WO2015106627A1 - 一株土芽孢乳杆菌及其应用 - Google Patents

一株土芽孢乳杆菌及其应用 Download PDF

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WO2015106627A1
WO2015106627A1 PCT/CN2014/095311 CN2014095311W WO2015106627A1 WO 2015106627 A1 WO2015106627 A1 WO 2015106627A1 CN 2014095311 W CN2014095311 W CN 2014095311W WO 2015106627 A1 WO2015106627 A1 WO 2015106627A1
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fermentation
lactic acid
medium
hkm
cultured
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PCT/CN2014/095311
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French (fr)
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许平
黄开明
唐鸿志
陶飞
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上海交通大学
许平
黄开明
唐鸿志
陶飞
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/56—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

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  • the invention discloses a strain of D-lactic acid preparation technology and a application method thereof, in particular to preparing a high optical purity and high concentration D-lactic acid of Lactobacillus sphaericus using an inexpensive nitrogen source-a corn syrup dry powder as an organic nitrogen source. And its application methods.
  • Lactic acid also known as ⁇ -hydroxypropionic acid
  • the production methods mainly include chemical synthesis method and microbial fermentation method.
  • the chemical method can only synthesize DL-lactic acid, and the fermentation method can synthesize a single L-lactic acid, D-lactic acid or DL-lactic acid depending on the strain used.
  • Currently, about 90% of lactic acid is produced by microbial fermentation.
  • L-lactic acid Since the human body only has L-lactate dehydrogenase which metabolizes L-lactic acid, L-lactic acid is widely used as a food additive, and the application of L-lactic acid in polylactic acid, the breeding of L-lactic acid high-yield bacteria and efficient production process have been Highly developed, while D-lactic acid research is relatively rare.
  • D-lactic acid is widely used in the synthesis of various chiral substances in medicine, pesticides and chemicals.
  • D-lactic acid can be used as a monomer for the next generation of high-strength biodegradable plastic polylactic acid, which has attracted the attention of large companies and scientists around the world.
  • the global demand for D-lactic acid is increasing at a rate of 6 to 8% per year.
  • the world's D-lactic acid production is 16,000 tons, while the demand for D-lactic acid is about 26,000 tons.
  • D-lactic acid can be seen.
  • the market has broad prospects.
  • aryloxypropionic acid herbicides have become more and more widely used in agriculture. These herbicides are the first kind of optically active herbicides in the world to achieve industrial production. There is a chiral carbon in such herbicides.
  • the atom, in which the D(-) type is 6 to 12 times more potent than the L(+) type, the most important raw material for the synthesis of such herbicides is R-(+)-2-chloropropionic acid.
  • the optically active R-(+)-2-chloropropionic acid can be prepared by using D-lactic acid as a raw material, that is, D-lactic acid is an optically active precursor for preparing such a herbicide.
  • polylactic acid (PLA) biodegradable plastics are produced using lactic acid as a raw material.
  • Polylactic acid is considered to be one of the ideal biomaterials to replace traditional plastics because of its good biodegradability and other excellent use characteristics (such as transparency, thermoplasticity, product safety, etc.).
  • Polylactic acid (PLA) is classified into poly D-lactic acid (PDLA), poly-L-lactic acid (PLLA), and poly-DL-lactic acid (PDLLA).
  • PDLA poly-lactic acid
  • Polylactic acid obtained by polymerizing pure D-lactic acid, L-lactic acid or DL-lactic acid has better stability, and PDLA has better thermal stability.
  • Polylactic acid has good biodegradability and can be completely degraded by microorganisms in nature after use, and does not pollute the environment, which is very beneficial to environmental protection.
  • Ding Zijian of Nanjing University of Technology was the first to report the process of producing D-lactic acid from glucose by using Sporolactobacillus sp. in 2004. After 72h of fermentation, the acid yield was 40.7g/L, and the optical purity was 96%. Yang Wenge, Nanjing University of Technology, etc.
  • the combination fermentation production D-lactic acid process (Chinese Patent Application No. CN200610097453.6) was carried out, and the aerobic, micro-oxygen and anaerobic three-stage combined fermentation technology was carried out by Lactobacillus. The fermentation time was 25-38 h, and the acid production was achieved. 75-131g/L; Chinese invention patent No.
  • CN201010101421.5 discloses a method for preparing D-lactic acid by using recombinant Escherichia coli to ferment glycerol as a raw material, but the strain has poor acid tolerance and low productivity, so that Escherichia coli is The practical application of D-lactic acid is limited;
  • the Chinese invention with application number CN201010247826.X discloses the use of genetic engineering means to successfully construct genetically engineered bacteria producing high optical purity D-lactic acid and use it for lactic acid fermentation production, D- The lactic acid production reaches 40g/L or more, and the purity is 99% or more;
  • the Chinese invention patent No. CN201010235511.3 discloses the use of homologous recombination.
  • the method of obtaining genetically engineered bacteria producing pure D-lactic acid and using it for lactic acid fermentation production has a D-lactic acid yield of only 20 g/L or more and a purity of 99% or more;
  • the Chinese invention patent No. CN201010235511.3 discloses the use of the same
  • the method of source recombination obtains a genetically engineered strain producing pure D-lactic acid and is used for lactic acid fermentation production, and the D-lactic acid yield is only 20 g/L or more, and the purity is 99% or more.
  • the above-mentioned prior art D-lactic acid production is generally not high. Continuing the search and found that the Chinese invention patent No.
  • CN200810098908.5 discloses a high optical purity D-lactic acid producing bacteria and a process for producing D-lactic acid by fermentation
  • the Chinese invention patent No. CN201210472413.0 discloses a strain. Lactobacillus and a method for producing D-lactic acid by fermentation thereof
  • Chinese Patent Application No. CN201010208148.6 discloses a method for producing high-concentration D-lactic acid by simultaneous enzymatic hydrolysis of peanut meal and a special medium thereof.
  • the above prior art fermentation time is too long, both of which are above 70h.
  • the present invention is directed to the above-mentioned deficiencies of the prior art, and provides a method for preparing D-lactic acid, and a method for using the same, which can directly utilize a six-carbon sugar such as glucose, maltose, lactose, sucrose or the like as a carbon source; Powder, peptone, peanut meal, corn syrup dry powder as a nitrogen source for the production of D-lactic acid, fermentation to produce high concentration, high optical purity of D-lactic acid, to improve production efficiency while saving costs, suitable for industrial applications.
  • a six-carbon sugar such as glucose, maltose, lactose, sucrose or the like
  • Powder, peptone, peanut meal, corn syrup dry powder as a nitrogen source for the production of D-lactic acid, fermentation to produce high concentration, high optical purity of D-lactic acid, to improve production efficiency while saving costs, suitable for industrial applications.
  • CCTCC Sporolactobacillus terrae HKM-1, deposited in the China Center for Type Culture Collection, referred to as CCTCC, with the accession number CCTCC NO: M2013389, and the deposit date is: September 2, 2013, and the deposit is Address: Wuhan University, Wuhan, China, classified as: Sporolactobacillus terrae.
  • the present invention relates to the above-mentioned application method for preparing D-lactic acid for Lactobacillus: firstly, seed culture of Bacillus licheniformis to obtain a seed culture liquid, and then Glucose, maltose, lactose, and sucrose are used as a carbon source, and yeast powder, peptone, peanut meal, and corn syrup dry powder are fermented and cultured as a nitrogen source to obtain D-lactic acid.
  • the strain of Lactobacillus subtilis was inoculated on a solid slant medium containing 20 g/L agar, and cultured at 40 to 45 ° C for 24 to 48 hours;
  • the slanted cultured Lactobacillus sp. is inoculated into the seed culture medium under aseptic conditions, and cultured at 35 to 45 ° C for 24 to 36 hours, and a neutralization agent is added to control the pH of the fermentation broth to prepare a seed culture solution;
  • the inoculation amount is 5 to 20% by volume, and is introduced into the fermentation medium, and cultured in an environment of 35 to 45 ° C for 48 to 72 hours, and the temperature is preferably 42 ° C.
  • the seed culture medium according to the step 2) contains: 100-120 g of glucose, 8-12 g of yeast powder, 3-8 g of peptone, 50 g of calcium carbonate, and the balance is water, preferably containing: glucose 100 g, yeast powder per liter. 10 g, peptone 5 g, calcium carbonate 50 g, the balance being water; the pH of the seed medium was 6.0. Sterilization at 115 ° C for 15 min.
  • the neutralizing agent includes one or more of NaOH, NH 4 OH, and Ca(OH) 2 .
  • the components of the fermentation medium and the content thereof in the step 3) are: a carbon source of 40 to 120 g/L, and a nitrogen source addition amount of 5 to 20 g/L.
