WO2010143323A1 - Novel d-lactic acid-producing strains and method for producing d-lactic acid - Google Patents
Novel d-lactic acid-producing strains and method for producing d-lactic acid Download PDFInfo
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- WO2010143323A1 WO2010143323A1 PCT/JP2009/070410 JP2009070410W WO2010143323A1 WO 2010143323 A1 WO2010143323 A1 WO 2010143323A1 JP 2009070410 W JP2009070410 W JP 2009070410W WO 2010143323 A1 WO2010143323 A1 WO 2010143323A1
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- lactic acid
- strain
- glucose
- lactobacillus
- nucleotide sequence
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- JVTAAEKCZFNVCJ-UWTATZPHSA-N D-lactic acid Chemical compound C[C@@H](O)C(O)=O JVTAAEKCZFNVCJ-UWTATZPHSA-N 0.000 title claims abstract description 122
- 229940022769 d- lactic acid Drugs 0.000 title claims abstract description 122
- 229930182843 D-Lactic acid Natural products 0.000 title claims abstract description 120
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 41
- 230000003287 optical effect Effects 0.000 claims abstract description 29
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 132
- 239000004310 lactic acid Substances 0.000 claims description 66
- 235000014655 lactic acid Nutrition 0.000 claims description 66
- 241000894006 Bacteria Species 0.000 claims description 52
- 239000008103 glucose Substances 0.000 claims description 48
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 46
- 241000186660 Lactobacillus Species 0.000 claims description 46
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- 239000002773 nucleotide Substances 0.000 claims description 24
- 125000003729 nucleotide group Chemical group 0.000 claims description 24
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- 230000004151 fermentation Effects 0.000 claims description 12
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 9
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 8
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 8
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- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 5
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- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 claims description 4
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 4
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Images
Classifications
-
- 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
- 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
- C12R2001/225—Lactobacillus
Definitions
- the present invention relates to a novel D-lactic acid-producing bacterium, and in particular, to a Lactobacillus delbrueckii QU41 strain. Furthermore, the present invention relates to a method for producing D-lactic acid using the D-lactic acid-producing bacterium.
- poly L-lactic acid a biodegradable plastic made from plant biomass
- Poly-L-lactic acid has the same advantage as plastics, which have a tensile strength and the like, and has the advantages of being transparent while being crystalline, and thus can be used as an alternative to polypropylene and polystyrene.
- poly-L-lactic acid its properties such as strength and heat resistance depend greatly on the optical purity of the L-lactic acid monomer, which is a raw material. It becomes.
- poly L-lactic acid has the advantages of high strength and biodegradability, but its low heat resistance of 170 ° C. hinders its widespread use.
- a stereocomplex polylactic acid that can blend poly L-lactic acid and poly D-lactic acid and improve heat resistance up to 220 ° C. has attracted attention.
- Lactic acid bacteria are gram-positive cocci or bacilli and are defined as bacteria that are catalase negative, assimilate saccharides, produce lactic acid in a yield of 50% or more, and do not form spores.
- JP-A-62-44188 reports that D-lactic acid is produced using Sport Lactobacillus inulinus ATCC 15538 strain.
- the Sport Lactobacillus inulinus ATCC 15538 strain was cultured at 37 ° C. for 40 hours in the presence of 100 g / L glucose, resulting in an optical purity of about 99.5%. Yields 101 g / L of D-lactic acid.
- JP-A-2-76592 reports that L-lactic acid is produced using Lactobacillus lactis ATCC 12314 strain. Lactobacillus lactis ATCC 12314 strain produces 123.3 g / L D-lactic acid by culturing at 40 ° C. for 45 hours in a medium neutralized with calcium carbonate using a amylase degradation product of wheat starch as a carbon source. To do.
- JP 2007-215427 A it is reported that D-lactic acid is produced using Lactobacillus delbricki IFO 3202 strain. Lactobacillus delbricki IFO 3202 strain in a medium at pH 7 produces 106.9 g / L D-lactic acid by culturing at 37 ° C. for 96 hours in the presence of 100 g / L glucose.
- Ammonia is added to control pH after 24 hours from the start of culture. Therefore, in the method of Japanese Patent Application Laid-Open No. 2007-215427, two-stage culture is performed, which includes a first stage in which pH adjustment is not performed and a second stage in which pH adjustment is performed.
- An object of the present invention is to provide a novel D-lactic acid-producing bacterium and a method for efficiently producing high optical purity D-lactic acid using the D-lactic acid-producing bacterium in order to obtain D-lactic acid. To do.
- the present inventors have isolated and obtained a novel D-lactic acid-producing bacterium, in particular, Lactobacillus delbricki QUI41 strain, and completed the present invention. Highly pure D-lactic acid can be efficiently produced using the D-lactic acid-producing bacterium of the present invention.
- the present invention provides the following: 1) (1) D-lactic acid can be produced with an optical purity of 99% or more using glucose as a substrate, And (2) when the glucose concentration starts at 20 g / L and pH 6.0 and is cultured at a temperature of 43 ° C. for 12 hours, it is 12.0 g / L or more, preferably 16.0 g / L or more, more preferably Can produce 20.0 g / L or more of D-lactic acid, Lactic acid bacteria belonging to Lactobacillus del Bricky. 2) Furthermore, (3) The lactic acid bacterium according to 1), which can produce D-lactic acid at a temperature of 49 ° C. 3) Lactic acid bacteria according to 1) or 2) having the following mycological properties: 1.
- D-lactic acid is produced by homolactic fermentation using glucose as a substrate, and Glucose, fructose, mannose, N-acetylglucosamine, maltose, lactose, sucrose and trehalose can be assimilated, and xylose and arabinose cannot be assimilated.
- the lactic acid bacterium according to any one of 1) to 4 which is a Lactobacillus delbricki QUA41 strain deposited under the NITE BP-679 accession number.
- a method for producing lactic acid comprising the step of culturing the lactic acid bacterium according to any one of 1) to 5).
- the production method according to 6 wherein the lactic acid contains D-lactic acid with an optical purity of 99% or more.
- Lactobacillus delbricky QU41 strain, QU42 strain, and QU43 strain which are novel D-lactic acid producing bacteria, are provided.
- the microbial strain of the present invention enables efficient production of D-lactic acid.
- FIG. 1 is a view showing the nucleotide sequence of 16S rRNA gene of QUI41 strain.
- FIG. 2 is a view showing the nucleotide sequence of the 16S rRNA gene of the QU42 strain.
- FIG. 3 is a diagram showing the nucleotide sequence of the 16S rRNA gene of QUI41 strain.
- FIG. 4 is a graph showing the results of test tube culture of Lactobacillus delbricky QU41 strain, QU42 strain, and QU43 strain.
- FIG. 5 is a graph showing the influence of the culture temperature on the D-lactic acid producing ability of the QUI41 strain.
- FIG. 6 is a graph showing the effect of culture pH on the D-lactic acid producing ability of the QUI41 strain.
- FIG. 7 is a graph showing the effect of the initial glucose concentration on the D-lactic acid producing ability of the QUI41 strain.
- FIG. 8 is a graph comparing the D-lactic acid producing ability in the QUI41 strain, the JCM1166 strain, and the JCM1246 strain.
- FIG. 9 is a graph comparing the influence of the neutralizing agent of the medium between when NaOH is used and when NH 4 OH is used.
- the lactic acid bacteria of the present invention have an initial glucose concentration of 20 g / L, pH 6.0, and glucose as a substrate at an optimal temperature (specifically, 43 ° C.) for 8 to 12 hours. When cultured, 12.0 g / L or more of D-lactic acid can be produced with an optical purity of 99% or more. Lactic acid bacteria having the above-mentioned properties and genetically belonging to Lactobacillus delbrick are within the scope of the present invention.
- the lactic acid bacterium of the present invention can produce D-lactic acid of preferably 16.0 g / L or more, more preferably 20.0 g / L or more under the above conditions.
- Such lactic acid bacteria are suitable for the purpose of efficiently producing high optical purity D-lactic acid.
- the lactic acid bacterium of the present invention can produce D-lactic acid with an optical purity of 99% or higher, preferably 99.5% or higher, more preferably 99.9% or higher.
- Lactic acid bacteria of the present invention are 10 g of peptone, 8 g of beef extract, 4 g of yeast extract, 20 g of glucose, 1 g of Tween 80, 2 g of K 2 HPO 4 and 5 g of sodium acetate trihydrate in 1 L of distilled water.
- citric acid diammonium hydrogen 2g, the MgSO 4 ⁇ H 2 O 0.2g, MnSO 4 ⁇ nH to 2 O in MRS- glucose medium containing 0.05 g stand at a temperature ranging from at least 30 ° C. of 49 ° C. By culturing, it can be cultured well.
- the medium used for culturing the lactic acid bacterium of the present invention is not limited to the MRS-glucose medium, and the MRS-glucose medium can be modified as appropriate, and the bacterium can grow well and produce D-lactic acid.
- a medium having any composition may be used as long as it is produced.
- Lactobacillus delbrikkii QU41 strain isolated from the drainage channel on the campus of Kyushu University.
- Lactobacillus delbricky QUI41 strain is a novel strain, and it was established as NITE BP-679 in January 2009 at the National Institute of Technology and Evaluation of Microorganisms (2-5-8, Kazusa Kamashichi, Kisarazu City, Chiba Prefecture). Deposited on the 19th.
- Lactobacillus delbricki QP41 The mycological properties of Lactobacillus delbricki QP41 are as follows. 1. Form: Neisseria gonorrhoeae. 2. Biochemical properties: Catalase negative. 3. Motility: None Oxygen demand: It is facultative anaerobic. 5). D-lactic acid is produced by homolactic fermentation using glucose as a substrate. 6). Glucose, fructose, mannose, N-acetylglucosamine, maltose, lactose, sucrose and trehalose can be assimilated, and xylose and arabinose cannot be assimilated.
- the lactic acid bacteria of the present invention also include lactic acid bacteria belonging to Lactobacillus delbricky and having the same bacteriological properties as the QU41 strain.
- suitable strains of the present invention there can be mentioned Lactobacillus delbriquie QU42 strain and Lactobacillus delbrixi QU43 strain.
- Lactobacillus delbricki QU42 strain and QU43 strain are also novel strains and can produce D-lactic acid with high efficiency.
- the strain of the present invention is not limited to Lactobacillus delbrickey QU41 strain, QU42 strain, and QU43 strain, and is capable of producing D-lactic acid with an optical purity of 99% or more using glucose as a substrate, and the glucose concentration. Is within the scope of the present invention as long as it can produce 1-2.0 g / L or more of D-lactic acid when cultured at 43 ° C. for 12 hours under the conditions of 20 g / L and pH 6.0. .
- the nucleotide sequences of the 16S rRNA genes of Lactobacillus delbrikkii QU41 strain, QU42 strain and QU43 strain are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
- Lactobacillus delbrikkii QU41 strain, QU42 strain, and QU43 strain showed high identity.
- the present invention provides a Lactobacillus delbrickey having a 16S rRNA gene comprising a sequence having 99.5% or more identity with the sequence shown in SEQ ID NO: 1 in the table consisting of any of the following sequences: (A) a nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing, or a nucleotide sequence having 99.7% or more identity thereto; (B) a nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing, or a nucleotide sequence having 99.75% or more identity thereto; or (c) a nucleotide sequence represented by SEQ ID NO: 3 in the sequence listing; or A nucleotide sequence having an identity of 99.8% or more.
- the identity of a nucleic acid sequence can be determined using, for example, a widely used program such as BLAST, but is not limited thereto, and the identity of a nucleic acid sequence is determined using another program. You can also
- Lactobacillus delbrikkii strains QU41, QU42 and QU43 are very similar in their genetic and biochemical properties, so these three strains are different strains. However, many properties including the ability to produce D-lactic acid are expected to be similar or identical.
- the lactic acid bacteria of the present invention can grow and produce D-lactic acid at a temperature in the range of at least 30 ° C. to 49 ° C.
- a preferable temperature for growth of the strain and production of D-lactic acid is 37 ° C to 43 ° C, and a particularly preferable temperature is 43 ° C.
- the lactic acid bacterium of the present invention can produce D-lactic acid even at 49 ° C. 49 ° C. is a considerably high temperature for lactic acid bacteria. Lactic acid fermentation at 49 ° C. can reduce contamination by various bacteria, and can reduce the need for cooling by fermentation heat.
- lactic acid can be produced it means that D-lactic acid can be produced at an industrially effective level. It was started under the conditions of a glucose concentration of 20 g / L and pH 6.0, and cultured for 12 hours. Sometimes, if it can produce 1-2.0 g / L or more of D-lactic acid, it can be said that “production is possible”.
- the lactic acid bacterium of the present invention can produce about 100 g / L of D-lactic acid from 100 g / L of glucose even in high-temperature culture at 49 ° C.
- the Lactobacillus delbricky QUI41 strain of the present invention can grow and produce D-lactic acid in a pH range of at least 5.5 to 6.5.
- a particularly preferred pH for the growth of the strain and the production of D-lactic acid is 6.0.
- Lactobacillus delbricky QUI41 of the present invention increases as the initial glucose concentration in the medium increases, and the productivity of D-lactic acid increases.
- a decrease in the maximum cell growth rate accompanying an increase in the initial glucose concentration and a decrease in the lactic acid production rate in the long-term culture occur. For this reason, batch culture is not suitable when D-lactic acid is efficiently produced using the strain.
- a method for producing D-lactic acid and a method for producing lactic acid by culturing the Lactobacillus delbrikkii strain of the present invention are also within the scope of the present invention.
- a medium in which the Lactobacillus delbrickey strain of the present invention can grow and produce lactic acid such as MRS-glucose medium
- the strain is grown under conditions suitable for its growth and lactic acid production (temperature and pH). Lactic acid can be produced by culturing in the above.
- D-lactic acid can be produced with high optical purity.
- D-lactic acid can be produced with an optical purity of 99% or more, preferably 99.5% or more, and more preferably 99.9% or more. it can.
- the concentration of lactic acid obtained by the present invention can be measured by a technique generally known in this technical field.
- the culture solution of the Lactobacillus delbricki strain of the present invention is centrifuged to obtain a supernatant, and the supernatant is filtered through a filter and then subjected to column chromatography (for example, a shodex Sugar S series etc.
- the amount of lactic acid can be measured with an exchange column, and the concentration of produced lactic acid can be measured by detecting lactic acid with a suggestive refractometer.
- the concentration of L-lactic acid can be measured by a biosensor (for example, enzyme electrode type biosensor BF-5), and the optical value of D-lactic acid is determined from the measured L-lactic acid concentration and total lactic acid concentration value. Purity can be calculated.
- the optical purity of D-lactic acid means a value calculated by the following formula, unless otherwise specified.
- the method for producing lactic acid of the present invention is preferably performed by subjecting the Lactobacillus delbrikkii strain of the present invention to pH 5.5 to pH 6.5 at a temperature of 37 ° C. to 43 ° C. for 12 hours in a medium containing glucose. For 72 hours to prepare a culture solution, and a step of centrifuging the culture solution and collecting a supernatant.
