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WO2011143800A1 - 用于制备l-乳酸的凝结芽孢杆菌及其应用方法 - Google Patents

用于制备l-乳酸的凝结芽孢杆菌及其应用方法 Download PDF

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Publication number
WO2011143800A1
WO2011143800A1 PCT/CN2010/001871 CN2010001871W WO2011143800A1 WO 2011143800 A1 WO2011143800 A1 WO 2011143800A1 CN 2010001871 W CN2010001871 W CN 2010001871W WO 2011143800 A1 WO2011143800 A1 WO 2011143800A1
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lactic acid
fermentation
dsm
bacillus coagulans
coagulans
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English (en)
French (fr)
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许平
王丽敏
赵博
马翠卿
苏斐
陶飞
唐鸿志
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上海交通大学
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Priority to US13/698,708 priority Critical patent/US8492127B2/en
Publication of WO2011143800A1 publication Critical patent/WO2011143800A1/zh

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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • 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
    • C12P7/56—Lactic 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
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
    • 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
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus

Definitions

  • Bacillus coagulans for preparing L-lactic acid and application method thereof Bacillus coagulans for preparing L-lactic acid and application method thereof
  • the invention relates to a strain in the technical field of lactic acid preparation and a method for applying the same, in particular to a method for preparing a B. coagulans for preparing a bismuth concentration L-lactic acid by using a five-carbon sugar/hexasaccharide sugar and an application thereof.
  • Lactic acid (Lactk Acid) is an important, versatile organic acid widely used in food, chemical, pharmaceutical and other fields.
  • the most important and most widely used industrial application is the monomer synthesized by polylactic acid.
  • the polylactic acid produced by the polymerization of lactic acid has good biocompatibility and biodegradability, and is therefore considered by the industry to be the most promising new renewable material in the new century.
  • the methods of lactic acid mainly include chemical synthesis, enzymatic catalysis and fermentation.
  • the microbial fermentation method can produce not only glucose, which is obtained by decomposing renewable resources such as starch or cellulose, but also fermentative production by fermentation of the strain and culture conditions, and optically pure L can be obtained.
  • Streptococcus ⁇ Enterococci CEnterococcus rhizopus and other bacteria have lactic acid-producing ability, such as Chinese Patent No. 200480036931.1.
  • Thermophilic Bacillus is a new L-lactic acid fermentation production. Microbial. Because Bacillus has low nutrient requirements and has a relatively low fermentation temperature (45 ⁇ 60 ⁇ ), it can realize open fermentation, and greatly reduce the chance of contaminating bacteria during fermentation, and improve the optical purity of L-lactic acid. Therefore, in recent years, studies on the production of lactic acid by Bacillus have been gradually increased.
  • Chinese Patent Application No. 200810098908.5 reports the production of lactic acid by Bacillus.
  • U.S. Patent No. 2005/0250192 A1 describes the production of L-lactic acid by a plurality of Bacillus coagulans capable of producing lactic acid using glucose and xylose.
  • Bacillus sp. 36m has the strongest ability to utilize hexose and pentose sugars.
  • the strains described in this technique produce lower lactic acid yields, such as Bacillus sp. 36D1 using pure glucose L.
  • the yield of lactic acid is about 25.2 g/L; the yield of pure xylose L-lactic acid is about 23.4 g/L.
  • L-lactic acid is fermented by reducing sugar (mainly five-carbon sugar-xylose, six-carbon sugar-glucose) in bagasse, and the reported yield of L-lactic acid is about 55.5 gL, and the fermentation time is about Very long, more than 190 hours.
  • reducing sugar mainly five-carbon sugar-xylose, six-carbon sugar-glucose
  • the main raw material for the industrial production of lactic acid is glucose, or crop products with a relatively high starch content such as corn and rice.
  • the main problem is the production cost. Fermentation of lactic acid using carbohydrate-rich organic waste not only reduces the production cost of lactic acid, but also solves the problem of recycling of waste. As a part of environmental protection and energy reduction that is promoted worldwide, organic waste is being fully reused at home and abroad.
  • the above substances contain five carbon sugars (such as xylose, arabinose, etc.) and six carbon sugars (such as glucose), and most of the lactic acid producing strains cannot utilize the five carbon sugars therein, thus limiting their use in lactic acid production.
  • xylitol is prepared by using xylose extracted from corncob hydrolysate as raw material; in this process, a large amount of by-products are produced, and the by-product of xylitol production is 50% ⁇ 70% of carbohydrates, if not used, cause both waste and pollution.
  • the present invention is directed to the above-mentioned deficiencies of the prior art, and provides a method for preparing L-lactic acid, Bacillus coagulans, and a method for using the same, which can directly produce L-lactic acid by using various reducing sugars in a by-product of xylitol production. Fermentation to obtain bismuth concentration of L-lactic acid, while saving cost and improving production efficiency, is suitable for popularization and application in industrial production.
  • the invention relates to a Bacillus coagulans for preparing L-lactic acid, which is a Bacillus coagulans ⁇ XZL4 and XZL9, deposited on December 18, 2009, and deposited in the Budapest Treaty of Microorganisms International Collection of German Microorganisms Collection (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH), with accession numbers DSM No. 23183 and DSM No. 23184.
  • Bacillus coagulans CR coagulans XZL4 DSM No. 23183 is a Gram-positive bacterium, the nucleotide sequence of which is shown in Seq. ID No. 1, fine
  • Bacillus coagulans CB. coagulans) XZL9 DSM No. 23184 is a Gram-positive bacterium, the nucleotide sequence of which is shown in Seq. ID No. 2, the diameter of the nucleus cells is (0.8 ⁇ 0.9). ⁇ x (3.0 ⁇ 5.0) ⁇ . Endophytes are formed. Small, pale milky white, neatly rounded colonies were formed on agar plates containing xylose, yeast powder and peptone, separated from the soil near the 3 ⁇ 45 m core processing plant in Shandong.
  • the present invention relates to the above-mentioned application method for preparing L-lactic acid of Bacillus coagulans, which firstly seed-cultures Bacillus coagulans to obtain a seed culture liquid, and then uses agricultural and industrial waste containing five-carbon sugar or six-carbon sugar or a combination thereof.
  • xylitol production by-products such as molasses, syrup, bagasse, etc.
  • molasses molasses
  • syrup bagasse, etc.
  • Fermentation refers to the use of fermentation medium, 10% by volume of the inoculum to access the seed culture, at 50 ⁇ ⁇ 60 ⁇ Incubate in an environment for 48 to 72 hours, preferably at a temperature of 50 to 55.
  • the fermentation includes a feed stream addition process, and the feed stream addition process refers to: adding a carbon source when the total reducing sugar content in the fermentation liquid is less than 20 to 30 g/L, so that the total reducing sugar content is maintained at 30 ⁇ 70 g/L, or 50 ⁇ 70 g/L.
  • composition of the fermentation medium and the content thereof are: a carbon source of 40 to 200 g/L, a nitrogen source of 5 to 12 g/L, and a neutralizing agent for regulating the pH of the medium 50 to: lOO g/L,
  • the balance is water.
  • the pH of the fermentation medium of ⁇ 3 ⁇ 4 is 5.5 to 6.2.
  • the carbon source of ⁇ 3 ⁇ 4 is a five- or six-carbon sugar or a raw material rich in five-carbon sugar or six-carbon sugar, which is any one of the following three types:
  • 03 ⁇ 4 xylitol by-products are commercially available, such as Shandong Longli Biotechnology Co., Ltd., Shandong Futian Pharmaceutical Co., Ltd., Shandong Xingdian Biotechnology Co., Ltd., etc., but different batches of products from different companies.
  • the total sugar content is different.
  • the xylitol production by-product used in the present invention contains 50% to 70% of carbohydrates, wherein the glucose content is 5% to 10% (w/v), and the content is 40% to 50% ( w/v), the arabinose content is 10% ⁇ 25% (w/v).
  • the nitrogen source is yeast powder, specifically yeast powder 5 ⁇ 12 g/L ;
  • the neutralizing agent is calcium carbonate, specifically calcium carbonate 50 ⁇ : 100 g/L.
  • the seed culture solution of Bacillus coagulans refers to a culture obtained by sequentially incubating and seeding the Bacillus coagulans ⁇ XZL4 DSM No. 23183 or XZL9 DSM No. 23184.
  • Inclined culture of 03 ⁇ 4E means: inoculation of Bacillus coagulans (X coagulans) XZ 4 DSM No. 23183 or XZL9 DSM No. 23184 on solid slant medium containing 15 g/L agar, 45 ⁇ 55*C Next, culture for 24 to 48 hours;
  • the seed culture refers to: inoculation of the slanted Bacillus in aseptic conditions into 30 ml of seed medium, 45 ⁇ 55 Torr, static culture for 10 to: 24 hours, adding a neutralizing agent to control the fermentation broth pH, seed culture solution, seed culture contains: glucose 40 ⁇ 60g, yeast powder 5 ⁇ 10g, calcium carbonate 20-40g, balance is water, preferably contains: glucose 50 /L, yeast powder 10 /L, CaCO 3 20 /L, the balance is water; the pH of the seed medium is 6.5.
  • the main raw materials of the invention are glucose, «and xylitol production by-products, which can directly metabolize the five-carbon sugar and the six-carbon sugar in the xylitol by-product to form L-lactic acid, thereby eliminating the process steps of separately performing five-carbon sugar metabolism. .
