the order from the first long-chain composition to the n-th long-chain composition.
  • the long-chain composition (also referred to as fed-batch) is continuously added according to the reaction time.
  • the continuous addition may be a constant flow rate or a non-constant flow rate, preferably a constant flow rate.
  • R is the mass ratio of the long-chain alkane to the long-chain carboxylic acid
  • t is the reaction time of the conversion step
  • f() represents any non-increasing function, preferably any decreasing function, and any monotonic Decreasing function or arbitrary linear decreasing function.
  • the manufacturing method further includes a step of separating the long-chain dibasic acid from the reaction product of the fermentation reaction (referred to as fermentation broth).
  • the long-chain dibasic acid product is obtained after demulsification, membrane filtration, acid precipitation, filtration, and drying treatments.
  • the demulsification can be carried out in any manner conventionally known in the art, and is not particularly limited.
  • the pH of the fermentation broth is adjusted to 8.5 to 10, preferably 9 to 9.5, and the temperature is increased to 75 to 90° C. and maintained for 20 to 40 minutes.
  • the membrane filtration can be performed in any manner conventionally known in the art, and is not particularly limited.
  • specific examples include filtering the fermentation broth after demulsification through a membrane to perform solid-liquid separation to obtain a filtered clear liquid.
  • the filtration temperature is generally 30 to 50°C, preferably 40 to 50°C.
  • the diameter of the membrane pores is generally 10 to 50 nm, preferably 20 to 25 nm.
  • the acid precipitation can be performed in any manner conventionally known in the art, and is not particularly limited.
  • the acidic pH adjustment of the filtered supernatant can be specifically mentioned, and the pH is controlled to 3 to 5, preferably 3 to 4, so that the long-chain dibasic acid is precipitated.
  • the acidic pH adjuster is generally a strong acid, such as at least one of sulfuric acid, hydrochloric acid, and nitric acid.
  • the filtration can be performed in any manner conventionally known in the art, and is not particularly limited.
  • the filtering plate and frame filtering can be specifically mentioned.
  • the filtration pressure is generally 0.5 to 1.0 MPa
  • the filtration temperature is generally room temperature, such as 20 to 30°C.
  • the drying can be performed in any manner conventionally known in the art, and is not particularly limited.
  • the drying temperature is 80 to 105° C.
  • the drying time is 5 to 20 hours.
  • the experimental methods in the following examples and comparative examples, unless otherwise specified, are conventional methods in the art.
  • the experimental materials used in the following examples and comparative examples, unless otherwise specified, can be purchased from biochemical reagent stores.
  • V is the volume of the clear liquid obtained after the long-chain dibasic acid fermentation broth is filtered through a membrane to remove unreacted alkanes, L; M is the dry weight of the extracted long-chain dibasic acid, g; C is the long-chain dibasic acid Concentration in the lower tank, g/L.
  • the formula for calculating the molar conversion rate K of the mixed substrate is:
  • M is the dry weight of the extracted long-chain dibasic acid, g; M 1 is the mass of alkane, g; M 2 is the mass of lauric acid, g; m is the molecular weight of the long-chain dibasic acid; m 1 is the molecular weight of the alkane; m 2 Is the molecular weight of lauric acid.
  • C is the lower tank concentration of the long-chain dibasic acid, g/L; H is the fermentation period, h.
  • the Candida tropicalis mutant strain PF-UV-56 is selected as the fermentation strain for the fermentation of long-chain alkanes to produce long-chain dibasic acids, and the mutant strain is preserved in the Chinese Microbial Species Collection Management Committee. Center, the deposit number is CGMCC No. 0356.
  • Fermentation seed liquid production inoculate the slant preserved strains in 4 5L shake flasks (300mL shake flasks) for strain activation culture, the culture temperature is 32°C, and the rotation speed of the culture shaker is 200 rpm. After 48 hours of incubation, 1.2L of the activated seed liquid was taken as the fermented seed liquid.
  • Fermentation medium formula: sucrose 20g/L, corn steep liquor 0.8g/L, yeast extract 2.0g/L, sodium chloride 0.8g/L, potassium dihydrogen phosphate 3.0g/L, magnesium sulfate 1.2g/L, urea 1.2g/L, ammonium sulfate 1.5g/L, sodium acetate 1.5g/L.
  • the total fermentation period is 145h.
  • the pH of the fermentation broth was adjusted to 9.5, and the temperature was raised to 90°C for 20 minutes; the demulsified fermentation broth was separated by membrane filtration to obtain a clear water phase.
  • the filtration condition was that the membrane pore diameter was 20nm.
  • the filtration temperature is 40°C; the filtered water phase clear liquid stream is added with concentrated sulfuric acid, the pH is adjusted to 3, and the long-chain dibasic acid in the water phase clear liquid crystallizes out.
  • the precipitated long-chain dibasic acid is filtered and dried to obtain the long-chain dibasic acid crude acid product.
  • Filtration conditions filtration pressure 0.5MPa, filtration temperature 30°C. Drying conditions are 80°C temperature and 20h drying time.
  • the fermentation result is: the fermentation concentration of long-chain dibasic acid in step (2) is 152.0g/L, in step (3), 13.4L of fermentation clear liquid is obtained by membrane filtration, and the product quality of crude acid with long-chain dibasic acid is 1914.5g obtained by acid precipitation.
  • the extraction yield of crude acid is 94%, and the molar conversion rate of the mixed substrate of alkane and lauric acid is 62.4%.
  • the fermentation period is 150h in total. After the fermentation, the pH of the fermentation broth was adjusted to 9, and the temperature was raised to 75°C for 40 minutes; the demulsified fermentation broth was separated by membrane filtration to obtain a clear water phase.
  • the filtration condition was that the membrane pore diameter was 25nm.
  • the filtration temperature is 50°C; the filtered water phase clear liquid stream is added with concentrated sulfuric acid, the pH is adjusted to 4, and the long-chain dibasic acid in the water phase clear liquid crystallizes out.
  • the precipitated long-chain dibasic acid is filtered and dried to obtain the crude dibasic acid product. Filtration conditions: filtration pressure 1.0MPa, filtration temperature 20°C. Drying conditions are 105°C temperature and 5h drying time.
  • the fermentation result is: the fermentation concentration of long-chain dibasic acid in step (2) is 156.0g/L, in step (3), 13.6L of fermentation clear liquid is obtained by membrane filtration, and the product quality of long-chain dibasic acid crude acid is 1973.0g obtained by acid precipitation.
  • the extraction yield of crude acid is 93%, and the molar conversion rate of the mixed substrate of alkane and lauric acid is 60.2%.
  • the total fermentation period is 144h.
  • the pH of the fermentation broth was adjusted to 9.5, and the temperature was raised to 90°C for 20 minutes; the demulsified fermentation broth was separated by membrane filtration to obtain a clear water phase.
  • the filtration condition was that the membrane pore diameter was 20nm.
  • the filtration temperature is 40°C; the filtered water phase clear liquid stream is added with concentrated sulfuric acid, the pH is adjusted to 3, and the long-chain dibasic acid in the water phase clear liquid crystallizes out.
  • the precipitated long-chain dibasic acid is filtered and dried to obtain the long-chain dibasic acid crude acid product.
  • Filtration conditions filtration pressure 0.5MPa, filtration temperature 30°C. Drying conditions are 80°C temperature and 20h drying time.
  • the fermentation result is: the fermentation concentration of long-chain dibasic acid in step (2) is 153.0g/L, in step (3), 13.5L of fermentation clear liquid is obtained by membrane filtration, and the product quality of long-chain dibasic acid crude acid is 1941.6g obtained by acid precipitation.
  • the extraction yield of crude acid is 94%, and the molar conversion rate of the mixed substrate of alkane and lauric acid is 63.3%.
  • step (1) does not use a mixed substrate of alkanes and lauric acid
  • step (2) a batch of alkane with a total mass of 1980 g is added during the fermentation process.
  • the total fermentation period is 158h
  • the fermentation result is: in step (2), the fermentation concentration of long-chain dibasic acid is 150.0g/L, in step (3), 13.0L of fermentation clear liquid is obtained by membrane filtration, and the crude long-chain dibasic acid is obtained by acid precipitation.
  • the mass of the acid product is 1833g
  • the extraction yield of crude acid is 94%
  • the molar conversion rate of the mixed substrate of alkane and lauric acid is 58.2%.
  • step (1) does not use alkane and lauric acid as a mixed substrate, but only in step (2) batch addition of lauric acid with a total mass of 1980 g during the fermentation process.
  • the total fermentation period is 155h, and the fermentation result is: the fermentation concentration of long-chain dibasic acid in step (2) is 15.0g/L, in step (3), 12.0L of fermentation clear liquid is obtained by membrane filtration, and the crude long-chain dibasic acid is obtained by acid precipitation.
  • the acid product mass is 169.2g
  • the crude acid extraction yield is 94%
  • the molar conversion rate of the mixed substrate of alkane and lauric acid is 6.3%.
  • step (1) does not use the mixed substrate of alkanes and lauric acid
  • step (2) a batch of alkane with a total mass of 1848 g is added during the fermentation process.
  • the fermentation cycle is 150h in total.
