WO2020042481A1 - 一种黑曲霉种子连续培养及其生产柠檬酸的方法 - Google Patents

一种黑曲霉种子连续培养及其生产柠檬酸的方法 Download PDF

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WO2020042481A1
WO2020042481A1 PCT/CN2018/123053 CN2018123053W WO2020042481A1 WO 2020042481 A1 WO2020042481 A1 WO 2020042481A1 CN 2018123053 W CN2018123053 W CN 2018123053W WO 2020042481 A1 WO2020042481 A1 WO 2020042481A1
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seed
fermentation
medium
liquid
continuous
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English (en)
French (fr)
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石贵阳
胡志杰
李由然
蒋小东
金赛
孙福新
张�成
周东姣
卢佳伟
苗茂栋
范子豪
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江苏国信协联能源有限公司
江南大学
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Priority to KR1020197006893A priority Critical patent/KR102174885B1/ko
Priority to BR112019008405-3A priority patent/BR112019008405A2/pt
Priority to JP2019512299A priority patent/JP6937366B2/ja
Priority to US16/467,990 priority patent/US11434467B2/en
Priority to EP18852710.5A priority patent/EP3640337B1/en
Publication of WO2020042481A1 publication Critical patent/WO2020042481A1/zh

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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
    • 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
    • C12P7/48—Tricarboxylic acids, e.g. citric acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/685—Aspergillus niger

Definitions

  • the invention relates to the technical field of microbial culture, in particular to a continuous culture of Aspergillus niger seeds and a method for producing citric acid.
  • Citric acid is the largest organic acid produced and used in the world. It is an important chemical product with a wide range of uses. Citric acid is mainly used in the food industry, such as acidulants, antioxidants, gelling agents, etc., and has a wide range of applications in the pharmaceutical, feed, chemical, electronics, textile and other industrial fields, and the market demand is increasing year by year. China is the largest citric acid producer and exporter with advanced production technology and strong market competitiveness. In particular, the original deep fermentation index based on corn starch, dried potato and other starchy raw materials ranks among the highest in the world.
  • the traditional method of citric acid fermentation is still used, that is, a large number of Aspergillus niger spores are prepared first; then the spores are inserted into the seed medium and cultivated to obtain mature seed liquid; and then the seed liquid is transferred into the fermentation medium to obtain citric acid Fermentation broth.
  • the traditional citric acid fermentation method has the following problems:
  • the batch batch culture of seeds is unstable: In batch batch culture, the growth environment of the bacteria (such as sugar concentration, pH, etc.) is constantly changing, resulting in that the growth of the bacteria cannot be in an optimal environment for a long time.
  • the growth environment of the bacteria such as sugar concentration, pH, etc.
  • Patent CN201410329652.X discloses a method for continuous cultivation of Aspergillus citric acid seeds based on the mycelial ball dispersion technology. Although it is called “continuous” cultivation, its essence is “circular” cultivation. The dispersed mycelium is inoculated into a new seed medium and cultivated into a seed liquid to realize the circulating culture of the seeds.
  • the applicant of the present invention provides a continuous culture of Aspergillus niger seeds and a method for producing citric acid.
  • the method of the invention breakthroughly solves the problems of slow growth of multicellular filamentous bacteria and the easy loss of hyphae in continuous culture, completely realizes continuous seed cultivation, keeps the bacteria constant in the most suitable growth environment, and avoids bacteria
  • the seed degradation caused the seed liquid to be in a continuous and stable high vigor state, and the corresponding fermentation index was significantly improved.
  • a method for continuous cultivation of Aspergillus niger seeds includes the following steps:
  • Start-up stage inoculate Aspergillus niger spores into the seed medium and culture for 16 to 36 hours to obtain seed liquid;
  • step (2) Continuous seed cultivation stage: The seed liquid obtained in step (1) is subjected to continuous dispersion treatment, and the seed liquid obtained from the dispersion is continuously cultured. During the cultivation process, the seed liquid continuously flows out to the fermentation medium for fermentation and cultivation, and the The seed liquid is supplemented with fresh seed feed medium at the same rate;
  • Stop phase stop supplementing the fresh seed feed medium and dispersing treatment, continue the cultivation to obtain the seed liquid, and then transfer the seed liquid into the fermentation medium for fermentation and culture.
  • the final concentration after inoculation of Aspergillus niger in step (1) is 1 to 9 * 10 5 cells / mL; the total sugar in the seed medium in step (1) is 100 to 180 g / L, and the C / N is 20 to 40; step (1)
  • the medium cultivation conditions are: the temperature is 35-39 ° C, the air volume is 0.2-0.4 vvm, the tank pressure is 0.05-0.1Mpa, and the stirring speed is 100-200 rpm.
  • the total sugar of the fresh seed feed medium supplemented in step (2) is 150-200g / L, and the C / N is 15-30; the continuous culture conditions in step (2) are: the temperature is 35-37 ° C, and the air volume is 0.3 to 0.6 vvm, the tank pressure is 0.05 to 0.07 MPa, and the stirring speed is 150 to 200 rpm.
  • the culture conditions for the continuous cultivation in step (3) are: the temperature is 35 to 39 ° C., the air volume is 0.3 to 0.6 vvm, the tank pressure is 0.05 to 0.1 MPa, and the stirring speed is 150 to 200 rpm.
  • the method for continuously dispersing the seed liquid in step (2) is: using a disperser to disperse the mycelium balls in the seed liquid into flocculent hyphae of 10-80 ⁇ m.
  • the fresh seed feed medium replenishment speed and the seed liquid outflow speed F V / t, where V is the volume of the seed liquid in the seed tank, which is determined in different tanks; t is the residence time of the seed liquid, according to The cell concentration of flocculent mycelium is adjusted by feedback, usually 6-24h.
  • the proportion of fermentation inoculation in steps (2) and (3) is 5-25% (v / v).
  • the total sugar in the fermentation medium in step (2) and step (3) is 160-200g / L, and the C / N is 50-90; the fermentation culture conditions in step (2) and step (3) are: the temperature is 35 ⁇ At 39 ° C, the air volume is 0.1 to 0.4 vvm, the tank pressure is 0.05 to 0.1 Mpa, the stirring speed is 100 to 200 rpm, and the fermentation is terminated when the reducing sugar concentration is lower than 5 g / L.