  • the L-lactic acid production process is not only easy to obtain raw materials, low in cost, but also achieves a relatively low acid production capacity.
  • the R coagulans XZL4 DSM No. 23183 and XZL9 DSM No. 23184 provided by the present invention can utilize both glucose and xylose as a carbon source for producing lactic acid, and have a strong ability to produce L-lactic acid.
  • glucose L- lactic acid as a carbon source yield up Gao most 173 g / L, greater than 99% optical purity, most Gao sugar acid conversion rate of up to 0.98, the production capacity of the fermentation 2.4 g / L / h; L- using xylose as the carbon source
  • the yield of lactic acid is up to 195 g/L, the optical purity is more than 99%, the conversion rate of sugar acid is up to 0.98, and the fermentation capacity is 2.7 g/L/h.
  • the reducing sugar in the by-product of xylitol production is used as substrate.
  • L-lactic acid is produced by fermentation.
  • the highest yield of L-lactic acid is 106 g/L, the optical purity is more than 99%, and the fermentation production capacity is 2.08 g/L/h.
  • the strain of Bacillus coagulans provided by the present invention does not degrade after repeated cycles of fermentation. It can still maintain a relatively vigorous vitality, and the strain can directly produce L-lactic acid by using glucose, and various reducing sugars in the by-product of xylitol production, and fermenting to obtain the concentration of strontium.
  • L-lactic acid Therefore, the production of L-lactic acid by the method of the invention can save cost and improve production efficiency, and is suitable for popularization and application in industrial production.
  • Figure 1 is a 16S r phylogenetic tree analysis diagram of coag lans' XZL4 DSM No. 23183 and XZL9 DSM No. 23184;
  • the horizontal distance (sum of length) of the solid line represents the evolution distance of the strain
  • the selected reference bacteria are strains of Bacillus coagulans which are closely related to the strain provided by the present invention, and 11 strains thereof, among which CR coagulans ⁇ 2-6 is a Chinese patent strain (CN 200710176060.9), whose 16S rDNA sequence is obtained by sequencing (see SEQ ID NO: 3); Bacillus coagulans CB. coagulans ⁇ 36D1 is a US patent strain (Pub.
  • composition of the medium used in this example is as follows:
  • Nutrient liquid medium xylose 10 g/L, yeast powder 10 g/L, pH 6.
  • Nutrition agar medium 50 g/L, yeast powder 10 g/L, CaCO 3 20 g/L, agar powder 20 g/L, pH 6.
  • the strain screening medium xylose 100 g/L, yeast powder 20 g/L, CaC0 3 75 g/L, pH 6.
  • the strain was subcultured several times, and then subjected to 10 cycles of fermentation test.
  • the L-lactic acid production and conversion rate produced by the 10th cycle fermentation were basically maintained at the original level, which proved that the above strain was the target strain, and the name was XZL4 and XZL9, II. Identification of strains
  • the two strains of the eagle are Gram-positive bacteria, the cells are straight, and the size of the vegetative cells is (0.8 ⁇ 0.9) ⁇ x (3.0 ⁇ 5.0) ⁇ . Endophytes are formed.
  • XZL4 forms round colonies with smooth, milky white edges and neat edges;
  • XZL9 forms small, pale milky white, rounded colonies with neat edges.
  • the fatty acid composition of XZL4 was similar to that of B. coagulans ⁇ . coagulans ⁇ with Wei ( (0.12) by database TSBA40 4.10 Library.
  • the nucleotide sequence of the 16S rDNA of this bacterium (see SEQ ID NO: 1) is different from all strains 16S rDNA that have been reported or submitted to the public database, and has the highest homology with B. baumannii ⁇ B. coagulans ⁇ (99 %), indicating that the strain is a brand new strain.
  • the L-lactic acid producing strain was identified as Bacillus coagulans dR coagulans XZL4, and it was mixed with the German Collection of Microorganisms, No. 23183.
  • the fatty acid composition of XZL9 has a similarity with coagulans (0.69).
  • the nucleotide sequence of the 16S rDNA of this bacterium (see SEQ ID NO: 2) is different from all strains 16S rDNA that have been reported or submitted to public databases, and has the highest homology with B. coagu!ans NRIC 1526. (99%), indicating that the strain is a brand new strain.
  • the strain L-lactic acid producing bacteria was identified as R coagulans XZL9, and deposited in the center of the German microbiology city, the job number is DSM No. 23184.
  • Coagulans-XLZ9 16S rDNA and 36D1 have base differences at positions 830, 890, 1262, 1267, 1268, 1286, 1308, 1315, 1344, 1345, 1362, and 2-6 at positions 340, 1212, 1225, 1276, 1309, 1345 , 1346 is different from the corresponding position (position is based on XLZ9 and the corresponding position is taken).
  • XLZ4 and 36D1 at 340, 830, 890, 1214, 1225, 1264, 1268, 1269, 1275 , 1316, 1345, 1346 are different, and there is a difference between the 2 and 6 in the 3rd position (the position is taken as the XLZ4 and the corresponding position is taken).
  • Bacillus coagulans (A g «/fl w) XLZ9 differs from XLZ4 in positions 1, 340, 1212, 1224, 1274, 1374, 1375 (positions are based on XLZ9 and the corresponding positions are taken).
  • Bacillus coagulans iB. coagulans) XZL4 DSM No. 23183 and XZL9 DSM No. 23184 were separated from the soil near a 33 ⁇ 4 core processing plant in Shandong.
  • Inclined culture Bacillus coagulans iB. coagulans') XZL4 DSM No. 23183 and XZL9 DSM No. 23184 were inoculated on solid slant medium containing 15 g/L agar, cultured under 50 to 60 , conditions. 24 to 48 hours.
  • step (1) Seed culture: The slant culture of step (1) is inoculated into 30 ml of seed medium under aseptic conditions.
  • the culture medium is statically cultured for 10 to 24 hours, and the pH of the fermentation liquid is controlled by adding a neutralizing agent to prepare a seed culture liquid.
  • Fermentation culture The seed culture solution is inoculated into the fermentation medium at a dose of 10% by volume, 50 ⁇ 60 Incubate for 48 to 72 hours under conditions.
  • the bacterial culture of the carved in steps (1), (2), and (3) is preferably 50 ⁇ SSO.
  • the neutralizing agent added during the cultivation of steps (2) and (3) is calcium carbonate, and the pH is controlled.
  • the fermentation broth is taken every 3 hours, first heated to 80 ⁇ 100, and then centrifuged at 6,000 rpm for 5 minutes. The supernatant is taken to detect the concentration of L-lactic acid, D-lactic acid and glucose in the fermentation broth. ⁇
  • the method for measuring total reducing sugar is the DNS method.
  • the method for measuring glucose is that the fermentation liquid is diluted and centrifuged, and measured by a biosensor analyzer SBA-40C (Shandong Academy of Sciences).
  • Biosensing Analyzer SBA-80 is an analytical instrument using an immobilized enzyme as a sensor. Glucose, oxygen and water are used to generate hydrogen peroxide under the catalysis of enzymes. The hydrogen peroxide released by the reaction is in contact with the platinum-silver electrode and generates a current signal which is linearly proportional to the glucose concentration. The glucose concentration can be determined by measuring the current signal intensity.
  • the method for measuring xylose is to use a xylose assay kit (Nanjing Jiancheng Technology Co., Ltd.).
  • the yield of L-lactic acid and D-lactic acid (lactic acid content or concentration of fermentation broth, g/L) is determined by the m Agilent 1100 liquid chromatograph equipped with a chiral separation column (Mitsubishi Chemical Co., Ltd., MCI GEL-CRS10 W ( 3 ⁇ ) 4.6 ID X 50 mm, for optical separation).
  • the specific operating conditions are: 0.002 mol/L copper sulfate as the mobile phase, flow rate 0.5 ml/min, injection volume 20 H L, UV detector, detection wavelength 254 nm, operation 25 .
  • a standard curve was prepared using L-lactic acid and D-lactic acid standards, and the contents of L-lactic acid and D-lactic acid in the fermentation broth were calculated according to the standard curve.
  • D-lactic acid as a standard product is a product of Sigma-Aldrich, Germany, and its product number is L0625-25MG;
  • L-lactic acid as a standard product is a product of Sigma-Aldrich, Germany, and its article number is L175 (0G).
  • the D-lactic acid retention time was 10.150 minutes.
  • optical purity is a measure of the amount of one enantiomer in an optically active sample over the other enantiomer. It can be expressed as an enantiomeric excess (ee).
  • the optical purity (ee) of L-lactic acid in the present invention is calculated by the following formula: ((L-lactic acid production - D-lactic acid production) ⁇ (L-lactic acid production + D-lactic acid production)) X 100%.
  • the conversion of sugar acid is defined as (g/g): (L-lactic acid production (g/L) ⁇ substrate consumption (g/L, glucose or xylose or total sugar consumption (g/L)) X 100 %.
  • the L-lactic acid fermentation capacity (g/L/h) is: L-lactic acid production (g/L) ⁇ Fermentation time (h).
  • Example 1 The invention is further illustrated by the following examples.
  • Example 1 The invention is further illustrated by the following examples.
  • the composition of each medium used in this example is as follows:
  • the slant medium contains: 30 g of xylose, 10 g of yeast powder, 10 g of CaCO 3 , 15 g of agar powder, and the balance is water.