  • the fermentation result is: the fermentation concentration of long-chain dibasic acid in step (2) is 141.0g/L, in step (3), 12.9L of fermentation clear liquid is obtained by membrane filtration, and the crude long-chain dibasic acid is obtained by acid precipitation.
  • the mass of the acid product is 1709.8g
  • the extraction yield of crude acid is 94%
  • the molar conversion rate of the mixed substrate of alkane and lauric acid is 58.2%.
  • step (1) alkane and lauric acid are stirred and mixed at room temperature, 1980 g of water is added, and after thorough mixing, solid sodium hydroxide is added to adjust the pH of the system to 8 to obtain a mixed substrate.
  • the fermentation period is 144h in total, and the fermentation result is: after detection and calculation, the fermentation concentration of long-chain dibasic acid in step (2) is 142.0g/L, and in step (3), 13.2L of fermentation clear liquid is obtained by membrane filtration, and long-chain dibasic acid is obtained by acid precipitation.
  • the quality of the crude acid of chain dibasic acid is 1762g, the extraction yield of crude acid is 94%, and the molar conversion rate of the mixed substrate of alkane and lauric acid is 57.4%.
  • step (1) after the alkane and lauric acid are stirred and dissolved at 60° C., the aqueous solution is not prepared by adding water, and the pH is not adjusted by alkalinity, and it is directly used in step (2) fermentation process.
  • the total fermentation period is 144h.
  • the fermentation result is that the fermentation concentration of long-chain dibasic acid in step (2) is 135.0g/L, and in step (3), 13.0L of fermentation clear liquid is obtained by membrane filtration, and the crude long-chain dibasic acid is obtained by acid precipitation.
  • the mass of the acid product is 1650g
  • the crude acid extraction yield is 94%
  • the molar conversion rate of the mixed substrate of alkane and lauric acid is 53.8%.
  • step (1) after stirring and dissolving the alkane and lauric acid at 60°C, water is added to prepare an aqueous solution, which is directly used in step (2) fermentation process without pH alkaline adjustment.
  • the total fermentation period is 145 hours.
  • the fermentation result is that the fermentation concentration of long-chain dibasic acid in step (2) is 142.0g/L, and in step (3), 13.5L of fermentation clear liquid is obtained by membrane filtration, and the crude long-chain dibasic acid is obtained by acid precipitation.
  • the mass of the acid product is 1802g
  • the crude acid extraction yield is 94%
  • the molar conversion rate of the mixed substrate of alkane and lauric acid is 58.7%.
  • the fermentation result is: the fermentation concentration of long-chain dibasic acid in step (2) is 160.0g/L, in step (3), 13.6L of fermentation clear liquid is obtained by membrane filtration, and the product quality of crude acid with long-chain dibasic acid is 2030.4g obtained by acid precipitation. , The extraction yield of crude acid is 94%, and the molar conversion rate of the mixed substrate of alkane and lauric acid is 62.2%.
  • the fermentation period is 142h in total. After the fermentation, the long-chain dibasic acid was extracted according to the method of Example 1 step (1).
  • the fermentation result is: the fermentation concentration of long-chain dibasic acid in step (2) is 148.0g/L, in step (3), 12.9L of fermentation clear liquid is obtained through membrane filtration, and the product quality of long-chain dibasic acid crude acid is 2048.9g by acid precipitation. , The extraction yield of crude acid is 94%, and the molar conversion rate of the mixed substrate of alkane and lauric acid is 62.1%.
  • step (1) the combination of tetradecane and myristic acid is used to replace the combination of alkane and lauric acid in the original embodiment.
  • the total fermentation period is 144h.
  • the fermentation result is that the fermentation concentration of long-chain dibasic acid in step (2) is 115.0g/L, and in step (3), 13.0L of fermentation clear liquid is obtained by membrane filtration, and the crude long-chain dibasic acid is obtained by acid precipitation.
  • the mass of the acid product is 1405.3g
  • the crude acid extraction yield is 94%
  • the molar conversion rate of the mixed substrate of alkane and lauric acid is 53.2%.
  • step (1) uses a combination of hexadecane and palmitic acid to replace the combination of alkane and lauric acid in the original embodiment.
  • the total fermentation period is 144h.
  • the fermentation result is that the fermentation concentration of long-chain dibasic acid in step (2) is 98.0g/L, and in step (3), 13.0L of fermentation clear liquid is obtained by membrane filtration, and the crude long-chain dibasic acid is obtained by acid precipitation.
  • the mass of the acid product is 1197.6g
  • the crude acid extraction yield is 94%
  • the molar conversion rate of the mixed substrate of alkane and lauric acid is 51.6%.
  • step (2) of Example 2 Carry out the long-chain dibasic acid fermentation process according to the method of step (2) of Example 1, the difference is that the alkane/lauric acid produced in step (1) is mixed when the fermentation is 24h, 48h, 72h, 96h, 120h
  • the substrates are sequentially added to the fermentation system in the order numbered 1-5.
  • the fermentation result is: the fermentation concentration of long-chain dibasic acid in step (2) is 149.0g/L, in step (3), 13.0L of fermentation clear liquid is obtained by membrane filtration, and the product quality of crude acid with long-chain dibasic acid is 1820.8g by acid precipitation.
  • the extraction yield of crude acid is 94%, and the molar conversion rate of the mixed substrate of alkane and lauric acid is 63.0%.
  • step (2) of Example 2 Carry out the long-chain dibasic acid fermentation process according to the method of step (2) of Example 1, the difference is that the alkane/lauric acid produced in step (1) is mixed when the fermentation is 24h, 48h, 72h, 96h, 120h
  • the substrates are sequentially added to the fermentation system in the order numbered 1-5.
  • the fermentation result is: the fermentation concentration of long-chain dibasic acid in step (2) is 154.0g/L, in step (3), 13.4L of fermentation clear liquid is obtained by membrane filtration, and the product quality of crude acid with long-chain dibasic acid is 1939.8g by acid precipitation.
  • the extraction yield of crude acid is 94%, and the molar conversion rate of the mixed substrate of alkane and lauric acid is 64.2%.
  • step (2) Carry out the long-chain dibasic acid fermentation process according to step (2) of Example 1, except that the fermentation is within the time range of 24h-48h, 48h-72h, 72h-96h, 96h-120h, and 120h-144h.
  • the alkane/lauric acid mixed substrate produced in step (1) is added to the fermentation system in a constant-rate flow-feeding manner according to the sequence numbered 1-5, and the flow rate is controlled to 32 g/h.
  • the fermentation result is: the fermentation concentration of long-chain dibasic acid in step (2) is 152.0g/L, in step (3), 13.2L of fermentation clear liquid is obtained by membrane filtration, and the product mass of long-chain dibasic acid crude acid is 1886.0g obtained by acid precipitation.
  • the extraction yield of crude acid is 94%, and the molar conversion rate of the mixed substrate of alkane and lauric acid is 65.3%.