- a suitable medium for culturing the Lactobacillus delbrikkii strain of the present invention is not limited thereto, but is preferably the MRS-glucose medium described above. However, the medium used in the method of the present invention is not limited thereto, and can be appropriately modified.
- the conditions such as the pH of the medium, the culture temperature, and the culture time can also be appropriately modified as long as the Lactobacillus delbriqui strain of the present invention can grow and produce D-lactic acid.
- the most preferred strain used in the method of the present invention is Lactobacillus delbrikkii QU41 strain.
- the method of the present invention can also be carried out using Lactobacillus delbricky QU42 strain and QU43 strain.
- D-lactic acid produced by using the method of the present invention can be used as a raw material for producing a stereocomplex of poly-L-lactic acid and poly-D-lactic acid, for example.
- the stereocomplex of poly L-lactic acid and poly D-lactic acid can be a biodegradable plastic having high heat resistance.
- the use of D-lactic acid is not limited to a stereocomplex with poly L-lactic acid.
- D-lactic acid is known to be used as an agricultural intermediate.
- Example 1 Isolation and Identification of New D-Lactic Acid-Producing Bacteria
- MRS-glucose medium and MRS-glucose agar medium from 137 sources such as plants, animals, soil, and insects
- D-lactic acid-producing bacteria were isolated.
- enrichment culture was performed before plating.
- prokaryote preferentially sodium azide and cycloheximide that inhibit the growth of eukaryotes were added to the accumulation medium.
- This enrichment culture medium was prepared by adding MRS-glucose medium having the composition described above, 100 ppm sodium azide, 100 ppm cycloheximide, dissolving in distilled water, adjusting the pH to 5.5 and 6.5, and causing brown reaction. In order to prevent this, autoclaving was performed at 115 ° C. for 15 minutes.
- the MRS-glucose agar medium is 1.5% agar and 0.5% CaCO 2 added to the enrichment culture medium, dissolved in distilled water, adjusted to pH 6.5, and autoclaved at 115 ° C for 15 minutes. After performing, it poured into a sterilization petri dish and produced.
- the culture conditions for isolation are temperatures of 30 ° C., 37 ° C., and 43 ° C., and initial pH values of 5.5 and 6.5. Under these conditions, the bacteria were anaerobically cultured and isolated, and again cultured in the MRS medium. Then, the supernatant was analyzed, and the bacteria producing high optical purity D-lactic acid were isolated by homofermentation.
- the nucleotide sequence of the 16S rRNA gene of the QUI41 strain is shown in SEQ ID NO: 1 in the sequence listing and FIG.
- the nucleotide sequence of the 16S rRNA gene of the QU42 strain is shown in SEQ ID NO: 2 in the sequence listing and FIG.
- the nucleotide sequence of the 16S rRNA gene of the QU43 strain is shown in SEQ ID NO: 3 in the sequence listing and FIG.
- Table 1 shows the results of the saccharide utilization test using the API50CH kit. For comparison, Lactobacillus delbrueckii subsp. Described in the API50CH kit. delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. The saccharide utilization pattern of lactis is also shown in Table 1.
- each number indicates the proportion of strains having the ability to assimilate among strains held by BioMerieux. Even for the same species, Lactobacillus delbrueckii subsp. Some have grouped saccharide utilization properties, such as lactis 1 and 2.
- the QU41 strain, the QU42 strain, and the QU43 strain showed the same sugar fermentability pattern. That is, these strains assimilated glucose, fructose, mannose, N-acetylglucosamine, maltose, lactose, sucrose, and trehalose, but not pentoses such as xylose and arabinose. Lactobacillus delbrueckii ssp lactis was most consistent with the sugar fermentability pattern.
- FIG. 4 (a) shows the results of the study with the QUI41 strain
- FIG. 4 (b) shows the results of the study with the QUI41 strain
- FIG. 4 (c) shows the results of the study with the QUI41 strain.
- the left vertical axis indicates the concentrations of glucose, lactic acid, and acetic acid
- the right axis indicates OD
- the horizontal axis indicates time.
- the black circle is the glucose concentration
- the white circle is the lactic acid concentration
- the white triangle is the OD.
- the three strains of the QU41 strain, the QU42 strain, and the QU43 strain showed similar fermentation behaviors when cultured in the MRS medium.
- the QUI41 strain showed the highest value of D-lactic acid production at 16.8 g / L at 72 hours.
- Lactobacillus delbricky QUI41 strain, QUI42 strain, and QUI43 strain showed very similar fermentation characteristics in terms of yield, production rate, etc.
- the optimum conditions for D-lactic acid production described below were examined.
- Example 2 Examination of Optimal Conditions (1) Examination of Optimal Temperature With regard to QUI41 strain, the ability to produce D-lactic acid at 30 ° C., 37 ° C., 43 ° C., and 49 ° C. was examined using 20 g / L glucose MRS medium. . The pH was adjusted to 5.5 with 10 N NaOH and the experiment was conducted in an anaerobic state by nitrogen gas replacement. The culture was performed for 6 hours on a 40 ml scale and 12 hours on a 400 ml scale for main culture.
- FIG. 5 (a) shows the result of the examination at 30 ° C.
- FIG. 5 (b) shows the result of the examination at 37 ° C.
- FIG. 5 (c) shows the result of the examination at 43 ° C.
- FIG. 5 (d) shows the result of examination at 49 ° C.
- the left vertical axis indicates glucose, lactic acid, and acetic acid concentrations
- the right vertical axis indicates OD
- the horizontal axis indicates time.
- the black circle is the glucose concentration
- the white circle is the lactic acid concentration
- the black triangle is the acetic acid concentration
- the white triangle is the OD.
- Table 3 The values of the culture parameters obtained from this result are shown in Table 3 below.
- Table 3 from the left, values of maximum cell growth rate, D-lactic acid production, maximum yield, maximum production, and D-lactic acid optical purity are shown.
- the highest maximum growth rate and the maximum production rate were exhibited at 43 ° C., and the optimum temperature for growth and D-lactic acid production of the QUI41 strain was 43 ° C.
- the QUI41 strain was able to grow and produce D-lactic acid even at 55 ° C.
- FIG. 6 (a) shows the results of investigation at pH 5.0
- FIG. 6 (b) shows the results of examination at pH 5.5
- FIG. 6 (c) shows the results at pH 6.0
- FIG. 6 (d) shows the results of investigation at pH 6.5.
- the left vertical axis indicates glucose, lactic acid, and acetic acid concentrations
- the right vertical axis indicates OD
- the horizontal axis indicates time.
- the black circle is the glucose concentration
- the white circle is the lactic acid concentration
- the black triangle is the acetic acid concentration
- the white triangle is the OD.
- Table 4 The values of the culture parameters obtained from this result are shown in Table 4 below.
- Table 4 From the left, the maximum cell growth rate, D-lactic acid production amount, maximum yield, maximum production amount, and D-lactic acid optical purity values are shown. Since the cultivation was completed in 12 hours at pH 6.0 and the maximum cell growth rate and lactic acid production were shown, the optimum pH was 6.0.
- Example 3 Examination of Influence of Initial Glucose Concentration Under the conditions of a temperature of 43 ° C. and pH 6.0, the influence of the glucose concentration on the D-lactic acid production ability of QUI41 strain was examined. The experiment was performed under the same conditions as those for the optimum temperature and optimum pH except that the pH was changed.
- FIG. 7 shows the results of investigations with the concentration of glucose added at the start of culture, that is, the initial glucose concentration set to 20, 50, and 100 g / L.
- FIG. 7A shows data with an initial glucose concentration of 20 g / L
- FIG. 7B shows data with an initial glucose concentration of 50 g / L (b)
- FIG. 7C shows the initial glucose concentration.
- the data is 100 g / L.
- the left vertical axis indicates glucose, lactic acid, and acetic acid concentrations
- the right vertical axis indicates OD
- the horizontal axis indicates time.
- the black circle is the glucose concentration
- the white circle is the lactic acid concentration
- the black triangle is the acetic acid concentration
- the white triangle is the OD.
- the values of the culture parameters obtained from this result are shown in Table 5 below.
- Table 5 the maximum cell growth rate, D-lactic acid production amount, maximum yield, maximum production amount, and D-lactic acid optical purity values are shown from the left.
- the maximum cell growth rate decreased and the lactic acid production rate also decreased with the lapse of culture time. From these results, it was found that QUI41 is inhibited by high concentrations of glucose and lactic acid, so that D-lactic acid cannot be efficiently produced by batch culture affected by changes in the medium environment accompanying bacterial growth.
- Example 4 Comparison of Lactobacillus delbrikkii QUA41 strain and existing D-lactic acid producing bacteria Comparison of D-lactic acid production between Lactobacillus delbrikkii QUI41 strain and existing D-lactic acid producing bacteria JCM1166 and JCM1248 It was. The results are shown in FIG. 8A shows data measured at 37 ° C., FIG. 8B shows data measured at 43 ° C., and FIG. 8C shows data measured at 50 ° C. In FIG. 8, the left vertical axis indicates the concentration of lactic acid, the right vertical axis indicates OD, and the horizontal axis indicates time.
- Lactobacillus del Bricky QU41 strain was also measured at 55 ° C. as shown in FIG.
- the right column shows the amount of D-lactic acid produced 24 hours after culturing, and the left column shows the amount of D-lactic acid produced 48 hours after culturing.
- Table 6 shows the values of D-lactic acid production of the three strains obtained from the results.
- Example 5 Examination of Neutralizing Agent for Medium As a neutralizing agent for the medium used for culturing QUA41 strain, a comparison was made between the case of using NaOH and the case of using NH 4 OH. The results are shown in FIG. FIG. 9A shows data when NaOH is used as a neutralizing agent, and FIG. 9B shows data when NH 4 OH is used as a neutralizing agent.
- the left vertical axis indicates the concentration of lactic acid
- the right vertical axis indicates OD
- the horizontal axis indicates time.
- the black circle is the glucose concentration
- the white circle is the lactic acid concentration
- the black triangle is the acetic acid concentration
- the white triangle is the OD.
- Table 7 The values of the culture parameters obtained from this result are shown in Table 7 below.
- Table 7 the maximum cell growth rate, D-lactic acid production amount, maximum yield, maximum production amount, and D-lactic acid optical purity values are shown from the left.
- the results were similar when NH 4 OH was used and when NaOH was used.
- the amount of D-lactic acid produced and the maximum D-lactic acid production rate were higher when NH 4 OH was used than when NaOH was used at 8 hours after culturing.
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Abstract
Disclosed are a novel D-lactic acid-producing strain and a method for producing D-lactic acid by using the D-lactic acid-producing strain.
Lactobacillus delbrueckii strains QU41, QU42 and QU43 that are novel D-lactic acid-producing strains can be provided. These microbial strains enable efficient production of D-lactic acid having a high optical purity.
Description
本発明は新規なＤ－乳酸生産菌に関し、特にラクトバシラス・デルブリッキー（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ）ＱＵ４１株に関する。さらに本発明は、当該Ｄ－乳酸生産菌を用いてＤ－乳酸を生産する方法に関する。
The present invention relates to a novel D-lactic acid-producing bacterium, and in particular, to a Lactobacillus delbrueckii QU41 strain. Furthermore, the present invention relates to a method for producing D-lactic acid using the D-lactic acid-producing bacterium.
現在地球温暖化などの環境問題が深刻化することにより、植物バイオマスを原料とした生分解性プラスチックであるポリＬ－乳酸が注目されている。ポリＬ－乳酸は引っ張り強度などが汎用されているプラスチックと同程度であり、結晶性でありながら透明であるという長所を有するために、ポリプロピレンやポリスチレンなどの代替物としての利用が可能である。ポリＬ－乳酸を作製する際に、その強度、耐熱性などの性質は原料であるＬ－乳酸のモノマーの光学純度に大きく依存するために、一般的に光学純度９９％以上の乳酸モノマーが必要となる。
Currently, as environmental problems such as global warming become more serious, poly L-lactic acid, a biodegradable plastic made from plant biomass, has attracted attention. Poly-L-lactic acid has the same advantage as plastics, which have a tensile strength and the like, and has the advantages of being transparent while being crystalline, and thus can be used as an alternative to polypropylene and polystyrene. When producing poly-L-lactic acid, its properties such as strength and heat resistance depend greatly on the optical purity of the L-lactic acid monomer, which is a raw material. It becomes.
一方、ポリＬ－乳酸は強度が高く、生分解性を有するという利点を有するが、耐熱性が１７０℃と低いことが普及の妨げとなっている。この問題の解決策として、ポリＬ－乳酸とポリＤ－乳酸とをブレンドし、２２０℃まで耐熱性を向上させることが可能なステレオコンプレックスポリ乳酸が注目されている。
On the other hand, poly L-lactic acid has the advantages of high strength and biodegradability, but its low heat resistance of 170 ° C. hinders its widespread use. As a solution to this problem, a stereocomplex polylactic acid that can blend poly L-lactic acid and poly D-lactic acid and improve heat resistance up to 220 ° C. has attracted attention.
ポリＤ－乳酸生産のためのＤ－乳酸モノマーは、乳酸菌を用いて生産する技術の研究が行われてきた。乳酸菌はグラム陽性の球菌又は桿菌であり、カタラーゼ陰性で、糖類を資化して５０％以上の収率で乳酸を生産し、胞子を形成しない細菌であると定義されている。
Research has been conducted on techniques for producing D-lactic acid monomers for producing poly-D-lactic acid using lactic acid bacteria. Lactic acid bacteria are gram-positive cocci or bacilli and are defined as bacteria that are catalase negative, assimilate saccharides, produce lactic acid in a yield of 50% or more, and do not form spores.
これまでＬ－乳酸は需要が高かったことから、Ｌ－乳酸の発酵生産に関する研究は盛んに行われてきた。一方Ｄ－乳酸に関しては、これまで需要が低かったことから、その発酵生産に関する報告は少なかった。以下にＤ－乳酸の発酵生産に関する知見の例を示す。
Since the demand for L-lactic acid has been high so far, research on fermentation production of L-lactic acid has been actively conducted. On the other hand, since the demand for D-lactic acid has been low so far, there have been few reports on its fermentative production. The following is an example of knowledge regarding fermentative production of D-lactic acid.
特開昭６２－４４１８８号公報では、スポルトラクトバシルス・イヌリヌス ＡＴＣＣ １５５３８株を用いてＤ－乳酸を生産することが報告されている。炭酸カルシウムでｐＨを中和した培地中で、スポルトラクトバシルス・イヌリヌス ＡＴＣＣ １５５３８株は、１００ｇ／Ｌのグルコースの存在下、３７℃で４０時間培養することにより、約９９．５％の光学純度で１０１ｇ／ＬのＤ－乳酸を産生する。
JP-A-62-44188 reports that D-lactic acid is produced using Sport Lactobacillus inulinus ATCC 15538 strain. In a medium neutralized with calcium carbonate, the Sport Lactobacillus inulinus ATCC 15538 strain was cultured at 37 ° C. for 40 hours in the presence of 100 g / L glucose, resulting in an optical purity of about 99.5%. Yields 101 g / L of D-lactic acid.