  • the components of the fermentation medium and the content thereof in the step 3) are: glucose/sucrose/maltose/fructose 40-120 g/L, corn syrup dry powder 10-20 g/L, yeast powder 3-10 g/L , peptone 3 ⁇ 10g / L, peanut meal 5 ⁇ 20g / L, neutralizing agent (NH 4 OH, NaOH, Ca (OH) 2) used to regulate the pH of the medium, the balance is water. Sterilize at 115 ° C for 15 min.
  • the fermentation process described in the step 3) is a feed flow addition process, and the feed flow addition process refers to: adding a carbon source when the total reducing sugar content in the fermentation liquid is less than 20 to 30 g/L, so that The total reducing sugar content is maintained at 30 to 70 g/L, or 50 to 70 g/L.
  • the fermentation medium has a pH of 5.0 to 7.0.
  • Glucose, sucrose, fructose, and maltose are used as carbon sources, and yeast powder, peptone, peanut meal, and corn syrup are used as nitrogen sources.
  • yeast powder, peptone, peanut meal, and corn syrup are used as nitrogen sources.
  • the above materials are commercially available.
  • the invention selects glucose, sucrose, maltose and fructose as carbon sources, yeast powder, peptone, peanut meal and corn syrup dry powder as nitrogen source, and the corresponding fermentation process conditions make the D-lactic acid production process raw material easy and low in cost.
  • the yield of D-lactic acid is up to 197g/L, the optical purity is more than 99.9%, the conversion rate of sugar acid is up to 0.94g/g, and the fermentation production capacity is 4.19g/L/h. Therefore, the production of D-lactic acid by the method of the invention can save cost and simplify the operation process, and has broad industrial application prospects.
  • Figure 1 is a phylogenetic tree analysis of the 16S rRNA system of Lactobacillus sp. (HKM-1).
  • the horizontal distance (sum of length) of the solid line represents the evolution distance of the strain
  • the selected reference bacteria are strains which are closely related to the Lactobacillus sp. (HKM-1) provided by the present invention, and 10 strains are closely related.
  • composition of the medium used in this example is as follows:
  • Nutrient liquid medium glucose 80g/L, yeast powder 8g/L, peptone 3g/L, calcium carbonate 35g/L, pH 6.
  • Nutrition agar medium glucose 40g / L, yeast powder 10g / L, peptone 5%, calcium carbonate 2g / L, agar powder 20g / L, pH of 6. sterilized at 115 ° C for 15min.
  • the strain screening medium glucose 120 g/L, yeast powder 10 g/L, peptone 5 g/L, calcium carbonate 50 g/L, pH 6. sterilized at 115 ° C for 15 min.
  • the soil collected from Yunnan was weighed and dissolved in 40 mL of nutrient liquid medium, and enriched and cultured at 42 ° C for 24 h. Then, the culture solution was diluted with sterile physiological saline, and diluted 10 times, 100 times, 1000 times, and 10000 times, respectively, and then applied to a culture dish containing nutrient agar medium, and cultured at 42 ° C for 48 hours. After growing out of single bacteria, select colonies with large colony area and acid-producing transparent circle, inoculate into fermentation medium, and culture at 42 °C for 48 h, measure the yield of D-lactic acid. After multiple screenings, pick a D - Strains with higher lactic acid production.
  • the above strains were streaked and purified on the nutrient agar plate several times, and then subjected to 10 cycles of fermentation test.
  • the D-lactic acid production and conversion rate produced by the 10 cycles of fermentation were basically maintained at the original level, which proved that the above strain was the target.
  • the strain is named HKM-1.
  • CTCC China Center for Type Culture Collection
  • the strain HKM-1 was cultured, and when the OD 600 was 2 to 5, the whole genome of the strain HKM-1 was extracted, and then the DNA sequence of the 16S rRNA of the strain HKM-1 was amplified.
  • the selected reference bacteria are strains closely related to the strains provided by the present invention, a total of 10 strains, and the phylogenetic tree analysis diagram of the strain HKM-1 is shown in Fig. 1.
  • the strain HKM-1 was identified as Sporalactobacillus terrae and deposited in the German Collection of Microorganisms and Cultures (DSMZ) under the accession number DSM No.27555. Deposited in the China Center for Type Culture Collection, the deposit number is CCTCC NO: M 2013389.
  • the gyrB gene is a gene of B subunit protein in DNA gyrase, which has higher resolution than the non-protein-coding gene 16S rDNA in distinguishing and identifying bacterial related species.
  • the gyrB conserved gene sequence of Lactobacillus subsp. HKM-1, as shown in the sequence listing (II), is 1,920 bp long, and has two base differences compared to the gyrB gene sequence of DSM 11697.
  • Lactobacillus sphaericus HKM-1 is different from the previously reported model strain DSM 11697 of Lactobacillus licheniformis, which is a new strain of Lactobacillus sp.
  • the strain is a Gram-positive bacterium, and the cell size of the cytotrophic cells is (0.8-0.9) ⁇ m ⁇ (3.0-5.0) ⁇ m. Endophytes are formed. On the agar plates containing glucose, yeast powder and peptone, round colonies with smooth, milky white edges and neat edges were formed; the bacteria formed small, pale milky white, rounded colonies with neat edges.
  • the steps of the method for producing D-lactic acid by fermentation using Lactobacillus subtilis HKM-1 in the following examples are as follows: (1) Inclined culture: Inoculation of Lactobacillus subtilis HKM-1 strain on solid slant culture containing 20 g/L agar The substrate was cultured at 40 to 45 ° C for 24 to 48 hours.
  • Seed culture the slant culture of step (1) is inoculated into 40 mL of seed culture medium under aseptic conditions, and cultured at 35-45 ° C for 24 to 36 hours, and a neutralizing agent is added to control the pH of the fermentation broth. , to prepare a seed culture solution.
  • Fermentation culture The seed culture solution is inoculated into the fermentation medium at a seeding rate of 5 to 20% by volume, and cultured at 35 to 45 ° C for 48 to 72 hours.
  • the cell culture temperature described in the steps (1), (2), and (3) is preferably 42 °C.
  • the neutralizing agent added during the culture process described in the steps (2) and (3) is calcium carbonate, and the pH is controlled.
  • the fermentation broth is taken every 5 hours, first centrifuged at 12,000 rpm for 5 min, the supernatant is taken, heated in a boiling water bath for 10 min, and then centrifuged at 12,000 rpm for 5 min, and the supernatant is taken to detect the concentration of L-lactic acid in the fermentation broth.
  • D-lactic acid concentration, glucose concentration, calculation of sugar acid conversion rate, D-lactic acid fermentation productivity and D-lactic acid optical purity is taken every 5 hours, first centrifuged at 12,000 rpm for 5 min, the supernatant is taken, heated in a boiling water bath for 10 min, and then centrifuged at 12,000 rpm for 5 min, and the supernatant is taken to detect the concentration of L-lactic acid in the fermentation broth.
  • D-lactic acid concentration, glucose concentration, calculation of sugar acid conversion rate, D-lactic acid fermentation productivity and D-lactic acid optical purity are examples of D-lactic acid concentration, glucose concentration, calculation of sugar acid conversion rate, D-lactic acid fermentation productivity and D-lactic acid optical purity.
  • the method for determining total reducing sugar is the DNS method.
  • Glucose is measured by diluting the fermentation broth and centrifuging it, and measuring it with a biosensor analyzer SBA-40D (Shandong Academy of Sciences).
  • the biosensor analyzer SBA-40D is an analytical instrument using an immobilized enzyme as a sensor.
  • Glucose, oxygen and water are used to generate hydrogen peroxide under the catalysis of an enzyme.
  • the hydrogen peroxide released by the reaction is in contact with the platinum-silver electrode and generates a current signal which is linearly proportional to the glucose concentration.
  • the glucose concentration can be obtained by measuring the current signal intensity.
  • L-lactic acid is determined by using biosensor analyzer SBA-40D, diluting the fermentation broth 100 times, and injecting 25 ⁇ L of the diluent into the biosensor analyzer SBA-40D with a syringe to directly read L-lactic acid. content.
  • the D-lactic acid was measured using an Agilent 1100 liquid chromatograph equipped with a chiral separation column (Mitsubishi Chemical Co., Ltd., MCI GEL-CRS10W (3 ⁇ ) 4.6 ID ⁇ 50 mm, for optical separation).
  • the specific operating conditions are: 0.005mol/L copper sulfate as mobile phase, flow rate 0.7mL/min, injection volume 5 ⁇ l, UV detector, detection wavelength 254nm, operating temperature 25°C.