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Abstract

提供一种长链组合物或长链组合物套组、其制造方法、及其在通过发酵法制造长链二元酸中的应用。所述长链组合物包含选自C9-18直链或支链烷烃中的至少一种长链烷烃和选自C9-18直链或支链、饱和或不饱和的脂肪族一元羧酸中的至少一种长链羧酸，其中所述长链烷烃与所述长链羧酸的质量比为1:1至40:1。所述长链组合物在作为原料通过发酵法来制造长链二元酸时，表现出发酵水平较高或底物利用率较高等优点。

Description

一种长链组合物、长链组合物套组、其制造方法及其应用 技术领域
本发明属于生物化工技术领域，具体涉及一种长链组合物或长链组合物套组。本发明还涉及一种长链组合物或长链组合物套组的制造方法，及其在长链二元酸制造中的应用。
背景技术
十二碳二元酸(简称DDDA)是指碳链中含有12个碳原子的脂肪族二羧酸，是一类有着重要和广泛工业用途的精细化工产品，同时也是化学工业中合成高级香料、高性能尼龙工程塑料、高档尼龙热熔胶、高温电介质、高级油漆和涂料、高级润滑油、耐寒性增塑剂、树脂、医药和农药等重要原料。
发酵法生产长链二元酸是利用微生物特有的氧化能力和微生物胞内酶的作用，在常温常压下分别通过α、ω-氧化，将长链正烷烃两端的甲基氧化成为羧基，生成相应链长的各种长链二元酸。能通过发酵生产长链二元酸的细菌、霉菌和放线菌的种类很多，其中假丝酵母属(candida)的酵母菌是正烷烃发酵生产二元酸的高产微生物。
现有技术一般将酵母菌作为发酵菌株，以正构烷烃作为底物，进行相应碳数的长链二元酸的生产，如CN102115767A、CN102115768A等，分别形成了十一碳二元酸和十六碳二元酸的制造方案。另外，CN103805643A公开了一种生产长链二元酸的方法，是将二元酸菌种扩大培养获得种子液，种子液进行扩大发酵，发酵过程中加入乳化烷烃，反应结束后除去菌体蛋白，结晶获得长链二元酸产品。
发明内容
本发明的发明人发现，现有技术在使用正构烷烃作为底物来制造长链二元酸时，仍然存在发酵水平较低或底物利用率较低等问题。本发明基于该发现而完成。
具体而言，本发明涉及以下方面的内容。
1.一种长链组合物(优选发酵用)，包含选自C9-18直链或支链(优选直 链)烷烃中的至少一种长链烷烃(优选选自正十二烷、正十四烷和正十六烷中的至少一种长链烷烃，特别是正十二烷)和选自C9-18直链或支链(优选直链)、饱和或不饱和(优选饱和)的脂肪族一元羧酸中的至少一种长链羧酸(优选选自月桂酸、肉豆蔻酸和棕榈酸中的至少一种长链羧酸，特别是月桂酸)，其中所述长链烷烃与所述长链羧酸的质量比为1:1至40:1(优选2:1至20:1或5:1至10:1)。
2.根据前述或后述任一方面所述的长链组合物，还包含水，其中所述水的质量是所述长链烷烃和所述长链羧酸的质量之和的0.5-10倍(优选1-5倍或1-3倍)。
3.根据前述或后述任一方面所述的长链组合物，其pH值为5-12(优选7-10、7.5-9或7.5-8.0)，和/或，在32℃下呈现为液体或固液混合物的形态(优选液体形态，特别是含水液体形态)，和/或，所述长链烷烃与所述长链羧酸具有相同的碳原子数。
4.一种长链组合物套组，包括彼此独立存在(比如彼此独立包装或彼此独立分隔)的n个根据前述或后述任一方面所述的长链组合物，其中n为2-40(优选4-20或5-10)的正整数，其中设在第i个(i为从2至n的任意正整数)长链组合物中，所述长链烷烃与所述长链羧酸的质量比为R i，在第1个长链组合物中，所述长链烷烃与所述长链羧酸的质量比为R 1，在第n个长链组合物中，所述长链烷烃与所述长链羧酸的质量比为R n，则R i-1/R i≥1(优选R i-1/R i＝1-20、R i-1/R i＝1.0001-10、R i-1/R i＝1.001-10、R i-1/R i＝1.01-10、R i-1/R i＝1.1-5或R i-1/R i＝1.5-2)并且R 1/R n＞1(优选R 1/R n＝1.0001-30、R 1/R n＝1.001-20、R 1/R n＝1.01-10、R 1/R n＝1.1-5或R 1/R n＝1.5-2)。
5.根据前述或后述任一方面所述的长链组合物套组，其中所述n个长链组合物是分别单独制造的，和/或，任意两个所述n个长链组合物彼此之间的重量比(以所述长链组合物的量计算)相同或不同(优选相同或基本上相同)，和/或，任意两个所述n个长链组合物彼此之间的重量比(以所述长链烷烃的量计算)相同或基本上相同。
6.一种长链组合物的制造方法，其中所述长链组合物(优选发酵用)包含选自C9-18直链或支链(优选直链)烷烃中的至少一种长链烷烃(优选选自正十二烷、正十四烷和正十六烷中的至少一种长链烷烃，特别是正十二烷) 和选自C9-18直链或支链(优选直链)、饱和或不饱和(优选饱和)的脂肪族一元羧酸中的至少一种长链羧酸(优选选自月桂酸、肉豆蔻酸和棕榈酸中的至少一种长链羧酸，特别是月桂酸)，其中所述长链烷烃与所述长链羧酸的质量比为1:1至40:1(优选2:1至20:1或5:1至10:1)，所述制造方法至少包括混合步骤：将所述长链烷烃与所述长链羧酸任选在加热(优选加热温度为45至70℃或50至60℃)和搅拌(优选搅拌转速为50至250rpm或150至250rpm)的情况下按照所述质量比进行混合(优选直至混合均匀)，获得所述长链组合物。
7.根据前述或后述任一方面所述的制造方法，其中在所述混合步骤中还加入水和pH值调节剂(比如碱，特别是选自氢氧化钠和氢氧化钾中的至少一种)，和/或，所述制造方法还包括任选在加热(优选加热温度为45至70℃或50至60℃)和搅拌(优选搅拌转速为50至250rpm或150至250rpm)的情况下，向所述长链组合物中加入水和pH值调节剂(比如碱，特别是选自氢氧化钠和氢氧化钾中的至少一种)的步骤，其中所述水的质量是所述长链烷烃和所述长链羧酸的质量之和的0.5-10倍(优选1-5倍或1-3倍)，并且所述pH值调节剂的用量使得所述长链组合物的pH值达到5-12(优选7-10、7.5-9或7.5-8.0)。
8.一种长链二元酸的制造方法，其中所述长链二元酸是选自C9-18直链或支链(优选直链)、饱和或不饱和(优选饱和)的脂肪族二元羧酸中的至少一种长链二元酸(优选选自十二碳二元酸、十四碳二元酸和十六碳二元酸中的至少一种长链二元酸，特别是十二碳二元酸)，
所述制造方法包括以下步骤：
(1)按照根据前述或后述任一方面所述的制造方法来制造长链组合物、提供根据前述或后述任一方面所述的长链组合物、或者提供根据前述或后述任一方面所述的长链组合物套组，统称为长链组合物；
(2)使所述长链组合物在发酵菌和发酵培养基的存在下进行发酵反应而转化为所述长链二元酸。
9.根据前述或后述任一方面所述的制造方法，其中所述发酵菌是具有完整α,ω-氧化途径的酵母菌(优选选自假丝酵母属、隐球酵母属、内孢霉属、汉逊氏酵母属、毕赤氏酵母属、红酵母属、球拟酵母属和丝孢酵母属中的至少一种酵母菌，更优选选自假丝酵母属中的至少一种酵母菌，特别是热带假丝酵母菌，更特别是热带假丝酵母菌突变株PF-UV-56(中国微生物菌种保藏 管理委员会普通微生物中心保藏，保藏编号为CGMCC No.0356))，和/或，所述发酵培养基包括：蔗糖20至28g/L、玉米浆0.8至1.5g/L、酵母膏2.0至4.0g/L、氯化钠0.8至1.2g/L、磷酸二氢钾3.0至3.5g/L、硫酸镁1.2至1.8g/L、尿素1.2至4.8g/L，硫酸铵1.5至2g/L、醋酸钠1.5至1.8g/L。
10.根据前述或后述任一方面所述的制造方法，其中所述发酵反应包括以下步骤：
预备步骤：混合所述发酵菌和所述发酵培养基，进行所述发酵反应5-60小时(优选10-40小时，更优选20-30小时或24小时)，获得发酵基础液，
转化步骤：向所述发酵基础液中加入所述长链组合物，进行所述发酵反应60-400小时(优选100-300小时，更优选100至160小时或100至140小时)，以将所述长链组合物转化为所述长链二元酸。
11.根据前述或后述任一方面所述的制造方法，其中发酵菌种子液的添加量(以体积计)为总装液控制体积的2-30％(优选5至20％或10至15％)，和/或，所述发酵反应的反应温度为25至37℃，优选28至32℃，和/或，所述发酵反应的搅拌转速为100至1000rpm(优选120至500rpm或150至300rpm)，和/或，所述发酵反应的通气量为0.2至10.0VVM(优选0.2至2.0VVM或0.5至1.0VVM)，和/或，所述发酵反应的反应时间为65小时以上(优选大于120小时，更优选130至400小时、130至300小时、130至200小时或138至160小时)，和/或，所述长链组合物的添加量为100至1000g/L总装液控制体积(优选100至500g/L总装液控制体积或150至250g/L总装液控制体积)，和/或，所述发酵培养基的添加量为12至80g/L总装液控制体积(优选32至50g/L总装液控制体积、35至45g/L总装液控制体积、或者37至42g/L总装液控制体积)。