  • the seed medium, fresh seed supplement medium, and fermentation medium are respectively prepared from a starchy raw material liquefaction liquid and a nitrogen source;
  • the starchy raw material includes at least one of corn flour, tapioca flour, sorghum flour, and wheat starch.
  • the nitrogen source includes at least one of ammonium sulfate, urea, soybean meal meal, and corn pulp.
  • the invention relates to a method for producing citric acid.
  • the method adopts fermentation of Aspergillus niger obtained by continuous seed cultivation, and is used for producing citric acid.
  • the method of the present invention completely realizes the continuous culture of the filamentous fungus seeds.
  • the equipment system is greatly simplified, and the amount of seed tanks is reduced to 1/3; the operation steps are reduced, and the production efficiency is significantly improved.
  • the auxiliary time of seed culture corresponding to the fermentation tank saves 12h; the automation level of seed culture is improved.
  • the method for continuously cultivating seeds of the present invention reduces the number of passages of bacteria, can effectively prevent the degradation of bacteria, and can continuously run for 15 days to keep the seeds viable and the fermentation index stable.
  • High-efficiency continuous operation not only reduces the start-stop assistance time, but also reduces the use of Aspergillus niger spores by 1/3, which greatly reduces the cost of Aspergillus niger spore culture.
  • the outstanding advantage of the method of the present invention is that the bacterial cells are constantly in the most suitable growth environment, so that the seeds are always in a stable state of high vigor and maturity, which ensures the stability and consistency of the quality of the seed liquid.
  • the seeds are cultivated in batches.
  • the pH in the tank is continuously reduced and the nutrients are gradually consumed.
  • the cultivation conditions are gradually detrimental to the growth and viability of the bacteria, which affects the quality of the seed liquid, and the quality of the seed liquid varies between batches. Larger; the bigger drawback is that the seed liquid moved into the fermenter is the dispersed hypha after the disperser treatment, and it needs to resume growth in the early stage of the fermenter, which affects the fermentation index.
  • the initial sugar concentration is 16%
  • the fermentation intensity is increased by 27.5%
  • the fermentation conversion rate is increased by 3.4 percentage points, and the fermentation stability is significantly improved.
  • the method of the present invention has a greater advantage than the patent CN201410329652.X under high-concentration fermentation conditions.
  • the initial sugar concentration is increased to 18%
  • the acid production by fermentation can exceed 18%
  • the fermentation cycle is less than 60h
  • the fermentation conversion rate exceeds 100%
  • the fermentation intensity is increased by 31.6%
  • the fermentation conversion rate is increased by 4.8 percentage points. Therefore, the method of the present invention can completely realize the high-concentration, high-conversion, and high-efficiency fermentation production of citric acid, which has an important promotion effect on the technological upgrading of the citric acid industry.
  • the invention adopts the method of continuous feeding coupled with continuous dispersion for the first time to realize continuous culture of Aspergillus niger seeds, determines the optimal residence time and hyphae size, and optimizes the culture parameters of different stages of seed culture to solve the multicellular filamentous bacteria.
  • Slow growth and the problem of easy loss of mycelium balls in continuous culture completely realize continuous seed cultivation, keep the bacteria constant in the most suitable growth environment, avoid the degradation of bacteria, and keep the seed liquid in a continuous and stable high activity
  • the state, corresponding to the fermentation index was significantly improved.
  • Figure 1 is a schematic diagram of the cultivation method of CN201410329652.X.
  • FIG. 2 is a schematic diagram of a culture method of the present invention.
  • the raw materials and reagents involved in the following examples and comparative examples are all commercially available products; Aspergillus niger is derived from the China Industrial Microbial Collection and Management Center (CICC), and the deposit number is CICC 40021.
  • the total sugar and reducing sugar were measured by film titration method, the nitrogen source was measured by Kjeldahl method, the citric acid was measured by 0.1429mol / L NaOH titration, and the spore count was measured by blood cell counting plate. Unless otherwise specified, equipment and processes commonly used in the art are used.
  • Corn flour and tap water are mixed evenly at a mass ratio of 1: 3.
  • the pH of the slurry is adjusted to 6.0 with Ca (OH) 2 and ⁇ -high temperature amylase is added at an amount of 20 U / g of corn flour.
  • iodine test After being light brown, a qualified corn liquefaction liquid was obtained; 80% of the corn liquefaction liquid was filtered through a plate and frame to remove the filter residue to obtain a corn sugar liquid.
  • Start-up phase The corn liquefaction solution and ammonium sulfate are formulated into a seed medium (100g / L of total sugar, C / N is 20), and the aspergillus niger spores are inoculated into the seed medium after sterilization and cooling, and the final spore concentration is 9 * 10 5 seeds / mL, cultured at a temperature of 35 ° C., an air volume of 0.2 vvm, a tank pressure of 0.05 MPa, and a stirring speed of 100 rpm for 16 h to obtain a seed liquid.
  • a seed medium 100g / L of total sugar, C / N is 20
  • the aspergillus niger spores are inoculated into the seed medium after sterilization and cooling, and the final spore concentration is 9 * 10 5 seeds / mL, cultured at a temperature of 35 ° C., an air volume of 0.2 vvm, a tank pressure of 0.05 MPa, and
  • Continuous stage The corn liquefaction liquid and ammonium sulfate are formulated into a seed feed medium (total sugar 150g / L, C / N is 15).
  • a disperser to continuously disperse the obtained seed liquid to disperse the hyphae into 10 ⁇ m flocculent mycelia; at the same time, control the rate of continuous supplementation of fresh seed feed medium according to the residence time of 6h, and the seed liquid is continuously at the same speed Flow out to the fermentation medium for fermentation.
  • the seed cultivation temperature was 35 ° C
  • the air volume was 0.3vvm
  • the tank pressure was 0.05Mpa
  • the stirring speed was 150rpm.
  • Stopping phase When it is detected that the seed vigor is decreasing, stop supplementing the fresh seed feed medium and dispersing treatment, and transfer the remaining seed liquid into the fermentation medium for fermentation and culture. Seed cultivation conditions at this stage: the temperature is 35 ° C., the air volume is 0.3 vvm, the tank pressure is 0.05 Mpa, and the stirring speed is 150 rpm.