  • a standard curve was prepared using D-lactic acid standard, and the content of D-lactic acid in the fermentation broth was calculated according to the standard curve.
  • D-lactic acid as a standard product is a product of Sigma-Aldrich, Germany, and its article number is L0625-25MG. Under the above chromatographic conditions, the D-lactic acid retention time was 6.5 min.
  • optical purity is a measure of the amount of one enantiomer in an optically active sample over the other enantiomer. It can be expressed as an enantiomeric excess (ee).
  • the optical purity (ee) of L-lactic acid in the present invention is calculated by the following formula: [(D-lactic acid production (g/L)-L-lactic acid production (g/L)) ⁇ (D-lactic acid production + L-lactic acid production) )] ⁇ 100%.
  • the sugar acid conversion rate is defined as (g/g): D-lactic acid yield (g) ⁇ substrate consumption (g).
  • the D-lactic acid production rate (g/L/h) is: L-lactic acid production (g/L) ⁇ fermentation time (h).
  • composition of each medium used in this example is as follows:
  • the slant medium contains: 30-50 g of glucose, 5-10 g of yeast powder, 2-8 g of peptone, 50 g of calcium carbonate, 15-25 g of agar powder, and the balance is water.
  • the pH of the slant medium was 6.0 and sterilized at 115 ° C for 15 min.
  • the seed culture medium contains: 40-120 g of glucose, 5-10 g of yeast powder, 2-8 g of peptone, 50 g of calcium carbonate, and the balance is water.
  • the seed culture medium has a pH of 5.0 to 7.0 and is sterilized at 115 ° C for 15 minutes.
  • the fermentation medium contains: 60-120 g of glucose, 20 g of corn syrup dry powder, 50 g of calcium carbonate, and the balance is water; the pH of the fermentation medium is 5.5-6.5.
  • the sterilization is carried out at 115 ° C for 15 min.
  • Inclined culture Bacillus licheniformis (HKM-1) was inoculated on a slant medium, and cultured at 42 ° C for 24 h;
  • Seed culture The strain cultured in the step (1) is cultured under sterile conditions in a 100 mL flask containing 40 mL of seed culture medium, and cultured at 42 ° C for 24 hours to prepare a seed culture solution. ;
  • Fermentation culture 5 mL of the seed culture solution prepared in the step (2) is placed in a 100 mL flask containing 40 mL of fermentation medium, and cultured at 42 ° C. When the rate of change of D-lactic acid is 0, it is regarded as The fermentation is over.
  • the D-lactic acid concentration and the total reducing sugar concentration in the fermentation broth were measured according to the detection and calculation methods described in the above specific embodiments, and the sugar acid conversion rate and the production rate were calculated.
  • Table 4 shows the formation of D-lactic acid with glucose as the carbon source.
  • the slant medium and the seed medium were the same as in Example 1.
  • the fermentation medium contains: sucrose 60-120 g, corn syrup dry powder 20 g, calcium carbonate 50 g, and the balance is water; the fermentation medium has a pH of 5.0-7.0 at 115 ° C for 15 min.
  • the method for producing D-lactic acid by fermentation comprises the following steps:
  • Example 1 Inclined culture: the same as in Example 1;
  • Fermentation culture 5 mL of the seed culture solution prepared in the step (2) is placed in a 100 mL flask containing 40 mL of fermentation medium, and cultured at 42 ° C. When the rate of change of D-lactic acid is 0, it is regarded as The fermentation is over.
  • the D-lactic acid concentration and the total reducing sugar concentration in the fermentation broth were measured according to the detection and calculation methods described in the above specific embodiments, and the sugar acid conversion rate and the production rate were calculated.
  • Lactobacillus sp. (HKM-1), maltose was used as a carbon source in a triangular flask, and the dry powder of corn syrup was used as a nitrogen source, and D-lactic acid was produced by static fermentation at 42 ° C in a shake flask. When the glucose and D-lactic acid contents remained stable, the fermentation was terminated.
  • composition of each medium used in this example is as follows:
  • the slant medium and the seed medium were the same as in Example 1.
  • the fermentation medium contains: maltose 60-120 g, corn syrup dry powder 20 g, calcium carbonate 100 g, and the balance is water; the fermentation medium has a pH of 5.0-7.0. It is sterilized at 115 ° C for 15 min.
  • the method for producing D-lactic acid by fermentation comprises the following steps:
  • Fermentation culture 5 mL of the seed culture solution prepared in the step (2) is placed in a 100 mL flask containing 40 mL of fermentation medium, and cultured at 42 ° C. When the rate of change of D-lactic acid is 0, it is regarded as The fermentation is over.
  • the D-lactic acid concentration and the total reducing sugar concentration in the fermentation broth were measured according to the detection and calculation methods described in the above specific embodiments, and the sugar acid conversion rate and the production rate were calculated.
  • Table 6 shows the formation of D-lactic acid using xylose as a carbon source
  • fructose was used as a carbon source in a triangular flask, and the corn syrup dry powder was used as a nitrogen source, and D-lactic acid was produced by static fermentation at 42 ° C shake flask. When the glucose and D-lactic acid contents remained stable, the fermentation was terminated.
  • composition of each medium used in this example is as follows:
  • the slant medium and the seed medium were the same as in Example 1.
  • the fermentation medium contains: 60-120 g of arabinose, 20 g of corn syrup dry powder, 100 g of calcium carbonate, and the balance is water; the pH of the fermentation medium is 5.0-7.0. Sterilization at 115 ° C for 15 min.
  • the method for producing D-lactic acid by fermentation comprises the following steps:
  • Fermentation culture 5 mL of the seed culture solution prepared in the step (2) is connected to a fermentation tank containing 40 mL. In a 100 mL flask of the culture medium, the culture was static at 42 ° C. When the rate of change of D-lactic acid was 0, it was regarded as the end of fermentation.
  • the D-lactic acid concentration and the total reducing sugar concentration in the fermentation broth were measured according to the detection and calculation methods described in the above specific embodiments, and the sugar acid conversion rate and the production rate were calculated.
  • Table 7 shows the formation of D-lactic acid with fructose as carbon source
  • Glucose, sucrose, maltose and fructose were used as carbon sources in the flasks, and yeast powder, peptone, peanut meal and corn syrup were used as nitrogen sources, and the flask was allowed to stand at 42 ° C for fermentation. Production of D-lactic acid. When the D-lactic acid content remained stable, the fermentation was terminated.
  • composition of each medium used in this example is as follows:
  • the slant medium and the seed medium were the same as in Example 1.
  • the fermentation medium contains: glucose, sucrose, maltose, fructose 60-120g, yeast powder 3-10g, peptone 3-10g, peanut meal 5-20g, corn syrup dry powder 5-20g, calcium carbonate 100g, balance It is water; the pH of the fermentation medium is 5.0 to 7.0. It is sterilized for 15 minutes at 115 °C.
  • the method for producing D-lactic acid by fermentation comprises the following steps:
  • Fermentation culture 5 mL of the seed culture solution prepared in the step (2) is placed in a 100 mL flask containing 40 mL of fermentation medium, and cultured at 42 ° C. When the rate of change of D-lactic acid is 0, it is regarded as The fermentation is over.
  • the D-lactic acid concentration in the fermentation broth was measured according to the detection and calculation method described in the above specific embodiment.
  • Table 8 uses glucose, sucrose, maltose, and fructose as carbon sources; yeast powder, peptone, corn syrup, and peanut meal as the nitrogen source D-lactic acid.
  • HMM-1 Bacillus thuringiensis
  • a 5 liter fully automatic fermenter with 120 g/L glucose as carbon source, 20 g/L corn syrup dry powder, 10 g/L yeast powder, 10 g/L peptone, 40 g/L Peanut meal is a nitrogen source, and feed (sugar) is fed to ferment to produce D-lactic acid.
  • composition of each medium used in this example is as follows:
  • the slant medium and the seed medium were the same as in Example 1.
  • the fermentation medium contains: 120g of glucose, 20g of dried corn syrup, 10g of yeast powder, 10g of yeast powder, 10g of peptone, 40g of peanut meal, and the balance is water; the pH of the fermentation medium is 5.5-6.5. Bacteria for 15 min.
  • the method for producing D-lactic acid by fermentation comprises the following steps:
  • Example 1 Inclined culture: the same as in Example 1;
  • Seed culture The strain cultured in the step (1) was inoculated with a loop in a 40 mL flask containing 40 mL of seed medium under sterile conditions, and cultured at 42 ° C for 24 hours to prepare a seed culture solution 1 . 5 mL of the seed culture solution 1 was placed under sterile conditions into a 500 mL flask containing 100 mL of seed medium, and cultured at 42 ° C for 24 hours to prepare a seed culture solution 2.