12.根据前述或后述任一方面所述的制造方法，其中在所述预备步骤中(优选在所述预备步骤从开始至24小时之内)，不添加所述长链组合物，并且所述发酵反应的pH值为自生状态(即，不进行pH值控制)，和/或，在所述转化步骤开始时(优选在所述预备步骤开始24小时之后)，将所述发酵反应的pH值调节至6.0至7.5之间(优选6.8至7.0之间)，随后每隔5-60小时(优选每隔10-40小时或每隔20-30小时或每隔24小时)，使所述发酵反应的pH值提高0.05至0.4(优选0.1至0.3)，直至所述发酵反应结束。
13.根据前述或后述任一方面所述的制造方法，其中在所述转化步骤中， 分n个批次加入所述长链组合物，相邻两个批次之间的时间间隔可以相同或不同(优选时间间隔为5-60小时、10-40小时、20-30小时或24小时)，其中n为2-40(优选4-20或5-10)的正整数，或者根据反应时间连续加入所述长链组合物。
14.根据前述或后述任一方面所述的制造方法，其中使用根据前述或后述任一方面所述的长链组合物套组，分所述n个批次分别加入所述n个长链组合物(优选按照从第1个长链组合物至第n个长链组合物的次序)，或者按照函数R＝f(t)连续加入所述长链组合物，其中R是所述长链烷烃与所述长链羧酸的质量比，t是所述转化步骤的反应时间，f()代表任意的非递增函数(优选任意的递减函数、任意的单调递减函数或任意的线性递减函数)。
15.根据前述或后述任一方面所述的制造方法，还包括将所述长链二元酸从所述发酵反应的反应产物中分离的步骤。
另外，本发明还涉及以下方面的内容。
1.一种制备十二碳二元酸的方法，其特征在于包括以下步骤：
(1)将十二碳烷烃与月桂酸按比例混合，升温搅拌使月桂酸充分溶解，随后在混合物中加入水，混匀后加碱调节pH值为7-9，得到混合底物；
(2)将发酵菌和发酵培养基加入发酵罐进行发酵，发酵过程中补加步骤(1)制备的混合底物，直至发酵结束；
(3)发酵结束后，经破乳、膜过滤、酸析、过滤、烘干处理后，获得长链二元酸产品。
2.根据前述或后述任一方面所述的方法，其特征在于：步骤(1)所述的十二碳烷烃与月桂酸的质量比为2:1-10:1，优选5:1-10:1。
3.根据前述或后述任一方面所述的方法，其特征在于：步骤(1)升温至45-70℃，优选50-60℃；搅拌转速为50-250rpm，优选150-250rpm。
4.根据前述或后述任一方面所述的方法，其特征在于：步骤(1)加入混合物质量1-3倍的水。
5.根据前述或后述任一方面所述的方法，其特征在于：步骤(1)混匀后加碱将pH值调至7-9，优选7.5-8.0；所述的碱为氢氧化钠、氢氧化钾中的至少一种。
6.根据前述或后述任一方面所述的方法，其特征在于：步骤(2)所述 的十二碳发酵菌为具有完整α、ω-氧化途径的酵母菌，优选假丝酵母属、隐球酵母属、内孢霉属、汉逊氏酵母属、毕赤氏酵母属、红酵母属、球拟酵母属或丝孢酵母属中的至少一种。
7.根据前述或后述任一方面所述的方法，其特征在于：步骤(2)所述的发酵培养基配方为：蔗糖20-28g/L、玉米浆0.8-1.5g/L、酵母膏2.0-4.0g/L、氯化钠0.8-1.2g/L、磷酸二氢钾3.0-3.5g/L、硫酸镁1.2-1.8g/L、尿素1.2-4.8g/L，硫酸铵1.5-2g/L、醋酸钠1.5-1.8g/L。
8.根据前述或后述任一方面所述的方法，其特征在于：步骤(2)所述的发酵菌种子液的添加量为发酵液体积的5％-20％，优选10％-15％。
9.根据前述或后述任一方面所述的方法，其特征在于：步骤(2)的发酵温度为25-37℃，优选28-32℃；搅拌转速为120-500rpm，优选150-300rpm；通气量为0.2-1.0VVM，优选0.5-1.0VVM；发酵时间为138-144小时。
10.根据前述或后述任一方面所述的方法，其特征在于：步骤(2)pH的控制方式具体为：在发酵0-24h内，不进行pH控制；24h后将pH调整至6.8-7.0的中性范围；随后每间隔24h将pH控制值提高0.1-0.3，直至反应结束。
11.根据前述或后述任一方面所述的方法，其特征在于：步骤(2)发酵过程采用批次补料或流加补料方式补加步骤(1)的混合底物。
12.根据前述或后述任一方面所述的方法，其特征在于：步骤(2)采用批次补料，分5-10个批次补加完；采用流加补料，根据发酵时间计算流加速度。
13.根据前述或后述任一方面所述的方法，其特征在于：步骤(3)所述破乳是将发酵液pH调至8.5-10，优选9-9.5，并升温至75-90℃，维持20-40min。
14.根据前述或后述任一方面所述的方法，其特征在于：步骤(3)所述膜过滤是将破乳液经膜过滤，进行固液分离，获得过滤清液；过滤温度30-50℃，优选40-50℃，膜孔直径为10-50nm，优选20-25nm。
15.根据前述或后述任一方面所述的方法，其特征在于：步骤(3)所述酸析是将过滤清液进行酸性pH调节，控制pH为3-5，优选3-4，使长链二元酸析出。
16.根据前述或后述任一方面所述的方法，其特征在于：步骤(3)所述过滤操作采用板框过滤，过滤压力为0.5-1.0MPa，过滤温度为20-30℃；所述 的烘干温度为80-105℃，烘干时间为5-20h。
技术效果
同现有技术相比，本发明能够实现如下技术效果中的至少一个，优选全部实现。
(1)根据本发明的长链组合物，长链烷烃与长链羧酸的质量比满足本发明的规定，在用于发酵过程时，既可以提高发酵水平，又可以解决长链羧酸的水溶性差、在发酵体系中难以利用的问题。
(2)根据本发明的长链组合物，在优选的情况下，采用物理溶解和化学解离两步工序，既增加了长链羧酸的溶解效果，又促使长链烷烃和长链羧酸形成极性可溶性胶团，进一步提高发酵水平。
(3)根据本发明的长链组合物的制造方法，在优选的情况下，采用碱性调节方案，减少了由于补料造成发酵过程pH波动问题，进一步采用流加方式补料，既提高了底物利用率，又提高了发酵水平。
(4)根据本发明的发酵方法，长链羧酸可以兼做碳源，能够使部分长链羧酸直接通过脂肪酸代谢，获得重要的中间代谢产物乙酰辅酶A，以利于生物量的快速累积，缩短发酵时间，并且提高长链二元羧酸的生产强度。
(5)根据本发明的发酵方法，在优选的情况下，通过在发酵过程中增加长链脂肪酸的后续添加量，可以进一步提高发酵水平和长链二元羧酸的生产强度。
具体实施方式
下面对本发明的具体实施方式进行详细说明，但是需要指出的是，本发明的保护范围并不受这些具体实施方式的限制，而是由附录的权利要求书来确定。
本说明书提到的所有出版物、专利申请、专利和其它参考文献全都引于此供参考。除非另有定义，本说明书所用的所有技术和科学术语都具有本领域技术人员常规理解的含义。在有冲突的情况下，以本说明书的定义为准。
当本说明书以词头“本领域技术人员公知”、“现有技术”或其类似 用语来导出材料、物质、方法、步骤、装置或部件等时，该词头导出的对象涵盖本申请提出时本领域常规使用的那些，但也包括目前还不常用，却将变成本领域公认为适用于类似目的的那些。
在本说明书的上下文中，所谓“基本上”指的是允许存在对于本领域技术人员而言可以接受或认为合理的偏差，比如偏差在±10％以内、±5％以内、±1％以内、±0.5％以内或者±0.1％以内。
在本说明书的上下文中，术语“总装液控制体积”的含义是发酵罐液相总体积，通常为发酵罐体积的70％-80％。
在没有明确指明的情况下，本说明书内所提到的所有百分数、份数、比率等都是以重量为基准的，而且压力是表压。
在本说明书的上下文中，本发明的任何两个或多个实施方式都可以任意组合，由此而形成的技术方案属于本说明书原始公开内容的一部分，同时也落入本发明的保护范围。
根据本发明的一个实施方式，涉及一种长链组合物。所述长链组合物优选是发酵用长链组合物，特别适合用于作为原料通过发酵法来制造长链二元酸。
根据本发明的一个实施方式，所述长链组合物包含选自C9-18直链或支链烷烃中的至少一种长链烷烃和选自C9-18直链或支链、饱和或不饱和的脂肪族一元羧酸中的至少一种长链羧酸。在此，作为所述长链烷烃，优选选自C9-18直链烷烃中的至少一种长链烷烃，更优选选自正十二烷、正十四烷和正十六烷中的至少一种长链烷烃，特别是正十二烷。另外，作为所述长链羧酸，优选选自C9-18直链且饱和的脂肪族一元羧酸中的至少一种长链羧酸，特别优选选自月桂酸、肉豆蔻酸和棕榈酸中的至少一种长链羧酸，特别是月桂酸。
根据本发明的一个实施方式，所述长链烷烃与所述长链羧酸的质量比一般为1:1至40:1，优选2:1至20:1或5:1至10:1。