  • Fermentation medium is prepared from corn liquefaction liquid, corn sugar liquid and ammonium sulfate (total sugar 160g / L, C / N is 50); fermentation inoculation ratio is 25% (v / v); fermentation culture conditions are: temperature 35 ° C , The air volume is 0.1vvm, the tank pressure is 0.05Mpa, the stirring speed is 100rpm, the fermentation is terminated when the reducing sugar concentration is lower than 5g / L, the fermentation acidity and residual sugar are measured for each batch, and the conversion rate, fermentation intensity and other indicators are calculated.
  • Corn flour and tap water are mixed evenly at a mass ratio of 1: 3.
  • the pH of the slurry is adjusted to 6.0 with Ca (OH) 2 and ⁇ -high temperature amylase is added at an amount of 20 U / g of corn flour.
  • iodine test After being light brown, a qualified corn liquefaction liquid was obtained; 80% of the corn liquefaction liquid was filtered through a plate and frame to remove the filter residue to obtain a corn sugar liquid.
  • Cassava flour and tap water are mixed evenly at a mass ratio of 1: 3.
  • the pH of the slurry is adjusted to 6.0 with Ca (OH) 2 and ⁇ -high temperature amylase is added at an amount of 20U / g of cassava flour.
  • iodine test After being light brown, qualified cassava sugar solution was obtained.
  • Start-up stage The corn liquefaction solution and urea are formulated into a seed medium (total sugar 140g / L, C / N is 30). After sterilization and cooling, Aspergillus niger spores are inoculated into the seed medium, and the final spore concentration is 4.5 * 10 5 Cells / mL, cultured at a temperature of 37 ° C, an air volume of 0.3 vvm, a tank pressure of 0.075 MPa, and a stirring speed of 150 rpm for 26 hours to obtain a seed liquid.
  • Continuous stage The corn liquefaction liquid and urea were formulated into a seed feed medium (total sugar 175g / L, C / N was 23). Use a disperser to continuously disperse the obtained seed liquid to disperse the hyphae into 45 ⁇ m flocculent hyphae; at the same time, control the rate of continuous supplementation of fresh seed feed medium according to the residence time of 15h, and the seed liquid is continuously at the same speed Flow out to the fermentation medium for fermentation.
  • the seed cultivation temperature was 36 ° C
  • the air volume was 0.45vvm
  • the tank pressure was 0.06Mpa
  • the stirring speed was 175rpm.
  • Stopping phase When it is detected that the seed vigor is decreasing, stop supplementing the fresh seed feed medium and dispersing treatment, and transfer the remaining seed liquid into the fermentation medium for fermentation and culture. Seed cultivation conditions at this stage: temperature is 37 ° C, air volume is 0.45vvm, tank pressure is 0.075Mpa, and stirring speed is 175rpm.
  • Fermentation medium is made of corn liquefaction liquid, corn sugar liquid, cassava sugar liquid and urea (total sugar 180g / L, C / N is 70); fermentation inoculation ratio is 15% (v / v); fermentation culture conditions are: The temperature is 37 ° C, the air volume is 0.25vvm, the tank pressure is 0.075Mpa, and the stirring speed is 150rpm. When the reducing sugar concentration is lower than 5g / L, the fermentation is ended. The fermentation acidity and residual sugar are measured for each batch, and the conversion rate and fermentation intensity are calculated.
  • Corn flour and tap water are mixed evenly at a mass ratio of 1: 3.
  • the pH of the slurry is adjusted to 6.0 with Ca (OH) 2 and ⁇ -high temperature amylase is added at an amount of 20 U / g of corn flour.
  • iodine test After being light brown, a qualified corn liquefaction liquid was obtained; 80% of the corn liquefaction liquid was filtered through a plate and frame to remove the filter residue to obtain a corn sugar liquid.
  • Wheat starch and tap water are mixed evenly at a mass ratio of 1: 3.
  • the pH of the powder slurry is adjusted to 6.0 with Ca (OH) 2 and ⁇ -high temperature amylase is added at an amount of 20U / g wheat starch; after spray liquefaction, iodine test After being light brown, qualified wheat sugar solution was obtained.
  • Start-up stage The corn liquefaction liquid and soybean meal powder are formulated into a seed medium (180g / L of total sugar, C / N is 40), and the aspergillus niger spores are inoculated into the seed medium after sterilization and cooling, and the final spore concentration is 1 * 10 5 cells / mL, cultured at a temperature of 39 ° C., an air volume of 0.4 vvm, a tank pressure of 0.1 MPa, and a stirring speed of 200 rpm for 36 h to obtain a seed liquid.
  • a seed medium 180g / L of total sugar, C / N is 40
  • the aspergillus niger spores are inoculated into the seed medium after sterilization and cooling, and the final spore concentration is 1 * 10 5 cells / mL, cultured at a temperature of 39 ° C., an air volume of 0.4 vvm, a tank pressure of 0.1 MPa, and a stirring speed of 200
  • Continuous stage The corn liquefaction liquid and soybean meal powder are formulated into a seed feed medium (200g / L of total sugar, C / N is 30).
  • a disperser to continuously disperse the obtained seed liquid to disperse the hyphae into 80 ⁇ m flocculent hyphae; at the same time, control the rate of continuous supplementation of fresh seed feed medium according to the residence time of 24h, and the seed liquid is continuously at the same speed Flow out to the fermentation medium for fermentation.
  • the seed cultivation temperature was 37 ° C
  • the air volume was 0.6vvm
  • the tank pressure was 0.07Mpa
  • the stirring speed was 200rpm.
  • Stopping phase When it is detected that the seed vigor is decreasing, stop supplementing the fresh seed feed medium and dispersing treatment, and transfer the remaining seed liquid into the fermentation medium for fermentation and culture. Seed cultivation conditions at this stage: the temperature is 39 ° C., the air volume is 0.6 vvm, the tank pressure is 0.1 MPa, and the stirring speed is 200 rpm.
  • Fermentation medium is prepared from corn liquefaction liquid, corn sugar liquid, wheat sugar liquid and soybean meal powder (total sugar 200g / L, C / N is 90); fermentation inoculation ratio is 5% (v / v); fermentation culture conditions are: : The temperature is 39 ° C, the air volume is 0.4vvm, the tank pressure is 0.1Mpa, the stirring speed is 200rpm, and the fermentation is terminated when the reducing sugar concentration is lower than 5g / L. The fermentation acidity and residual sugar are measured for each batch, and the conversion rate and fermentation intensity are calculated.