  • Fermentation culture 400 mL of the seed culture solution 2 prepared in the step (2) is placed under sterile conditions into a fermenter containing 3.6 L of fermentation medium, and cultured at 42 ° C, 70 rpm, and sampled every 10 hours. Once, the amount of residual sugar in the fermentation broth was measured. When the glucose concentration was lowered to 20 to 30 g/L, glucose was added to a glucose concentration of 50 to 70 g/L, and the total amount of sugar was increased twice. When the rate of glucose consumption during fermentation tends to zero, the fermentation is terminated.
  • the D-lactic acid concentration in the fermentation broth was measured according to the detection and calculation method described in the above specific embodiment.
  • Table 9 shows the formation of D-lactic acid, sugar conversion rate and production rate when glucose is used as carbon source, peanut meal, yeast powder, peptone, and corn syrup as nitrogen source.
  • HMM-1 Bacillus thuringiensis
  • a 5 liter fully automatic fermenter with 120 g/L glucose as the carbon source, 5 g/L corn syrup dry powder, 0.9 g/L dipotassium hydrogen phosphate, 3.5 g/L
  • the triamine citrate is inoculated in an amount of 20%, and the feed (sugar) is fed to ferment to produce D-lactic acid.
  • composition of each medium used in this example is as follows:
  • the slant medium and the seed medium were the same as in Example 1.
  • the fermentation medium contains: 120 g of glucose, 5 g of corn syrup dry powder, 0.9 g of dipotassium hydrogen phosphate, 3.5 g of triamine citrate, and the balance being water; the pH of the fermentation medium is 5.5-6.5. 115 ° C conditions Sterilize for 15 min.
  • the method for producing D-lactic acid by fermentation comprises the following steps:
  • Example 1 Inclined culture: the same as in Example 1;
  • Seed culture The strain cultured in the step (1) was inoculated with a loop in a 40 mL flask containing 40 mL of seed medium under sterile conditions, and cultured at 42 ° C for 24 hours to prepare a seed culture solution 1 . 5 mL of the seed culture solution 1 was placed under sterile conditions into a 500 mL flask containing 100 mL of seed medium, and cultured at 42 ° C for 24 hours to prepare a seed culture solution 2.
  • Fermentation culture 800 mL of the seed culture solution 2 prepared in the step (2) is placed under sterile conditions into a fermenter containing 3.2 L of fermentation medium, and cultured at 42 ° C, 70 rpm, and sampled every 10 hours. Once, the amount of residual sugar in the fermentation broth was measured. When the glucose concentration was lowered to 20 to 30 g/L, glucose was added to a glucose concentration of 50 to 70 g/L, and the total amount of sugar was increased twice. When the rate of glucose consumption during fermentation tends to zero, the fermentation is terminated.
  • the D-lactic acid concentration in the fermentation broth was measured according to the detection and calculation method described in the above specific embodiment.
  • the fermentation experiment was carried out in three repetitions, and the fermentation at the end of 74 hours was completed, the D-lactic acid yield was 152 ⁇ 3 g/L, the production rate was 2.05 g/L/h, and the sugar acid conversion rate was 0.79 g/g.
  • a spore of 15 g/L of glucose, sucrose, xylose, arabinose, lactose, maltose as a carbon source and 20 g/L of corn syrup in a 5 liter fully automatic fermentor using a Lactobacillus plantarum (HKM-1)
  • the dry powder is a nitrogen source, 10 mol/L NaOH, 250 g/L NH 4 OH, 250 g/L calcium carbonate is used as a neutralizing agent, and fed (sugar) is fed to ferment to produce D-lactic acid.
  • composition of each medium used in this example is as follows:
  • the slant medium and the seed medium were the same as in Example 1.
  • the fermentation medium contains: glucose, sucrose, fructose, maltose 120g, corn syrup dry powder 20g, the balance is water; 20g/L corn syrup dry powder is nitrogen source, and the fermentation medium has a pH of 5.0-7.0. Sterilize at 115 ° C for 15 min.
  • the method for producing D-lactic acid by fermentation comprises the following steps:
  • Example 1 Inclined culture: the same as in Example 1;