根据本发明的一个实施方式，所述长链组合物还包含水。在此，所述水的质量一般是所述长链烷烃和所述长链羧酸的质量之和的0.5-10倍，优选1-5倍或1-3倍。
根据本发明的一个实施方式，所述长链组合物的pH值一般为5-12，优选7-10、7.5-9或7.5-8.0。在本说明书的上下文中，所述长链组合物的pH 值测量方法可采用pH试纸法或玻璃电极法。
根据本发明的一个实施方式，所述长链组合物在32℃下呈现为液体或固液混合物的形态，优选液体形态，特别是含水液体形态。在本说明书的上下文中，液体包含均相液体和非均相液体，后者比如两种或多种液体所形成的多相液体状混合物。
根据本发明的一个实施方式，在所述长链组合物中，所述长链烷烃与所述长链羧酸具有相同的碳原子数，比如正十二烷与月桂酸的组合。
根据本发明的一个实施方式，还涉及一种长链组合物套组，其包括多个本说明书任意一个实施方式所述的长链组合物。特别是，所述长链组合物套组包括彼此独立存在的n个本说明书任意一个实施方式所述的长链组合物。在此，n一般为2-40的正整数，优选4-20或5-10的正整数。另外，所谓彼此独立存在，特别可以举出彼此独立包装，或者彼此独立分隔，也即同一个包装内的多个隔间。
根据本发明的该实施方式，设在第i个长链组合物中，所述长链烷烃与所述长链羧酸的质量比为R i，在第1个长链组合物中，所述长链烷烃与所述长链羧酸的质量比为R 1，在第n个长链组合物中，所述长链烷烃与所述长链羧酸的质量比为R n，则一般R i-1/R i≥1，优选R i-1/R i＝1-20、R i-1/R i＝1.0001-10、R i-1/R i＝1.001-10、R i-1/R i＝1.01-10、R i-1/R i＝1.1-5或R i-1/R i＝1.5-2。在此，i为从2至n的任意正整数。
根据本发明的该实施方式，优选的是，R 1/R n＞1，优选R 1/R n＝1.0001-30、R 1/R n＝1.001-20、R 1/R n＝1.01-10、R 1/R n＝1.1-5或R 1/R n＝1.5-2。
根据本发明的一个实施方式，在所述长链组合物套组中，所述n个长链组合物是分别单独制造的。这些长链组合物的制造方法可以参考本说明书下文所述。
根据本发明的一个实施方式，在所述长链组合物套组中，任意两个所述n个长链组合物彼此之间的重量比(以所述长链组合物的量计算)相同或不同，优选相同或基本上相同。
根据本发明的另一个实施方式，在所述长链组合物套组中，任意两个所述n个长链组合物彼此之间的重量比(以所述长链烷烃的量计算)相同或基本上相同。
根据本发明的一个实施方式，所述长链组合物可以通过一种制造方法进行制造。具体举例而言，所述制造方法至少包括如下的混合步骤。
混合步骤：将所述长链烷烃与所述长链羧酸按照所述质量比进行混合，获得所述长链组合物。在此，所述混合任选在加热和搅拌的情况下进行。作为所述加热，特别可以举出加热温度为45至70℃或50至60℃。另外，作为所述搅拌，特别可以举出搅拌转速为50至250rpm或150至250rpm。再者，作为所述混合，优选直至混合均匀，直至获得均匀的液体混合物。
根据本发明的一个实施方式，根据所述制造方法，在所述混合步骤中还加入水和pH值调节剂。在此，作为所述pH值调节剂，比如可以举出碱，特别是选自氢氧化钠和氢氧化钾中的至少一种。
根据本发明的一个实施方式，所述制造方法还包括向所述长链组合物中加入水和pH值调节剂的步骤。在此，所述步骤任选在加热和搅拌的情况下进行。作为所述加热，特别可以举出加热温度为45至70℃或50至60℃。另外，作为所述搅拌，特别可以举出搅拌转速为50至250rpm或150至250rpm。再者，作为所述pH值调节剂，比如可以举出碱，特别是选自氢氧化钠和氢氧化钾中的至少一种。
根据本发明的一个实施方式，在加入时，所述水的质量一般是所述长链烷烃和所述长链羧酸的质量之和的0.5-10倍，优选1-5倍或1-3倍。
根据本发明的一个实施方式，在加入时，所述pH值调节剂的用量一般使得所述长链组合物的pH值达到5-12，优选7-10、7.5-9或7.5-8.0。
根据本发明的一个实施方式，还涉及一种长链二元酸的制造方法。在此，所述长链二元酸是选自C9-18直链或支链、饱和或不饱和的脂肪族二元羧酸中的至少一种长链二元酸，优选选自C9-18直链、饱和的脂肪族二元羧酸中的至少一种长链二元酸，优选选自十二碳二元酸、十四碳二元酸和十六碳二元酸中的至少一种长链二元酸，特别是十二碳二元酸。
根据本发明的一个实施方式，所述制造方法包括以下步骤：
(1)提供本说明书任意一个实施方式所述的长链组合物或长链组合物套组(统称为长链组合物)；
(2)使所述长链组合物在发酵菌和发酵培养基的存在下进行发酵反应而转化为所述长链二元酸。
根据本发明的一个实施方式，在所述制造方法中，所述发酵菌是具有完整α,ω-氧化途径的酵母菌，优选选自假丝酵母属、隐球酵母属、内孢霉属、汉逊氏酵母属、毕赤氏酵母属、红酵母属、球拟酵母属和丝孢酵母属中的至少一种酵母菌，更优选选自假丝酵母属中的至少一种酵母菌，特别是热带假丝酵母菌，更特别是热带假丝酵母菌突变株PF-UV-56(中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏编号为CGMCC No.0356，保藏日期为1998年8月31日)。
根据本发明的一个实施方式，在所述制造方法中，所述发酵培养基可以是本领域在通过发酵法制造长链二元酸时常规使用的发酵培养基，一般包括碳源10-60g/L、氮源1-10g/L、磷源1-10g/L和微量金属元素源0.1-50ppm等。在此，作为所述碳源，比如可以举出选自葡萄糖、蔗糖、麦芽糖、果糖、糖蜜、甘油、山梨醇、阿拉伯糖、鼠李糖、甲醇和乙醇中的一种或多种。作为所述氮源，比如可以举出选自酵母膏、蛋白胨、玉米浆、尿素、铵盐和硝酸盐中的一种或多种。作为所述磷源，比如可以举出选自磷酸正盐、磷酸一氢盐和磷酸二氢盐中的一种或多种，优选选自磷酸二氢钾、磷酸氢二钾、磷酸二氢铵、磷酸氢二铵、磷酸二氢钠和磷酸氢二钠中的一种或多种。作为所述微量金属元素源，比如可以举出选自钠、钾、钙、镁、铁、铜、锌和锰的硫酸盐、盐酸盐、醋酸盐和硝酸盐中的一种或多种，优选选自氯化钠、醋酸钠、氯化钾、硫酸镁、氯化钙、氯化铁和硫酸铜中的一种或多种。特别地，作为所述发酵培养基的组成，优选包括：蔗糖20至28g/L、玉米浆0.8至1.5g/L、酵母膏2.0至4.0g/L、氯化钠0.8至1.2g/L、磷酸二氢钾3.0至3.5g/L、硫酸镁1.2至1.8g/L、尿素1.2至4.8g/L，硫酸铵1.5至2g/L、醋酸钠1.5至1.8g/L。
根据本发明的一个实施方式，在所述制造方法中，所述发酵反应至少包括以下两个步骤。
预备步骤：混合所述发酵菌和所述发酵培养基，进行所述发酵反应5-60小时，获得发酵基础液。在此，作为所述预备步骤的反应时间，优选10-40小时，更优选20-30小时或24小时
转化步骤：向所述发酵基础液中加入所述长链组合物，进行所述发酵反应60-400小时，以将所述长链组合物转化为所述长链二元酸。在此，作为所述转化步骤的反应时间，优选100-300小时，更优选100至160小时或100至140 小时。
根据本发明的一个实施方式，在所述制造方法中，发酵菌种子液的添加量(以体积计)一般为总装液控制体积的2-30％，优选5至20％或10至15％。
根据本发明的一个实施方式，在所述制造方法中，所述发酵反应的反应温度一般为25至37℃，优选28至32℃。
根据本发明的一个实施方式，在所述制造方法中，所述发酵反应的搅拌转速一般为100至1000rpm，优选120至500rpm或150至300rpm。
根据本发明的一个实施方式，在所述制造方法中，所述发酵反应的通气量一般为0.2至10.0VVM，优选0.2至2.0VVM或0.5至1.0VVM。
根据本发明的一个实施方式，在所述制造方法中，所述发酵反应的反应时间一般为65小时以上，优选大于120小时，更优选130至400小时、130至300小时、130至200小时或138至160小时。在此，所述反应时间包括所述预备步骤的反应时间和所述转化步骤的反应时间。
根据本发明的一个实施方式，在所述制造方法中，所述长链组合物的添加量一般为100至1000g/L总装液控制体积，优选100至500g/L总装液控制体积或150至250g/L总装液控制体积。
根据本发明的一个实施方式，在所述制造方法中，所述发酵培养基的添加量一般为12至80g/L总装液控制体积，优选32至50g/L总装液控制体积、35至45g/L总装液控制体积、或者37至42g/L总装液控制体积。