特開平２－７６５９２号公報では、Ｌａｃｔｏｂａｃｉｌｌｕｓ ｌａｃｔｉｓ ＡＴＣＣ １２３１４株を用いてＤ－乳酸を生産することが報告されている。Ｌａｃｔｏｂａｃｉｌｌｕｓ ｌａｃｔｉｓ ＡＴＣＣ １２３１４株は、コムギ澱粉のアミラーゼ分解物を炭素源とし炭酸カルシウムでｐＨを中和した培地中で、４０℃で４５時間培養することにより、１２３．３ｇ／ＬのＤ－乳酸を産生する。
JP-A-2-76592 reports that L-lactic acid is produced using Lactobacillus lactis ATCC 12314 strain. Lactobacillus lactis ATCC 12314 strain produces 123.3 g / L D-lactic acid by culturing at 40 ° C. for 45 hours in a medium neutralized with calcium carbonate using a amylase degradation product of wheat starch as a carbon source. To do.
特開２００７－２１５４２７号公報では、ラクトバシラス・デルブリッキー ＩＦＯ ３２０２株を用いてＤ－乳酸を生産することが報告されている。ｐＨ７の培地中で、ラクトバシラス・デルブリッキー ＩＦＯ ３２０２株は１００ｇ／Ｌのグルコースの存在下、３７℃で９６時間培養することにより、１０６．９ｇ／ＬのＤ－乳酸を産生する。なお特開２００７－２１５４２７号においては培養を開始して２４時間以降は、アンモニアを添加してｐＨを制御している。よって特開２００７－２１５４２７号の方法では、ｐＨの調節を行なわない第１段階とｐＨの調節を行なう第２段階からなる、２段階の培養を行っている。
In JP 2007-215427 A, it is reported that D-lactic acid is produced using Lactobacillus delbricki IFO 3202 strain. Lactobacillus delbricki IFO 3202 strain in a medium at pH 7 produces 106.9 g / L D-lactic acid by culturing at 37 ° C. for 96 hours in the presence of 100 g / L glucose. In JP 2007-215427 A, ammonia is added to control pH after 24 hours from the start of culture. Therefore, in the method of Japanese Patent Application Laid-Open No. 2007-215427, two-stage culture is performed, which includes a first stage in which pH adjustment is not performed and a second stage in which pH adjustment is performed.
Ｌｅｅは、ラクトバシラス・デルブリッキーの標準株（ＡＴＣＣ １２３１５）を用いてＭＲＳ培地中で培地する際にバッチ培養を繰り返し、その間に当該菌株を次第に酸性条件に適応させることにより、Ｄ－乳酸が生産性を向上することを報告している。ラクトバシラス・デルブリッキーの標準株を、１００ｇ／Ｌのグルコースの存在下４２℃で４８時間培養してＤ－乳酸の生産性の改善を検討したところ、最初のバッチでのＤ－乳酸の産生量は７１．７ｇ／Ｌであるが、８回目のバッチでのＤ－乳酸の産生量は９０ｇ／ＬのＤ－乳酸である。
Lee repeats batch culture when using a standard strain of Lactobacillus delbricky (ATCC 12315) in MRS medium, and gradually adapts the strain to acidic conditions, thereby increasing the productivity of D-lactic acid. Reported improvement. A standard strain of Lactobacillus delbricky was cultured at 42 ° C. for 48 hours in the presence of 100 g / L glucose to examine the improvement of D-lactic acid productivity. As a result, the amount of D-lactic acid produced in the first batch was 71. Although the amount of D-lactic acid in the eighth batch is 90 g / L, it is 90 g / L.
Ｃａｌａｂｉａらは、ラクトバシラス・デルブリッキー ＪＣＭ １１４８株を用いてＤ－乳酸を生産することを報告している。ラクトバシラス・デルブリッキー ＪＣＭ １１４８株は４０℃で７２時間培養することにより、コウモロコシのジュース（当初の糖類１３３ｇ／Ｌ）から１１８ｇ／ＬのＤ－乳酸と２ｇ／ＬのＬ－乳酸を産生する（Ｄ－乳酸の光学純度：９８．３％）。
Calabia et al. Have reported that D-lactic acid is produced using Lactobacillus del Bricky JCM 1148 strain. Lactobacillus delbricky JCM 1148 strain is cultured at 40 ° C for 72 hours to produce 118 g / L D-lactic acid and 2 g / L L-lactic acid from corn juice (original sugar 133 g / L) (D- Optical purity of lactic acid: 98.3%).
さらにＸｕらは、低エネルギーのイオンビーム・インプランテーションにより突然変異したスポロラクトバチルス・ｓｐ．ＤＸ１２株が、Ｄ－乳酸を高産生することを報告している。この変異株は炭酸カルシウムで中和した培地中で７日間培養することにより、１５０ｇ／Ｌのグルコースのから１４３．６ｇ/ＬのＬ－乳酸を、９９．０８％の光学純度で産生する。
Xu et al., Sporolactobacillus sp. Mutated by low energy ion beam implantation. The DX12 strain has been reported to produce high amounts of D-lactic acid. This mutant strain is cultured in a medium neutralized with calcium carbonate for 7 days to produce 143.6 g / L L-lactic acid from 150 g / L glucose with an optical purity of 99.08%.
さらにＯｋｉｎｏらは、コリネバクテリウム・グルタミカムにラクトバシラス・デルブリッキー由来のＤ－ラクテート・デヒドロゲナーゼの遺伝子を導入する遺伝子改変を行い、変異体株を得たことを報告している。当該変異体株は、間歇的なグルコース添加の条件下、３０時間の培養で１２０ｇ／ＬのＤ－乳酸を、９９．９％の光学純度で産生する。
Furthermore, Okino et al. Reported that a mutant strain was obtained by carrying out a genetic modification in which a gene of D-lactate dehydrogenase derived from Lactobacillus delbricky was introduced into Corynebacterium glutamicum. The mutant strain produces 120 g / L D-lactic acid with an optical purity of 99.9% after 30 hours of culture under intermittent glucose addition conditions.
しかしこれらにおいて報告されたＤ－乳酸の発酵生産の効率や光学純度は必ずしも高くなく、Ｌ－乳酸と比較するとＤ－乳酸の発酵生産量は少ない。よってより効率的に光学純度が高いＤ－乳酸を生産するために、Ｄ－乳酸を高生産する菌株、及び当該菌株を用いてＤ－乳酸を生産する技術が求められている。
However, the efficiency and optical purity of the fermentation production of D-lactic acid reported in these are not necessarily high, and the fermentation production of D-lactic acid is small compared to L-lactic acid. Therefore, in order to more efficiently produce D-lactic acid with high optical purity, a strain that produces D-lactic acid at a high level and a technique for producing D-lactic acid using the strain are required.
本発明はＤ－乳酸を得るために、新規なＤ－乳酸生産菌、及び当該Ｄ－乳酸生産菌を用いて高い光学純度のＤ－乳酸を効率的に生産する方法を提供することを目的とする。
An object of the present invention is to provide a novel D-lactic acid-producing bacterium and a method for efficiently producing high optical purity D-lactic acid using the D-lactic acid-producing bacterium in order to obtain D-lactic acid. To do.
本発明者らは、上記課題を解決するために鋭意研究に努めた結果、新規なＤ－乳酸生産菌、特にラクトバシラス・デルブリッキーＱＵ４１株を分離して得て、本発明を完成するに至った。本発明のＤ－乳酸生産菌を用いて高い光学純度のＤ－乳酸を効率良く生産することができる。
As a result of diligent research to solve the above-mentioned problems, the present inventors have isolated and obtained a novel D-lactic acid-producing bacterium, in particular, Lactobacillus delbricki QUI41 strain, and completed the present invention. Highly pure D-lactic acid can be efficiently produced using the D-lactic acid-producing bacterium of the present invention.
本発明は以下を提供する：
１）（１）グルコースを基質として９９％以上の光学純度でＤ－乳酸を生産可能であり、
かつ
（２）グルコース濃度が２０ｇ／Ｌ、ｐＨ６．０の条件で開始し、４３℃の温度で１２時間培養したときに、１２．０ｇ／Ｌ以上、好ましくは１６．０ｇ／Ｌ以上、さらに好ましくは２０．０ｇ／Ｌ以上のＤ－乳酸を生産可能である、
ラクトバシラス・デルブリッキーに属する乳酸菌。
２）さらに、
（３）４９℃の温度でＤ－乳酸を生産可能である、１）に記載の乳酸菌。
３）下記の菌学的性質を有する、１）又は２）記載の乳酸菌：
１．形態：桿菌、
２．生化学的性質：カタラーゼ陰性、
３．運動性：なし、
４．酸素要求性：通性嫌気性、
５．グルコースを基質としてホモ乳酸発酵によりＤ－乳酸を産生する、及び
６．グルコース、フルクトース、マンノース、Ｎ－アセチルグルコサミン、マルトース、ラクトース、スクロース、トレハロースを資化することができ、キシロース、アラビノースは資化することができない。
４）下記のいずれかの配列からなる１６ＳｒＲＮＡ遺伝子を有する、１）～３）記載の乳酸菌：
（ａ）配列表の配列番号：１で示されるヌクレオチド配列、又はそれと９９．７％以上の同一性を有するヌクレオチド配列；
（ｂ）配列表の配列番号：２で示されるヌクレオチド配列、又はそれと９９．７５％以上の同一性を有するヌクレオチド配列；又は
（ｃ）配列表の配列番号：３で示されるヌクレオチド配列、又はそれと９９．８％以上の同一性を有するヌクレオチド配列。
５）ＮＩＴＥ ＢＰ－６７９の受託番号で寄託されたラクトバシラス・デルブリッキーＱＵ４１株である、１）～４）のいずれか１に記載の乳酸菌。
６）１）～５）のいずれか１に記載の乳酸菌を培養する工程を含む、乳酸を製造する方法。
７）乳酸が、Ｄ－乳酸を９９％以上の光学純度で含む、６）に記載の製造方法。 The present invention provides the following:
1) (1) D-lactic acid can be produced with an optical purity of 99% or more using glucose as a substrate,
And (2) when the glucose concentration starts at 20 g / L and pH 6.0 and is cultured at a temperature of 43 ° C. for 12 hours, it is 12.0 g / L or more, preferably 16.0 g / L or more, more preferably Can produce 20.0 g / L or more of D-lactic acid,
Lactic acid bacteria belonging to Lactobacillus del Bricky.
2) Furthermore,
(3) The lactic acid bacterium according to 1), which can produce D-lactic acid at a temperature of 49 ° C.
3) Lactic acid bacteria according to 1) or 2) having the following mycological properties:
1. Form: Neisseria gonorrhoeae
2. Biochemical properties: Catalase negative,
3. Mobility: None,
4). Oxygen demand: facultative anaerobic,
5). 5. D-lactic acid is produced by homolactic fermentation using glucose as a substrate, and Glucose, fructose, mannose, N-acetylglucosamine, maltose, lactose, sucrose and trehalose can be assimilated, and xylose and arabinose cannot be assimilated.
4) A lactic acid bacterium according to 1) to 3) having a 16S rRNA gene having any of the following sequences:
(A) a nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing, or a nucleotide sequence having 99.7% or more identity thereto;
(B) a nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing, or a nucleotide sequence having 99.75% or more identity thereto; or (c) a nucleotide sequence represented by SEQ ID NO: 3 in the sequence listing, or A nucleotide sequence having an identity of 99.8% or more.
5) The lactic acid bacterium according to any one of 1) to 4), which is a Lactobacillus delbricki QUA41 strain deposited under the NITE BP-679 accession number.
6) A method for producing lactic acid, comprising the step of culturing the lactic acid bacterium according to any one of 1) to 5).
7) The production method according to 6), wherein the lactic acid contains D-lactic acid with an optical purity of 99% or more.
１）（１）グルコースを基質として９９％以上の光学純度でＤ－乳酸を生産可能であり、
かつ
（２）グルコース濃度が２０ｇ／Ｌ、ｐＨ６．０の条件で開始し、４３℃の温度で１２時間培養したときに、１２．０ｇ／Ｌ以上、好ましくは１６．０ｇ／Ｌ以上、さらに好ましくは２０．０ｇ／Ｌ以上のＤ－乳酸を生産可能である、
ラクトバシラス・デルブリッキーに属する乳酸菌。
２）さらに、
（３）４９℃の温度でＤ－乳酸を生産可能である、１）に記載の乳酸菌。
３）下記の菌学的性質を有する、１）又は２）記載の乳酸菌：
１．形態：桿菌、
２．生化学的性質：カタラーゼ陰性、
３．運動性：なし、
４．酸素要求性：通性嫌気性、
５．グルコースを基質としてホモ乳酸発酵によりＤ－乳酸を産生する、及び
６．グルコース、フルクトース、マンノース、Ｎ－アセチルグルコサミン、マルトース、ラクトース、スクロース、トレハロースを資化することができ、キシロース、アラビノースは資化することができない。
４）下記のいずれかの配列からなる１６ＳｒＲＮＡ遺伝子を有する、１）～３）記載の乳酸菌：
（ａ）配列表の配列番号：１で示されるヌクレオチド配列、又はそれと９９．７％以上の同一性を有するヌクレオチド配列；
（ｂ）配列表の配列番号：２で示されるヌクレオチド配列、又はそれと９９．７５％以上の同一性を有するヌクレオチド配列；又は
（ｃ）配列表の配列番号：３で示されるヌクレオチド配列、又はそれと９９．８％以上の同一性を有するヌクレオチド配列。
５）ＮＩＴＥ ＢＰ－６７９の受託番号で寄託されたラクトバシラス・デルブリッキーＱＵ４１株である、１）～４）のいずれか１に記載の乳酸菌。
６）１）～５）のいずれか１に記載の乳酸菌を培養する工程を含む、乳酸を製造する方法。
７）乳酸が、Ｄ－乳酸を９９％以上の光学純度で含む、６）に記載の製造方法。 The present invention provides the following:
1) (1) D-lactic acid can be produced with an optical purity of 99% or more using glucose as a substrate,
And (2) when the glucose concentration starts at 20 g / L and pH 6.0 and is cultured at a temperature of 43 ° C. for 12 hours, it is 12.0 g / L or more, preferably 16.0 g / L or more, more preferably Can produce 20.0 g / L or more of D-lactic acid,
Lactic acid bacteria belonging to Lactobacillus del Bricky.
2) Furthermore,
(3) The lactic acid bacterium according to 1), which can produce D-lactic acid at a temperature of 49 ° C.