  • Glucose and soybean meal powder were prepared as seed medium (total sugar 80g / L, total nitrogen 1.5g / L) and fermentation medium (total sugar 160g / L, total nitrogen 1.5g / L); the spore concentration after transplantation was 550,000 cells / mL inoculation; cultured for 20 hours to obtain mature seed liquid, the seed liquid mycelium is dispersed by a disperser, the average diameter of the treated mycelia is 100 ⁇ m, and the seed culture medium and the fermentation medium of the next stage are respectively connected; When the fermentation medium is connected to the next level, the fermentation ends when the reducing sugar concentration in the fermentation medium drops below 5 g / L. This cycle is stopped until the seed vigor declines significantly. The fermentation acidity and residual sugar were measured for each batch, and the conversion rate and fermentation intensity were calculated.
  • the total sugar in the fermentation medium was 180 g / L, and the total nitrogen was 1.5 g / L; other conditions were the same as those in Comparative Example 1.
  • the corn flour liquid and corn sugar liquid were prepared in the same manner as in Example 1.
  • the corn liquefaction liquid and ammonium sulfate were used to prepare a seed medium (total sugar 120 g / L, total nitrogen 2 g / L) and a fermentation medium (total sugar 160 g / L, total nitrogen 1.0 g / L).
  • Aspergillus niger spores were inoculated into the seed medium with a final spore concentration of 4 * 10 5 cells / mL and cultured for 20 hours to obtain a first-stage mature seed solution. 70% of the mature seed solution was transferred to the first batch of fermentation medium for cultivation, and the fermentation was terminated when the reducing sugar concentration was lower than 5g / L.
  • the remaining 30% of the seed solution was processed by a disperser to obtain an average diameter of 62 ⁇ m of mycelium balls or fungal pieces, transferred to a seed medium, and cultured for 16 hours to obtain a secondary mature seed solution. This cycle is stopped until the seed vigor declines significantly.
  • the fermentation acidity and residual sugar were measured for each batch, and the conversion rate and fermentation intensity were calculated.
  • the corn flour liquid and corn sugar liquid were prepared in the same manner as in Example 1.
  • the corn liquefaction liquid and ammonium sulfate were used to prepare a seed medium (total sugar 120 g / L, total nitrogen 2 g / L) and a fermentation medium (total sugar 160 g / L, total nitrogen 1.0 g / L).
  • Aspergillus niger spores were inoculated into the seed medium with a final spore concentration of 4 * 10 5 cells / mL and cultured for 20 hours to obtain a first-stage mature seed solution. 70% of the mature seed solution was transferred to the first batch of fermentation medium for cultivation, and the fermentation was terminated when the reducing sugar concentration was lower than 5g / L.
  • the remaining 30% of the seed solution was treated with a disperser to obtain an average diameter of 50 ⁇ m of hyphae or fungus.
  • Fresh seed medium was added to the remaining seed solution, and cultured for 16 hours to obtain a secondary mature seed solution. This cycle is stopped until the seed vigor declines significantly.
  • the fermentation acidity and residual sugar were measured for each batch, and the conversion rate and fermentation intensity were calculated.
  • the speed of continuously supplementing the fresh seed feed medium in the continuous seed culture stage was controlled according to the residence time of 4 h; other conditions were the same as in Example 1.
  • the C / N of the seed feed medium in the continuous seed culture stage was 40; other conditions were the same as in Example 1.
  • Example 1 The technical effects of Example 1 and Comparative Examples 1, 3, and 4 are compared, as shown in Table 1.
  • Example 1 and Comparative Examples 1, 3, and 4 are compared and displayed: (1) Compared with the patent CN201410329652.X, the method of the present invention simplifies the equipment system, reduces the use of seed tanks to 1/3; and reduces operations This step significantly improves the production efficiency, and saves 12h of seed cultivation assistance time corresponding to each batch of fermenters. (2) The method of the present invention has a longer continuous running time, the amount of spores used in Aspergillus niger can be reduced by 1/3, and the cost of spore culture of Aspergillus niger can be greatly reduced. (3) Compared with the patent CN201410329652.X, when the initial sugar concentration is 16%, the fermentation intensity is increased by 27.5%, and the fermentation conversion rate is increased by 3.4 percentage points.
  • Example 2 The technical effects of Example 2 and Comparative Example 2 are compared, as shown in Table 2.
  • Example 2 The comparison of the technical effects of Example 2 and Comparative Example 2 shows that the method of the present invention has a greater advantage than the patent CN201410329652.X under high-concentration fermentation conditions.
  • the initial sugar concentration of the fermentation is increased to 18%, acid production by fermentation It can exceed 18%, the fermentation cycle is less than 60h, the fermentation conversion rate exceeds 100%, the fermentation intensity is increased by 31.6%, and the fermentation conversion rate is increased by 4.8 percentage points. Therefore, the method of the present invention can completely realize the high-concentration, high-conversion, and high-efficiency fermentation production of citric acid, which has an important promotion effect on the technological upgrading of the citric acid industry.
  • Test Example 3 The performance comparison between Example 1 of the present invention and Comparative Examples 5 and 6 is shown in Table 3.
  • Example 1 of the present invention shows that when the parameters such as the residence time of the continuous cultivation stage and the feed medium C / N are changed, the continuous running time of the seed will be significantly affected, the running time will be shortened, and the seed The vitality decreases rapidly, and the corresponding indexes such as fermentation conversion rate and fermentation intensity are also significantly reduced.