根据本发明的一个实施方式，根据所述制造方法，在所述预备步骤中，优选在所述预备步骤从开始至24小时之内，不添加所述长链组合物，并且所述发酵反应的pH值为自生状态，也即在该步骤中或者在该时间段内不进行pH值控制。
根据本发明的一个实施方式，根据所述制造方法，在所述转化步骤开始时，优选在所述预备步骤开始24小时之后，将所述发酵反应的pH值调节至6.0至7.5之间，优选6.8至7.0之间。更优选的是，随后每隔5-60小时，优选每隔10-40小时或每隔20-30小时或每隔24小时，使所述发酵反应的pH值提高0.05至0.4，优选提高0.1至0.3，直至所述发酵反应结束。
根据本发明的一个实施方式，根据所述制造方法，在所述转化步骤中，分n个批次加入所述长链组合物(也称为批次补料)。在此，相邻两个批次之 间的时间间隔可以相同或不同，优选相同。作为所述时间间隔，比如可以举出5-60小时、10-40小时、20-30小时或24小时。另外，n为2-40的正整数，优选4-20或5-10的正整数。
根据本发明的一个实施方式，根据所述制造方法，在所述转化步骤中，使用本说明书任意一个实施方式所述的长链组合物套组，分所述n个批次分别加入所述n个长链组合物，优选按照从第1个长链组合物至第n个长链组合物的次序分别加入所述n个长链组合物。
根据本发明的一个实施方式，根据所述制造方法，在所述转化步骤中，根据反应时间连续加入所述长链组合物(也称为流加补料)。在此，所述连续加入可以是恒定流量，也可以是不恒定流量，优选恒定流量。
根据本发明的一个实施方式，根据所述制造方法，在所述转化步骤中，按照函数R＝f(t)连续加入所述长链组合物。在此，R是所述长链烷烃与所述长链羧酸的质量比，t是所述转化步骤的反应时间，f()代表任意的非递增函数，优选任意的递减函数、任意的单调递减函数或任意的线性递减函数。
根据本发明的一个实施方式，所述制造方法还包括将所述长链二元酸从所述发酵反应的反应产物(称为发酵液)中分离的步骤。具体举例而言，在发酵反应结束后，经破乳、膜过滤、酸析、过滤、烘干处理后，获得长链二元酸产品。
根据本发明的一个实施方式，所述破乳可以按照本领域常规已知的任何方式进行，并没有特别的限定。作为举例，作为所述破乳，具体可以举出将发酵液pH调至8.5至10，优选9至9.5，并升温至75至90℃，维持20至40min。
根据本发明的一个实施方式，所述膜过滤可以按照本领域常规已知的任何方式进行，并没有特别的限定。作为举例，作为所述膜过滤，具体可以举出将破乳之后的发酵液经膜过滤，进行固液分离，获得过滤清液。在此，过滤温度一般为30至50℃，优选40至50℃。膜孔直径一般为10至50nm，优选20至25nm。
根据本发明的一个实施方式，所述酸析可以按照本领域常规已知的任何方式进行，并没有特别的限定。作为举例，作为所述酸析，具体可以举出将所述过滤清液进行酸性pH调节，控制pH为3至5，优选3至4，使长链二元 酸析出。在此，所述酸性pH调节剂一般为强酸，如可以是硫酸、盐酸、硝酸等中的至少一种。
根据本发明的一个实施方式，所述过滤可以按照本领域常规已知的任何方式进行，并没有特别的限定。作为举例，作为所述过滤，具体可以举出板框过滤。在此，过滤压力一般为0.5至1.0MPa，过滤温度一般为室温，比如20至30℃。
根据本发明的一个实施方式，所述烘干可以按照本领域常规已知的任何方式进行，并没有特别的限定。作为举例，作为所述烘干，具体可以举出烘干温度为80至105℃，烘干时间为5至20h。
实施例
以下采用实施例进一步详细地说明本发明，但本发明并不限于这些实施例。
以下实施例和对比例中的实验方法，如无特殊说明，均为本领域常规方法。下述实施例和对比例中所用的实验材料，如无特殊说明，均可从生化试剂商店购买得到。
长链二元酸的提取总收率T的计算公式为：
Figure PCTCN2020123700-appb-000001
其中，V为长链二元酸发酵液经膜过滤，除去未反应烷烃后获得的清液体积，L；M为提取的长链二元酸干重，g；C为长链二元酸的下罐浓度，g/L。
混合底物摩尔转化率K计算公式为：
Figure PCTCN2020123700-appb-000002
其中，M为提取的长链二元酸干重，g；M 1为烷烃质量，g；M 2为月桂酸质量，g；m为长链二元酸分子量；m 1为烷烃分子量；m 2为月桂酸分子量。
长链二元羧酸的生产强度Q的计算公式为：
Figure PCTCN2020123700-appb-000003
其中，C为长链二元酸的下罐浓度，g/L；H为发酵周期，h。
本发明实施例中选用热带假丝酵母(Candida tropicalis)突变株 PF-UV-56作为发酵菌株进行长链烷烃发酵生产长链二元酸，该突变株保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.0356。
发酵种子液制造：将斜面保藏菌种接种于4个5L摇瓶(摇瓶装液量300mL)进行菌种活化培养，培养温度为32℃，培养摇床转数为200rpm。培养48小时后，取活化种子液1.2L，作为发酵种子液。
发酵培养基配方为：蔗糖20g/L、玉米浆0.8g/L、酵母膏2.0g/L、氯化钠0.8g/L、磷酸二氢钾3.0g/L、硫酸镁1.2g/L、尿素1.2g/L，硫酸铵1.5g/L、醋酸钠1.5g/L。
实施例1
(1)取十二碳烷烃1650g，加入月桂酸330g，升温至60℃，搅拌转速150rpm进行混合溶解，随后加入1980g水，充分混合后，加入固体氢氧化钠将体系pH调至8，得到混合底物。
(2)于15L发酵罐中进行长链二元酸发酵，总装液控制体积12L，其中发酵菌种子液1.2L，发酵温度32℃、通气量1.0VVM、搅拌转速为250rpm，在发酵0至24h内，不进行pH控制，24h后将pH调整至7.0，随后每间隔24h将pH控制值提高0.15，并且于发酵24h、48h、72h、96h、120h，将步骤(1)制造的烷烃/月桂酸混合底物分5批次加入发酵体系中。
(3)发酵周期共计145h。发酵结束后，将发酵液pH调至9.5，并升温至90℃，维持20min；经破乳的发酵液通过膜过滤进行固液分离，获得水相清液，过滤条件为膜孔直径为20nm，过滤温度40℃；将过滤水相清液流加浓硫酸，pH调至3，水相清液中长链二元酸结晶析出。析出的长链二元酸经板框过滤及烘干后获得长链二元酸粗酸产品。过滤条件：过滤压力0.5MPa，过滤温度30℃。烘干条件是温度80℃，烘干时间20h。
发酵结果为：步骤(2)中长链二元酸发酵浓度152.0g/L，步骤(3)中经膜过滤获得发酵清液13.4L，酸析获得长链二元酸粗酸产品质量1914.5g，粗酸提取收率94％，烷烃和月桂酸的混合底物摩尔转化率62.4％。
实施例2
(1)取十二碳烷烃1900g，加入月桂酸190g，升温至60℃，搅拌转速250rpm进行混合溶解，随后加入2090g水，充分混合后，加入固体氢氧化钠将体系pH调至7.5，得到混合底物；
(2)于15L发酵罐进行长链二元酸发酵，总装液控制体积12L，其中发酵菌种子液1.2L，发酵温度32℃、通气量1.0VVM、搅拌转速为250rpm，在发酵0至24h内，不进行pH控制，24h后将pH调整至7.0，随后每间隔24h将pH控制值提高0.2，并且于发酵24h、48h、72h、96h、120h，将步骤(1)制造的烷烃/月桂酸混合液分5批次分别加入发酵体系中。
(3)发酵周期共计150h。发酵结束后，将发酵液pH调至9，并升温至75℃，维持40min；经破乳的发酵液通过膜过滤进行固液分离，获得水相清液，过滤条件为膜孔直径为25nm，过滤温度50℃；将过滤水相清液流加浓硫酸，pH调至4，水相清液中长链二元酸结晶析出。析出的长链二元酸经板框过滤及烘干后获得二元酸粗酸产品。过滤条件：过滤压力1.0MPa，过滤温度20℃。烘干条件是温度105℃，烘干时间5h。
发酵结果为：步骤(2)中长链二元酸发酵浓度156.0g/L，步骤(3)中经膜过滤获得发酵清液13.6L，酸析获得长链二元酸粗酸产品质量1973.0g，粗酸提取收率93％，烷烃和月桂酸的混合底物摩尔转化率60.2％。
实施例3
(1)取十二碳烷烃1650g，加入月桂酸330g，升温至60℃，搅拌转速150rpm进行混合溶解，随后加入5940g水，充分混合后，加入固体氢氧化钠将体系pH调至8，得到混合底物；
(2)于15L发酵罐进行长链二元酸发酵，总装液控制体积12L，其中发酵菌种子液1.2L，发酵温度32℃、通气量1.0VVM、搅拌转速为250rpm，在发酵0至24h内，不进行pH控制，24h后将pH调整至7.0，随后每间隔24h将pH控制值提高0.15，并且于发酵24h开始，将步骤(1)制造的烷烃/月桂酸混合液采用流加方式加入发酵体系中，流速控制为66g/h。