  • Seed culture The strain cultured in the step (1) is sterilized under an aseptic condition in a 100 mL flask containing 40 mL of seed culture medium, and cultured at 42 ° C for 24 hours to prepare a seed culture solution 1 .
  • 40 mL of the seed culture solution 1 was placed under sterile conditions into a 500 mL flask containing 100 mL of seed culture medium, and cultured at 42 ° C for 24 hours to prepare a seed culture solution 2; the seed culture was further expanded in the same manner to obtain 400 mL of seeds.
  • Fermentation culture 4 mL of the seed culture solution 3 prepared in the step (2) is placed under aseptic conditions into a 5 liter fully automatic fermenter (Shanghai Bailun) equipped with 3.6 L of fermentation medium, 42 ° C, Incubate at 50 to 100 rpm. Samples were taken every 10 hours to determine the amount of residual sugar and D-lactic acid in the fermentation broth. When the concentration of the carbon source was reduced to 20-30 g/L, the sugar was added to make the sugar concentration 50-70 g/L. At 48 hours, the fermentation was terminated and the concentration of D-lactic acid was measured.
  • the D-lactic acid concentration in the fermentation broth was measured according to the detection and calculation method described in the above specific embodiment.
  • Table 10 uses glucose, sucrose, maltose, and fructose as carbon sources; 250 g/L of NH 4 OH, 10 mol/L of NaOH, 250 g/L of Ca(OH) 2 as a neutralizing agent, and the formation and production of D-lactic acid. rate:

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Abstract

提供了一种保藏编号为CCTCCNO：M2013389和DSMNo.27555的土芽孢乳杆菌（Sporolactobacillus terrae）HKM-1，及利用其发酵生产D-乳酸的方法。该土芽孢乳杆菌HKM-1能够利用氮源-玉米浆干粉发酵生产D-乳酸，最高D-乳酸发酵产量为197g/L，对应的发酵周期为47h，发酵生产能力达4.19g/L/h，所产的D-乳酸光学纯度达99.9%。

Description

一株土芽孢乳杆菌及其应用 技术领域
本发明公开了一株D-乳酸制备技术领域的菌株及其应用方法，具体是一种利用廉价氮源-玉米浆干粉作为有机氮源制备高光学纯度、高浓度D-乳酸的土芽孢乳杆菌及其应用方法。
背景技术
乳酸(lactic acid)又名α-羟基丙酸(α-hydroxypropionic acid)，因旋光性不同，可以分为D-乳酸、L-乳酸和DL-乳酸。其生产方法主要有化学合成法和微生物发酵法，化学法只能合成DL-乳酸，而发酵法根据所采用菌株的不同，可以合成单一的L-乳酸、D-乳酸或者DL-乳酸。目前，约90％的乳酸是通过微生物发酵法生产的。由于人体只有代谢L-乳酸的L-乳酸脱氢酶，L-乳酸作为食品添加剂等被广泛使用，加之L-乳酸在聚乳酸方面的应用，L-乳酸高产菌的选育及高效生产工艺已得到高度的开发，而D-乳酸的研究则相对较少。
D-乳酸作为一个重要的手性中间体，广泛的应用于医药、农药、化工等方面的多种手性物质合成。尤其是D-乳酸可作为下一代高强度生物可降解塑料聚乳酸的单体，引起全球大公司和科学家的高度关注。D-乳酸的全球需求量每年都以6～8％的速度增长，目前全世界D-乳酸的产量为1.6万吨，而D-乳酸的需求量约2.6万吨，由此可见，D-乳酸的市场前景广泛。
近年来，芳氧丙酸类除草剂在农业中的应用越来越广泛，这类除草剂是国际上最早实现工业化生产的一大类旋光性除草剂，此类除草剂中有一个手性碳原子，其中D(-)型的药性比L(+)型高出6～12倍，合成这类除草剂最重要的原料是R-(+)-2-氯丙酸。以D-乳酸为原料可以制备光学活性的R-(+)-2-氯丙酸，即D-乳酸是制备这类除草剂的光学活性前体。
随着新型生物材料的普及推广，L-乳酸、D-乳酸在新材料的应用方面有了很大的发展。例如，以乳酸为原料来制造聚乳酸类(PLA)生物降解塑料。聚乳酸以其良好的生物可降解性及其它优良的使用特性(如透明性、热塑性、产物安全性等)而被认为是理想的取代传统塑料的生物材料之一。聚乳酸(PLA)分为聚D-乳酸(PDLA)、聚L-乳酸(PLLA)和聚DL-乳酸(PDLLA)。由纯的D-乳酸、L-乳酸或者DL-乳酸聚合而成的聚乳酸具有更好的稳定性，其中，PDLA具有更好的热稳定性。聚乳酸具有良好的生物可降解性，在使用之后可以被自然界中微生物完全降解，不污染环境，对环境保护非常有利。
南京工业大学丁子建等于2004年首次报道利用芽孢乳杆菌(Sporolactobacillus sp.)从葡萄糖发酵产D-乳酸的工艺，发酵72h后产酸40.7g/L，光学纯度96％；南京工业大学杨文革等在2006年发表组合发酵生产D-乳酸工艺(中国专利申请号CN200610097453.6)以乳杆菌(Lactobacillus)进行有氧、微氧、厌氧三段组合发酵技术，发酵时间25～38h，产酸达到75～131g/L；申请号为CN201010101421.5的中国发明专利公开了利用重组大肠杆菌以甘油为原料发酵制备D-乳酸的方法，但是菌株对酸的耐受性差，生产率低，使得大肠杆菌在D-乳酸实际应用当中受到限制；申请号为CN201010247826.X的中国发明公开了利用基因工程手段，成功构建了产高光学纯度D-乳酸的基因工程菌并利用其进行乳酸发酵生产，其D-乳酸产量达到40g/L以上，纯度为99％以上；申请号为CN201010235511.3的中国发明专利公开了利用同源重组的方法得到产纯D-乳酸的基因工程菌并用其进行乳酸发酵生产，其D-乳酸产量仅为20g/L以上，纯度为99％以上；申请号为CN201010235511.3的中国发明专利公开了利用同源重组的方法得到产纯D-乳酸的基因工程菌并用其进行乳酸发酵生产，其D-乳酸产量仅为20g/L以上，纯度为99％以上。上述现有技术中D-乳酸的产量普遍不高。继续检索发现：申请号为CN200810098908.5的中国发明专利公开了一株高光学纯度D-乳酸生产菌及其发酵生产D-乳酸的工艺，申请号为CN201210472413.0的中国发明专利公开了一株乳杆菌及利用其发酵生产D-乳酸的方法，申请号为CN201010208148.6的中国发明专利公开了一种采用花生粕同步酶解发酵生产高浓度D-乳酸的方法及其专用培养基。上述现有技术发酵时间过长，均在70h以上。这些公开的技术，与L-乳酸生产菌株和方法相比，D-乳酸生产菌株和方法存在发酵时间过长或者产D-乳酸浓度相对较低的缺点，需要进一步开发基于高效D-乳酸生产菌株的生产方法。
发明内容
本发明针对现有技术存在的上述不足，提供一种用于制备D-乳酸的芽孢乳杆菌及其应用方法，可以直接利用六碳糖，如葡萄糖、麦芽糖、乳糖、蔗糖等作为碳源；酵母粉、蛋白胨、花生粕、玉米浆干粉作为氮源发酵生产D-乳酸，发酵生产高浓度、高光学纯度的D-乳酸，在节约成本的同时提高生产效率，适合于工业生产中推广应用。所述芽孢乳杆菌为土芽孢乳杆菌(Sporolactobacillus terrae)HKM-1，保藏于中国典型培养物保藏中心，简称CCTCC，保藏编号为CCTCC NO：M2013389，保藏日期为：2013年9月2日，保藏单位地址：中国武汉武汉大学，分类命名：土芽孢乳杆菌Sporolactobacillus terrae。
本发明是通过以下技术方案实现的：本发明涉及上述用于制备D-乳酸的芽孢乳杆菌的应用方法：首先对芽孢乳杆菌进行种子培养获得种子培养液，然后以 葡萄糖、麦芽糖、乳糖、蔗糖作为碳源，酵母粉、蛋白胨、花生粕、玉米浆干粉作为氮源的发酵培养基进行发酵培养，得到D-乳酸。
具体包括如下步骤：
1)斜面培养
将土芽孢乳杆菌(HKM-1)菌种接种于含有20g/L琼脂的固体斜面培养基上，40～45℃条件下，培养24～48h；
2)种子培养
将经过斜面培养的芽孢乳杆菌在无菌条件下接种到种子培养基中，35～45℃条件下，静止培养24～36h，加入中和剂控制发酵液pH，制得种子培养液；
3)发酵培养
按5～20％体积比的接种量接入到发酵培养基中，在35℃～45℃环境下培养48～72h，温度优选42℃。