  • the pH of the medium was 6.5. Sterilize for 115 minutes at 115 Torr.
  • Seed culture & «liter contains: glucose 50 g, yeast powder 10 g, CaCO 3 20 g, the balance is water. The pH of the seed medium was 6.5. 115 ⁇ sterilization 20 .
  • Fermentation culture Hesheng contains: glucose 55 ⁇ 150 g, yeast powder 10 g, CaC0 3 60 g, the balance is water; the pH of the fermentation medium is 5.5-7. Sterilize for 20 minutes under 115 conditions.
  • Inclined culture Bacillus coagulans R coagulans)
  • XZL4 DSM No. 23183 and XZL9 DSM No. 23184 were respectively inoculated on a slant medium, and cultured for 50 hours;
  • Seed culture The strain cultivated in the step (1) was cultured under sterile conditions with a loop of 2 loops in a 100 ml flask containing 30 ml of seed medium, and cultured at 50 ° for 20 hours to prepare seeds.
  • Culture medium
  • Fermentation culture 10 ml of the seed culture solution prepared in the step (2) is placed in a 300 ml flask containing 90 ml of fermentation medium, 50 quiescent culture, when the glucose and L-lactic acid contents are kept stable, End the fermentation.
  • the L-lactic acid concentration and the glucose concentration in the fermentation broth were measured according to the detection and calculation methods described in the above specific embodiments, and the L-lactic acid production rate was calculated.
  • Table 3 uses glucose as a carbon source to generate L-lactic acid.
  • L-lactic acid was fermented in batches using a mixture of R coagulans XZL4 DSM No. 23183 and XZL9 DSM No. 23184 in a triangular flask with a carbon source:
  • composition of each medium used in this example is as follows:
  • Fermentation culture 3 ⁇ 4 « liter contains: « 55-100 g, yeast powder 10 g, CaCO 3 60 g, the balance is water; the pH of the fermentation medium is 5.5 ⁇ 7. Sterilize for 20 minutes under 115 conditions.
  • the method for producing L-lactic acid by fermentation comprises the following steps:
  • Table 4 shows the formation of L-lactic acid as a carbon source
  • L-lactic acid was produced by batch fermentation of B. coagulans XZL4 DSM No. 23183 and XZL9 DSM No. 23184 in a flask with xylitol by-products as a carbon source:
  • the slant medium and the seed medium were the same as in Example 1.
  • Fermentation culture 3 ⁇ 4 « liter contains: xylitol production by-product 7S ⁇ 150 g, yeast powder 10 g, CaCO 3 60 g, the balance is water; the pH of the fermentation fermentation medium is 5.5 ⁇ 7. Sterilize for 20 minutes under conditions of 115.
  • the method for producing L-lactic acid by fermentation comprises the following steps:
  • Fermentation culture 10 ml of the seed culture solution prepared in the step (2) was placed in a 300 ml flask containing 90 ml of fermentation medium, and cultured at 50 Torr for 48 hours, and the fermentation was terminated.
  • the L-lactic acid concentration and the total reducing sugar concentration in the fermentation broth were measured according to the detection and calculation methods described in the above specific embodiments, and the L-lactic acid production rate was calculated.
  • Table 5 shows the formation of L-lactic acid with carbon as a by-product (g/L)
  • composition of each medium used in this example is as follows:
  • the slant medium and the seed medium were the same as in Example 1.
  • the fermentation medium contains: 150 g of by -product of xylitol production, 10 g of yeast powder, CaCO 3 100, and the balance is water; the pH of the fermentation medium is 5.5-7. Sterilize for 20 minutes under conditions of 115.
  • the method for producing L-lactic acid by fermentation comprises the following steps:
  • Fermentation culture 10 ml of the seed culture solution prepared in the step (2) was placed in a 300 ml flask containing 90 ml of fermentation medium, and cultured at 55 for 48 hours, and the fermentation was terminated.
  • the L-lactic acid concentration and the total reducing sugar concentration in the fermentation broth were measured according to the detection and calculation methods described in the above specific embodiments, and the L-lactic acid production rate was calculated.
  • composition of each medium used in this example is as follows:
  • the slant medium and the seed medium were the same as in Example 1.
  • the fermentation medium contains: 200 g of by -product of xylitol production, 5 g of yeast powder, and 100 g of CaC0 3 , and the balance is water; the pH of the fermentation medium is 5.5 to 7. Sterilize for 20 minutes under 115 conditions.
  • the method for producing L-lactic acid by fermentation comprises the following steps:
  • Seed culture the same as in Example 1;
  • Fermentation culture 10 ml of the seed culture solution prepared in the step (2) was placed in a 300 ml flask containing 90 ml of fermentation medium, and cultured at 60 Torr for 48 hours, and the fermentation was terminated.
  • the L-lactic acid concentration and the total reducing sugar concentration in the fermentation broth are detected according to the detection and calculation methods described in the specific embodiments, and the L-lactic acid production rate is calculated.
  • L-lactic acid was produced by feeding (sugar) with 50 g/L glucose as a carbon source in a 50-liter fully automatic fermentor using a coagulans of CR Coagulans) XZL4 DSM No. 23183 and XZL9 DSM No. 23184.
  • composition of each medium used in this example is as follows:
  • the slant medium and the seed medium were the same as in Example 1.
  • Fermentation culture 3 ⁇ 4 « liter contains: glucose 100 g, yeast powder 12 g , CaCO 3 100, the balance is water; the pH of the fermentation medium is 5.5 ⁇ 7. Sterilize for 20 minutes under conditions of 115.
  • the method for producing L-lactic acid by fermentation comprises the following steps:
  • Seed culture The strain cultured in the step (1) is cultured under sterile conditions in a 100 ml flask containing 30 ml of seed medium, and cultured for 20 hours at 50 rpm to prepare seed culture. Liquid 1; 30 ml of seed culture solution 1 was placed under sterile conditions into a 500 ml flask containing 300 ml of seed medium, and cultured at 50 ° for 20 hours to prepare seed culture solution 2; continue to expand seeds in the same manner The culture was carried out to obtain 4 liters of seed culture solution 3.
  • Fermentation culture 4 liters of seed culture solution 3 prepared in step (2) is placed under sterile conditions into a 50 liter fully automatic fermenter (Shanghai Baoxing BIOTECH) containing 36 liters of fermentation medium, S0 *C static culture. Sample every 3 hours, determine the amount of residual sugar and L-lactic acid concentration in the fermentation broth. When the glucose concentration drops to 20 ⁇ 30 g/L, add glucose to make the glucose concentration reach 50 ⁇ 70 g L. Sugar 2 times. When the total sugar consumption rate in the fermentation process tends to be stable, the fermentation is terminated.
  • the L-lactic acid concentration and the residual glucose concentration in the fermentation broth were measured according to the detection and calculation methods described in the above specific embodiments, and the L-lactic acid production rate was calculated.
  • the fermentation experiment was set up with 3 repetitions. The results showed that: XZL4 DSM No. 23183 produced L-lactic acid concentration of 173 ⁇ 3 g / L, fermentation time was 72 hours, L-lactic acid production rate was 2.40 g / L / h, sugar acid conversion rate was 0.98, optical purity was 99.2%; XZL9 DSM No. 23184 produced L-lactic acid concentration of 171 ⁇ 5 g / L, fermentation time was 72 hours, L-lactic acid production rate was 2.38 g / L / h, sugar acid conversion rate was 0.96, optical purity was 99.3%.
  • composition of each medium used in this example is as follows:
  • the slant medium and the seed medium were the same as in Example 1.
  • Fermentation culture 3 ⁇ 4 «liter contains: xylose 100 g , yeast powder 12 g , CaCO 3 100 g, the balance is water; the pH of the fermentation medium is 5.5-7. Sterilize for 20 minutes under 115 conditions.
  • the method for producing L-lactic acid by fermentation comprises the following steps:
  • Fermentation culture 4 liters of the seed culture solution 3 obtained in the step (2) is placed under aseptic conditions into a 50-liter fully automatic fermenter (Shanghai Baoxing BIOTECH) containing 36 liters of fermentation medium, 50 ⁇ Static culture. Sampling every 3 hours, measuring the amount of residual sugar and L-lactic acid in the fermentation broth.
  • concentration of xylose drops to 20 ⁇ 30 g/L
  • add xylose to make the concentration of xylose reach 50 ⁇ 70 g/ L, a total of 2 sugar supplements.
  • the rate of xylose consumption in the fermentation process tends to be stable, the fermentation is terminated.
  • the L-lactic acid concentration and the residual xylose concentration in the fermentation broth were measured according to the detection and calculation methods described in the specific embodiments, and the L-lactic acid production rate was calculated.
  • XZL4 DSM No. 23183 produced L-lactic acid concentration of 195 ⁇ 1 g / L, L-lactic acid production rate of 2.70 g L / h, sugar acid conversion of 0.98, optical purity of 99.3%;
  • XZL9 DSM No. 23184 The production of L-lactic acid was 186 ⁇ 4 g/L, the fermentation time was 72 hours, the L-lactic acid production rate was 2.58 g/L/h, the sugar acid conversion rate was 0.97, and the optical ⁇ g was 99.4%.
  • the slant medium and the seed medium were the same as in Example 1.