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Abstract

一种黑曲霉种子连续培养及其生产柠檬酸的方法，黑曲霉种子连续培养方法包括：（1）启动阶段，将黑曲霉孢子接种至种子培养基，得到种子液；（2）种子连续培养阶段，对步骤（1）得到的种子液进行连续分散处理，分散得到的种子液进行连续培养，并同时补充新鲜种子补料培养基；（3）停止阶段，停止补充新鲜种子补料培养基和分散，继续培养得到种子液，移入发酵培养基进行发酵培养。

Description

一种黑曲霉种子连续培养及其生产柠檬酸的方法 技术领域
本发明涉及微生物培养的技术领域，尤其是涉及一种黑曲霉种子连续培养及其生产柠檬酸的方法。
背景技术
柠檬酸是世界上生产量和使用量最大的有机酸，是一种重要的化工产品，具有广泛的用途。柠檬酸主要用于食品工业，如酸味剂、抗氧化剂、胶凝剂等，而且医药、饲料、化工、电子、纺织等工业领域都有十分广阔的应用，市场需求量逐年增加。我国是最大的柠檬酸生产和出口国，生产工艺先进，市场竞争力强，特别是独创以玉米、薯干等淀粉质原料的深层发酵指数居世界前列。
虽然近年来生物发酵技术不断快速发展，新的发酵技术不断涌现，多种高效率的连续培养方式在不同发酵产品上得到广泛应用，但由于柠檬酸生产菌种黑曲霉是多核丝状真菌，其生长特性和菌体形态完全不同于酵母和细菌等单细胞微生物，以酵母和细菌为生产菌株的高效成熟的发酵方式并不能很好的应用于黑曲霉柠檬酸发酵生产中，严重制约了柠檬酸等以丝状菌作为生产菌株的发酵产业发展。
当前，柠檬酸发酵生产仍然沿用传统的方式，即先制备大量的黑曲霉孢子；再将孢子接入种子培养基中培养获得成熟的种子液；然后将种子液移入发酵培养基中发酵得到柠檬酸发酵液。柠檬酸传统发酵方式存在以下问题：
(1)黑曲霉孢子制备繁琐、周期长、效率低：黑曲霉菌种需要经过复壮，平板筛选和三级扩培，制备周期超过30天；人工操作繁琐，染菌风险大。
(2)菌种稳定性、一致性差：当前孢子制备机械化程度低，麸曲培养容器小，在不同容器内一致性较差，导致种子培养质量和发酵结果不稳定。
(3)种子培养效率低：孢子接种至种子培养需要8～12h的萌发时间，降低了种子罐的利用效率。
(4)种子分批间歇培养不稳定：分批间歇培养中，菌体生长环境(如糖浓度、pH等)在不断变化，导致菌体生长无法长期处于最适的环境中。
目前行业内还没有丝状菌的种子连续培养方法，原因在于丝状菌一般为多细胞，生长慢，成块、成球，在连续培养中细胞容易流失。常见的连续培养主要集中在细菌、酵母等种类的微生物方面，它们都是单细胞，形态简单，容易控制。专利CN201410329652.X公布了一种基于菌丝球分散技术的柠檬酸黑曲霉种子连续培养的方法，虽然称为“连续”培养，但其本质为“循环”培养，该方法是将前一批剩余的分散菌丝接种至新的种子培养基培养成种子液来实现种子的循环培养。该方法虽然减少了孢子使用量和节省了孢子的萌发时间；但是种子需要在不同罐体内进行切换培养，设备系统增加，操作相对繁琐；而且每一批种子液仍然采用分批间歇培养的方式进行，培养环境在不断变化，无法保障种子在最适的环境条件下生长，不仅存在批次间的不稳定，更为重要的是容易导致菌种衰退，影响发酵水平。因此，目前行业内还没有产生真正的种子连续培养方法。
相比专利CN201410329652.X中分批间歇培养，黑曲霉种子连续培养需要克服以下技术难点：(1)菌体生长慢；菌丝球容易流失；(2)减少菌种传代次数，避免菌体退化；(3)确定合适的种子液停留时间，避免菌体流失过快，同时保障移种种子液中菌体量充足，且保证培养环境稳定；(4)确定合适的菌丝大小，使菌体生长速率和稀释速率达到平衡。
因此，如何突破多细胞丝状菌的局限，改进种子液制备过程，既能减少孢子使用量，提高种子培养效率，又能使种子的生长环境恒定在最适状态，培养环境根据菌体生长状态实时进行反馈调整，避免菌种退化，保证种子液质量稳定，提高发酵水平，是当前柠檬酸生产中亟待解决的一个问题。
发明内容
针对现有技术存在的上述问题，本发明申请人提供了一种黑曲霉种子连续培养及其生产柠檬酸的方法。本发明方法突破性解决了多细胞丝状菌生长缓慢，以及菌丝球在连续培养中容易流失的问题，完全实现了种子连续培养，使菌体恒定在最适的生长环境中，避免了菌种退化，使种子液处于连续稳定的高活力状态，对应发酵指标显著提高。
本发明的技术方案如下：
一种黑曲霉种子连续培养的方法，所述方法包括如下步骤：
(1)启动阶段：将黑曲霉孢子接种至种子培养基，培养16～36h，得到种子 液；
(2)种子连续培养阶段：对步骤(1)得到的种子液进行连续分散处理，分散得到的种子液进行连续培养，培养过程中种子液连续流出至发酵培养基进行发酵培养，同时以与流出种子液相同的速度补充新鲜种子补料培养基；
(3)停止阶段：停止补充新鲜种子补料培养基和分散处理，继续培养得到种子液，之后将种子液移入发酵培养基进行发酵培养。
步骤(1)中黑曲霉孢子接种后终浓度为1～9*10 5个/mL；步骤(1)中种子培养基的总糖为100～180g/L，C/N为20～40；步骤(1)中种子培养条件为：温度为35～39℃，风量为0.2～0.4vvm，罐压为0.05～0.1Mpa，搅拌转速为100～200rpm。
步骤(2)中补充的新鲜种子补料培养基的总糖为150～200g/L，C/N为15-30；步骤(2)中连续培养条件为：温度为35～37℃，风量为0.3～0.6vvm，罐压为0.05～0.07Mpa，搅拌转速为150～200rpm。
步骤(3)中继续培养的培养条件为：温度为35～39℃，风量为0.3～0.6vvm，罐压为0.05～0.1Mpa，搅拌转速为150～200rpm。
步骤(2)中所述种子液连续分散处理的方法为：采用分散器将种子液中的菌丝球分散成10～80μm的絮状菌丝。