优选地，步骤2)所述的种子培养基每升中含有：葡萄糖100～120g，酵母粉8～12g，蛋白胨3～8g，碳酸钙50g，余量为水，优选含有：葡萄糖100g，酵母粉10g，蛋白胨5g，碳酸钙50g，余量为水；该种子培养基的pH为6.0。115℃灭菌15min。中和剂包括NaOH、NH4OH和Ca(OH)2中的一种或几种。
优选地，步骤3)中所述的发酵培养基的组分及其含量为：碳源40～120g/L、氮源添加量5～20g/L。
优选地，步骤3)中所述的发酵培养基的组分及其含量为：葡萄糖/蔗糖/麦芽糖/果糖40～120g/L、玉米浆干粉10～20g/L、酵母粉3～10g/L、蛋白胨3～10g/L、花生粕5～20g/L，用于调控培养基pH的中和剂(NH4OH、NaOH、Ca(OH)2)，余量为水。115℃灭菌15min。
优选地，步骤3)中所述的发酵工艺为补料流加工艺，该补料流加工艺是指：当发酵液中总还原糖含量低于20～30g/L时补加碳源，使总还原糖含量维持在30～70g/L，或达到50～70g/L。
优选地，所述的发酵培养基的pH为5.0～7.0。
以葡萄糖、蔗糖、果糖、麦芽糖作为碳源，酵母粉、蛋白胨、花生粕、玉米浆干粉作为氮源。上述原料可从商业途径获得。
本发明选用葡萄糖、蔗糖、麦芽糖、果糖作为碳源，酵母粉、蛋白胨、花生粕、玉米浆干粉作为氮源，通过提供相应的发酵工艺条件，使得该D-乳酸生产工艺原料易得，成本低廉，D-乳酸产量最高可达197g/L，光学纯度大于99.9％，糖酸转化率最高可达0.94g/g，发酵生产能力4.19g/L/h。因此，利用本发明方法生产D-乳酸，能节约成本、简化操作流程，具有广阔的工业应用前景。
附图说明
图1为土芽孢乳杆菌(HKM-1)的16S rRNA系统进化树分析图。
图中：实线的水平距离(长度之和)代表菌株的进化距离，选取的参照菌是与本发明提供的土芽孢乳杆菌(HKM-1)亲缘关系较近的菌株，共10株。
具体实施方式
下面对本发明的实施例作详细说明，本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，本发明的保护范围包括但不限于下述的实施例。
以下实施例涉及的土芽孢乳杆菌(HKM-1)的分离、筛选和鉴定方法如下：
一、菌株的分离、筛选
该实施例中所用的培养基的组成如下：
营养液体培养基：葡萄糖80g/L，酵母粉8g/L，蛋白胨3g/L，碳酸钙35g/L，pH为6。
营养琼脂培养基：葡萄糖40g/L，酵母粉10g/L，蛋白胨5％，碳酸钙2g/L，琼脂粉20g/L，pH为6。115℃灭菌15min。
菌株筛选培养基：葡萄糖120g/L，酵母粉10g/L，蛋白胨5g/L，碳酸钙50g/L，pH为6。115℃灭菌15min。
该实施例的具体操作过程如下：
采集自云南的土壤，称取2g溶于40mL营养液体培养基中，42℃富集培养24h。然后用无菌的生理盐水稀释培养液，分别稀释10倍、100倍、1000倍、10000倍后涂布到含营养琼脂培养基的培养皿中，42℃培养48h。待长出单菌落后，挑选菌落面积和产酸透明圈面积大的菌落，接种到发酵培养基中，42℃静止培养48h，测定D-乳酸的产量，经过多次筛选，挑取一株D-乳酸产量较高的菌株。
将上述菌株多次在营养琼脂平板上划线分离纯化，然后再进行10个循环的发酵测试，10次循环发酵产生的D-乳酸产量和转化率基本保持原有水平，证明上述菌株即是目标菌株，名称为HKM-1。
二、菌株的鉴定
1.菌株HKM-1的生理生化鉴定：
生理生化鉴定在中国典型培养物保藏中心(CCTCC)完成，具体生理生化鉴定如表1、表2所示。
2.菌株HKM-116S rRNA系统进化树分析
对菌株HKM-1进行培养，当OD600为2～5时，抽提菌株HKM-1的全基因组，然后扩增菌株HKM-1的16S rRNA的DNA序列。选取的参照菌是与本发明提供的 菌株亲缘关系较近的菌株，共10株，菌株HKM-1的系统进化树分析图见图1。
表1土芽孢乳杆菌HKM-1(革兰氏阳性菌)生理生化特性-利用碳源产酸
Figure PCTCN2014095311-appb-000001
+：阳性，-：阴性，w：弱阳性
表2菌株HKM-1生理生化特性-酶活、碳源同化
Figure PCTCN2014095311-appb-000002
+：阳性反应；-：阴性反应；W：弱阳性反应
根据上述检测结果，结合16S rRNA诊断区分析，将菌株HKM-1鉴定为土芽孢乳杆菌(Sporolactobacillus terrae)，并将其保藏于德国微生物菌种保藏中心(DSMZ)，保藏号为DSM No.27555；保藏于中国典型培养物保藏中心，保藏号为CCTCC NO：M 2013389。
3.土芽孢乳杆菌HKM-1与已经公开的菌株DSM11697的比较
虽然土芽孢乳杆菌HKM-1与DSM11697两株菌经16SrRNA的DNA序列比对，相似性为100％，但根据文献Yanagida F，Suzuki K I，Kozaki M，et al.Proposal of Sporolactobacillus nakayamae subsp.nakayamae sp.nov.，subsp.nov.，Sporolactobacillus nakayamae subsp.racemicus subsp.nov.，Sporolactobacillus terrae sp.nov.，Sporolactobacillus kofuensis sp.nov.，and Sporolactobacillus  lactosus sp.nov[J].International journal of systematic bacteriology，1997，47(2)：499-504.报道的信息，与表1“菌株HKM-1生理生化特性-利用碳源产酸”部分进行对比，发现这两株菌有六种糖的利用存在明显的差异，如表3所示：
表3土芽孢乳杆菌HKM-1与菌株DSM11697生理生化特性-利用碳源产酸比较
Figure PCTCN2014095311-appb-000003
+：阳性，-：阴性
gyrB基因为DNA促旋酶中B亚单位蛋白的基因，该基因序列在区分和鉴定细菌近缘种方面，比非蛋白编码基因16S rDNA具有更高的分辨率。土芽孢乳杆菌HKM-1的gyrB保守基因序列如序列表(二)，长1,920bp，与DSM 11697的gyrB基因序列相比，有两个碱基的差异。
据此，可以认为土芽孢乳杆菌HKM-1不同于之前报道的土芽孢乳杆菌的模式菌DSM 11697，为一株新的土芽孢乳杆菌。
本实施例得到一株可以利用玉米浆干粉作为氮源生成D-乳酸的菌株。
所述的菌株为革兰氏阳性菌，细胞直杆，营养细胞的菌体大小为(0.8～0.9)μm×(3.0～5.0)μm。形成内生孢子。在含有葡萄糖、酵母粉和蛋白胨的琼脂平板上形成表面光滑、乳白色、边缘整齐的圆形菌落；该菌形成细小、淡乳白色、边缘整齐的圆形菌落。
以下实施例涉及的利用土芽孢乳杆菌HKM-1发酵生产D-乳酸方法的步骤如下：(1)斜面培养：将土芽孢乳杆菌HKM-1菌种接种于含有20g/L琼脂的固体斜面培养基上，40～45℃条件下，培养24～48h。
(2)种子培养：将步骤(1)的斜面培养物，在无菌条件下接种到40mL种子培养基中，35～45℃条件下，静止培养24～36h，加入中和剂控制发酵液pH，制得种子培养液。
(3)发酵培养：以5～20％体积比的接种量，将种子培养液接种于发酵培养基中，35～45℃条件下培养48～72h。
其中，步骤(1)、(2)、(3)中所述的菌体培养温度优选是42℃。
其中，步骤(2)、(3)所述的培养过程中加入的中和剂为碳酸钙，控制pH。
上述发酵培养过程中，每5个小时取发酵液，先用12,000rpm离心5min，取上清，沸水浴加热10min，再用12,000rpm离心5min，取上清液检测发酵液中L-乳酸浓度、D-乳酸浓度、葡萄糖浓度，计算糖酸转化率、D-乳酸发酵生产能力和D-乳酸光学纯度。
总还原糖的测定方法为DNS法。葡萄糖的测定方法为，发酵液稀释后离心，采用生物传感分析仪SBA-40D(山东省科学院)测定。生物传感分析仪SBA-40D是以固定化酶为传感器的分析仪器，葡萄糖与氧气、水在酶的催化下生成双氧水。反应放出的双氧水与白金-银电极接触，并产生电流信号，该电流信号与葡萄糖浓度成线性比例关系，通过测定电流信号强度可得出葡萄糖浓度。
L-乳酸的测定方法为，采用生物传感分析仪SBA-40D，发酵液稀释100倍，用针管吸取25μL稀释液快速注入生物传感分析仪SBA-40D中，可直接读出L-乳酸的含量。
D-乳酸的测定使用Agilent 1100液相色谱仪，配备手性分离柱(日本三菱化学公司，MCI GEL-CRS10W(3μ)4.6ID×50mm，光学异体分离用)。具体操作条件为：0.005mol/L硫酸铜作为流动相，流量0.7mL/min，进样量5μl，紫外检测器，检测波长254nm，操作温度25℃。利用D-乳酸标准品做出标准曲线，再根据标准曲线计算出发酵液中D-乳酸的含量。
本发明中，作为标准品的D-乳酸为德国Sigma-Aldrich公司的产品，其货号为L0625-25MG。在如上色谱条件下，D-乳酸保留时间为6.5min。
光学纯度(optical purity)是衡量旋光性样品中一个对映体超过另一个对映体的量的量度，它可用对映体过量值(enantiomeric excess，ee)表示。本发明中L-乳酸的光学纯度(ee)按以下公式计算：[(D-乳酸产量(g/L)-L-乳酸产量(g/L))÷(D-乳酸产量+L-乳酸产量)]×100％。
糖酸转化率定义为(g/g)：D-乳酸产量(g)÷底物消耗量(g)。
D-乳酸生产速率(g/L/h)为：L-乳酸产量(g/L)÷发酵时间(h)。
实施例1
利用土芽孢乳杆菌(HKM-1)在三角瓶中以葡萄糖为碳源，玉米浆干粉作为氮源分批发酵生产D-乳酸
本实施例中所使用的各培养基的组成如下：
斜面培养基每升中含有：葡萄糖30～50g，酵母粉5～10g，蛋白胨2～8g，碳酸钙50g，琼脂粉15～25g，余量为水。所述斜面培养基的pH为6.0。115℃灭菌15min。