  • Fermentation Nilsson contains: Production of xylitol byproduct 100 g, yeast extract 12 g, CaC0 3 100 g, sodium chloride 0.1 g, potassium dihydrogen phosphate 0.5 g, magnesium sulfate 0.2 g, balance water; the The pH of the fermentation medium is 5.5 to 7. Sterilize for 20 minutes under 115 conditions.
  • the method for producing L-lactic acid by fermentation comprises the following steps:
  • Seed culture the same as Example 6;
  • Fermentation culture 4 liters of seed culture solution 3 prepared in step (2) was placed under aseptic conditions into a 50-liter fully automatic fermenter (Shanghai Baoxing BIOTECH) containing 36 liters of fermentation medium, 50 ⁇ Static culture. Sample every 3 hours, determine the amount of residual sugar and L-lactic acid concentration in the fermentation broth. When the total sugar concentration drops to 20 ⁇ 30 g/L, add xylitol to produce by-products to achieve a substrate concentration of 50. ⁇ 70 L, total sugar 2 times. When the total sugar consumption rate in the fermentation process tends to be stable, the fermentation ends.
  • the L-lactic acid concentration and the total reducing sugar concentration in the fermentation broth were measured according to the detection and calculation methods described in the above specific embodiments, and the L-lactic acid rate was calculated.
  • Example 9 Using Co coagulans XZL4 DSM No. 23183 and XZL9 DSM No. 23184 in a 50 liter fully automatic fermentor with 200 g of x-litol as a by-product as a carbon source, feed (sugar) flow Fermentation to produce L-lactic acid
  • composition of each medium used in this example is as follows:
  • the slant medium and the seed medium were the same as in Example 1.
  • Fermentation culture «liter contains: 200 g of by-product of xylitol production, 12 g of yeast powder, CaCO 3 100, 0.1 g of sodium chloride, 0.5 g of potassium dihydrogen phosphate, 0.2 g of magnesium sulfate, and the balance is water; The pH of the fermentation medium is 5.5 to 7. Sterilize for 20 minutes under conditions of 115.
  • the method for producing L-lactic acid by fermentation comprises the following steps:
  • the L-lactic acid concentration and the total reducing sugar concentration in the fermentation broth were measured according to the detection and calculation methods described in the above specific embodiments, and the L-lactic acid production rate was calculated.
  • Example 10 Using Bacillus coagulans US. coagulans) XZL4 DSM No. 23183 and XZL9 DSM No. 23184 in a 50 liter fully automatic fermentor with 150 g/L xylitol by-product as a carbon source, feeding (sugar) Production of L-lactic acid by fed-batch fermentation
  • composition of each medium used in this example is as follows: The slant medium and the seed medium were the same as in Example 1.
  • Fermentation medium ⁇ alcohol byproduct 150g, yeast extract 12 g, CaCO 3 100 g, sodium chloride 0.1 g, potassium dihydrogen phosphate 0.5 g, magnesium sulfate 0.2 g, balance water; the pH of the fermentation medium Sterilize for 20 minutes under conditions of 5.5 ⁇ 7, 115 ⁇ "
  • the method for producing L-lactic acid by fermentation comprises the following steps:
  • the L-lactic acid concentration and the total reducing sugar concentration in the fermentation broth were measured according to the detection and calculation methods described in the specific embodiments, and the L-lactic acid rate was calculated.

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Description

说 明 书
用于制备 L-乳酸的凝结芽孢杆菌及其应用方法 领域
本发明涉及的是一种乳酸制备技术领域的菌株及其应用方法，具体是一种利用五碳糖 /六碳 糖的用于制备髙浓度 L-乳酸的凝结芽孢杆菌及其应用¾¾'
背景技术
乳酸（Lactk Acid)是一种重要的， 多用途的有机酸， 广泛应用于食品、 化工、 医药等领 域， 其中最重要、最广泛的工业应用为聚乳酸合成的单体。 由乳酸聚合生成的聚乳酸具有良好 的生物相容性和生物可降解性， 因此被产业界认为是新世纪最有发展前途的新型可再生材料。 乳酸分子中存在一个手性中心， 具有旋光性， 是一种手性分子。 根据旋光性的不同， 乳酸可分 为 L-乳酸、 D-乳酸和消旋乳酸。 由于聚 L-乳酸使用规模最为广泛， 因此光学纯 L-乳酸的生产 一直受到普遍关注。
^乳酸的方法主要有化学合成法、酶催化法和发酵法。 与化学法和酶催化法相比， 微生 物发酵法不仅能以淀粉、纤维素等可再生资源分解所得到的葡萄糖等为原料， 通过菌种和培养 条件的选择发酵生产乳酸， 能获得光学纯的 L-乳酸、 D-乳酸或是两者一定比例的混合物，而且 生产成本低， 产品安全性髙， 目前是大规模生产乳酸的主要方法。 乳杆菌 (.Lactobacillus^链 球菌 (.Streptococcus^ 肠球菌 CEnterococcus 根霉 Rhizopus 等菌具有乳酸生成能力， 如 中国专利文献号 200480036931.1。 嗜热的芽孢杆菌是一种新的可用于 L-乳酸发酵生产的微生 物。 由于芽孢杆菌具有营养要求低， 具有较髙的发酵温度（45〜60Ό )， 可实现开放式发酵， 同时大大减少了发酵过程中污染杂菌的机会，提髙了 L-乳酸的光学纯度，因此近年来关于利用 芽孢杆菌生产乳酸的研究逐渐增多。 日本专利文献号 JP5840093、 JP606200. JP32729K美国 专利文献号 US5079164. 中国专利申请号 200810098908.5均报道了芽孢杆菌生产乳酸。
经 ϋΧί现有技术的检索发现，中国专利申请号 200710176060.9、200910028930.7、02806664,2 都报道了凝结芽孢杆菌发酵葡萄糖生产 L-乳酸。但上述技术中所用凝结芽孢杆菌没有显示利用 五碳糖如 生产髙浓度髙纯度乳酸。尤其在 "一种生产 L-乳酸的方法及其专用凝结芽孢杆菌" 中使用的微生物为凝结芽孢杆菌， 根据该菌株的特性， 该菌株不能够利用 *«。
进一步检索发现，美国专利文献号 US 2005/0250192 A1中记载了能够利用葡萄糖和木糖生 产乳酸的多株凝结芽孢杆菌生产 L-乳酸等。 在分离得到的多株菌中， Bacillus sp. 36m具有最 强的利用六碳糖和五碳糖的能力' 但该技术所记载的菌株生产乳酸产量较低， 如 Bacillus sp. 36D1利用纯葡萄糖 L-乳酸报道产量约 25.2 g/L左右； 利用纯木糖 L-乳酸报道产量约 23.4 g/L 左右； 利用甘蔗渣中还原糖（主要为五碳糖-木糖， 六碳糖-葡萄糖）为底物发酵生产 L-乳酸， 报道的 L-乳酸最髙产量约 55.5 g L左右， 且发酵时间很长， 髙达 190多小时。
目前，乳酸的工业化生产主要原料是葡萄糖， 或者玉米、大米等淀粉含量较髙的农作物产 品，存在的主要问题是生产成本髙。利用富含碳水化合物的有机废弃物发酵生产乳酸，不仅能 够降低乳酸的生产成本，而且可以解决废弃物的资源化问题。作为世界范围内提倡的环境保护 和减少能源需求的一个环节， 当前在国内外都在充分地进行有机废弃物的再利用。然而上述物 质中含有五碳糖（如木糖、 阿拉伯糖等）和六碳糖（如葡萄糖)， 大多数乳酸生产菌株无法利 用其中的五碳糖， 因此限制其在乳酸生产中的应用。在我国，一部分木糖醇是利用从玉米芯水 解液中提取的木糖为原料制得的； 在该工艺中，产生了大量的副产物，且木糖醇生产的副产物 中含有 50%〜70%的碳水化合物， 如不利用既造成浪费又造成污染。
发明内容
本发明针对现有技术存在的上述不足，提供一种用于制备 L-乳酸的凝结芽孢杆菌及其应用 方法， 可以直接利用木糖醇生产副产物中的各种还原糖发酵生产 L-乳酸， 发酵获得髙浓度的 L-乳酸， 在节约成本的同时提髙生产效率， 适合于工业生产中推广应用。
本发明是通过以下技术方案实现的：
本发明涉及一种用于制备 L-乳酸的凝结芽孢杆菌为凝结芽孢杆菌 (Bacillus coagulans^ XZL4和 XZL9,于 2009年 12月 18日保藏于布达佩斯条约指定的微生物国际保藏单位德国微 生物菌种保藏中心 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH), 保 藏编号分别为 DSM No.23183和 DSM No.23184.