3) Lactic acid bacteria according to 1) or 2) having the following mycological properties:
1. Form: Neisseria gonorrhoeae
2. Biochemical properties: Catalase negative,
3. Mobility: None,
4). Oxygen demand: facultative anaerobic,
5). 5. D-lactic acid is produced by homolactic fermentation using glucose as a substrate, and Glucose, fructose, mannose, N-acetylglucosamine, maltose, lactose, sucrose and trehalose can be assimilated, and xylose and arabinose cannot be assimilated.
4) A lactic acid bacterium according to 1) to 3) having a 16S rRNA gene having any of the following sequences:
(A) a nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing, or a nucleotide sequence having 99.7% or more identity thereto;
(B) a nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing, or a nucleotide sequence having 99.75% or more identity thereto; or (c) a nucleotide sequence represented by SEQ ID NO: 3 in the sequence listing, or A nucleotide sequence having an identity of 99.8% or more.
5) The lactic acid bacterium according to any one of 1) to 4), which is a Lactobacillus delbricki QUA41 strain deposited under the NITE BP-679 accession number.
6) A method for producing lactic acid, comprising the step of culturing the lactic acid bacterium according to any one of 1) to 5).
7) The production method according to 6), wherein the lactic acid contains D-lactic acid with an optical purity of 99% or more.
本発明により、新規なＤ－乳酸生産菌であるラクトバシラス・デルブリッキーＱＵ４１株、ＱＵ４２株、及びＱＵ４３株が提供された。本発明の微生物株はＤ－乳酸の効率良い生産を可能とする。
According to the present invention, Lactobacillus delbricky QU41 strain, QU42 strain, and QU43 strain, which are novel D-lactic acid producing bacteria, are provided. The microbial strain of the present invention enables efficient production of D-lactic acid.
以下、本発明の詳細、並びにその他の特徴及び利点について、形態に基づいて詳しく説明する。
Hereinafter, details of the present invention and other features and advantages will be described in detail based on the embodiments.
１．新規乳酸菌
本発明の乳酸菌は、初発グルコース濃度が２０ｇ／Ｌ、ｐＨ６．０の条件下で、グルコースを基質として、至適な温度（具体的には、４３℃）の温度で、８～１２時間培養したときに、１２．０ｇ／Ｌ以上のＤ－乳酸を９９％以上の光学純度で生産することができる。上記の性質を有し、遺伝学的にラクトバシラス・デルブリッキーに属する乳酸菌は本発明の範囲内である。なお本発明の乳酸菌は、上記の条件下で、好ましくは１６．０ｇ／Ｌ以上、さらに好ましくは２０．０ｇ／Ｌ以上のＤ－乳酸を生産することができる。このような乳酸菌は、高い光学純度のＤ－乳酸を効率的に生産する目的に適している。また本発明の乳酸菌はＤ－乳酸を、９９％以上の光学純度、好ましくは９９．５％以上の光学純度、さらに好ましくは９９．９％以上の光学純度で生産することができる。 1. Novel Lactic Acid Bacteria The lactic acid bacteria of the present invention have an initial glucose concentration of 20 g / L, pH 6.0, and glucose as a substrate at an optimal temperature (specifically, 43 ° C.) for 8 to 12 hours. When cultured, 12.0 g / L or more of D-lactic acid can be produced with an optical purity of 99% or more. Lactic acid bacteria having the above-mentioned properties and genetically belonging to Lactobacillus delbrick are within the scope of the present invention. The lactic acid bacterium of the present invention can produce D-lactic acid of preferably 16.0 g / L or more, more preferably 20.0 g / L or more under the above conditions. Such lactic acid bacteria are suitable for the purpose of efficiently producing high optical purity D-lactic acid. The lactic acid bacterium of the present invention can produce D-lactic acid with an optical purity of 99% or higher, preferably 99.5% or higher, more preferably 99.9% or higher.
本発明の乳酸菌は、初発グルコース濃度が２０ｇ／Ｌ、ｐＨ６．０の条件下で、グルコースを基質として、至適な温度（具体的には、４３℃）の温度で、８～１２時間培養したときに、１２．０ｇ／Ｌ以上のＤ－乳酸を９９％以上の光学純度で生産することができる。上記の性質を有し、遺伝学的にラクトバシラス・デルブリッキーに属する乳酸菌は本発明の範囲内である。なお本発明の乳酸菌は、上記の条件下で、好ましくは１６．０ｇ／Ｌ以上、さらに好ましくは２０．０ｇ／Ｌ以上のＤ－乳酸を生産することができる。このような乳酸菌は、高い光学純度のＤ－乳酸を効率的に生産する目的に適している。また本発明の乳酸菌はＤ－乳酸を、９９％以上の光学純度、好ましくは９９．５％以上の光学純度、さらに好ましくは９９．９％以上の光学純度で生産することができる。 1. Novel Lactic Acid Bacteria The lactic acid bacteria of the present invention have an initial glucose concentration of 20 g / L, pH 6.0, and glucose as a substrate at an optimal temperature (specifically, 43 ° C.) for 8 to 12 hours. When cultured, 12.0 g / L or more of D-lactic acid can be produced with an optical purity of 99% or more. Lactic acid bacteria having the above-mentioned properties and genetically belonging to Lactobacillus delbrick are within the scope of the present invention. The lactic acid bacterium of the present invention can produce D-lactic acid of preferably 16.0 g / L or more, more preferably 20.0 g / L or more under the above conditions. Such lactic acid bacteria are suitable for the purpose of efficiently producing high optical purity D-lactic acid. The lactic acid bacterium of the present invention can produce D-lactic acid with an optical purity of 99% or higher, preferably 99.5% or higher, more preferably 99.9% or higher.
本発明の乳酸菌は、蒸留水１Ｌ中に、ペプトンを１０ｇ、牛肉エキスを８ｇ、酵母エキスを４ｇ、グルコースを２０ｇ、Ｔｗｅｅｎ８０を１ｇ、Ｋ２ＨＰＯ４を２ｇ、酢酸ナトリウム三水和物を５ｇ、クエン酸水素二アンモニウムを２ｇ、ＭｇＳＯ４・Ｈ２Ｏを０．２ｇ、ＭｎＳＯ４・ｎＨ２Ｏを０．０５ｇ含むＭＲＳ－グルコース培地中で、少なくとも３０℃から４９℃の範囲の温度で静置培養することにより、良好に培養することができる。しかし本発明の乳酸菌の培養に使用する培地はＭＲＳ－グルコース培地に限定されるものではなく、ＭＲＳ－グルコース培地を適宜改変することも可能であり、当該菌が良好に増殖してＤ－乳酸を産生する限り如何なる組成の培地であっても良い。
Lactic acid bacteria of the present invention are 10 g of peptone, 8 g of beef extract, 4 g of yeast extract, 20 g of glucose, 1 g of Tween 80, 2 g of K 2 HPO 4 and 5 g of sodium acetate trihydrate in 1 L of distilled water. citric acid diammonium hydrogen 2g, the MgSO 4 · H 2 O 0.2g, MnSO 4 · nH to 2 O in MRS- glucose medium containing 0.05 g, stand at a temperature ranging from at least 30 ° C. of 49 ° C. By culturing, it can be cultured well. However, the medium used for culturing the lactic acid bacterium of the present invention is not limited to the MRS-glucose medium, and the MRS-glucose medium can be modified as appropriate, and the bacterium can grow well and produce D-lactic acid. A medium having any composition may be used as long as it is produced.
本発明における最も好適な乳酸菌の一つは、九州大学構内の排水溝から分離されたラクトバシラス・デルブリッキーＱＵ４１株である。なおラクトバシラス・デルブリッキーＱＵ４１株は新規な菌株であり、独立行政法人製品評価技術基盤機構特許微生物寄託センター（千葉県木更津市かずさ鎌足２－５－８）に、ＮＩＴＥ ＢＰ－６７９として２００９年１月１９日に寄託されている。
One of the most preferred lactic acid bacteria according to the present invention is Lactobacillus delbrikkii QU41 strain isolated from the drainage channel on the campus of Kyushu University. In addition, Lactobacillus delbricky QUI41 strain is a novel strain, and it was established as NITE BP-679 in January 2009 at the National Institute of Technology and Evaluation of Microorganisms (2-5-8, Kazusa Kamashichi, Kisarazu City, Chiba Prefecture). Deposited on the 19th.
ラクトバシラス・デルブリッキーＱＵ４１株の菌学的性質は以下の通りである。
１．形態：桿菌である。
２．生化学的性質：カタラーゼ陰性である。
３．運動性：なし
４．酸素要求性：通性嫌気性である。
５．グルコースを基質としてホモ乳酸発酵によりＤ－乳酸を産生する。
６．グルコース、フルクトース、マンノース、Ｎ－アセチルグルコサミン、マルトース、ラクトース、スクロース、トレハロースを資化することができ、キシロース、アラビノースは資化することができない。 The mycological properties of Lactobacillus delbricki QP41 are as follows.
1. Form: Neisseria gonorrhoeae.
2. Biochemical properties: Catalase negative.
3. Motility: None Oxygen demand: It is facultative anaerobic.
5). D-lactic acid is produced by homolactic fermentation using glucose as a substrate.
6). Glucose, fructose, mannose, N-acetylglucosamine, maltose, lactose, sucrose and trehalose can be assimilated, and xylose and arabinose cannot be assimilated.
１．形態：桿菌である。
２．生化学的性質：カタラーゼ陰性である。
３．運動性：なし
４．酸素要求性：通性嫌気性である。
５．グルコースを基質としてホモ乳酸発酵によりＤ－乳酸を産生する。
６．グルコース、フルクトース、マンノース、Ｎ－アセチルグルコサミン、マルトース、ラクトース、スクロース、トレハロースを資化することができ、キシロース、アラビノースは資化することができない。 The mycological properties of Lactobacillus delbricki QP41 are as follows.
1. Form: Neisseria gonorrhoeae.
2. Biochemical properties: Catalase negative.
3. Motility: None Oxygen demand: It is facultative anaerobic.
5). D-lactic acid is produced by homolactic fermentation using glucose as a substrate.
6). Glucose, fructose, mannose, N-acetylglucosamine, maltose, lactose, sucrose and trehalose can be assimilated, and xylose and arabinose cannot be assimilated.
本発明の乳酸菌には、ラクトバシラス・デルブリッキーに属し、ＱＵ４１株と同じ菌学的性質を有する乳酸菌も含まれる。本発明の他の好適な菌株として、ラクトバシラス・デルブリッキーＱＵ４２株及びラクトバシラス・デルブリッキーＱＵ４３株を挙げることができる。ラクトバシラス・デルブリッキーＱＵ４２株、ＱＵ４３株もまた新規な菌株であり、高い効率でＤ－乳酸を産生能することができる。なお本発明の菌株は、ラクトバシラス・デルブリッキーＱＵ４１株、ＱＵ４２株、及びＱＵ４３株に限定されるものではなく、グルコースを基質として９９％以上の光学純度でＤ－乳酸を生産可能であり、かつグルコース濃度が２０ｇ／Ｌ、ｐＨ６．０の条件で開始し、４３℃の温度で１２時間培養したときに、１２．０ｇ／Ｌ以上のＤ－乳酸を生産可能であるかぎり、本発明の範囲内である。
The lactic acid bacteria of the present invention also include lactic acid bacteria belonging to Lactobacillus delbricky and having the same bacteriological properties as the QU41 strain. As other suitable strains of the present invention, there can be mentioned Lactobacillus delbriquie QU42 strain and Lactobacillus delbrixi QU43 strain. Lactobacillus delbricki QU42 strain and QU43 strain are also novel strains and can produce D-lactic acid with high efficiency. Note that the strain of the present invention is not limited to Lactobacillus delbrickey QU41 strain, QU42 strain, and QU43 strain, and is capable of producing D-lactic acid with an optical purity of 99% or more using glucose as a substrate, and the glucose concentration. Is within the scope of the present invention as long as it can produce 1-2.0 g / L or more of D-lactic acid when cultured at 43 ° C. for 12 hours under the conditions of 20 g / L and pH 6.0. .
ラクトバシラス・デルブリッキーＱＵ４１株、ＱＵ４２株及びＱＵ４３株の１６ＳｒＲＮＡ遺伝子のヌクレオチド配列をそれぞれ、配列表の配列番号：１、配列番号：２及び配列番号：３に示す。１６ＳｒＲＮＡ遺伝子解析において、ラクトバシラス・デルブリッキーＱＵ４１株、ＱＵ４２株、ＱＵ４３株は高い同一性を示した。すなわち本発明は、下記の何れかの配列からなる表の配列番号１で示される配列と９９．５％以上の同一性を有する配列を含んでなる１６ＳｒＲＮＡ遺伝子を有する、ラクトバシラス・デルブリッキーを提供する：
（ａ）配列表の配列番号：１で示されるヌクレオチド配列、又はそれと９９．７％以上の同一性を有するヌクレオチド配列；
（ｂ）配列表の配列番号：２で示されるヌクレオチド配列、又はそれと９９．７５％以上の同一性を有するヌクレオチド配列；又は
（ｃ）配列表の配列番号：３で示されるヌクレオチド配列、又はそれと９９．８％以上の同一性を有するヌクレオチド配列。
本明細書において核酸配列の同一性は、例えば汎用されているＢＬＡＳＴなどのプログラム用いて決定することができるが、それに限定されるものではなく、他のプログラムを用いて核酸配列の同一性を決定することもできる。 The nucleotide sequences of the 16S rRNA genes of Lactobacillus delbrikkii QU41 strain, QU42 strain and QU43 strain are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively. In the 16S rRNA gene analysis, Lactobacillus delbrikkii QU41 strain, QU42 strain, and QU43 strain showed high identity. That is, the present invention provides a Lactobacillus delbrickey having a 16S rRNA gene comprising a sequence having 99.5% or more identity with the sequence shown in SEQ ID NO: 1 in the table consisting of any of the following sequences:
(A) a nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing, or a nucleotide sequence having 99.7% or more identity thereto;
(B) a nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing, or a nucleotide sequence having 99.75% or more identity thereto; or (c) a nucleotide sequence represented by SEQ ID NO: 3 in the sequence listing; or A nucleotide sequence having an identity of 99.8% or more.
In this specification, the identity of a nucleic acid sequence can be determined using, for example, a widely used program such as BLAST, but is not limited thereto, and the identity of a nucleic acid sequence is determined using another program. You can also
（ａ）配列表の配列番号：１で示されるヌクレオチド配列、又はそれと９９．７％以上の同一性を有するヌクレオチド配列；
（ｂ）配列表の配列番号：２で示されるヌクレオチド配列、又はそれと９９．７５％以上の同一性を有するヌクレオチド配列；又は
（ｃ）配列表の配列番号：３で示されるヌクレオチド配列、又はそれと９９．８％以上の同一性を有するヌクレオチド配列。
本明細書において核酸配列の同一性は、例えば汎用されているＢＬＡＳＴなどのプログラム用いて決定することができるが、それに限定されるものではなく、他のプログラムを用いて核酸配列の同一性を決定することもできる。 The nucleotide sequences of the 16S rRNA genes of Lactobacillus delbrikkii QU41 strain, QU42 strain and QU43 strain are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively. In the 16S rRNA gene analysis, Lactobacillus delbrikkii QU41 strain, QU42 strain, and QU43 strain showed high identity. That is, the present invention provides a Lactobacillus delbrickey having a 16S rRNA gene comprising a sequence having 99.5% or more identity with the sequence shown in SEQ ID NO: 1 in the table consisting of any of the following sequences:
(A) a nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing, or a nucleotide sequence having 99.7% or more identity thereto;
(B) a nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing, or a nucleotide sequence having 99.75% or more identity thereto; or (c) a nucleotide sequence represented by SEQ ID NO: 3 in the sequence listing; or A nucleotide sequence having an identity of 99.8% or more.