(3)发酵周期共计144h。发酵结束后，将发酵液pH调至9.5，并升温至90℃，维持20min；经破乳的发酵液通过膜过滤进行固液分离，获得水相清液，过滤条件为膜孔直径为20nm，过滤温度40℃；将过滤水相清液流加浓 硫酸，pH调至3，水相清液中长链二元酸结晶析出。析出的长链二元酸经板框过滤及烘干后获得长链二元酸粗酸产品。过滤条件：过滤压力0.5MPa，过滤温度30℃。烘干条件是温度80℃，烘干时间20h。
发酵结果为：步骤(2)中长链二元酸发酵浓度153.0g/L，步骤(3)中经膜过滤获得发酵清液13.5L，酸析获得长链二元酸粗酸产品质量1941.6g，粗酸提取收率94％，烷烃和月桂酸的混合底物摩尔转化率63.3％。
比较例1
同实施例1，不同在于步骤(1)不采用烷烃与月桂酸混合底物，仅在步骤(2)发酵过程中批次添加总质量为1980g的烷烃。发酵周期共计158h，发酵结果为：步骤(2)中长链二元酸发酵浓度150.0g/L，步骤(3)中经膜过滤获得发酵清液13.0L，酸析获得长链二元酸粗酸产品质量1833g，粗酸提取收率94％，烷烃和月桂酸的混合底物摩尔转化率58.2％。
比较例2
同实施例1，不同在于步骤(1)不采用烷烃与月桂酸混合底物，仅在步骤(2)发酵过程中批次添加总质量为1980g的月桂酸。发酵周期共计155h，发酵结果为：步骤(2)中长链二元酸发酵浓度15.0g/L，步骤(3)中经膜过滤获得发酵清液12.0L，酸析获得长链二元酸粗酸产品质量169.2g，粗酸提取收率94％，烷烃和月桂酸的混合底物摩尔转化率6.3％。
比较例3
同实施例1，不同在于步骤(1)不采用烷烃与月桂酸混合底物，仅在步骤(2)发酵过程中批次添加总质量为1848g的烷烃。发酵周期共计150h，发酵结果为：步骤(2)中长链二元酸发酵浓度141.0g/L，步骤(3)中经膜过滤获得发酵清液12.9L，酸析获得长链二元酸粗酸产品质量1709.8g，粗酸提取收率94％，烷烃和月桂酸的混合底物摩尔转化率58.2％。
实施例4
同实施例1，不同在于步骤(1)烷烃与月桂酸常温搅拌混合，加入1980g 水，充分混合后，加入固体氢氧化钠将体系pH调至8，得到混合底物。发酵周期共计144h，发酵结果为：经检测和计算，步骤(2)中长链二元酸发酵浓度142.0g/L，步骤(3)中经膜过滤获得发酵清液13.2L，酸析获得长链二元酸粗酸产品质量1762g，粗酸提取收率94％，烷烃和月桂酸的混合底物摩尔转化率57.4％。
实施例5
同实施例1，不同在于步骤(1)烷烃与月桂酸与60℃搅拌溶解后，不通过加水配制水溶液，不经过pH碱性调节，直接用于步骤(2)发酵过程。发酵周期共计144h，发酵结果为，步骤(2)中长链二元酸发酵浓度135.0g/L，步骤(3)中经膜过滤获得发酵清液13.0L，酸析获得长链二元酸粗酸产品质量1650g，粗酸提取收率94％，烷烃和月桂酸的混合底物摩尔转化率53.8％。
实施例6
同实施例1，不同在于步骤(1)烷烃与月桂酸与60℃搅拌溶解后，加水配制水溶液，不经过pH碱性调节，直接用于步骤(2)发酵过程。发酵周期共计145h，发酵结果为，步骤(2)中长链二元酸发酵浓度142.0g/L，步骤(3)中经膜过滤获得发酵清液13.5L，酸析获得长链二元酸粗酸产品质量1802g，粗酸提取收率94％，烷烃和月桂酸的混合底物摩尔转化率58.7％。
实施例7
(1)取十二碳烷烃1800g，加入月桂酸300g，升温至60℃，搅拌转速150rpm进行混合溶解，随后加入4200g水，充分混合后，加入固体氢氧化钠将体系pH调至7.5，得到混合底物。
(2)于15L发酵罐中进行长链二元酸发酵，总装液控制体积12L，其中发酵菌种子液1.2L，发酵温度32℃、通气量1.0VVM、搅拌转速为250rpm，在发酵0至24h内，不进行pH控制，24h后将pH调整至7.0，随后每间隔24h将pH控制值提高0.2，并且于发酵24h、48h、72h、96h、120h，将步骤(1)制造的烷烃/月桂酸混合底物分5批次加入发酵体系中。
(3)发酵周期共计153h。发酵结束后，按照实施例1步骤(1)的方法 进行长链二元酸提取。
发酵结果为：步骤(2)中长链二元酸发酵浓度160.0g/L，步骤(3)中经膜过滤获得发酵清液13.6L，酸析获得长链二元酸粗酸产品质量2030.4g，粗酸提取收率94％，烷烃和月桂酸的混合底物摩尔转化率62.2％。
实施例8
(1)取十二碳烷烃1650g，加入月桂酸198g，升温至60℃，搅拌转速150rpm进行混合溶解，随后加入1848g水，充分混合后，加入固体氢氧化钠将体系pH调至8，得到混合底物。
(2)于15L发酵罐中进行长链二元酸发酵，总装液控制体积12L，其中发酵菌种子液1.2L，发酵温度32℃、通气量1.0VVM、搅拌转速为250rpm，在发酵0至24h内，不进行pH控制，24h后将pH调整至7.0，随后每间隔24h将pH控制值提高0.2，并且于发酵24h、48h、72h、96h、120h，将步骤(1)制造的烷烃/月桂酸混合底物分5批次加入发酵体系中。
(3)发酵周期共计142h。发酵结束后，按照实施例1步骤(1)的方法进行长链二元酸提取。
发酵结果为：步骤(2)中长链二元酸发酵浓度148.0g/L，步骤(3)中经膜过滤获得发酵清液12.9L，酸析获得长链二元酸粗酸产品质量2048.9g，粗酸提取收率94％，烷烃和月桂酸的混合底物摩尔转化率62.1％。
实施例9
同实施例1，不同在于步骤(1)用十四碳烷烃与肉豆蔻酸组合替代原实施例中烷烃与月桂酸的组合。发酵周期共计144h，发酵结果为，步骤(2)中长链二元酸发酵浓度115.0g/L，步骤(3)中经膜过滤获得发酵清液13.0L，酸析获得长链二元酸粗酸产品质量1405.3g，粗酸提取收率94％，烷烃和月桂酸的混合底物摩尔转化率53.2％。
实施例10
同实施例1，不同在于步骤(1)用十六碳烷烃与棕榈酸组合替代原实施例中烷烃与月桂酸的组合。发酵周期共计144h，发酵结果为，步骤(2)中 长链二元酸发酵浓度98.0g/L，步骤(3)中经膜过滤获得发酵清液13.0L，酸析获得长链二元酸粗酸产品质量1197.6g，粗酸提取收率94％，烷烃和月桂酸的混合底物摩尔转化率51.6％。
实施例11
(1)取十二碳烷烃1650g，分成5份，每份330g，然后分别加入月桂酸33.0g、36.3g、39.6g、42.9g、46.2g，组成含有不同比例的烷烃与月桂酸混合物，编号1-5。并按照实施例1步骤(1)的处理方式进行烷烃与月桂酸混合物的预处理。
(2)按照实施例1步骤(2)的方式进行长链二元酸发酵过程，不同之处在于发酵24h、48h、72h、96h、120h时，将步骤(1)制造的烷烃/月桂酸混合底物按照编号1-5的顺序依次加入发酵体系中。
(3)发酵周期共计140h。发酵结束后，按照实施例1步骤(3)进行长链二元酸粗酸提取。
发酵结果为：步骤(2)中长链二元酸发酵浓度149.0g/L，步骤(3)中经膜过滤获得发酵清液13.0L，酸析获得长链二元酸粗酸产品质量1820.8g，粗酸提取收率94％，烷烃和月桂酸的混合底物摩尔转化率63.0％。
实施例12
(1)取十二碳烷烃1650g，分成5份，每份330g，然后分别加入月桂酸52.8g、56.1g、59.4g、62.7g、66.0g，组成含有不同比例的烷烃与月桂酸混合物，编号1-5。并按照实施例1步骤(1)的处理方式进行烷烃与月桂酸混合物的预处理。
(2)按照实施例1步骤(2)的方式进行长链二元酸发酵过程，不同之处在于发酵24h、48h、72h、96h、120h时，将步骤(1)制造的烷烃/月桂酸混合底物按照编号1-5的顺序依次加入发酵体系中。
(3)发酵周期共计140h。发酵结束后，按照实施例1步骤(3)进行长链二元酸粗酸提取。
发酵结果为：步骤(2)中长链二元酸发酵浓度154.0g/L，步骤(3)中经膜过滤获得发酵清液13.4L，酸析获得长链二元酸粗酸产品质量1939.8g， 粗酸提取收率94％，烷烃和月桂酸的混合底物摩尔转化率64.2％。
实施例13
(1)取十二碳烷烃1650g，分成5份，每份330g，然后分别加入月桂酸33.0g、36.3g、39.6g、42.9g、46.2g，按照实施例1步骤(1)的处理方式加热溶解后，分别加水控制总质量为至768g。进一步加入固体氢氧化钠将体系pH调至7.5，得到含有不同比例的烷烃与月桂酸混合物，编号1-5。
(2)按照实施例1步骤(2)的方式进行长链二元酸发酵过程，不同之处在于发酵24h-48h、48h-72h、72h-96h、96h-120h、120h-144h时间范围内，分别将步骤(1)制造的烷烃/月桂酸混合底物按照编号1-5的顺序依次以定速流加的方式加入发酵体系中，流速控制为32g/h。
(3)发酵周期共计144h。发酵结束后，按照实施例1步骤(3)进行长链二元酸粗酸提取。