的凝结芽孢杆菌 CR coagulans XZL4 DSM No. 23183为革兰氏阳性菌，其核苷酸序 列如 Seq. ID No.l所示，细 |&¾杆，营养细胞的菌体大小为（0.8〜0.9) μπι (3.0〜5.0) μιη。 形成内生孢子。在含有木糖、酵母粉和蛋白胨的琼脂平 ¾±形成表面光滑、乳白色、边缘整齐 的圆形菌落， 由山东某 芯加工厂附近的土壤中分离得到。
的凝结芽孢杆菌 CB. coagulans) XZL9 DSM No. 23184为革兰氏阳性菌，其核苷酸序 列如 Seq. ID No.2所示，细 杆，营养细胞的菌体大小为（0.8〜0.9) μιη x (3.0〜5.0) μιη。 形成内生孢子。在含有木糖、酵母粉和蛋白胨的琼脂平板上形成细小、淡乳白色、边缘整齐的 圆形菌落， 由山东 ¾5米芯加工厂附近的土壤中分离得到。
本发明涉及上述用于制备 L-乳酸的凝结芽孢杆菌的应用方法,首先对凝结芽孢杆菌进行种 子培养获得种子培养液，然后以含有五碳糖或六碳糖或其组合的农业和工废弃物（如糖蜜、糖 浆、甘蔗渣等木糖醇生产副产物）作为发酵培养基的碳源进行发酵后得到 L-乳酸。
的发酵是指采用发酵培养基,按 10%体积比的接种量接入种子培养液，在 50Χ〜 60Ό 环境下培养 48〜72小时， 温度优选 50 〜55 。
所述的发酵中包含补料流加工艺， 该补料流加工艺是指： 当发酵液中总还原糖含量低于 20〜30 g/L时补加碳源， 使总还原糖含量维持在 30^70 g/L， 或达到 50〜70 g/L。
所述的发酵培养基的组分及其含量为：碳源 40〜200 g/L、氮源 5〜12 g/L和用于调控培养 基 pH的中和剂 50〜： lOO g/L, 余量为水。
^¾的发酵培养基的 pH为 5.5〜6.2。
^¾的碳源为五碳或六碳糖或富含五碳糖、 六碳糖的原料， 为以下三种中的任意一种：
(1)葡萄糖 40〜150 g/L;
(2)木糖 40〜100 g/L;
(3)木糖醇生产副产物 100〜200 g/L。
0¾的木糖醇副产物可从商业途径获得， 如山东龙力生物科技有限公司、 山东福田药业有 限公司、 山东邢店生物科技有限公司等公司直接购得， 但不同公司不同批次的产品总糖含量有 所不同， 本发明所用的木糖醇生产副产物含有 50%〜70%的碳水化合物， 其中葡萄糖含量为 5%〜10% (w/v), 含量为 40%〜50% (w/v)， 阿拉伯糖含量为 10%〜25% (w/v). 的氮源为酵母粉， 具体为酵母粉 5〜12 g/L;
所述的中和剂为碳酸钙， 具体为碳酸钙 50〜： 100 g/L。
的凝结芽孢杆菌的种子培养液是指：将芽孢杆菌 coagulans^XZL4 DSM No. 23183 或 XZL9 DSM No. 23184依次进行斜面培养和种子培养后获得培养物。
0¾E的斜面培养是指:将凝结芽孢杆菌 (β coagulans)XZ 4 DSM No. 23183或 XZL9 DSM No. 23184菌种接种于含有 15 g/L琼脂的固体斜面培养基上， 45〜55*C条件下，培养 24〜48小 时；
所述的种子培养是指： 将经过斜面培养的芽孢杆菌在无菌条件下接种到 30 ml种子培养基 中， 45〜55Ό条件下， 静止培养 10〜： 24小时， 加入中和剂控制发酵液 pH, 制得种子培养液， 的种子培养 升中含有： 葡萄糖 40〜60g, 酵母粉 5〜10 g, 碳酸钙 20-40 g, 余 量为水， 优选含有： 葡萄糖 50 /L， 酵母粉 10 /L， CaCO3 20 /L， 余量为水； 该种子培养基 的 pH为 6.5。
本发明主要原料为葡萄糖、 «和木糖醇生产副产物， 可以直接代谢木糖醇副产物中的五 碳糖和六碳糖生成 L-乳酸，免去了单独进行五碳糖代谢的工艺步骤。通过提供合适的发酵工艺 条件， 使得该 L-乳酸生产工艺不仅原料易得， 成本低廉， 而且达到较髙的产酸能力。
本发明所提供的凝结芽孢杆菌 (R coagulans) XZL4 DSM No. 23183和 XZL9 DSM No. 23184既能利用葡萄糖又能利用木糖作为乳酸生产碳源， 且生产 L-乳酸的能力强。利用葡萄糖 作碳源 L-乳酸产量最髙可达 173 g/L， 光学纯度大于 99%, 糖酸转化率最髙可达 0.98, 发酵生 产能力 2.4 g/L/h; 利用木糖作碳源 L-乳酸产量最髙可达 195 g/L,光学纯度大于 99%,糖酸转 化率最髙可达 0.98,发酵生产能力 2.7 g/L/h;利用木糖醇生产副产物中还原糖为底物发酵生产 L-乳酸， L-乳酸最高产量 106 g/L，光学纯度大于 99%,发酵生产能力 2.08 g/L/h„本发明所提 供的凝结芽孢杆菌经过多次循环发酵后菌株不退化，仍能保持较髙的活力。且该菌株可以直接 利用葡萄糖、 以及木糖醇生产副产物中的各种还原糖发酵生产 L-乳酸，发酵获得髙浓度的
L-乳酸。因此，利用本发明方法生产 L-乳酸，能节约成本、提髙生产效率，适合于工业生产中 推广应用。
附图说明
图 1为凝结芽孢杆菌 ( coag lans')XZL4 DSM No. 23183和 XZL9 DSM No. 23184的 16S r賺进化树分析图；
图中： 实线的水平距离（长度之和）代表菌株的进化距离，选取的参照菌是与本发明提供 的菌株亲缘关系较近的凝结芽孢杆菌菌株， 共 11株， 其中凝结芽孢杆菌 CR coagulans^ 2-6 是中国专利菌株 (CN 200710176060.9), 其 16S rDNA序列为测序得到（见 SEQ ID NO: 3)； 凝结芽孢杆菌 CB. coagulans^ 36D1是美国专利菌株（Pub. No: US 2005/0250192 A1 ), 其 16S rDNA序列根据参考文献 "Patel, M. A.; Ou， M. S.; Harbrucker, R.; Aldrich, H. ; Buszko, M. L.; Ingram, L. O.; Shanmugam, K. T. Isolation and characterization of acid-tolerant, thermophilic bacteria for effective fermentation of biomass-derived sugars to lactic acid. AppL Environ. Microbiol 2006, 72, 3228-3235"获得（见 SEQ ID NO: 4)。
具体实施方式
下面对本发明的实施例作详细说明，本实施例在以本发明技术方案为前提下进行实施，给 出了详细的实施方式和具体的操作过程， 但本发明的保护范围不限于下述的实施例。
以下实施例涉及的凝结芽孢杆菌 coagulans KZlA DSM No. 23183和 XZL9 DSM No. 23184的分离、筛选和鉴定方法如下:
―、菌株的分离、筛选
该实施例中所用的培养基的组成如下：
营养液体培养基： 木糖 10 g/L, 酵母粉 10 g/L, pH为 6。
营养琼脂培养基： 50 g/L, 酵母粉 10 g/L， CaCO3 2 0g/L, 琼脂粉 20 g/L, pH为 6。 菌株筛选培养基： 木糖 100 g/L， 酵母粉 20 g/L， CaC03 75 g/L, pH为 6。
该实施例的具体操作过程如下：
. 采集山东省某 芯加工厂附近的土壤， 称取 2 g溶于 50 ml营养液体培养基中， 50Ό富 集培养 6〜10小时，然后稀释涂布到含营养琼脂培养基的培养皿中， 50Ό培养 24小时。待长 出单菌落后，挑选菌落面积和产酸透明圈面积大的菌落，接种到发酵培养基中， 501C静止培养 48小时， 测定 L-乳酸的产量， 经过多次筛选， 挑取两株 L-乳酸产量较髙的菌株。
将 菌株多次传代培养，然后再进行 10个循环的发酵测试，其第 10次循环发酵产生的 L-乳酸产量和转化率基本保持原有水平，证明上述菌株即是目的菌株，名称为 XZL4和 XZL9， 二、菌株的鉴定
±¾菌株的形态特征观察和生理生化特性鉴定在德国微生物保藏中心（DSMZ)完成。 16S rDNA序列鉴定和比对参照文献" Altschul, S. R; Gish, W.; Miller, W.; Myers, E. W.; Lipman, D. J. Basic local alignment search tool. J. MoL BioL 1990, 215, 403~410，，提供的方法。具体结果如 下：
本实施例得到两株以 为碳源^^ L-乳酸的菌株。
雕的两株菌均为革兰氏阳性菌，细胞直杆，营养细胞的菌体大小为 (0.8〜0.9)μιη x(3.0〜 5.0) μιη。形成内生孢子。在含有木糖、酵母粉和蛋白胨的琼脂平板上 XZL4形成表面光滑、 乳白色、边缘整齐的圆形菌落； XZL9形成细小、淡乳白色、边缘整齐的圆形菌落。具体的生 理生化特征如表 1和表 2所示。
表 1 XZL4生理生化特性
检测指标 XZL4 XZL9 厌氧生长 + +
60 生长 + +
65 生长 - -
V.P. + +
pH in V.P. 4.6 4.5
利用葡萄糖、木糖、 阿拉伯糖、 果糖产酸 + +
利用甘露醇产酸 - - 葡萄糖发酵产气 - - 代 »ff檬酸、 丙酸 ― ―
降解酪氨酸 - - , 苯丙氨酸脱氨酶 - - 卵磷脂酶 ― -
Arginindihydrolase 一 - 还原硝酸盐 + - 形成吲哚 - 一 明胶液化 - - 水解酪蛋白 +
水解淀粉、 七叶苷 + +
水解 Tween 80 - 一 同型发酵产生 L-乳酸 + +
表中结果" +"表示结果为阳性， "- "表示结果为阴性， "w"表示菌株弱生长， "V.P."为 Voges proskauer縮写。
通过数据库 TSBA40 4.10 Library 检索， XZL4 的脂肪酸组成与凝结芽孢杆菌 ίΒ. coagulans^具魏髙的相似性（0.12)。 该菌的 16S rDNA 的核苷酸序列 (见 SEQ ID NO: 1)不 同于所有已报导或者提交到公共数据库的菌株 16S rDNA, 与芽抱杆菌 {B. coagulans^的同源 性最髙 (99%), 说明该菌是一个全新的菌株。 基于以上特征， 结合 16S rDNA诊断区分析， 将 本株 L-乳酸生产菌鉴定为凝结芽孢杆菌 dR coagulans XZL4, 并将其雜于德国微生物保藏 中心， 号为 DSM No. 23183。
表 2 XZL9 生理生化特性
Figure imgf000008_0001
水解酷蛋白 + w
水解淀粉、 七叶苷 + +
水解 Tween 80 - 一
同型发酵产生 L-乳酸 + + 表中结果" +"表示结果为阳性， "-"表示结果为阴性， "w"表示菌株弱生长， "V.P."为 Voges proskauer縮写
通过数据库 TSBA40 4.10 Library检索， XZL9 的脂肪酸组成与凝结芽抱杆菌 coagulans^具有较髙的相似性（0.69)。该菌的 16S rDNA的核苷酸序列 (见 SEQ ID NO: 2)不 同于所有已报导或者提交到公共数据库的菌株 16S rDNA, 与芽孢杆菌 (B. coagu!ans) NRIC 1526的同源性最高 (99%)， 说明该菌是一个全新的菌株。基于以上特征， 结合 16S rDNA诊断 区分析，将本株 L-乳酸生产菌鉴定为凝结芽孢杆菌 (R coagulans) XZL9,并将其保藏于德国 微生物城中心， 職号为 DSM No. 23184.