In this specification, the identity of a nucleic acid sequence can be determined using, for example, a widely used program such as BLAST, but is not limited thereto, and the identity of a nucleic acid sequence is determined using another program. You can also
またＡＰＩ５０ＣＨＬ糖類資化試験において、それらの３株は同一の糖発酵性パターンを示した。そのように、ラクトバシラス・デルブリッキーＱＵ４１株、ＱＵ４２株、及びＱＵ４３株は、遺伝学的にも生化学的性質も相互に非常に類似しているために、これら３つの菌株は異なった菌株であっても、Ｄ－乳酸の産生能を含む多くの性質が類似又は同一であることが予想される。
In the API50CHL saccharide utilization test, these three strains showed the same sugar fermentability pattern. As such, the Lactobacillus delbrikkii strains QU41, QU42 and QU43 are very similar in their genetic and biochemical properties, so these three strains are different strains. However, many properties including the ability to produce D-lactic acid are expected to be similar or identical.
本発明の乳酸菌は少なくとも３０℃から４９℃の範囲の温度で、増殖及びＤ－乳酸の産生が可能である。当該菌株の増殖及びＤ－乳酸の産生において好ましい温度は３７℃から４３℃であり、特に好ましい温度は４３℃である。
The lactic acid bacteria of the present invention can grow and produce D-lactic acid at a temperature in the range of at least 30 ° C. to 49 ° C. A preferable temperature for growth of the strain and production of D-lactic acid is 37 ° C to 43 ° C, and a particularly preferable temperature is 43 ° C.
本発明の乳酸菌は、４９℃においてもＤ－乳酸を生産可能である。４９℃は乳酸菌にとってかなり高い温度である。４９℃での乳酸発酵により、雑菌汚染を低減することができ、また発酵熱による冷却の必要性を低減することができる。なお、本発明で乳酸を「生産可能」というときは、産業上有効なレベルでＤ－乳酸が生産できることをいい、グルコース濃度が２０ｇ／Ｌ、ｐＨ６．０の条件で開始し、１２時間培養したときに、１２．０ｇ／Ｌ以上のＤ－乳酸を生産することができれば、「生産可能」といえる。本発明の乳酸菌は、４９℃の高温培養でも、グルコース１００ｇ／ＬからＤ－乳酸を約１００ｇ／Ｌ産生することができる。
The lactic acid bacterium of the present invention can produce D-lactic acid even at 49 ° C. 49 ° C. is a considerably high temperature for lactic acid bacteria. Lactic acid fermentation at 49 ° C. can reduce contamination by various bacteria, and can reduce the need for cooling by fermentation heat. In the present invention, when “lactic acid can be produced”, it means that D-lactic acid can be produced at an industrially effective level. It was started under the conditions of a glucose concentration of 20 g / L and pH 6.0, and cultured for 12 hours. Sometimes, if it can produce 1-2.0 g / L or more of D-lactic acid, it can be said that “production is possible”. The lactic acid bacterium of the present invention can produce about 100 g / L of D-lactic acid from 100 g / L of glucose even in high-temperature culture at 49 ° C.
本発明のラクトバシラス・デルブリッキーＱＵ４１株は少なくとも５．５から６．５のｐＨの範囲で、増殖及びＤ－乳酸の産生が可能である。当該菌株の増殖及びＤ－乳酸の産生において特に好ましいｐＨは６．０である。
The Lactobacillus delbricky QUI41 strain of the present invention can grow and produce D-lactic acid in a pH range of at least 5.5 to 6.5. A particularly preferred pH for the growth of the strain and the production of D-lactic acid is 6.0.
本発明のラクトバシラス・デルブリッキーＱＵ４１株は、培地中の初発グルコース濃度が増加すると増殖及びＤ－乳酸の産生性が高くなる。その一方、初発グルコース濃度の増加に伴う最大菌体増殖速度の低下と、長時間の培養における乳酸生産速度の低下が起こる。このために、当該菌株を用いてＤ－乳酸を効率的に産生する場合には、回分培養は適さない。
The growth of Lactobacillus delbricky QUI41 of the present invention increases as the initial glucose concentration in the medium increases, and the productivity of D-lactic acid increases. On the other hand, a decrease in the maximum cell growth rate accompanying an increase in the initial glucose concentration and a decrease in the lactic acid production rate in the long-term culture occur. For this reason, batch culture is not suitable when D-lactic acid is efficiently produced using the strain.
２．Ｄ－乳酸を産生する方法
また本発明のラクトバシラス・デルブリッキー株を培養することにより乳酸を製造する方法も、本発明の範囲内である。ＭＲＳ－グルコース培地など、本発明のラクトバシラス・デルブリッキー株が増殖して乳酸を産生することができる培地を用いて、当該菌株を、それの増殖及び乳酸の産生に適した条件下（温度やｐＨ）で培養することにより、乳酸を製造することができる。 2. A method for producing D-lactic acid and a method for producing lactic acid by culturing the Lactobacillus delbrikkii strain of the present invention are also within the scope of the present invention. Using a medium in which the Lactobacillus delbrickey strain of the present invention can grow and produce lactic acid, such as MRS-glucose medium, the strain is grown under conditions suitable for its growth and lactic acid production (temperature and pH). Lactic acid can be produced by culturing in the above.
また本発明のラクトバシラス・デルブリッキー株を培養することにより乳酸を製造する方法も、本発明の範囲内である。ＭＲＳ－グルコース培地など、本発明のラクトバシラス・デルブリッキー株が増殖して乳酸を産生することができる培地を用いて、当該菌株を、それの増殖及び乳酸の産生に適した条件下（温度やｐＨ）で培養することにより、乳酸を製造することができる。 2. A method for producing D-lactic acid and a method for producing lactic acid by culturing the Lactobacillus delbrikkii strain of the present invention are also within the scope of the present invention. Using a medium in which the Lactobacillus delbrickey strain of the present invention can grow and produce lactic acid, such as MRS-glucose medium, the strain is grown under conditions suitable for its growth and lactic acid production (temperature and pH). Lactic acid can be produced by culturing in the above.
本発明の方法によれば、高い光学純度でＤ－乳酸を産生することができる。具体的には本発明の方法によれば、Ｄ－乳酸を９９％以上の光学純度、好ましくは９９．５％以上の光学純度、さらに好ましくは９９．９％以上の光学純度で製造することができる。
According to the method of the present invention, D-lactic acid can be produced with high optical purity. Specifically, according to the method of the present invention, D-lactic acid can be produced with an optical purity of 99% or more, preferably 99.5% or more, and more preferably 99.9% or more. it can.
本発明により得られる乳酸の濃度は、本技術分野で一般的に知られた技術により測定することができる。例えば、本発明のラクトバシラス・デルブリッキー株の培養液を遠心分離して上清を得、その上清をフィルターで濾過した後に、カラムクロマトグラフィー（例えば、Ｓｈｏｄｅｘ Ｓｕｇａｒ Ｓシリーズなどの糖分析用配意子交換カラム）により乳酸量を測定することができ、また示唆屈折計により乳酸を検出することにより、生産された乳酸の濃度を測定することができる。
The concentration of lactic acid obtained by the present invention can be measured by a technique generally known in this technical field. For example, the culture solution of the Lactobacillus delbricki strain of the present invention is centrifuged to obtain a supernatant, and the supernatant is filtered through a filter and then subjected to column chromatography (for example, a shodex Sugar S series etc. The amount of lactic acid can be measured with an exchange column, and the concentration of produced lactic acid can be measured by detecting lactic acid with a suggestive refractometer.
また、Ｄ－乳酸の光学純度は、本技術分野で知られた種々の手段を用いることができる。例えば、バイオセンサー（例えば酵素電極式バイオセンサーＢＦ－５）によりＬ－乳酸の濃度を測定することができ、そして測定したＬ－乳酸の濃度と全乳酸の濃度の値から、Ｄ－乳酸の光学純度を算出することができる。本発明でＤ－乳酸の光学純度をいうときは、特に示した場合を除き、下記の式により算出した値をいう。
Also, various means known in the art can be used for the optical purity of D-lactic acid. For example, the concentration of L-lactic acid can be measured by a biosensor (for example, enzyme electrode type biosensor BF-5), and the optical value of D-lactic acid is determined from the measured L-lactic acid concentration and total lactic acid concentration value. Purity can be calculated. In the present invention, the optical purity of D-lactic acid means a value calculated by the following formula, unless otherwise specified.
本発明の乳酸を製造する方法は、好ましくは、グルコースを含有する培地中で本発明のラクトバシラス・デルブリッキー株を、ｐＨ５．５からｐＨ６．５、温度３７℃から４３℃の条件下で、１２時間から７２時間培養して培養液を調製する工程、及び、上記培養液を遠心分離して上清を採取する工程からなる。本発明のラクトバシラス・デルブリッキー株を培養するのに適した培地は、それに限定されるものではないが、好ましくは上記で述べたＭＲＳ－グルコース培地である。しかし本発明の方法で使用される培地はそれに限定されるものではなく、適宜改変することができる。また培地のｐＨ、培養温度、培養時間などの条件もまた、本発明のラクトバシラス・デルブリッキー株が増殖し、Ｄ－乳酸を産生することができる限り、適宜改変することができる。
The method for producing lactic acid of the present invention is preferably performed by subjecting the Lactobacillus delbrikkii strain of the present invention to pH 5.5 to pH 6.5 at a temperature of 37 ° C. to 43 ° C. for 12 hours in a medium containing glucose. For 72 hours to prepare a culture solution, and a step of centrifuging the culture solution and collecting a supernatant. A suitable medium for culturing the Lactobacillus delbrikkii strain of the present invention is not limited thereto, but is preferably the MRS-glucose medium described above. However, the medium used in the method of the present invention is not limited thereto, and can be appropriately modified. The conditions such as the pH of the medium, the culture temperature, and the culture time can also be appropriately modified as long as the Lactobacillus delbriqui strain of the present invention can grow and produce D-lactic acid.
また遠心分離して得た上清から純粋なＤ－乳酸を得るために、例えばフィルターによる濾過やクロマトグラフィーによる精製にかけることも、本発明の好適な態様である。
In addition, in order to obtain pure D-lactic acid from the supernatant obtained by centrifugation, it is also a preferred embodiment of the present invention that it is subjected to, for example, filtration with a filter or purification by chromatography.
本発明の方法において用いられる最も好適な菌株はラクトバシラス・デルブリッキーＱＵ４１株である。その他に、ラクトバシラス・デルブリッキーＱＵ４２株、ＱＵ４３株を用いても、本発明の方法を実施することができる。
The most preferred strain used in the method of the present invention is Lactobacillus delbrikkii QU41 strain. In addition, the method of the present invention can also be carried out using Lactobacillus delbricky QU42 strain and QU43 strain.
３．用途
本発明の方法を用いて産生したＤ－乳酸を、例えば、ポリＬ－乳酸とポリＤ－乳酸とのステレオコンプレックスを製造するための原料として使用することができる。上記で述べたようにポリＬ－乳酸とポリＤ－乳酸とのステレオコンプレックスは、耐熱性が高い生分解性プラスチックとなり得る。しかしＤ－乳酸の用途はポリＬ－乳酸とのステレオコンプレックスに限定されるものではない。なおＤ－乳酸には、農業中間体としての用途もあることが知られている。 3. Applications D-lactic acid produced by using the method of the present invention can be used as a raw material for producing a stereocomplex of poly-L-lactic acid and poly-D-lactic acid, for example. As described above, the stereocomplex of poly L-lactic acid and poly D-lactic acid can be a biodegradable plastic having high heat resistance. However, the use of D-lactic acid is not limited to a stereocomplex with poly L-lactic acid. D-lactic acid is known to be used as an agricultural intermediate.
本発明の方法を用いて産生したＤ－乳酸を、例えば、ポリＬ－乳酸とポリＤ－乳酸とのステレオコンプレックスを製造するための原料として使用することができる。上記で述べたようにポリＬ－乳酸とポリＤ－乳酸とのステレオコンプレックスは、耐熱性が高い生分解性プラスチックとなり得る。しかしＤ－乳酸の用途はポリＬ－乳酸とのステレオコンプレックスに限定されるものではない。なおＤ－乳酸には、農業中間体としての用途もあることが知られている。 3. Applications D-lactic acid produced by using the method of the present invention can be used as a raw material for producing a stereocomplex of poly-L-lactic acid and poly-D-lactic acid, for example. As described above, the stereocomplex of poly L-lactic acid and poly D-lactic acid can be a biodegradable plastic having high heat resistance. However, the use of D-lactic acid is not limited to a stereocomplex with poly L-lactic acid. D-lactic acid is known to be used as an agricultural intermediate.
以下、実施例に基づいて本発明を詳細に説明するが、本発明はこれらの実施例に限定されるものではない。
Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.
実施例１ 新規Ｄ－乳酸生産菌の分離及び同定
（１）新規Ｄ－乳酸生産菌の分離
植物、動物、土壌、昆虫など１３７の分離源から、ＭＲＳ－グルコース培地及びＭＲＳ－グルコース寒天培地を用いてＤ－乳酸生産菌を単離した。分離条件として、酸生成菌を優先的に取得するため、プレーティングを行う前に集積培養を行った。また、原核生物を優先的に取得するため、真核生物の生育を阻害するアジ化ナトリウム、シクロヘキシミドを集積培地に添加した。この集積培養用培地は、上記で述べた組成のＭＲＳ－グルコース培地、１００ｐｐｍアジ化ナトリウム、１００ｐｐｍシクロヘキシミドを加え、蒸留水に溶解し、ｐＨを５．５と６．５に調整し、褐色反応を防ぐために１１５℃、１５分間オートクレーブ滅菌を行ったものである。またＭＲＳ－グルコース寒天培地は、集積培養用培地に１．５％寒天と０．５％ＣａＣＯ２を加え蒸留水に溶解し、ｐＨを６．５に調整し、１１５℃、１５分間オートクレーブ滅菌を行った後、滅菌シャーレに注いで作製したものである。 Example 1 Isolation and Identification of New D-Lactic Acid-Producing Bacteria (1) Isolation of New D-Lactic Acid-Producing Bacteria Using MRS-glucose medium and MRS-glucose agar medium from 137 sources such as plants, animals, soil, and insects Thus, D-lactic acid-producing bacteria were isolated. In order to obtain acid-producing bacteria preferentially as a separation condition, enrichment culture was performed before plating. Moreover, in order to obtain prokaryote preferentially, sodium azide and cycloheximide that inhibit the growth of eukaryotes were added to the accumulation medium. This enrichment culture medium was prepared by adding MRS-glucose medium having the composition described above, 100 ppm sodium azide, 100 ppm cycloheximide, dissolving in distilled water, adjusting the pH to 5.5 and 6.5, and causing brown reaction. In order to prevent this, autoclaving was performed at 115 ° C. for 15 minutes. The MRS-glucose agar medium is 1.5% agar and 0.5% CaCO 2 added to the enrichment culture medium, dissolved in distilled water, adjusted to pH 6.5, and autoclaved at 115 ° C for 15 minutes. After performing, it poured into a sterilization petri dish and produced.