步骤(2)中新鲜种子补料培养基补充速度和种子液流出的速度F＝V/t，其中V为种子罐内种子液体积，以不同罐体进行确定；t为种子液停留时间，根据絮状菌丝的细胞浓度反馈调节，通常取6-24h。
步骤(2)、步骤(3)中发酵接种比例为5～25％(v/v)。
步骤(2)、步骤(3)中发酵培养基的总糖为160～200g/L，C/N为50-90；步骤(2)、步骤(3)中发酵培养条件为：温度为35～39℃，风量为0.1～0.4vvm，罐压为0.05～0.1Mpa，搅拌转速为100～200rpm，还原糖浓度低于5g/L时结束发酵。
所述种子培养基、新鲜种子补料培养基、发酵培养基分别为淀粉质原料液化液和氮源配制而成；所述淀粉质原料至少包括玉米粉、木薯粉、高粱粉、小麦淀粉其中一种；所述氮源至少包括硫酸铵、尿素、豆粕粉、玉米浆其中一种。
一种生产柠檬酸的方法，该方法采用种子连续培养的方法所得的黑曲霉进行发酵，用于生产柠檬酸。
本发明有益的技术效果在于：
本发明方法完全实现了丝状菌的种子连续培养，相比专利CN201410329652.X，大幅度简化了设备系统，种子罐使用量减少至1/3；减少了操作步骤，显著提高生产效率，每批发酵罐对应的种子培养辅助时间节省12h；提高了种子培养自动化水平。
本发明种子连续培养方法，相比专利CN201410329652.X，减少了菌种传代次数，能有效避免菌种退化，可以连续运行15天保持种子活力不下降，发酵指标稳定。高效率的连续运行不仅减少启停的辅助时间，而且黑曲霉孢子的使用量可以减少1/3，大幅降低黑曲霉孢子培养成本。
本发明方法突出的优势是菌体恒定在最适的生长环境中，使种子始终处于连续稳定的高活力成熟状态，保证了种子液质量的稳定性和一致性。而专利CN201410329652.X中种子采用分批间歇培养，罐内pH不断下降和营养物质逐渐消耗，培养条件逐渐不利于菌体生长和活力保持，从而影响种子液质量，并且批次间种子液质量差异较大；更大的缺陷是移入发酵罐内的种子液是分散器处理后的分散菌丝，需要在发酵罐前期恢复生长，从而影响发酵指标。相比专利CN201410329652.X，在发酵初糖浓度为16％时，发酵强度提高了27.5％，发酵转化率提高了3.4个百分点，且发酵稳定性显著提高。
另外，由于种子活力的提高，产酸速率增加，本发明方法在高浓度发酵条件下相比专利CN201410329652.X具有更大的优势，将发酵初始糖浓度提高至18％时，发酵产酸可超过18％，发酵周期小于60h，发酵转化率超过100％，发酵强度提高了31.6％，发酵转化率提高了4.8个百分点。因此，利用本发明方法可以完全实现柠檬酸的高浓度、高转化率和高效率发酵生产，对柠檬酸产业技术提升具有重要促进作用。
本发明首次采用连续补料耦合连续分散来实现黑曲霉种子连续培养的方法，确定了最适的停留时间和菌丝大小，并且优化得到种子培养不同阶段的培养参数，解决了多细胞丝状菌生长缓慢，以及菌丝球在连续培养中容易流失的问题，完全实现了种子连续培养，使菌体恒定在最适的生长环境中，避免了菌种退化，使种子液处于连续稳定的高活力状态，对应发酵指标显著提高。
附图说明
图1为CN201410329652.X的培养方法示意图。
图2为本发明的培养方法示意图。
具体实施方式
下面结合附图和实施例，对本发明进行具体描述。
以下实施例和对比例所涉及原料和试剂均为市售商品；黑曲霉菌来源于中国工业微生物菌种保藏管理中心(CICC)，保藏编号CICC 40021。总糖、还原糖测定均采用菲林滴定法，氮源测定采用凯氏定氮法，柠檬酸测定采用0.1429mol/L的NaOH滴定，孢子计数采用血球计数板。如无特殊说明，均采用本领域常用设备和工艺方法。
实施例1
玉米粉与自来水按1:3的质量比混合均匀，用Ca(OH) 2将粉浆pH调至6.0，按20U/g玉米粉的添加量加入α-高温淀粉酶；经过喷射液化，碘试呈浅棕色后得到合格玉米液化液；将80％的玉米液化液经过板框过滤去掉滤渣，得到玉米糖液。
启动阶段：将玉米液化液与硫酸铵配制成种子培养基(总糖100g/L，C/N为20)，灭菌冷却后将黑曲霉孢子接种至种子培养基，孢子终浓度为9*10 5个/mL，在温度35℃，风量0.2vvm，罐压0.05Mpa，搅拌转速100rpm条件下培养16h，得到种子液。
连续阶段：将玉米液化液和硫酸铵配制成种子补料培养基(总糖150g/L，C/N为15)。利用分散器对得到的种子液进行连续分散处理，将菌丝球分散成10μm的絮状菌丝；同时按停留时间为6h控制连续补充新鲜种子补料培养基的速度，种子液以相同速度连续流出到发酵培养基进行发酵培养。种子连续培养阶段的培养温度为35℃，风量为0.3vvm，罐压为0.05Mpa，搅拌转速为150rpm。
停止阶段：检测到种子活力呈下降趋势时，停止补充新鲜种子补料培养基和分散处理，将剩余种子液移入发酵培养基进行发酵培养。该阶段种子培养条件：温度为35℃，风量为0.3vvm，罐压为0.05Mpa，搅拌转速为150rpm。
发酵培养基由玉米液化液、玉米糖液和硫酸铵配制成(总糖160g/L，C/N为50)；发酵接种比例为25％(v/v)；发酵培养条件为：温度35℃，风量0.1vvm，罐压0.05Mpa，搅拌转速100rpm，当还原糖浓度低于5g/L时结束发酵，测定每批发酵酸度、残糖，并计算转化率、发酵强度等指标。
实施例2
玉米粉与自来水按1:3的质量比混合均匀，用Ca(OH) 2将粉浆pH调至6.0，按20U/g玉米粉的添加量加入α-高温淀粉酶；经过喷射液化，碘试呈浅棕色后得到合格玉米液化液；将80％的玉米液化液经过板框过滤去掉滤渣，得到玉米糖液。木薯粉与自来水按1:3的质量比混合均匀，用Ca(OH) 2将粉浆pH调至6.0，按20U/g木薯粉的添加量加入α-高温淀粉酶；经过喷射液化，碘试呈浅棕色后得到合格木薯糖液。