种子培养基每升中含有：葡萄糖40～120g，酵母粉5～10g，蛋白胨2～8g，碳酸钙50g，余量为水。所述种子培养基的pH为5.0～7.0。115℃灭菌15min。
发酵培养基每升中含有：葡萄糖60～120g，玉米浆干粉20g，碳酸钙50g，余量为水；所述发酵培养基的pH为5.5～6.5。115℃条件下灭菌15min。
本实施例所述发酵生产D-乳酸的方法包括以下步骤：
(1)斜面培养：将土芽孢乳杆菌(HKM-1)接种于斜面培养基上，42℃培养24h；
(2)种子培养：将步骤(1)培养的菌株，在无菌条件下用接种环接2环于装有40mL种子培养基的100mL三角瓶中，42℃静止培养24h，制得种子培养液；
(3)发酵培养：将5mL步骤(2)制得的种子培养液接入装有40mL发酵培养基的100mL三角瓶中，42℃静止培养，当D-乳酸的变化速率为0时，视为发酵结束。
发酵结束后，根据上述具体实施方式中所述的检测和计算方法，检测发酵液中D-乳酸浓度和总还原糖浓度，计算糖酸转化率及生产速率。
该实验共设3次重复，结果见表4。
表4以葡萄糖为碳源D-乳酸的生成情况
Figure PCTCN2014095311-appb-000004
实施例2
利用土芽孢乳杆菌(HKM-1)在三角瓶中以蔗糖为碳源，玉米浆干粉为氮源，分批发酵生产D-乳酸：
斜面培养基和种子培养基同实施例1。
发酵培养基每升中含有：蔗糖60～120g，玉米浆干粉20g，碳酸钙50g，余量为水；所述发酵培养基的pH为5.0～7.0。115℃条件下灭菌15min。
该发酵生产D-乳酸的方法包括以下步骤：
(1)斜面培养：同实施例1；
(2)种子培养：同实施例1；
(3)发酵培养：将5mL步骤(2)制得的种子培养液接入装有40mL发酵培养基的100mL三角瓶中，42℃静止培养，当D-乳酸的变化速率为0时，视为发酵结束。
发酵结束后，根据上述具体实施方式中所述的检测和计算方法，检测发酵液中D-乳酸浓度和总还原糖浓度，计算糖酸转化率及生产速率。
该实验共设3次重复，结果见表5。
表5以蔗糖为碳源D-乳酸的生成情况
Figure PCTCN2014095311-appb-000005
实施例3
利用土芽孢乳杆菌(HKM-1)在三角瓶中以麦芽糖为碳源，玉米浆干粉为氮源，42℃摇瓶静止发酵生产D-乳酸。当葡萄糖和D-乳酸含量保持稳定时，结束发酵。
本实施例中所使用的各培养基的组成如下：
斜面培养基和种子培养基同实施例1。
发酵培养基每升中含有：麦芽糖60～120g，玉米浆干粉20g，碳酸钙100g，余量为水；所述发酵培养基的pH为5.0～7.0。115℃条件下灭菌15min。
该发酵生产D-乳酸的方法包括以下步骤：
(1)斜面培养：同实施例1。
(2)种子培养：同实施例1。
(3)发酵培养：将5mL步骤(2)制得的种子培养液接入装有40mL发酵培养基的100mL三角瓶中，42℃静止培养，当D-乳酸的变化速率为0时，视为发酵结束。
发酵结束后，根据上述具体实施方式中所述的检测和计算方法，检测发酵液中D-乳酸浓度和总还原糖浓度，计算糖酸转化率及生产速率。
该实验共设3次重复。结果如表6：
表6以木糖为碳源D-乳酸的生成情况
Figure PCTCN2014095311-appb-000006
实施例4
利用土芽孢乳杆菌(HKM-1)在三角瓶中以果糖为碳源，玉米浆干粉为氮源，42℃摇瓶静止发酵生产D-乳酸。当葡萄糖和D-乳酸含量保持稳定时，结束发酵。
本实施例中所使用的各培养基的组成如下：
斜面培养基和种子培养基同实施例1。
发酵培养基每升中含有：阿拉伯糖60～120g，玉米浆干粉20g，碳酸钙100g，余量为水；所述发酵培养基的pH为5.0～7.0。115℃条件下灭菌15min。
该发酵生产D-乳酸的方法包括以下步骤：
(1)斜面培养：同实施例1。
(2)种子培养：同实施例1。
(3)发酵培养：将5mL步骤(2)制得的种子培养液接入装有40mL发酵 培养基的100mL三角瓶中，42℃静止培养，当D-乳酸的变化速率为0时，视为发酵结束
发酵结束后，根据上述具体实施方式中所述的检测和计算方法，检测发酵液中D-乳酸浓度和总还原糖浓度，计算糖酸转化率及生产速率。
该实验共设3次重复。结果如表7：
表7以果糖为碳源D-乳酸的生成情况
Figure PCTCN2014095311-appb-000007
实施例5
利用土芽孢乳杆菌(HKM-1)在三角瓶中分别选用葡萄糖、蔗糖、麦芽糖、果糖为碳源，酵母粉、蛋白胨、花生粕粉、玉米浆干粉为氮源，42℃摇瓶静置发酵生产D-乳酸。当D-乳酸含量保持稳定时，结束发酵。
本实施例中所使用的各培养基的组成如下：
斜面培养基和种子培养基同实施例1。
发酵培养基每升中含有：葡萄糖、蔗糖、麦芽糖、果糖60～120g，酵母粉3～10g、蛋白胨3～10g、花生粕粉5～20g、玉米浆干粉5～20g，碳酸钙100g，余量为水；所述发酵培养基的pH为5.0～7.0。115℃条件下灭菌15min。
该发酵生产D-乳酸的方法包括以下步骤：
(1)斜面培养：同实施例1。
(2)种子培养：同实施例1。
(3)发酵培养：将5mL步骤(2)制得的种子培养液接入装有40mL发酵培养基的100mL三角瓶中，42℃静止培养，当D-乳酸的变化速率为0时，视为发酵结束。
发酵结束后，根据上述具体实施方式中所述的检测和计算方法，检测发酵液中D-乳酸浓度。
该实验共设3次重复。结果如表8：
表8以葡萄糖、蔗糖、麦芽糖、果糖为碳源；酵母粉、蛋白胨、玉米浆干粉、花生粕粉为氮源D-乳酸的生成情况
Figure PCTCN2014095311-appb-000008
Figure PCTCN2014095311-appb-000009
实施例6
利用土芽孢乳杆菌(HKM-1)在5升全自动发酵罐中以120g/L的葡萄糖为碳源，20g/L的玉米浆干粉，10g/L酵母粉，10g/L蛋白胨，40g/L花生粕粉为氮源，补料(糖)流加发酵生产D-乳酸。
本实施例中所使用的各培养基的组成如下：
斜面培养基和种子培养基同实施例1。
发酵培养基每升中含有：葡萄糖120g，玉米浆干粉20g，酵母粉10g，蛋白胨10g，花生粕粉40g，余量为水；所述发酵培养基的pH为5.5～6.5。115℃条件下灭菌15min。
该发酵生产D-乳酸的方法包括以下步骤：
(1)斜面培养：同实施例1；
(2)种子培养：将步骤(1)培养的菌株在无菌条件下用接种环接2环于装有40mL种子培养基的40mL三角瓶中，42℃静止培养24h，制得种子培养液1；将5mL种子培养液1在无菌条件下接入装有100mL种子培养基的500mL三角瓶中，42℃静止培养24h，制得种子培养液2。
(3)发酵培养：将400mL步骤(2)制得的种子培养液2在无菌条件下接入装有3.6L发酵培养基的发酵罐中，42℃、70rpm搅拌培养，每10个小时取样一次，测定发酵液中的残糖量，当葡萄糖浓度降到20～30g/L时，流加葡萄糖，使葡萄糖浓度达到50～70g/L，共计补糖2次。当发酵过程中葡萄糖消耗速率趋于0时，结束发酵。
发酵结束后，根据上述具体实施方式中所述的检测和计算方法，检测发酵液中D-乳酸浓度。
该发酵实验共设3次重复，结果见表9。
表9以葡萄糖为碳源，花生粕粉、酵母粉、蛋白胨、玉米浆干粉作为氮源时D-乳酸的生成情况、糖转化率及生产速率
Figure PCTCN2014095311-appb-000010
实施例7
利用土芽孢乳杆菌(HKM-1)在5升全自动发酵罐中以120g/L的葡萄糖为碳源，5g/L的玉米浆干粉，0.9g/L的磷酸氢二钾，3.5g/L的柠檬酸三胺，接种量20％，补料(糖)流加发酵生产D-乳酸。
本实施例中所使用的各培养基的组成如下：
斜面培养基和种子培养基同实施例1。
发酵培养基每升中含有：葡萄糖120g，玉米浆干粉5g，磷酸氢二钾0.9g，柠檬酸三胺3.5g，余量为水；所述发酵培养基的pH为5.5～6.5。115℃条件下灭菌15min。
该发酵生产D-乳酸的方法包括以下步骤：
(1)斜面培养：同实施例1；
(2)种子培养：将步骤(1)培养的菌株在无菌条件下用接种环接2环于装有40mL种子培养基的40mL三角瓶中，42℃静止培养24h，制得种子培养液1；将5mL种子培养液1在无菌条件下接入装有100mL种子培养基的500mL三角瓶中，42℃静止培养24h，制得种子培养液2。
(3)发酵培养：将800mL步骤(2)制得的种子培养液2在无菌条件下接入装有3.2L发酵培养基的发酵罐中，42℃、70rpm搅拌培养，每10个小时取样一次，测定发酵液中的残糖量，当葡萄糖浓度降到20～30g/L时，流加葡萄糖，使葡萄糖浓度达到50～70g/L，共计补糖2次。当发酵过程中葡萄糖消耗速率趋于0时，结束发酵。
发酵结束后，根据上述具体实施方式中所述的检测和计算方法，检测发酵液中D-乳酸浓度。
该发酵实验共设3次重复，74h发酵结束，D-乳酸产量为152±3g/L、生产速率2.05g/L/h、糖酸转化率0.79g/g。
实施例8
利用土芽孢乳杆菌(HKM-1)在5升全自动发酵罐中以初糖浓度为120g/L的葡萄糖、蔗糖、木糖、阿拉伯糖、乳糖、麦芽糖为碳源，20g/L的玉米浆干粉为氮源，10mol/L的NaOH、250g/L NH4OH、250g/L的碳酸钙为中和剂，补料(糖)流加发酵生产D-乳酸。
本实施例中所使用的各培养基的组成如下：
斜面培养基和种子培养基同实施例1。
发酵培养基每升中含有：葡萄糖、蔗糖、果糖、麦芽糖120g，玉米浆干粉20g，余量为水；20g/L的玉米浆干粉为氮源，所述发酵培养基的pH为5.0～7.0。115℃条件下灭菌15min。
该发酵生产D-乳酸的方法包括以下步骤：
(1)斜面培养：同实施例1；
(2)种子培养：将步骤(1)培养的菌株在无菌条件下用接种环接2环于装有40mL种子培养基的100mL三角瓶中，42℃静止培养24h，制得种子培养液1；将40mL种子培养液1在无菌条件下接入装有100mL种子培养基的500mL三角瓶中，42℃静止培养24h，制得种子培养液2；继续以相同方式扩大种子培养，得到400mL种子培养液3。