进一歩比较和分析了凝结芽孢杆菌（A coagulans^XUZA和 XLZ9以及 2-6和 36D1的 16S rDNA序列和在进化地位上的差异（图 1)。从进化地位的角度来说， 2-6， XLZ4和 XLZ9与标 准菌株 ATCC7050归为同一分支， 但 36D1则位于另一分支， 这说明 36D1与 2-6， XLZ4和 XLZ9可能是从不同的祖先进化而来。凝结芽孢杆菌 ( coagulans-)XLZ9的 16S rDNA与 36D1 在第 830, 890, 1262， 1267, 1268, 1286, 1308， 1315， 1344, 1345, 1362位存在碱基差异， 与 2-6在第 340， 1212, 1225, 1276, 1309, 1345， 1346位与相对应的位置存在不同（位置以 XLZ9为准其余取对应位置）。凝结芽孢杆菌 iB. coagulans) XLZ4与 36D1在 340, 830, 890, 1214， 1225, 1264, 1268， 1269, 1275, 1316, 1345, 1346位存在不同， 与 2·6在第 3位存在 差别（位置以 XLZ4为准其余取对应位置)。凝结芽孢杆菌（A g«/fl w) XLZ9与 XLZ4在 第 1， 340, 1212， 1224， 1274, 1374, 1375位存在不同（位置以 XLZ9为准其余取对应位置）。
凝结芽孢杆菌 iB. coagulans) XZL4 DSM No. 23183和 XZL9 DSM No. 23184均通过 山东某 3¾芯加工厂附近的土壤中分离得到。
以下实施例涉及的利用凝结芽孢杆菌 (B. coagulans") ZL4 DSM No. 23183和 XZL9 DSM No. 23184发酵生产 L-乳酸方法的步骤如下：
( 1 )斜面培养：将凝结芽孢杆菌 iB. coagulans') XZL4 DSM No. 23183和 XZL9 DSM No. 23184菌种接种于含有 15 g/L琼脂的固体斜面培养基上， 50〜60Ό条件下，培养 24〜48小时。
(2)种子培养： 将步骤（1 )的斜面培养物， 在无菌条件下接种到 30 ml种子培养基中，
50〜601C条件下， 静止培养 10〜24小时， 加入中和剂控制发酵液 pH, 制得种子培养液。
(3)发酵培养： 以 10%体积比的接种量， 将种子培养液接种于发酵培养基中， 50〜60 条件下培养 48〜72小时。
其中， 步骤（1)、 （2)、 （3) 中雕的菌体培 优选是 50~SSO。
其中， 步骤（2)、 (3) 的培养过程中加入的中和剂为碳酸钙， 控制 pH。
发酵培养过程中， 每 3个小时取发酵液， 先加热至 80〜100 , 再 6,000转 /分钟离心 5分钟， 取上清液检测发酵液中 L-乳酸浓度、 D-乳酸浓度、 葡萄糖浓度， ΤΜ|浓度， 计算糖酸 转化率、 L-乳酸发酵生产能力和 L-乳酸光学 ^。
其中， 总还原糖的测定方法为 DNS法。其中， 葡萄糖的测定方法为， 发酵液稀释后离心， 采用生物传感分析仪 SBA-40C (山东省科学院）测定。 生物传感分析仪 SBA-80是以固定化酶 为传感器的分析仪器， 葡萄糖与氧气、水在酶的催化下生成双氧水。反应放出的双氧水与白金 —银电极接触， 并产生电流信号， 该电流信号与葡萄糖浓度成线性比例关系， 通过测定电流信 号强度可得出葡萄糖浓度。
木糖测定方法为使用木糖测定试剂盒（南京建成科技有限公司）。
L-乳酸和 D-乳酸产量（发酵液乳酸含量或者浓度， g/L)的测定方法为， m Agilent 1100 液相色谱仪，配备手性分离柱（日本三菱化学公司， MCI GEL-CRS10 W(3 μ ) 4.6 ID X 50 mm, 光学异体分离用）。 具体操作条件为： 0.002 mol/L硫酸铜作为流动相， 流量 0.5 ml/min， 进样 量 20 H L, 紫外检测器， 检测波长 254 nm, 操作 25 。 利用 L-乳酸和 D-乳酸标准品做 出标准曲线， 再根据标准曲线计算出发酵液中 L-乳酸和 D-乳酸的含量。
本发明中， 作为标准品的 D-乳酸为德国 Sigma-Aldrich 公司的产品， 其货号为 L0625-25MG;作为标准品的 L-乳酸为德国 Sigma-Aldrich公司的产品，其货号为 L175( 0G。 在如上色谱条件下， D-乳酸保留时间为 10.150分钟。
光学纯度 (optical purity)是衡量旋光性样品中一个对映体超过另一个对映体的量的量度, 它可用对映体过量值 (enantiomeric excess, ee)表示。 本发明中 L-乳酸的光学纯度（ee)按 以下公式计算： （(L-乳酸产量— D-乳酸产量) ÷(L-乳酸产量 +D-乳酸产量)） X 100%。
糖酸转化率定义为 (g/g): (L-乳酸产量 (g/L)÷底物消耗量（g/L， 葡萄糖或者木糖或者总 糖的消耗量 (g/L)) X 100%。
L-乳酸发酵^能力 （g/L/h )为： L-乳酸产量（g/L ) ÷ 发酵时间 （h)。
下面通过实施例进一步阐述本发明。 实施例 1
利用凝结芽孢杆菌 (B. coagulans XZL4 DSM No. 23183和 XZL9 DSM No. 23184在三角 瓶中以葡萄糖为碳源分批发酵生产 L-乳酸
本实施例中所使用的各培养基的组成如下： 斜面培养基每升中含有： 木糖 30 g, 酵母粉 10 g, CaCO3 10 g, 琼脂粉 15 g, 余量为水。 所 面培养基的 pH为 6.5。 115Ό灭菌 20分钟。
种子培养&«升中含有： 葡萄糖 50 g,酵母粉 10 g， CaCO3 20 g, 余量为水。所述种子培 养基的 pH为 6.5。 115Ό灭菌 20 。
发酵培养鶴升中含有： 葡萄糖 55〜150 g, 酵母粉 10 g， CaC03 60 g, 余量为水； 所述 发酵培养基的 pH为 5.5〜7。 115Ό条件下灭菌 20分钟。
本实施例所述发酵生产 L-乳酸的方法包括以下步骤：
(1)斜面培养：将凝结芽孢杆菌 R coagulans) XZL4 DSM No. 23183和 XZL9 DSM No. 23184分别接种于斜面培养基上， 50 培养 24小时；
(2)种子培养： 将步骤（1)培养的菌株' 在无菌条件下用接种环接 2环于装有 30 ml种 子培养基的 100 ml三角瓶中， 50 静止培养 20小时， 制得种子培养液；
(3)发酵培养：将 10 ml步骤（2)制得的种子培养液接入装有 90 ml发酵培养基的 300 ml 三角瓶中， 50 静止培养， 当葡萄糖和 L-乳酸含量保持稳定时， 结束发酵。
发酵结束后，根据上述具体实施方式中所述的检测和计算方法，检测发酵液中 L-乳酸浓度 和葡萄糖浓度， 计算 L-乳酸生产速率。
该实验共设 3次重复， 结果见表 3。
表 3以葡萄糖为碳源 L-乳酸的生成情况
Figure imgf000011_0001
实施例 2
利用凝结芽孢杆菌 (R coagulans) XZL4 DSM No. 23183和 XZL9 DSM No. 23184在三角 瓶中以^ ¾为碳源分批发酵^ L-乳酸：
本实施例中所使用的各培养基的组成如下：
斜面培养基和种子培养基同实施例 1.