发酵结果为：步骤(2)中长链二元酸发酵浓度152.0g/L，步骤(3)中经膜过滤获得发酵清液13.2L，酸析获得长链二元酸粗酸产品质量1886.0g，粗酸提取收率94％，烷烃和月桂酸的混合底物摩尔转化率65.3％。

Claims (15)

  1. 一种长链组合物(优选发酵用)，包含选自C9-18直链或支链(优选直链)烷烃中的至少一种长链烷烃(优选选自正十二烷、正十四烷和正十六烷中的至少一种长链烷烃，特别是正十二烷)和选自C9-18直链或支链(优选直链)、饱和或不饱和(优选饱和)的脂肪族一元羧酸中的至少一种长链羧酸(优选选自月桂酸、肉豆蔻酸和棕榈酸中的至少一种长链羧酸，特别是月桂酸)，其中所述长链烷烃与所述长链羧酸的质量比为1:1至40:1(优选2:1至20:1或5:1至10:1)。
  2. 权利要求1所述的长链组合物，还包含水，其中所述水的质量是所述长链烷烃和所述长链羧酸的质量之和的0.5-10倍(优选1-5倍或1-3倍)。
  3. 权利要求1所述的长链组合物，其pH值为5-12(优选7-10、7.5-9或7.5-8.0)，和/或，在32℃下呈现为液体或固液混合物的形态(优选液体形态，特别是含水液体形态)，和/或，所述长链烷烃与所述长链羧酸具有相同的碳原子数。
  4. 一种长链组合物套组，包括彼此独立存在(比如彼此独立包装或彼此独立分隔)的n个权利要求1-3任一项所述的长链组合物，其中n为2-40(优选4-20或5-10)的正整数，其中设在第i个(i为从2至n的任意正整数)长链组合物中，所述长链烷烃与所述长链羧酸的质量比为R i，在第1个长链组合物中，所述长链烷烃与所述长链羧酸的质量比为R 1，在第n个长链组合物中，所述长链烷烃与所述长链羧酸的质量比为R n，则R i-1/R i≥1(优选R i-1/R i＝1-20、R i-1/R i＝1.0001-10、R i-1/R i＝1.001-10、R i-1/R i＝1.01-10、R i-1/R i＝1.1-5或R i-1/R i＝1.5-2)并且R 1/R n＞1(优选R 1/R n＝1.0001-30、R 1/R n＝1.001-20、R 1/R n＝1.01-10、R 1/R n＝1.1-5或R 1/R n＝1.5-2)。
  5. 权利要求4所述的长链组合物套组，其中所述n个长链组合物是分别单独制造的，和/或，任意两个所述n个长链组合物彼此之间的重量比(以所述长链组合物的量计算)相同或不同(优选相同或基本上相同)，和/或，任意两个所述n个长链组合物彼此之间的重量比(以所述长链烷烃的量计算)相同或基本上相同。
  6. 一种长链组合物的制造方法，其中所述长链组合物(优选发酵用)包含选自C9-18直链或支链(优选直链)烷烃中的至少一种长链烷烃(优选选自正十二烷、正十四烷和正十六烷中的至少一种长链烷烃，特别是正十二烷)和选自C9-18直链或支链(优选直链)、饱和或不饱和(优选饱和)的脂肪族一元羧酸中的至少一种长链羧酸(优选选自月桂酸、肉豆蔻酸和棕榈酸中的至少一种长链羧酸，特别是月桂酸)，其中所述长链烷烃与所述长链羧酸的质量比为1:1至40:1(优选2:1至20:1或5:1至10:1)，所述制造方法至少包括混合步骤：将所述长链烷烃与所述长链羧酸任选在加热(优选加热温度为45至70℃或50至60℃)和搅拌(优选搅拌转速为50至250rpm或150至250rpm)的情况下按照所述质量比进行混合(优选直至混合均匀)，获得所述长链组合物。
  7. 权利要求6所述的制造方法，其中在所述混合步骤中还加入水和pH值调节剂(比如碱，特别是选自氢氧化钠和氢氧化钾中的至少一种)，和/或，所述制造方法还包括任选在加热(优选加热温度为45至70℃或50至60℃)和搅拌(优选搅拌转速为50至250rpm或150至250rpm)的情况下，向所述长链组合物中加入水和pH值调节剂(比如碱，特别是选自氢氧化钠和氢氧化钾中的至少一种)的步骤，其中所述水的质量是所述长链烷烃和所述长链羧酸的质量之和的0.5-10倍(优选1-5倍或1-3倍)，并且所述pH值调节剂的用量使得所述长链组合物的pH值达到5-12(优选7-10、7.5-9或7.5-8.0)。
  8. 一种长链二元酸的制造方法，其中所述长链二元酸是选自C9-18直链或支链(优选直链)、饱和或不饱和(优选饱和)的脂肪族二元羧酸中的至少一种长链二元酸(优选选自十二碳二元酸、十四碳二元酸和十六碳二元酸中的至少一种长链二元酸，特别是十二碳二元酸)，
    所述制造方法包括以下步骤：
    (1)按照权利要求6所述的制造方法来制造长链组合物、提供权利要求1所述的长链组合物、或者提供权利要求4所述的长链组合物套组，统称为长链组合物；
    (2)使所述长链组合物在发酵菌和发酵培养基的存在下进行发酵反应而转化为所述长链二元酸。
  9. 权利要求8所述的制造方法，其中所述发酵菌是具有完整α,ω-氧化途径的酵母菌(优选选自假丝酵母属、隐球酵母属、内孢霉属、汉逊氏酵母 属、毕赤氏酵母属、红酵母属、球拟酵母属和丝孢酵母属中的至少一种酵母菌，更优选选自假丝酵母属中的至少一种酵母菌，特别是热带假丝酵母菌，更特别是热带假丝酵母菌突变株PF-UV-56(中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏编号为CGMCC No.0356))，和/或，所述发酵培养基包括：蔗糖20至28g/L、玉米浆0.8至1.5g/L、酵母膏2.0至4.0g/L、氯化钠0.8至1.2g/L、磷酸二氢钾3.0至3.5g/L、硫酸镁1.2至1.8g/L、尿素1.2至4.8g/L，硫酸铵1.5至2g/L、醋酸钠1.5至1.8g/L。
  10. 权利要求8所述的制造方法，其中所述发酵反应包括以下步骤：
    预备步骤：混合所述发酵菌和所述发酵培养基，进行所述发酵反应5-60小时(优选10-40小时，更优选20-30小时或24小时)，获得发酵基础液，
    转化步骤：向所述发酵基础液中加入所述长链组合物，进行所述发酵反应60-400小时(优选100-300小时，更优选100至160小时或100至140小时)，以将所述长链组合物转化为所述长链二元酸。
  11. 权利要求8所述的制造方法，其中发酵菌种子液的添加量(以体积计)为总装液控制体积的2-30％(优选5至20％或10至15％)，和/或，所述发酵反应的反应温度为25至37℃，优选28至32℃，和/或，所述发酵反应的搅拌转速为100至1000rpm(优选120至500rpm或150至300rpm)，和/或，所述发酵反应的通气量为0.2至10.0VVM(优选0.2至2.0VVM或0.5至1.0VVM)，和/或，所述发酵反应的反应时间为65小时以上(优选大于120小时，更优选130至400小时、130至300小时、130至200小时或138至160小时)，和/或，所述长链组合物的添加量为100至1000g/L总装液控制体积(优选100至500g/L总装液控制体积或150至250g/L总装液控制体积)，和/或，所述发酵培养基的添加量为12至80g/L总装液控制体积(优选32至50g/L总装液控制体积、35至45g/L总装液控制体积、或者37至42g/L总装液控制体积)。
  12. 权利要求10所述的制造方法，其中在所述预备步骤中(优选在所述预备步骤从开始至24小时之内)，不添加所述长链组合物，并且所述发酵反应的pH值为自生状态(即，不进行pH值控制)，和/或，在所述转化步骤开始时(优选在所述预备步骤开始24小时之后)，将所述发酵反应的pH值调节至6.0至7.5之间(优选6.8至7.0之间)，随后每隔5-60小时(优选每隔10-40小时或每隔20-30小时或每隔24小时)，使所述发酵反应的pH值提高0.05至0.4(优 选0.1至0.3)，直至所述发酵反应结束。
  13. 权利要求10所述的制造方法，其中在所述转化步骤中，分n个批次加入所述长链组合物，相邻两个批次之间的时间间隔可以相同或不同(优选时间间隔为5-60小时、10-40小时、20-30小时或24小时)，其中n为2-40(优选4-20或5-10)的正整数，或者根据反应时间连续加入所述长链组合物。
  14. 权利要求13所述的制造方法，其中使用权利要求4所述的长链组合物套组，分所述n个批次分别加入所述n个长链组合物(优选按照从第1个长链组合物至第n个长链组合物的次序)，或者按照函数R＝f(t)连续加入所述长链组合物，其中R是所述长链烷烃与所述长链羧酸的质量比，t是所述转化步骤的反应时间，f()代表任意的非递增函数(优选任意的递减函数、任意的单调递减函数或任意的线性递减函数)。
  15. 权利要求8所述的制造方法，还包括将所述长链二元酸从所述发酵反应的反应产物中分离的步骤。
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