启动阶段：将玉米液化液与尿素配制成种子培养基(总糖140g/L，C/N为30)，灭菌冷却后将黑曲霉孢子接种至种子培养基，孢子终浓度为4.5*10 5个/mL，在温度37℃，风量0.3vvm，罐压0.075Mpa，搅拌转速150rpm条件下培养26h，得到种子液。
连续阶段：将玉米液化液和尿素配制成种子补料培养基(总糖175g/L，C/N为23)。利用分散器对得到的种子液进行连续分散处理，将菌丝球分散成45μm的絮状菌丝；同时按停留时间为15h控制连续补充新鲜种子补料培养基的速度，种子液以相同速度连续流出到发酵培养基进行发酵培养。种子连续培养阶段的培养温度为36℃，风量为0.45vvm，罐压为0.06Mpa，搅拌转速为175rpm。
停止阶段：检测到种子活力呈下降趋势时，停止补充新鲜种子补料培养基和分散处理，将剩余种子液移入发酵培养基进行发酵培养。该阶段种子培养条件：温度为37℃，风量为0.45vvm，罐压为0.075Mpa，搅拌转速为175rpm。
发酵培养基由玉米液化液、玉米糖液、木薯糖液和尿素配制成(总糖180g/L，C/N为70)；发酵接种比例为15％(v/v)；发酵培养条件为：温度37℃，风量0.25vvm，罐压0.075Mpa，搅拌转速150rpm，当还原糖浓度低于5g/L时结束发酵，测定每批发酵酸度、残糖，并计算转化率、发酵强度等指标。
实施例3
玉米粉与自来水按1:3的质量比混合均匀，用Ca(OH) 2将粉浆pH调至6.0，按20U/g玉米粉的添加量加入α-高温淀粉酶；经过喷射液化，碘试呈浅棕色后得到合格玉米液化液；将80％的玉米液化液经过板框过滤去掉滤渣，得到玉米糖液。小麦淀粉与自来水按1:3的质量比混合均匀，用Ca(OH) 2将粉浆pH调至 6.0，按20U/g小麦淀粉的添加量加入α-高温淀粉酶；经过喷射液化，碘试呈浅棕色后得到合格小麦糖液。
启动阶段：将玉米液化液与豆粕粉配制成种子培养基(总糖180g/L，C/N为40)，灭菌冷却后将黑曲霉孢子接种至种子培养基，孢子终浓度为1*10 5个/mL，在温度39℃，风量0.4vvm，罐压0.1Mpa，搅拌转速200rpm条件下培养36h，得到种子液。
连续阶段：将玉米液化液和豆粕粉配制成种子补料培养基(总糖200g/L，C/N为30)。利用分散器对得到的种子液进行连续分散处理，将菌丝球分散成80μm的絮状菌丝；同时按停留时间为24h控制连续补充新鲜种子补料培养基的速度，种子液以相同速度连续流出到发酵培养基进行发酵培养。种子连续培养阶段的培养温度为37℃，风量为0.6vvm，罐压为0.07Mpa，搅拌转速为200rpm。
停止阶段：检测到种子活力呈下降趋势时，停止补充新鲜种子补料培养基和分散处理，将剩余种子液移入发酵培养基进行发酵培养。该阶段种子培养条件：温度为39℃，风量为0.6vvm，罐压为0.1Mpa，搅拌转速为200rpm。
发酵培养基由玉米液化液、玉米糖液、小麦糖液和豆粕粉配制成(总糖200g/L，C/N为90)；发酵接种比例为5％(v/v)；发酵培养条件为：温度39℃，风量0.4vvm，罐压0.1Mpa，搅拌转速200rpm，当还原糖浓度低于5g/L时结束发酵，测定每批发酵酸度、残糖，并计算转化率、发酵强度等指标。
对比例1(CN201410329652.X中实施例1)
葡萄糖与豆粕粉分别配制种子培养基(总糖为80g/L，总氮1.5g/L)与发酵培养基(总糖为160g/L，总氮1.5g/L)；移种后孢子浓度为55万个/mL接种；培养20h，得到成熟的种子液，种子液菌丝经分散器分散，处理后的菌球平均直径为100μm，分别接入下一级种子培养基与发酵培养基；其中接入下一级发酵培养基的，当发酵培养基中还原糖浓度降至5g/L以下时发酵结束。如此循环直到种子活力显著衰退时停止。测定每批发酵酸度、残糖，并计算转化率、发酵强度等指标。
对比例2
发酵培养基总糖为180g/L，总氮1.5g/L；其他条件与对比例1相同。
对比例3
玉米粉液化液和玉米糖液制备与实施例1相同。玉米液化液与硫酸铵配制种子培养基(总糖120g/L，总氮2g/L)和发酵培养基(总糖160g/L、总氮1.0g/L)。将黑曲霉孢子接种至种子培养基，孢子终浓度为4*10 5个/mL，培养20h，得到一级成熟种子液。将70％的成熟种子液转接至第一批发酵培养基内培养，当还原糖浓度低于5g/L时结束发酵。将剩余30％种子液经过分散器处理，得到菌丝球或菌块的平均直径为62μm，转接至种子培养基，培养16h重新得到二级成熟种子液。如此循环直到种子活力显著衰退时停止。测定每批发酵酸度、残糖，并计算转化率、发酵强度等指标。
对比例4
玉米粉液化液和玉米糖液制备与实施例1相同。玉米液化液与硫酸铵配制种子培养基(总糖120g/L，总氮2g/L)和发酵培养基(总糖160g/L、总氮1.0g/L)。将黑曲霉孢子接种至种子培养基，孢子终浓度为4*10 5个/mL，培养20h，得到一级成熟种子液。将70％的成熟种子液转接至第一批发酵培养基内培养，当还原糖浓度低于5g/L时结束发酵。将剩余30％种子液经过分散器处理，得到菌丝球或菌块的平均直径为50μm，将新鲜种子培养基补加至剩余种子液，培养16h重新得到二级成熟种子液。如此循环直到种子活力显著衰退时停止。测定每批发酵酸度、残糖，并计算转化率、发酵强度等指标。
对比例5
种子连续培养阶段中连续补充新鲜种子补料培养基的速度按停留时间4h控制；其他条件与实施例1相同。
对比例6
种子连续培养阶段中种子补料培养基的C/N为40；其他条件与实施例1相同。
测试例1
将实施例1和对比例1、3、4的技术效果进行对比，如表1所示。
表1
  实施例1 对比例1 对比例3 对比例4
种子连续运行时间(h) 288 260 260 260
种子罐使用个数 1 3 3 1
每批种子罐辅助时间(h) 0 12 9 1
麸曲使用量(桶/发酵罐) 0.22 0.33 0.33 0.33
对应发酵罐数量 18 12 12 12
对应发酵初始总糖平均(g/L) 160 160 160 160