发酵培养¾«升中含有： « 55-100 g,酵母粉 10 g， CaCO3 60 g,余量为水； 发酵 培养基的 pH为 5.5〜7。 115Ό条件下灭菌 20分钟。 该发酵生产 L-乳酸的方法包括以下步骤：
(1 )斜面培养： 同实施例 1 ;
(2)种子培养： 同实施例 1 ;
(3)发酵培养： 同实施例 1。
当木糖和 L-乳酸含量保持稳定时，发酵结束，根据上述具体实施方式中所述的检测和计算 方法， 检测发酵液中 L-乳酸浓度和木糖浓度' 计算 L-乳酸生产速率。该实验共设 3次重复， 结果见表 4。
表 4以 Τ «为碳源 L-乳酸的生成情况
Figure imgf000012_0001
实施例 3
利用凝结芽孢杆菌 (B. coagulans) XZL4 DSM No. 23183和 XZL9 DSM No. 23184在三角 瓶中以木糖醇生产副产物为碳源分批发酵生产 L-乳酸：
斜面培养基和种子培养基同实施例 1。
发酵培养¾«升中含有： 木糖醇生产副产物 7S~150 g，酵母粉 10 g， CaCO3 60 g,余量为 水； 雕发酵培养基的 pH为 5.5〜7。 115 条件下灭菌 20分钟。
该发酵生产 L-乳酸的方法包括以下歩骤：
( 1 )斜面培养： 同实施例 1 ;
(2)种子培养： 同实施例 1 ;
(3)发酵培养：将 10ml歩骤（2)制得的种子培养液接入装有 90 ml发酵培养基的 300 ml 三角瓶中， 50Ό静止培养 48小时， 结束发酵。
发酵结束后，根据上述具体实施方式中所述的检测和计算方法，检测发酵液中 L-乳酸浓度 和总还原糖浓度， 计算 L-乳酸生产速率。
该实验共设 3次重复， 结果见表 5。
表 5以^醇^副产物为碳源 L-乳酸的生成情况
Figure imgf000012_0002
(g/L )
75 31 ±1 0.41 0.7 33士 1 0.44 0.7
100 35±2 0.35 0.7 42 ± 1 0.42 0.9
125 50士 2 0.40 1.0 45 ±2 0.36 0.9
150 54 ±3 0.36 1.1 45 ±2 0.30 0.9 实施例 4
利用凝结芽孢杆菌 B. coagulans) XZL4 DSM No. 23183和 XZL9 DSM No. 23184在三角 瓶中以木糖醇生产副产物为碳源， 55Ό分批发酵生产 L-乳酸
本实施例中所使用的各培养基的组成如下：
斜面培养基和种子培养基同实施例 1。
发酵培养基每升中含有：木糖醇生产副产物 150 g,酵母粉 10 g， CaCO3 100 ,余量为水; 发酵培养基的 pH为 5.5〜7。 115 条件下灭菌 20分钟。
该发酵生产 L-乳酸的方法包括以下步骤：
( 1 )斜面培养： 同实施例 1。
(2)种子培养： 同实施例 1。
(3)发酵培养：将 10 ml步骤 (2)制得的种子培养液接入装有 90 ml发酵培养基的 300 ml 三角瓶中， 55 静止培养 48小时， 结束发酵。
发酵结束后，根据上述具体实施方式中所述的检测和计算方法，检测发酵液中 L-乳酸浓度 和总还原糖浓度， 计算 L-乳酸生产速率。
该实验共设 3次重复-结果表明： XZL4 DSM No. 23183 & L-乳酸浓度为 57±3 g/L, L-乳酸生产速率为 1.19 g/L/h; XZL9 DSM No. 23184生产 L-乳酸浓度为 53±2 g/L, L-乳酸生 产速率为 1.10 g/L/h.
实施例 5
利用凝结芽孢杆菌 CB. coagulans XZL4 DSM No. 23183和 XZL9 DSM No. 23184在三角 瓶中以木糖醇生产副产物为碳源， 60Ό分批发酵生产 L-乳酸
本实施例中所使用的各培养基的组成如下：
斜面培养基和种子培养基同实施例 1。
发酵培养基每升中含有： 木糖醇生产副产物 200 g， 酵母粉 5 g, CaC03 100 g, 余量为水; 所述发酵培养基的 pH为 5.5〜7。 115Ό条件下灭菌 20分钟。
该发酵生产 L-乳酸的方法包括以下步骤：
(1 )斜面培养： 同实施例 1 ;
(2)种子培养： 同实施例 1； (3)发酵培养：将 10 ml步骤（2 )制得的种子培养液接入装有 90 ml发酵培养基的 300 ml 三角瓶中， 60Ό静止培养 48小时， 结束发酵。
发酵结束后，根据 具体实施方式中所述的检测和计算方法，检测发酵液中 L-乳酸浓度 和总还原糖浓度， 计算 L-乳酸生产速率.
该实验共设 3次重复。 结果表明： XZL4 DSM No. 23183生产 L-乳酸浓度为 48 ±6 g/L, L-乳酸生产速率为 1.0 g/L/h; XZL9 DSM No. 23184生产 L-乳酸浓度为 44±2 g/L, L-乳酸生 产速率为 0.92 gfLlh
实施例 ό
利用凝结芽孢杆菌 CR coagulans) XZL4 DSM No. 23183和 XZL9 DSM No. 23184在 50 升全自动发酵罐中以 100 g/L葡萄糖为碳源， 补料（糖）流加发酵生产 L-乳酸
本实施例中所使用的各培养基的组成如下：
斜面培养基和种子培养基同实施例 1。
发酵培养¾«升中含有： 葡萄糖 100 g， 酵母粉 12 g， CaCO3 100 , 余量为水； 发酵 培养基的 pH为 5.5〜7。 115 条件下灭菌 20分钟。
该发酵生产 L-乳酸的方法包括以下步骤：
( 1 )斜面培养： 同实施例 1 ;
(2)种子培养： 将步骤（1 )培养的菌株在无菌条件下用接种环接 2环于装有 30 ml种子 培养基的 100 ml三角瓶中， 50Ό静止培养 20小时，制得种子培养液 1 ;将 30 ml种子培养液 1 在无菌条件下接入装有 300 ml种子培养基的 500 ml三角瓶中， 50 静止培养 20小时，制得种 子培养液 2; 继续以相同方式扩大种子培养，得到 4升种子培养液 3。
(3) 发酵培养： 将 4升步骤（2)制得的种子培养液 3在无菌条件下接入装有 36升发酵 培养基的 50升全自动发酵罐（上海保兴 BIOTECH) 中， S0*C静止培养。 每 3个小时取样一 次， 测定发酵液中的残糖量和 L-乳酸浓度， 当葡萄糖浓度降到 20〜30 g/L时， 流加葡萄糖， 使葡萄糖浓度达到 50〜70 g L， 共计补糖 2次。 当发酵过程中总糖消耗速率趋于平稳， 结束发 酵。
发酵结束后，根据上述具体实施方式中所述的检测和计算方法，检测发酵液中 L-乳酸浓度 和残留葡萄糖浓度， 计算 L-乳酸生产速率。
该发酵实验共设 3次重复。结果表明： XZL4 DSM No. 23183生产 L-乳酸浓度为 173 ±3 g/L， 发酵时间为 72小时， L-乳酸生产速率为 2.40 g/L/h, 糖酸转化率为 0.98， 光学纯度为 99.2%; XZL9 DSM No. 23184生产 L-乳酸浓度为 171 ±5 g/L, 发酵时间为 72小时， L-乳酸生产速率 为 2.38 g/L/h， 糖酸转化率为 0.96， 光学纯度为 99.3%。 实施例 7
利用凝结芽抱杆菌 (B. coagulans} XZL4 DSM No. 23183和 XZL9 DSM No. 23184在 50 升全自动发酵罐中以 100 g L的木糖为碳源， 补料（糖）流加发酵生产 L-乳酸
本实施例中所使用的各培养基的组成如下：
斜面培养基和种子培养基同实施例 1。
发酵培养¾«升中含有： 木糖 100 g, 酵母粉 12 g， CaCO3 100 g, 余量为水； 所述发酵培 养基的 pH为 5.5〜7。 115Ό条件下灭菌 20分钟。
该发酵生产 L-乳酸的方法包括以下步骤：
( 1 )斜面培养： 同实施例 1;
(2)种子培养： 同实施例 6；
(3)发酵培养： 将 4升步骤（2)制得的种子培养液 3在无菌条件下接入装有 36升发酵 培养基的 50升全自动发酵罐（上海保兴 BIOTECH) 中， 50Ό静止培养。 每 3个小时取样一 次， 测定发酵液中的残糖量和 L-乳酸浓度， 当木糖浓度降到 20〜30 g/L时， 流加木糖， 使木 糖浓度达到 50〜70 g/L， 共计补糖 2次。 当发酵过程中木糖消耗速率趋于平稳， 结束发酵。
发酵结束后，根据 具体实施方式中所述的检测和计算方法，检测发酵液中 L-乳酸浓度 和残留木糖浓度， 计算 L-乳酸生产速率。