(3)发酵培养：将4mL步骤(2)制得的种子培养液3在无菌条件下接入装有3.6L发酵培养基的5升全自动发酵罐(上海百伦)中，42℃、50～100rpm搅动培养。每10个小时取样一次，测定发酵液中的残糖量和D-乳酸浓度，当碳源浓度降到20～30g/L时，流加补糖，使糖浓度达到50～70g/L。48h时，结束发酵，测定D-乳酸的浓度。
发酵结束后，根据上述具体实施方式中所述的检测和计算方法，检测发酵液中D-乳酸浓度。
该发酵实验共设3次重复，结果见表10。
表10以葡萄糖、蔗糖、麦芽糖、果糖为碳源；250g/L的NH4OH、10mol/L的NaOH、250g/L的Ca(OH)2为中和剂，D-乳酸的生成情况、生产速率：
Figure PCTCN2014095311-appb-000011
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术人员无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。

Claims (10)

  1. 一种芽孢乳杆菌，其特征在于，所述芽孢乳杆菌为土芽孢乳杆菌(Sporolactobacillus terrae)HKM-1，保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2013389。
  2. 根据权利要求1所述的土芽孢乳杆菌HKM-1在发酵制备D-乳酸中的应用。
  3. 根据权利要求1所述的应用，其特征在于，以六碳糖作为碳源，以酵母粉、玉米浆干粉、蛋白胨和花生粕中的一种或多种作为氮源，进行发酵后得到D-乳酸。
  4. 根据权利要求1所述的应用，其特征在于，所述六碳糖为葡萄糖、麦芽糖、乳糖、蔗糖中的一种或多种。
  5. 一种制备D-乳酸的方法，包括如下步骤：
    1)斜面培养
    将土芽孢乳杆菌(HKM-1)菌种接种于含有20g/L琼脂的固体斜面培养基上，40～45℃条件下，培养24～48h；
    2)种子培养
    将经过斜面培养的芽孢乳杆菌在无菌条件下接种到种子培养基中，35～45℃条件下，静止培养24～36h，加入中和剂控制发酵液pH，制得种子培养液；
    3)发酵培养
    按5～20％体积比的接种量接入到发酵培养基中，在35℃～45℃环境下培养48～72h。
  6. 根据权利要求5所述的方法，其特征在于：步骤2)所述的种子培养基每升中含有：葡萄糖100～120g，酵母粉8～12g，蛋白胨3～8g，碳酸钙30～60g。
  7. 根据权利要求5所述的方法，其特征在于：步骤3)中所述的发酵培养基的组分及其含量包括：碳源40～120g/L、氮源添加量5～20g/L。
  8. 根据权利要求5所述的方法，其特征在于：步骤3)中所述的发酵培养基的组分及其含量包括：碳源40～120g/L、玉米浆干粉10～20g/L、酵母粉3～10g/L、蛋白胨3～10g/L、花生粕5～20g/L。
  9. 根据权利要求8所述的方法，其特征在于：步骤3)中所述的发酵工艺为补料流加工艺，该补料流加工艺是指：当发酵液中总还原糖含量低于20～30g/L时补加碳源，使总还原糖含量维持在30～70g/L，或达到50～70g/L。
  10. 根据权利要求5所述的方法，其特征在于，所述的中和剂包括NaOH、NH4OH和Ca(OH)2中的一种或几种，培养体系pH控制在5.0～7.0。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111378697A (zh) * 2020-06-01 2020-07-07 中粮营养健康研究院有限公司 以水溶性柠檬酸盐为中和剂提高乳酸发酵糖酸转化率的方法
CN114196713A (zh) * 2021-11-25 2022-03-18 山东润德生物科技有限公司 一种降低氨基葡萄糖发酵过程中二氧化碳排放量方法
CN115216419A (zh) * 2021-04-21 2022-10-21 宁夏金维制药股份有限公司 一种利用枯草芽孢杆菌工程菌发酵生产维生素b2的培养基

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103756939B (zh) * 2014-01-17 2015-07-15 上海交通大学 一株土芽孢乳杆菌及其应用
CN106190901B (zh) 2016-07-15 2020-06-26 上海交通大学 一种菌及其获取方法和应用
CN109906268A (zh) * 2016-11-01 2019-06-18 Ptt全球化学股份有限公司 用于生产d-乳酸或其盐的发酵方法
JP7444370B2 (ja) * 2017-08-04 2024-03-06 株式会社明治 乳酸菌の培養方法
JP6915932B1 (ja) * 2021-04-02 2021-08-11 ハイアマウント株式会社 新規微生物
CN114181980A (zh) * 2021-11-09 2022-03-15 吉林中粮生化有限公司 玉米浆及其制备方法和应用以及高光学纯度乳酸的生产方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3262862A (en) * 1962-05-10 1966-07-26 Kyowa Hakko Kogyo Kk Method for producing lactic acid with sporolactobacillus
CN101285047A (zh) * 2008-05-16 2008-10-15 南京工业大学 一株高光学纯度d-乳酸生产菌及其发酵生产d-乳酸的工艺
CN103756939A (zh) * 2014-01-17 2014-04-30 上海交通大学 一株土芽孢乳杆菌及其应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61293388A (ja) * 1985-06-21 1986-12-24 Daicel Chem Ind Ltd Ｄ−乳酸の製造方法
CN100485027C (zh) * 2007-10-18 2009-05-06 中国科学院微生物研究所 一种生产d-乳酸的方法及其专用芽孢乳杆菌
JP5217736B2 (ja) * 2008-07-30 2013-06-19 東レ株式会社 Ｄ−乳酸の製造方法
CN103114053B (zh) * 2012-12-28 2015-10-14 张健鹏 产高光学纯度d-乳酸的棒状乳杆菌陕西亚种及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3262862A (en) * 1962-05-10 1966-07-26 Kyowa Hakko Kogyo Kk Method for producing lactic acid with sporolactobacillus
CN101285047A (zh) * 2008-05-16 2008-10-15 南京工业大学 一株高光学纯度d-乳酸生产菌及其发酵生产d-乳酸的工艺
CN103756939A (zh) * 2014-01-17 2014-04-30 上海交通大学 一株土芽孢乳杆菌及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BUDSABATHIP, P. ET AL.: "Characterization of Lactic Acid Producing Bacteria from Thai Sources", JOURNAL OF APPLIED PHARMACEUTICAL SCIENCE, vol. 3, no. 1, 28 January 2013 (2013-01-28), pages 33 - 38, XP055213008 *
FUJITOSHI, Y. ET AL.: "Proposal of Sporolactobacillus nakayamae subsp. nakayamae sp. nov., subsp. nov., Sporolactobacillus nakayamae subsp. Racernicus subsp. nov., Sporolactobacillus terrae sp. nov., Sporolactobacillus kofiensis sp. nov., and Sporolactobacillus lactosus sp. nov.", INT.J. SYST. BACTERIOL, vol. 47, no. 2, 30 April 1997 (1997-04-30), pages 499 - 504, XP055212995 *
XU, TINGTING ET AL.: "Advances in the Preparation of D-Lactic Acid", CHEMICAL INDUSTRY AND ENGINEERING PROGRESS, vol. 28, no. 6, 31 December 2009 (2009-12-31), pages 991 - 996 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111378697A (zh) * 2020-06-01 2020-07-07 中粮营养健康研究院有限公司 以水溶性柠檬酸盐为中和剂提高乳酸发酵糖酸转化率的方法
CN111378697B (zh) * 2020-06-01 2020-08-28 中粮营养健康研究院有限公司 以水溶性柠檬酸盐为中和剂提高乳酸发酵糖酸转化率的方法
CN115216419A (zh) * 2021-04-21 2022-10-21 宁夏金维制药股份有限公司 一种利用枯草芽孢杆菌工程菌发酵生产维生素b2的培养基
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