对应发酵产酸平均(g/L) 162.5 157.0 159.0 158.5
对应发酵周期平均(h) 52.5 64.7 57.2 56.6
对应发酵转化率平均(％) 101.6 98.1 99.4 99.1
对应发酵强度平均(g/(h·L)) 3.10 2.43 2.78 2.80
将实施例1和对比例1、3、4的技术效果进行对比显示：(1)本发明方法相比专利CN201410329652.X，简化了设备系统，种子罐使用量减少至1/3；减少了操作步骤，显著提高生产效率，每批发酵罐对应的种子培养辅助时间节省12h。(2)本发明方法连续运行时间更长，黑曲霉孢子的使用量可以减少1/3，大幅降低黑曲霉孢子培养成本。(3)相比专利CN201410329652.X，在发酵初糖浓度为16％时，发酵强度提高了27.5％，发酵转化率提高了3.4个百分点。
测试例2
将实施例2和对比例2的技术效果进行对比，如表2所示。
表2
Figure PCTCN2018123053-appb-000001
Figure PCTCN2018123053-appb-000002
将实施例2和对比例2的技术效果进行对比显示，本发明方法在高浓度发酵条件下相比专利CN201410329652.X具有更大的优势，将发酵初始糖浓度提高至18％时，发酵产酸可超过18％，发酵周期小于60h，发酵转化率超过100％，发酵强度提高了31.6％，发酵转化率提高了4.8个百分点。因此，利用本发明方法可以完全实现柠檬酸的高浓度、高转化率和高效率发酵生产，对柠檬酸产业技术提升具有重要促进作用。
测试例3：本发明实施例1和对比例5、6的性能对比，如表3所示。
表3
  实施例1 对比例5 对比例6
种子连续运行时间(h) 288 136 152
种子罐使用个数 1 1 1
种子罐单罐辅助时间(h) 24 24 24
麸曲使用量(桶/发酵罐) 0.22 0.50 0.44
对应发酵罐数量 18 8 9
对应发酵初始总糖平均(g/L) 160 160 160
对应发酵产酸平均(g/L) 162.5 158.7 157.3
对应发酵周期平均(h) 52.5 55.9 58.1
对应发酵转化率平均(％) 101.6 99.2 98.3
对应发酵强度平均(g/(h·L)) 3.10 2.84 2.71
本发明实施例1和对比例5、6的性能比较显示，当改变种子连续培养阶段停留时间和补料培养基C/N等参数时，种子连续运行时间会受到显著影响，运行时间缩短，种子活力快速下降，对应发酵转化率、发酵强度等指标也明显降低。
以上所述仅是本发明的优选实施方式，本发明不限于以上实施例。可以理解，本领域技术人员在不脱离本发明的精神和构思的前提下直接导出或联想到的其他改进和变化，均应认为包含在本发明的保护范围之内。

Claims (10)

  1. 一种黑曲霉种子连续培养的方法，其特征在于，所述方法包括如下步骤：
    (1)启动阶段：将黑曲霉孢子接种至种子培养基，培养16～36h，得到种子液；
    (2)种子连续培养阶段：对步骤(1)得到的种子液进行连续分散处理，分散得到的种子液进行连续培养，培养过程中种子液连续流出至发酵培养基进行发酵培养，同时以与流出种子液相同的速度补充新鲜种子补料培养基；
    (3)停止阶段：停止补充新鲜种子补料培养基和分散处理，继续培养得到种子液，之后将种子液移入发酵培养基进行发酵培养。
  2. 根据权利要求1所述的方法，其特征在于，步骤(1)中黑曲霉孢子接种后终浓度为1～9*10 5个/mL；步骤(1)中种子培养基的总糖为100～180g/L，C/N为20～40；步骤(1)中种子培养条件为：温度为35～39℃，风量为0.2～0.4vvm，罐压为0.05～0.1Mpa，搅拌转速为100～200rpm。
  3. 根据权利要求1所述的方法，其特征在于，步骤(2)中补充的新鲜种子补料培养基的总糖为150～200g/L，C/N为15-30；步骤(2)中连续培养条件为：温度为35～37℃，风量为0.3～0.6vvm，罐压为0.05～0.07Mpa，搅拌转速为150～200rpm。
  4. 根据权利要求1所述的方法，其特征在于，步骤(3)中继续培养的培养条件为：温度为35～39℃，风量为0.3～0.6vvm，罐压为0.05～0.1Mpa，搅拌转速为150～200rpm。
  5. 根据权利要求1所述的方法，其特征在于，步骤(2)中所述种子液连续分散处理的方法为：采用分散器将种子液中的菌丝球分散成10～80μm的絮状菌丝。
  6. 根据权利要求1所述的方法，其特征在于，步骤(2)中新鲜种子补料培养基补充速度和种子液流出的速度F＝V/t，其中V为种子罐内种子液体积，以不同罐体进行确定；t为种子液停留时间，根据絮状菌丝的细胞浓度反馈调节，通常取6-24h。
  7. 根据权利要求1所述的方法，其特征在于，步骤(2)、步骤(3)中发酵接种比例为5～25％(v/v)。
  8. 根据权利要求1所述的方法，其特征在于，步骤(2)、步骤(3)中发酵培养基的总糖为160～200g/L，C/N为50-90；步骤(2)、步骤(3)中发酵培养条件为：温度为35～39℃，风量为0.1～0.4vvm，罐压为0.05～0.1Mpa，搅拌转速为100～200rpm，还原糖浓度低于5g/L时结束发酵。
  9. 根据权利要求1所述的方法，其特征在于，所述种子培养基、新鲜种子补料培养基、发酵培养基分别为淀粉质原料液化液和氮源配制而成；所述淀粉质原料至少包括玉米粉、木薯粉、高粱粉、小麦淀粉其中一种；所述氮源至少包括硫酸铵、尿素、豆粕粉、玉米浆其中一种。
  10. 一种生产柠檬酸的方法，其特征在于，该方法采用权利要求1所述种子连续培养的方法所得的黑曲霉进行发酵，用于生产柠檬酸。
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