该实验共设 3次重复。 结果表明： XZL4 DSM No. 23183生产 L-乳酸浓度为 195士 1 g/L， L-乳酸生产速率为 2.70 g L/h, 糖酸转化率为 0.98, 光学纯度为 99.3%; XZL9 DSM No. 23184 生产 L-乳酸浓度为 186±4 g/L，发酵时间为 72小时， L-乳酸生产速率为 2.58 g/L/h, 糖酸转化 率为 0.97， 光学^ g为 99.4%,
实施例 8
利用凝结芽孢杆菌 (B. coagulans) XZL4 DSM No. 23183和 XZL9 DSM No. 23184在 50 升全自动发酵罐中以 100 g/L木糖醇生产副产物为碳源， 补料（糖）流加发酵生产 L-乳酸 本实施例中所使用的各培养基的组成如下：
斜面培养基和种子培养基同实施例 1。
发酵培养麟升中含有：木糖醇生产副产物 100 g,酵母粉 12 g, CaC03 100 g,氯化钠 0.1 g， 磷酸二氢钾 0.5 g，硫酸镁 0.2 g， 余量为水； 所述发酵培养基的 pH为 5.5〜7。 115Ό条件下 灭菌 20分钟。
该发酵生产 L-乳酸的方法包括以下歩骤：
(1)斜面培养： 同实施例 1 :
(2)种子培养： 同实施例 6； (3)发酵培养： 将 4升步骤（2 )制得的种子培养液 3在无菌条件下接入装有 36升发酵 培养基的 50升全自动发酵罐（上海保兴 BIOTECH) 中， 50Ό静止培养。 每 3个小时取样一 次， 测定发酵液中的残糖量和 L-乳酸浓度， 当总糖浓度降到 20〜30 g/L时， 流加木糖醇生产 副产物， 使底物浓度达到 50〜70 L， 共计补糖 2次。 当发酵过程中总糖消耗速率趋于平稳， 结束发酵《
发酵结束后，根据上述具体实施方式中所述的检测和计算方法，检测发酵液中 L-乳酸浓度 和总还原糖浓度， 计算 L-乳酸^速率。
该实验共设 3次重复， 发酵时间为 48小时。 结果表明： XZL4 DSM NO. 23183生产 L-乳 酸浓度为 95±2 g/L, L-乳酸生产速率为 1.98 g/L/h，光学 ^为 99.1%; XZL9 DSM No. 23184 生产 L-乳酸浓度为 92±5 g/L， L-乳酸生产速率为 1.92 g/L/h, 光学纯度为 99.3%,
实施例 9 利用凝结芽孢杆菌 (R coagulans) XZL4 DSM No. 23183和 XZL9 DSM No. 23184在 50升全自动发酵罐中以 200 g L木糖醇生产副产物为碳源， 补料（糖）流加发酵生产 L-乳酸
本实施例中所使用的各培养基的组成如下：
斜面培养基和种子培养基同实施例 1。
发酵培养»«升中含有：木糖醇生产副产物 200 g,酵母粉 12 g， CaCO3 100 ,氯化钠 0.1 g, 磷酸二氢钾 0.5 g,硫酸镁 0.2 g， 余量为水； 所述发酵培养基的 pH为 5.5〜7。 115 条件下 灭菌 20分钟。
该发酵生产 L-乳酸的方法包括以下步骤：
(1 )斜面培养： 同实施例 1 ;
(2)种子培养： 同实施例 6;
(3)发酵培养： 同实施例 8。
发酵结束后，根据上述具体实施方式中所述的检测和计算方法，检测发酵液中 L-乳酸浓度 和总还原糖浓度， 计算 L-乳酸生产速率。
该实验共设 3次重复， 发酵时间为 72小时。 结果表明： XZL4 DSM No. 23183生产 L-乳 酸浓度为 98±5 g L， L-乳酸^速率为 1.36 g/L/h， 光学纯度为 99.2%; XZL9 DSM No. 23184 生产 L-乳酸浓度为 94±5 g/L, L-乳酸生产速率为 1.31 g/L/h， 光学纯度为 99.1%。
实施例 10 利用凝结芽孢杆菌 US. coagulans) XZL4 DSM No. 23183和 XZL9 DSM No. 23184在 50升全自动发酵罐中以 150 g/L木糖醇生产副产物为碳源， 补料（糖）流加发酵生产 L-乳酸
本实施例中所使用的各培养基的组成如下: 斜面培养基和种子培养基同实施例 1。
发酵培养基： 醇^副产物 150g,酵母粉 12 g, CaCO3 100 g,氯化钠 0.1 g,磷酸二 氢钾 0.5 g，硫酸镁 0.2 g, 余量为水； 所述发酵培养基的 pH为 5.5〜7, 115Ό条件下灭菌 20 分钟》
该发酵生产 L-乳酸的方法包括以下步骤：
(1)斜面培养： 同实施例 1；
(2)种子培养： 同实施例 6;
(3)发酵培养： 同实施例 8。
发酵结束后，根据 具体实施方式中所述的检测和计算方法，检测发酵液中 L-乳酸浓度 和总还原糖浓度， 计算 L-乳酸^速率。
该实验共设 3次重复， 发酵时间为 51小时。结果表明： XZL4 DSM NO. 23183生产 L-乳 酸浓度为 106±3 g/L, L-乳酸生产速率为 2.08 g/L/h,光学纯度为 99.1%; XZL9 DSM No. 23184 生产 L-乳酸浓度为 10fti6 g/L, L-乳酸生产速率为 1.96 g/L/h, 光学纯度为 99.3%。

Claims

权 利 要 求 书
1、一种用于制备 L-乳酸的凝结芽孢杆菌,其特征在于，具体为凝结芽孢杆菌 ( coagulans XZL4和 XZL9, 其中：
繊的凝结芽孢杆菌 R coagulans) XZL4 DSM No. 23183为革兰氏阳性菌，其 16S rR A 基因的核苷酸序列如 Seq. ID No.l所示；
¾E的凝结芽孢杆菌 CR coagulans XZL9 DSM No. 23184为革兰氏阳性菌，其 16S rRNA 基因的核苷酸序列如 Seq. ID No.2所示。
2、一种根据权利要求 1所述的用于制备 L-乳酸的凝结芽孢杆菌的应用方法，其特征在于， 以含有五碳糖或六碳糖或其组合的农业和工业产品和废弃物作为发酵培养基的碳源，进行发酵 后得到 L-乳酸。
3、根据权利要求 2所述的用于制备 L-乳酸的凝结芽孢杆菌的应用方法， 其特征是， 所述 的发酵是指采用发酵培养基按 10%体积比的接种量在 50 〜60 环境下培养 48〜72小时。
4、根据权利要求 2或 3所述的用于制备 L-乳酸的凝结芽孢杆菌的应用方法， 其特征是， 所述的发酵中包含补料流加工艺，该补料流加工艺是指：当发酵液中总还原糖含量低于 20〜30 g/L时补加碳源， 使总还原糖含量维持在 30-70 g L，或达到 50〜70 g/L。
5、根据权利要求 2所述的用于制备 L-乳酸的凝结芽孢杆菌的应用方法， 其特征是， 所述 的发酵培养基的组分及其含量为：碳源 40〜200 g/L、氮源 S〜12 g/L和用于调控培养基 pH的 中和剂 50〜： 100 g/L, 余量为水。
6、根据权利要求 2所述的用于制备 L-乳酸的凝结芽孢杆菌的应用方法， 其特征是， 所述 的碳源为以五碳糖或六碳糖或其组合为有效组分的葡萄糖、木糖或农业和工业废弃物， 其中： 葡萄糖浓度为 40〜150 g/L,木糖浓度为 40〜100 g/L ,木糖醇生产副产物浓度为 100〜200 g/L。
7、根据权利要求 2所述的用于制备 L-乳酸的凝结芽孢杆菌的应用方法， 其特征是， m. 的芽孢杆菌的种子培养液是指： 将芽孢杆菌 iB. coagulans XZL4 DSM No. 23183和 XZL9 DSM No. 23184依次进行斜面培养和种子培养后获得的培养物。
8、 根据权利要求 7所述的用于制备 L-乳酸的凝结芽孢杆菌的应用方法， 其特征是, 所述 的斜面培养是指： 将凝结芽孢杆菌 CB. coagulans') XZL4 DSM No. 23183和 XZL9 DSM No. 23184菌种接种于含有 15 g/L琼脂的固体斜面培养基上， 50〜60 条件下， 培养 24〜48小时。
9、 根据权利要求 7 的用于制备 L-乳酸的凝结芽孢杆菌的应用方法， 其特征是， ff 的种子培养是指： 将经过斜面培养的芽孢杆菌在无菌条件下接种到 30ml种子培养基中， 50〜 60Ό条件下， 静止培养 10〜24小时， 加入中和剂控制发酵液 pH, 制得种子培养液。
10、根据权利要求 9所述的用于制备 L-乳酸的凝结芽孢杆菌的应用方法，其特征是，所述 的种子培养基每升中含有： 葡萄糖 40〜60 g， 酵母粉 5〜10g， CaCO3 20〜40 g， 余量为水， 优 选含有：葡萄糖 50 g/L,酵母粉 10 g/L, CaC03 20 g/L,余量为水；该种子培养基的 pH为 6.5。
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