（１）新規Ｄ－乳酸生産菌の分離
植物、動物、土壌、昆虫など１３７の分離源から、ＭＲＳ－グルコース培地及びＭＲＳ－グルコース寒天培地を用いてＤ－乳酸生産菌を単離した。分離条件として、酸生成菌を優先的に取得するため、プレーティングを行う前に集積培養を行った。また、原核生物を優先的に取得するため、真核生物の生育を阻害するアジ化ナトリウム、シクロヘキシミドを集積培地に添加した。この集積培養用培地は、上記で述べた組成のＭＲＳ－グルコース培地、１００ｐｐｍアジ化ナトリウム、１００ｐｐｍシクロヘキシミドを加え、蒸留水に溶解し、ｐＨを５．５と６．５に調整し、褐色反応を防ぐために１１５℃、１５分間オートクレーブ滅菌を行ったものである。またＭＲＳ－グルコース寒天培地は、集積培養用培地に１．５％寒天と０．５％ＣａＣＯ２を加え蒸留水に溶解し、ｐＨを６．５に調整し、１１５℃、１５分間オートクレーブ滅菌を行った後、滅菌シャーレに注いで作製したものである。 Example 1 Isolation and Identification of New D-Lactic Acid-Producing Bacteria (1) Isolation of New D-Lactic Acid-Producing Bacteria Using MRS-glucose medium and MRS-glucose agar medium from 137 sources such as plants, animals, soil, and insects Thus, D-lactic acid-producing bacteria were isolated. In order to obtain acid-producing bacteria preferentially as a separation condition, enrichment culture was performed before plating. Moreover, in order to obtain prokaryote preferentially, sodium azide and cycloheximide that inhibit the growth of eukaryotes were added to the accumulation medium. This enrichment culture medium was prepared by adding MRS-glucose medium having the composition described above, 100 ppm sodium azide, 100 ppm cycloheximide, dissolving in distilled water, adjusting the pH to 5.5 and 6.5, and causing brown reaction. In order to prevent this, autoclaving was performed at 115 ° C. for 15 minutes. The MRS-glucose agar medium is 1.5% agar and 0.5% CaCO 2 added to the enrichment culture medium, dissolved in distilled water, adjusted to pH 6.5, and autoclaved at 115 ° C for 15 minutes. After performing, it poured into a sterilization petri dish and produced.
単離の際の培養条件は、温度が３０℃、３７℃、４３℃であり、当初ｐＨが５．５と６．５である。この条件下で嫌気的に菌を培養・単離し、再度ＭＲＳ培地で培養した後、上清を分析し、ホモ発酵により光学純度の高いＤ－乳酸を生産する菌を分離した。
The culture conditions for isolation are temperatures of 30 ° C., 37 ° C., and 43 ° C., and initial pH values of 5.5 and 6.5. Under these conditions, the bacteria were anaerobically cultured and isolated, and again cultured in the MRS medium. Then, the supernatant was analyzed, and the bacteria producing high optical purity D-lactic acid were isolated by homofermentation.
その結果、約３５０株の乳酸生産菌を分離した。得られたＤ－乳酸生産菌のうち殆どはヘテロ発酵型の菌であったが、４０の菌株が９９％以上の光学純度でＤ－乳酸を生産した。とりわけ九州大学構内の排水溝から分離されたＱＵ４１株、ＱＵ４２株、及びＱＵ４３株が、収率約１００％、光学純度９９．９％以上でＤ－乳酸を生産したので、以下でさらに検討を行った。
As a result, about 350 lactic acid-producing bacteria were isolated. Most of the D-lactic acid-producing bacteria obtained were heterofermentative, but 40 strains produced D-lactic acid with an optical purity of 99% or more. In particular, the QU41, QU42, and QU43 strains isolated from the drainage channel on the campus of Kyushu University produced D-lactic acid with a yield of about 100% and an optical purity of 99.9% or more. It was.
（２）得られたＤ－乳酸生産菌の同定
ＱＵ４１株、ＱＵ４２株、及びＱＵ４３株につき、分離した株の１６ＳｒＲＮＡ遺伝子のポジション２８－１４９１について解析した。ゲノム抽出キット（ＭａｇＥｘｔｒａｃｔｏｒ Ｇｅｎｏｍｅ）を用いて分離株のＤＮＡを抽出し、それを鋳型としてプライマーとＴａｑ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ（Ｐｒｏｍｅｇａ，ＵＳＡ）を用いてＰＣＲを行った。ＰＣＲ産物は、精製キット（Ｈｉｇｈ ｐｕｒｅ ｐｒｏｄｕｃｔ ｐｕｒｉｆｉｃａｔｉｏｎ ｋｉｔ，Ｒｏｃｈｅ，Ｓｗｉｔｅｒｌａｎｄ）を用いて精製した。定法に従って、それをベクター（ｐＧＥＭ-Ｔ，Ｐｒｏｍｅｇａ）にライゲーションし、大腸菌ＤＨ５αにクローニングした後、シークエンス解析を行った。データベース（ＢＬＡＳＴ ｐｒｏｇｒａｍ ｏｆ ｔｈｅ Ｎａｔｉｏｎａｌ Ｃｅｎｔｅｒ ｆｏｒ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ｉｎｆｏｒｍａｔｉｏｎ）で、得られた配列について相同性検索を行った。 (2) Identification of D-Lactic Acid-Producing Bacteria Obtained QS41 strain, QU42 strain, and QU43 strain were analyzed for positions 28-1491 of the 16S rRNA gene of the isolated strains. The DNA of the isolate was extracted using a genome extraction kit (MagExtractor Genome), and PCR was performed using the primer and Taq DNA polymerase (Promega, USA) as a template. The PCR product was purified using a purification kit (High pure product purification kit, Roche, Switzerland). According to a conventional method, it was ligated to a vector (pGEM-T, Promega), cloned into E. coli DH5α, and then subjected to sequence analysis. A homology search was performed on the obtained sequences in a database (BLAST program of the National Center for Biotechnology Information).
ＱＵ４１株、ＱＵ４２株、及びＱＵ４３株につき、分離した株の１６ＳｒＲＮＡ遺伝子のポジション２８－１４９１について解析した。ゲノム抽出キット（ＭａｇＥｘｔｒａｃｔｏｒ Ｇｅｎｏｍｅ）を用いて分離株のＤＮＡを抽出し、それを鋳型としてプライマーとＴａｑ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ（Ｐｒｏｍｅｇａ，ＵＳＡ）を用いてＰＣＲを行った。ＰＣＲ産物は、精製キット（Ｈｉｇｈ ｐｕｒｅ ｐｒｏｄｕｃｔ ｐｕｒｉｆｉｃａｔｉｏｎ ｋｉｔ，Ｒｏｃｈｅ，Ｓｗｉｔｅｒｌａｎｄ）を用いて精製した。定法に従って、それをベクター（ｐＧＥＭ-Ｔ，Ｐｒｏｍｅｇａ）にライゲーションし、大腸菌ＤＨ５αにクローニングした後、シークエンス解析を行った。データベース（ＢＬＡＳＴ ｐｒｏｇｒａｍ ｏｆ ｔｈｅ Ｎａｔｉｏｎａｌ Ｃｅｎｔｅｒ ｆｏｒ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ｉｎｆｏｒｍａｔｉｏｎ）で、得られた配列について相同性検索を行った。 (2) Identification of D-Lactic Acid-Producing Bacteria Obtained QS41 strain, QU42 strain, and QU43 strain were analyzed for positions 28-1491 of the 16S rRNA gene of the isolated strains. The DNA of the isolate was extracted using a genome extraction kit (MagExtractor Genome), and PCR was performed using the primer and Taq DNA polymerase (Promega, USA) as a template. The PCR product was purified using a purification kit (High pure product purification kit, Roche, Switzerland). According to a conventional method, it was ligated to a vector (pGEM-T, Promega), cloned into E. coli DH5α, and then subjected to sequence analysis. A homology search was performed on the obtained sequences in a database (BLAST program of the National Center for Biotechnology Information).
１６ＳｒＲＮＡ遺伝子配列解析の結果、それらの菌株はＬａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ ｓｓｐ．ｌａｃｔｉｓ（基準株）と最も高い同一性を示した。ＱＵ４１株、ＱＵ４２株、ＱＵ４３株と、基準株の間の同一性はそれぞれ、９９．５９６％（全長１４８６ｂｐにおいて、６塩基異なる）、９９．６６４％（５塩基異なる）、９９．８０％（３塩基異なる）であった。また、ＱＵ４１株とＱＵ４２株の間の同一性は９９．６６４％（５塩基異なる）、ＱＵ４１株とＱＵ４３株の間の同一性は９９．５９６％（６塩基異なる）、ＱＵ４２株とＱＵ４３株の間の同一性は９９．６６４％（５塩基異なる）であった。さらにＱＵ４２株とＱＵ４３株の１６ＳｒＲＮＡ遺伝子配列間の同一性は、９９．６６４％（５塩基異なる）であった。
As a result of 16S rRNA gene sequence analysis, those strains were found to be Lactobacillus delbrueckii ssp. Lactis (reference strain) showed the highest identity. The identity between the QUI41 strain, the QUI42 strain, the QUI43 strain and the reference strain is 99.596% (6 bases are different at the total length of 1486 bp), 99.664% (5 bases are different), 99.80% (3 Different base). Moreover, the identity between the CU41 strain and the QUI42 strain is 99.664% (5 bases different), the identity between the CU41 strain and the KU43 strain is 99.596% (6 bases different), The identity between them was 99.664% (5 bases different). Furthermore, the identity between the 16S rRNA gene sequences of the QU42 and QU43 strains was 99.664% (5 bases different).
なおＱＵ４１株の１６ＳｒＲＮＡ遺伝子のヌクレオチド配列を、配列表の配列番号：１と図１に示す。ＱＵ４２株の１６ＳｒＲＮＡ遺伝子のヌクレオチド配列を、配列表の配列番号：２と図２に示す。ＱＵ４３株の１６ＳｒＲＮＡ遺伝子のヌクレオチド配列を、配列表の配列番号：３と図３に示す。
The nucleotide sequence of the 16S rRNA gene of the QUI41 strain is shown in SEQ ID NO: 1 in the sequence listing and FIG. The nucleotide sequence of the 16S rRNA gene of the QU42 strain is shown in SEQ ID NO: 2 in the sequence listing and FIG. The nucleotide sequence of the 16S rRNA gene of the QU43 strain is shown in SEQ ID NO: 3 in the sequence listing and FIG.
さらにＡＰＩ５０ＣＨキット（ＢｉｏＭｅｒｉｅｕｘ、フランス）を用いて、糖類資化性試験を行なった。詳細は本キットのマニュアルに従った。２４時間と４８時間培養後の糖類資化性パターンをアピウェブサイトにて解析した（https://apiweb.biomerieux.com）。
Furthermore, a saccharide utilization test was performed using an API50CH kit (BioMerieux, France). Details followed the kit manual. The sugar utilization pattern after 24 and 48 hours of culture was analyzed on the API website (https://apiweb.biomerieux.com).
ＡＰＩ５０ＣＨキットを用いた糖類資化性試験の結果を、表１に示す。比較対象として、ＡＰＩ５０ＣＨキットに記載のあるＬａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ ｓｕｂｓｐ．ｄｅｌｂｒｕｅｃｋｉｉ、Ｌａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ ｓｕｂｓｐ．ｂｕｌｇａｒｉｃｕｓ、Ｌａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ ｓｕｂｓｐ．ｌａｃｔｉｓの糖類資化性のパターンも併せて、表１に示す。
Table 1 shows the results of the saccharide utilization test using the API50CH kit. For comparison, Lactobacillus delbrueckii subsp. Described in the API50CH kit. delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. The saccharide utilization pattern of lactis is also shown in Table 1.
表１において各数字は、ＢｉｏＭｅｒｉｅｕｘ社が保有する菌株のうち、資化能を有する菌株の割合を示す。同一種でも、Ｌａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ ｓｕｂｓｐ．ｌａｃｔｉｓ１，２のように、糖類資化性をグループ化しているものもある。
In Table 1, each number indicates the proportion of strains having the ability to assimilate among strains held by BioMerieux. Even for the same species, Lactobacillus delbrueckii subsp. Some have grouped saccharide utilization properties, such as lactis 1 and 2.
この試験においてＱＵ４１株、ＱＵ４２株、及びＱＵ４３株は同一の糖発酵性パターンを示した。すなわちこれらの菌株は、グルコース、フルクトース、マンノース、Ｎ－アセチルグルコサミン、マルトース、ラクトース、スクロース、トレハロースを資化したが、五単糖であるキシロース、アラビノースは資化できなかった。その糖発酵性パターンと最も一致するのは、Ｌａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ ｓｓｐ ｌａｃｔｉｓであった。
In this test, the QU41 strain, the QU42 strain, and the QU43 strain showed the same sugar fermentability pattern. That is, these strains assimilated glucose, fructose, mannose, N-acetylglucosamine, maltose, lactose, sucrose, and trehalose, but not pentoses such as xylose and arabinose. Lactobacillus delbrueckii ssp lactis was most consistent with the sugar fermentability pattern.
よってこれらの結果から、ＱＵ４１株、ＱＵ４２株、及びＱＵ４３株の３株ともラクトバシラス・デルブリッキーであると同定された。
Therefore, from these results, the three strains, QU41 strain, QU42 strain, and QU43 strain, were identified as Lactobacillus delbricki.
（３）試験管培養
ラクトバシラス・デルブリッキーＱＵ４１株、ＱＵ４２株、及びＱＵ４３株の３つの菌株を、１０ｍｌのＭＲＳ－グルコース培地中で、嫌気条件下、３７℃で培養を行った。図４（ａ）はＱＵ４１株で検討を行なった結果であり、図４（ｂ）はＱＵ４１株で検討を行なった結果であり、図４（ｃ）はＱＵ４１株で検討を行なった結果である。図４において左の縦軸はグルコース、乳酸、酢酸の濃度を示し、右軸がＯＤを示し、横軸が時間を示す。図４のプロットにおいて、黒丸がグルコース濃度、白丸が乳酸濃度であり、白三角がＯＤである。さらに３つの株における試験官培養の結果を比較した数値データを、下の表２に示す。 (3) Test-tube culture Three strains of Lactobacillus delbrikkii QU41, QU42 and QU43 were cultured in 10 ml of MRS-glucose medium at 37 ° C under anaerobic conditions. FIG. 4 (a) shows the results of the study with the QUI41 strain, FIG. 4 (b) shows the results of the study with the QUI41 strain, and FIG. 4 (c) shows the results of the study with the QUI41 strain. . In FIG. 4, the left vertical axis indicates the concentrations of glucose, lactic acid, and acetic acid, the right axis indicates OD, and the horizontal axis indicates time. In the plot of FIG. 4, the black circle is the glucose concentration, the white circle is the lactic acid concentration, and the white triangle is the OD. Further numerical data comparing the results of investigator culture in three strains is shown in Table 2 below.
ラクトバシラス・デルブリッキーＱＵ４１株、ＱＵ４２株、及びＱＵ４３株の３つの菌株を、１０ｍｌのＭＲＳ－グルコース培地中で、嫌気条件下、３７℃で培養を行った。図４（ａ）はＱＵ４１株で検討を行なった結果であり、図４（ｂ）はＱＵ４１株で検討を行なった結果であり、図４（ｃ）はＱＵ４１株で検討を行なった結果である。図４において左の縦軸はグルコース、乳酸、酢酸の濃度を示し、右軸がＯＤを示し、横軸が時間を示す。図４のプロットにおいて、黒丸がグルコース濃度、白丸が乳酸濃度であり、白三角がＯＤである。さらに３つの株における試験官培養の結果を比較した数値データを、下の表２に示す。 (3) Test-tube culture Three strains of Lactobacillus delbrikkii QU41, QU42 and QU43 were cultured in 10 ml of MRS-glucose medium at 37 ° C under anaerobic conditions. FIG. 4 (a) shows the results of the study with the QUI41 strain, FIG. 4 (b) shows the results of the study with the QUI41 strain, and FIG. 4 (c) shows the results of the study with the QUI41 strain. . In FIG. 4, the left vertical axis indicates the concentrations of glucose, lactic acid, and acetic acid, the right axis indicates OD, and the horizontal axis indicates time. In the plot of FIG. 4, the black circle is the glucose concentration, the white circle is the lactic acid concentration, and the white triangle is the OD. Further numerical data comparing the results of investigator culture in three strains is shown in Table 2 below.
図４と表２に示されるように、ＱＵ４１株、ＱＵ４２株、及びＱＵ４３株の３つの菌株は、ＭＲＳ培地での培養において、類似した発酵挙動を示した。ＱＵ４１株は７２時間目においてＤ－乳酸生産量が１６．８ｇ／Ｌであり、最も高い値を示した。
As shown in FIG. 4 and Table 2, the three strains of the QU41 strain, the QU42 strain, and the QU43 strain showed similar fermentation behaviors when cultured in the MRS medium. The QUI41 strain showed the highest value of D-lactic acid production at 16.8 g / L at 72 hours.
試験管培養において、ラクトバシラス・デルブリッキーＱＵ４１株、ＱＵ４２株、及びＱＵ４３株の３株が収率や生産速度などの点で極めて類似した発酵特性を示したことから、ＱＵ４１株を代表株として用いて、以下に述べるＤ－乳酸生産における最適条件の検討を行なった。
In the test tube culture, Lactobacillus delbricky QUI41 strain, QUI42 strain, and QUI43 strain showed very similar fermentation characteristics in terms of yield, production rate, etc. The optimum conditions for D-lactic acid production described below were examined.
実施例２ 最適条件の検討
（１）最適温度の検討
ＱＵ４１株について、グルコース２０ｇ／ＬのＭＲＳ培地を用いて、３０℃、３７℃、４３℃、及び４９℃におけるＤ－乳酸生産能を検討した。なお１０規定のＮａＯＨによりｐＨを５．５に調整し、窒素ガス置換により嫌気的状態で実験を行った。培養は前培養を４０ｍｌスケールで６時間、主培養を４００ｍｌスケールで１２時間行った。 Example 2 Examination of Optimal Conditions (1) Examination of Optimal Temperature With regard to QUI41 strain, the ability to produce D-lactic acid at 30 ° C., 37 ° C., 43 ° C., and 49 ° C. was examined using 20 g / L glucose MRS medium. . The pH was adjusted to 5.5 with 10 N NaOH and the experiment was conducted in an anaerobic state by nitrogen gas replacement. The culture was performed for 6 hours on a 40 ml scale and 12 hours on a 400 ml scale for main culture.
（１）最適温度の検討
ＱＵ４１株について、グルコース２０ｇ／ＬのＭＲＳ培地を用いて、３０℃、３７℃、４３℃、及び４９℃におけるＤ－乳酸生産能を検討した。なお１０規定のＮａＯＨによりｐＨを５．５に調整し、窒素ガス置換により嫌気的状態で実験を行った。培養は前培養を４０ｍｌスケールで６時間、主培養を４００ｍｌスケールで１２時間行った。 Example 2 Examination of Optimal Conditions (1) Examination of Optimal Temperature With regard to QUI41 strain, the ability to produce D-lactic acid at 30 ° C., 37 ° C., 43 ° C., and 49 ° C. was examined using 20 g / L glucose MRS medium. . The pH was adjusted to 5.5 with 10 N NaOH and the experiment was conducted in an anaerobic state by nitrogen gas replacement. The culture was performed for 6 hours on a 40 ml scale and 12 hours on a 400 ml scale for main culture.
その結果を図５に示す。図５（ａ）は３０℃で検討を行なった結果であり、図５（ｂ）は３７℃で検討を行なった結果であり、図５（ｃ）は４３℃で検討を行なった結果であり、図５（ｄ）は４９℃で検討を行なった結果である。図５において左の縦軸はグルコース、乳酸、酢酸の濃度を示し、右の縦軸がＯＤを示し、横軸が時間を示す。また図５のプロットにおいて、黒丸がグルコース濃度、白丸が乳酸濃度、黒三角が酢酸の濃度であり、白三角がＯＤである。
The result is shown in FIG. FIG. 5 (a) shows the result of the examination at 30 ° C., FIG. 5 (b) shows the result of the examination at 37 ° C., and FIG. 5 (c) shows the result of the examination at 43 ° C. FIG. 5 (d) shows the result of examination at 49 ° C. In FIG. 5, the left vertical axis indicates glucose, lactic acid, and acetic acid concentrations, the right vertical axis indicates OD, and the horizontal axis indicates time. In the plot of FIG. 5, the black circle is the glucose concentration, the white circle is the lactic acid concentration, the black triangle is the acetic acid concentration, and the white triangle is the OD.
この結果から得た培養のパラメーターの値を、下の表３に示す。表３において左から、最大菌体増殖速度、Ｄ－乳酸生産量、最大収率、最大生産量、Ｄ－乳酸光学純度の値をそれぞれ示す。表３に示すように、４３℃の時に最も高い最大増殖速度と最大生産速度を示し、ＱＵ４１株の増殖とＤ－乳酸生産の最適温度は４３℃であった。なおこのデータには示さないが、ＱＵ４１株は５５℃でも増殖及びＤ－乳酸の産生が可能であった。
The values of the culture parameters obtained from this result are shown in Table 3 below. In Table 3, from the left, values of maximum cell growth rate, D-lactic acid production, maximum yield, maximum production, and D-lactic acid optical purity are shown. As shown in Table 3, the highest maximum growth rate and the maximum production rate were exhibited at 43 ° C., and the optimum temperature for growth and D-lactic acid production of the QUI41 strain was 43 ° C. Although not shown in this data, the QUI41 strain was able to grow and produce D-lactic acid even at 55 ° C.
（２）最適ｐＨの検討
最適温度を４３℃として、ＱＵ４１株のＤ－乳酸生産へｐＨが及ぼす影響を４つのｐＨ（ｐＨ５．０、５．５、６．０、６．５）で検討した。ｐＨを変えた以外は、上記の最適温度の検討と同じ条件で実験を行った。 (2) Examination of optimum pH The optimum temperature was set to 43 ° C., and the influence of pH on the D-lactic acid production of QUI41 strain was examined at four pHs (pH 5.0, 5.5, 6.0, 6.5). . The experiment was performed under the same conditions as those in the examination of the optimum temperature except that the pH was changed.
最適温度を４３℃として、ＱＵ４１株のＤ－乳酸生産へｐＨが及ぼす影響を４つのｐＨ（ｐＨ５．０、５．５、６．０、６．５）で検討した。ｐＨを変えた以外は、上記の最適温度の検討と同じ条件で実験を行った。 (2) Examination of optimum pH The optimum temperature was set to 43 ° C., and the influence of pH on the D-lactic acid production of QUI41 strain was examined at four pHs (pH 5.0, 5.5, 6.0, 6.5). . The experiment was performed under the same conditions as those in the examination of the optimum temperature except that the pH was changed.
その結果を図６に示す。なお図６（ａ）はｐＨ５．０で検討を行なった結果であり、図６（ｂ）はｐＨ５．５で検討を行なった結果であり、図６（ｃ）はｐＨ６．０で検討を行なった結果であり、図６（ｄ）はｐＨ６．５で検討を行なった結果である。図６において左の縦軸はグルコース、乳酸、酢酸の濃度を示し、右の縦軸がＯＤを示し、横軸が時間を示す。また図６のプロットにおいて、黒丸がグルコース濃度、白丸が乳酸濃度、黒三角が酢酸の濃度であり、白三角がＯＤである。
The result is shown in FIG. 6 (a) shows the results of investigation at pH 5.0, FIG. 6 (b) shows the results of examination at pH 5.5, and FIG. 6 (c) shows the results at pH 6.0. FIG. 6 (d) shows the results of investigation at pH 6.5. In FIG. 6, the left vertical axis indicates glucose, lactic acid, and acetic acid concentrations, the right vertical axis indicates OD, and the horizontal axis indicates time. In the plot of FIG. 6, the black circle is the glucose concentration, the white circle is the lactic acid concentration, the black triangle is the acetic acid concentration, and the white triangle is the OD.
この結果から得た培養のパラメーターの値を、下の表４に示す。表４において左から、最大菌体増殖速度、Ｄ－乳酸生産量、最大収率、最大生産量、Ｄ－乳酸光学純度の値をそれぞれ示す。ｐＨ６．０の時に１２時間で培養が終了し、且つ最大の菌体増殖速度と乳酸生産量を示したので、最適ｐＨは６．０であった。
The values of the culture parameters obtained from this result are shown in Table 4 below. In Table 4, from the left, the maximum cell growth rate, D-lactic acid production amount, maximum yield, maximum production amount, and D-lactic acid optical purity values are shown. Since the cultivation was completed in 12 hours at pH 6.0 and the maximum cell growth rate and lactic acid production were shown, the optimum pH was 6.0.
実施例３ 初発グルコース濃度が及ぼす影響の検討
温度が４３℃、ｐＨ６．０の条件下で、ＱＵ４１株のＤ－乳酸生産能にグルコース濃度が及ぼす影響を検討した。ｐＨを変えた以外は、最適温度及び最適ｐＨの検討と同じ条件で実験を行った。 Example 3 Examination of Influence of Initial Glucose Concentration Under the conditions of a temperature of 43 ° C. and pH 6.0, the influence of the glucose concentration on the D-lactic acid production ability of QUI41 strain was examined. The experiment was performed under the same conditions as those for the optimum temperature and optimum pH except that the pH was changed.
温度が４３℃、ｐＨ６．０の条件下で、ＱＵ４１株のＤ－乳酸生産能にグルコース濃度が及ぼす影響を検討した。ｐＨを変えた以外は、最適温度及び最適ｐＨの検討と同じ条件で実験を行った。 Example 3 Examination of Influence of Initial Glucose Concentration Under the conditions of a temperature of 43 ° C. and pH 6.0, the influence of the glucose concentration on the D-lactic acid production ability of QUI41 strain was examined. The experiment was performed under the same conditions as those for the optimum temperature and optimum pH except that the pH was changed.
培養開始時に添加したグルコースの濃度、即ち初発グルコース濃度を２０、５０、１００ｇ／Ｌとして検討を行なった結果を図７に示す。なお図７（ａ）は初発グルコース濃度が２０ｇ／Ｌのデータであり、図７（ｂ）は初発グルコース濃度が５０ｇ／Ｌのデータ（ｂ）であり、図７（ｃ）は初発グルコース濃度が１００ｇ／Ｌのデータである。図７において左の縦軸はグルコース、乳酸、酢酸の濃度を示し、右の縦軸がＯＤを示し、横軸が時間を示す。また図７のプロットにおいて、黒丸がグルコース濃度、白丸が乳酸濃度、黒三角が酢酸の濃度であり、白三角がＯＤである。
FIG. 7 shows the results of investigations with the concentration of glucose added at the start of culture, that is, the initial glucose concentration set to 20, 50, and 100 g / L. FIG. 7A shows data with an initial glucose concentration of 20 g / L, FIG. 7B shows data with an initial glucose concentration of 50 g / L (b), and FIG. 7C shows the initial glucose concentration. The data is 100 g / L. In FIG. 7, the left vertical axis indicates glucose, lactic acid, and acetic acid concentrations, the right vertical axis indicates OD, and the horizontal axis indicates time. In the plot of FIG. 7, the black circle is the glucose concentration, the white circle is the lactic acid concentration, the black triangle is the acetic acid concentration, and the white triangle is the OD.
この結果から得た培養のパラメーターの値を、下の表５に示す。表５において左から、最大菌体増殖速度、Ｄ－乳酸生産量、最大収率、最大生産量、Ｄ－乳酸光学純度の値をそれぞれ示す。初発グルコース濃度の増加に伴い、最大ＯＤや乳酸生産性は高まったが、一方、最大菌体増殖速度は低下し、培養時間経過に伴って乳酸生産速度も低下した。この結果から、ＱＵ４１は高濃度のグルコースや、乳酸により阻害を受けるので、細菌の増殖に伴う培地環境の変化に影響される回分培養では、効率的にＤ－乳酸を生産できないことが判った。
The values of the culture parameters obtained from this result are shown in Table 5 below. In Table 5, the maximum cell growth rate, D-lactic acid production amount, maximum yield, maximum production amount, and D-lactic acid optical purity values are shown from the left. As the initial glucose concentration increased, the maximum OD and lactic acid productivity increased, but on the other hand, the maximum cell growth rate decreased and the lactic acid production rate also decreased with the lapse of culture time. From these results, it was found that QUI41 is inhibited by high concentrations of glucose and lactic acid, so that D-lactic acid cannot be efficiently produced by batch culture affected by changes in the medium environment accompanying bacterial growth.
実施例４ ラクトバシラス・デルブリッキーＱＵ４１株と既存のＤ－乳酸生産菌の比較
ラクトバシラス・デルブリッキーＱＵ４１株と、既存のＤ－乳酸生産菌であるＪＣＭ１１６６及びＪＣＭ１２４８の間で、Ｄ－乳酸生産量の比較を行った。図８に結果を示す。図８（ａ）は３７℃で測定したデータであり、図８（ｂ）は４３℃で測定したデータであり、図８（ｃ）は５０℃で測定したデータである。図８において左の縦軸は乳酸の濃度を示し、右の縦軸はＯＤを示し、横軸は時間を示す。 Example 4 Comparison of Lactobacillus delbrikkii QUA41 strain and existing D-lactic acid producing bacteria Comparison of D-lactic acid production between Lactobacillus delbrikkii QUI41 strain and existing D-lactic acid producing bacteria JCM1166 and JCM1248 It was. The results are shown in FIG. 8A shows data measured at 37 ° C., FIG. 8B shows data measured at 43 ° C., and FIG. 8C shows data measured at 50 ° C. In FIG. 8, the left vertical axis indicates the concentration of lactic acid, the right vertical axis indicates OD, and the horizontal axis indicates time.
ラクトバシラス・デルブリッキーＱＵ４１株と、既存のＤ－乳酸生産菌であるＪＣＭ１１６６及びＪＣＭ１２４８の間で、Ｄ－乳酸生産量の比較を行った。図８に結果を示す。図８（ａ）は３７℃で測定したデータであり、図８（ｂ）は４３℃で測定したデータであり、図８（ｃ）は５０℃で測定したデータである。図８において左の縦軸は乳酸の濃度を示し、右の縦軸はＯＤを示し、横軸は時間を示す。 Example 4 Comparison of Lactobacillus delbrikkii QUA41 strain and existing D-lactic acid producing bacteria Comparison of D-lactic acid production between Lactobacillus delbrikkii QUI41 strain and existing D-lactic acid producing bacteria JCM1166 and JCM1248 It was. The results are shown in FIG. 8A shows data measured at 37 ° C., FIG. 8B shows data measured at 43 ° C., and FIG. 8C shows data measured at 50 ° C. In FIG. 8, the left vertical axis indicates the concentration of lactic acid, the right vertical axis indicates OD, and the horizontal axis indicates time.
ラクトバシラス・デルブリッキーＱＵ４１株のみ、図８（ｄ）に示すように、５５℃においても測定を行なった。なお右カラムは培養後２４時間におけるＤ－乳酸生産量であり、左カラムは培養後４８時間におけるＤ－乳酸生産量である。さらに、この結果から得た３つの菌株のＤ－乳酸生産量の値を表６に示す。
Only Lactobacillus del Bricky QU41 strain was also measured at 55 ° C. as shown in FIG. The right column shows the amount of D-lactic acid produced 24 hours after culturing, and the left column shows the amount of D-lactic acid produced 48 hours after culturing. Furthermore, Table 6 shows the values of D-lactic acid production of the three strains obtained from the results.
図８と表６から判るように、各温度でラクトバシラス・デルブリッキーＱＵ４１株は、既存のＤ－乳酸生産菌よりも高いＤ－乳酸生産能を示した。
As can be seen from FIG. 8 and Table 6, at each temperature, the Lactobacillus delbrikkii QUI41 strain exhibited higher D-lactic acid-producing ability than existing D-lactic acid-producing bacteria.
実施例５ 培地の中和剤の検討
ＱＵ４１株の培養に用いる培地の中和剤として、ＮａＯＨを用いた場合とＮＨ４ＯＨを用いた場合の比較を行なった。結果を図９に示す。なお図９（ａ）は中和剤としてＮａＯＨを用いた場合のデータであり、図９（ｂ）は中和剤としてＮＨ４ＯＨを用いた場合のデータである。図９において左の縦軸は乳酸の濃度を示し、右の縦軸がＯＤを示し、横軸が時間を示す。また図９のプロットにおいて、黒丸がグルコース濃度、白丸が乳酸濃度、黒三角が酢酸の濃度であり、白三角がＯＤである。 Example 5 Examination of Neutralizing Agent for Medium As a neutralizing agent for the medium used for culturing QUA41 strain, a comparison was made between the case of using NaOH and the case of using NH 4 OH. The results are shown in FIG. FIG. 9A shows data when NaOH is used as a neutralizing agent, and FIG. 9B shows data when NH 4 OH is used as a neutralizing agent. In FIG. 9, the left vertical axis indicates the concentration of lactic acid, the right vertical axis indicates OD, and the horizontal axis indicates time. In the plot of FIG. 9, the black circle is the glucose concentration, the white circle is the lactic acid concentration, the black triangle is the acetic acid concentration, and the white triangle is the OD.
ＱＵ４１株の培養に用いる培地の中和剤として、ＮａＯＨを用いた場合とＮＨ４ＯＨを用いた場合の比較を行なった。結果を図９に示す。なお図９（ａ）は中和剤としてＮａＯＨを用いた場合のデータであり、図９（ｂ）は中和剤としてＮＨ４ＯＨを用いた場合のデータである。図９において左の縦軸は乳酸の濃度を示し、右の縦軸がＯＤを示し、横軸が時間を示す。また図９のプロットにおいて、黒丸がグルコース濃度、白丸が乳酸濃度、黒三角が酢酸の濃度であり、白三角がＯＤである。 Example 5 Examination of Neutralizing Agent for Medium As a neutralizing agent for the medium used for culturing QUA41 strain, a comparison was made between the case of using NaOH and the case of using NH 4 OH. The results are shown in FIG. FIG. 9A shows data when NaOH is used as a neutralizing agent, and FIG. 9B shows data when NH 4 OH is used as a neutralizing agent. In FIG. 9, the left vertical axis indicates the concentration of lactic acid, the right vertical axis indicates OD, and the horizontal axis indicates time. In the plot of FIG. 9, the black circle is the glucose concentration, the white circle is the lactic acid concentration, the black triangle is the acetic acid concentration, and the white triangle is the OD.
この結果から得た培養のパラメーターの値を、下の表７に示す。表７において左から、最大菌体増殖速度、Ｄ－乳酸生産量、最大収率、最大生産量、Ｄ－乳酸光学純度の値をそれぞれ示す。
The values of the culture parameters obtained from this result are shown in Table 7 below. In Table 7, the maximum cell growth rate, D-lactic acid production amount, maximum yield, maximum production amount, and D-lactic acid optical purity values are shown from the left.
ＯＤと菌体増殖速度に関しては、ＮＨ４ＯＨを用いた場合とＮａＯＨを用いた場合において、同程度の結果であった。一方Ｄ－乳酸生産量と最大Ｄ－乳酸生産速度については培養後８時間において、ＮＨ４ＯＨを用いた場合には、ＮａＯＨを用いた場合と比較して高い値を示した。
Regarding the OD and the cell growth rate, the results were similar when NH 4 OH was used and when NaOH was used. On the other hand, the amount of D-lactic acid produced and the maximum D-lactic acid production rate were higher when NH 4 OH was used than when NaOH was used at 8 hours after culturing.
これまでＬａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ ｓｕｂｓｐ．ｄｅｌｂｒｕｅｃｋｉｉを用いたＤ－乳酸生産において、中和剤にＮＨ４ＯＨ、ＮａＯＨを用いた場合と比較して炭酸カルシウムを用いた方が、Ｄ－乳酸産生量が高いことが報告されている。しかし中和剤にＮＨ４ＯＨを用いるＮＨ３回収－ブチルエステル化プロセス（ＢＵＬ）精製法は、炭酸カルシウムによる中和と比較して、廃棄物の発生が少なく、中和剤の回収も可能であるといった長所を有している。この結果から判るようにＱＵ４１株はアンモニアによる乳酸発酵への悪影響が見られなかったので、ＢＵＬ精製法と組み合わせることにより環境に対する負荷が低い乳酸発酵が可能となる。
Until now, Lactobacillus delbrueckii subsp. In D-lactic acid production using delbrueckii, it has been reported that the amount of D-lactic acid produced is higher when calcium carbonate is used than when NH 4 OH or NaOH is used as a neutralizing agent. However, the NH 3 recovery-butyl esterification process (BUL) purification method using NH 4 OH as the neutralizing agent generates less waste and can recover the neutralizing agent compared to neutralization with calcium carbonate. It has the advantage of being. As can be seen from this result, the QUA41 strain did not have an adverse effect on the lactic acid fermentation by ammonia, so that it can be combined with the BUL purification method to perform lactic acid fermentation with a low environmental load.
Claims (7)
- （１）グルコースを基質として９９％以上の光学純度でＤ－乳酸を生産可能であり、かつ
（２）グルコース濃度が２０ｇ／Ｌ、ｐＨ６．０の条件で開始し、４３℃の温度で１２時間培養したときに、１２．０ｇ／Ｌ以上、好ましくは１６．０ｇ／Ｌ以上、さらに好ましくは２０．０ｇ／Ｌ以上のＤ－乳酸を生産可能である、
ラクトバシラス・デルブリッキーに属する乳酸菌。 (1) D-lactic acid can be produced with an optical purity of 99% or more using glucose as a substrate, and (2) starting at the glucose concentration of 20 g / L and pH 6.0, and at a temperature of 43 ° C. for 12 hours When cultured, it can produce 1-2.0 g / L or more, preferably 16.0 g / L or more, more preferably 20.0 g / L or more of D-lactic acid.
Lactic acid bacteria belonging to Lactobacillus del Bricky. - さらに、
（３）４９℃の温度でＤ－乳酸を生産可能である、
請求項１に記載の乳酸菌。 further,
(3) D-lactic acid can be produced at a temperature of 49 ° C.
The lactic acid bacterium according to claim 1. - 下記の菌学的性質を有する、請求項１記載の乳酸菌：
１．形態：桿菌、
２．生化学的性質：カタラーゼ陰性、
３．運動性：なし、
４．酸素要求性：通性嫌気性、
５．グルコースを基質としてホモ乳酸発酵によりＤ－乳酸を産生する、及び
６．グルコース、フルクトース、マンノース、Ｎ－アセチルグルコサミン、マルトース、ラクトース、スクロース、トレハロースを資化することができ、キシロース、アラビノースは資化することができない。 Lactic acid bacteria according to claim 1, having the following mycological properties:
1. Form: Neisseria gonorrhoeae
2. Biochemical properties: Catalase negative,
3. Mobility: None,
4). Oxygen demand: facultative anaerobic,
5). 5. D-lactic acid is produced by homolactic fermentation using glucose as a substrate, and Glucose, fructose, mannose, N-acetylglucosamine, maltose, lactose, sucrose and trehalose can be assimilated, and xylose and arabinose cannot be assimilated. - 下記のいずれかの配列からなる１６ＳｒＲＮＡ遺伝子を有する、請求項１記載の乳酸菌：
（ａ）配列表の配列番号：１で示されるヌクレオチド配列、又はそれと９９．７％以上の同一性を有するヌクレオチド配列；
（ｂ）配列表の配列番号：２で示されるヌクレオチド配列、又はそれと９９．７５％以上の同一性を有するヌクレオチド配列；又は
（ｃ）配列表の配列番号：３で示されるヌクレオチド配列、又はそれと９９．８％以上の同一性を有するヌクレオチド配列。 The lactic acid bacterium according to claim 1, which has a 16S rRNA gene consisting of any of the following sequences:
(A) a nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing, or a nucleotide sequence having 99.7% or more identity thereto;
(B) a nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing, or a nucleotide sequence having 99.75% or more identity thereto; or (c) a nucleotide sequence represented by SEQ ID NO: 3 in the sequence listing; or A nucleotide sequence having an identity of 99.8% or more. - ＮＩＴＥ ＢＰ－６７９の受託番号で寄託されたラクトバシラス・デルブリッキーＱＵ４１株である、請求項１～４のいずれか１項に記載の乳酸菌。 The lactic acid bacterium according to any one of claims 1 to 4, which is the Lactobacillus delbricki QUI41 strain deposited under the deposit number of NITE BP-679.
- 請求項１～５のいずれか１項に記載の乳酸菌を培養する工程を含む、乳酸を製造する方法。 A method for producing lactic acid, comprising a step of culturing the lactic acid bacterium according to any one of claims 1 to 5.
- 乳酸が、Ｄ－乳酸を９９％以上の光学純度で含む、請求項６に記載の製造方法。 The production method according to claim 6, wherein the lactic acid contains D-lactic acid with an optical purity of 99% or more.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103589668A (en) * | 2013-10-24 | 2014-02-19 | 天津大学 | Method for producing D-lactic acid bacteria strain by fermenting cane molasses and fermentation method of cane molasses |
| CN103787561A (en) * | 2014-01-23 | 2014-05-14 | 杭州师范大学 | Application of trehalose in preservation of anaerobic ammonia oxidation granular sludge |
| WO2021032896A1 (en) * | 2019-08-22 | 2021-02-25 | Brainboost Solutions | Food, food precursors or beverages comprising d-lactic acid and/or a salt thereof and a method of producing the same |
| CN114381393A (en) * | 2021-12-21 | 2022-04-22 | 西藏安琪生物科技有限公司 | Lactobacillus delbrueckii subsp lactis strain and application thereof |
| CN118599735A (en) * | 2024-07-12 | 2024-09-06 | 南京工业大学 | A kind of lactic acid bacteria resistant to low pH and producing D-lactic acid and its application |
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| KR101764095B1 (en) * | 2011-04-11 | 2017-08-14 | 동국대학교 산학협력단 | Method for preparation of lactic acid bacteria powder |
| JP2015080430A (en) * | 2013-10-22 | 2015-04-27 | 王子ホールディングス株式会社 | Method for producing d-lactic acid |
| JP6331327B2 (en) * | 2013-10-22 | 2018-05-30 | 王子ホールディングス株式会社 | Method for producing D-lactic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103589668A (en) * | 2013-10-24 | 2014-02-19 | 天津大学 | Method for producing D-lactic acid bacteria strain by fermenting cane molasses and fermentation method of cane molasses |
| CN103787561A (en) * | 2014-01-23 | 2014-05-14 | 杭州师范大学 | Application of trehalose in preservation of anaerobic ammonia oxidation granular sludge |
| CN103787561B (en) * | 2014-01-23 | 2016-04-20 | 杭州师范大学 | The application of trehalose in anaerobic ammonium oxidation granular sludge is preserved |
| WO2021032896A1 (en) * | 2019-08-22 | 2021-02-25 | Brainboost Solutions | Food, food precursors or beverages comprising d-lactic acid and/or a salt thereof and a method of producing the same |
| CN114381393A (en) * | 2021-12-21 | 2022-04-22 | 西藏安琪生物科技有限公司 | Lactobacillus delbrueckii subsp lactis strain and application thereof |
| CN114381393B (en) * | 2021-12-21 | 2024-03-29 | 西藏安琪生物科技有限公司 | Lactobacillus delbrueckii subspecies lactis strain and application thereof |
| CN118599735A (en) * | 2024-07-12 | 2024-09-06 | 南京工业大学 | A kind of lactic acid bacteria resistant to low pH and producing D-lactic acid and its application |
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