WO2015068645A1 - Procédé de production d'acide lactique - Google Patents

Procédé de production d'acide lactique Download PDF

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WO2015068645A1
WO2015068645A1 PCT/JP2014/079030 JP2014079030W WO2015068645A1 WO 2015068645 A1 WO2015068645 A1 WO 2015068645A1 JP 2014079030 W JP2014079030 W JP 2014079030W WO 2015068645 A1 WO2015068645 A1 WO 2015068645A1
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lactic acid
fermentation
hours
concentration
culture
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PCT/JP2014/079030
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Japanese (ja)
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野田　秀夫
真司 ▲濱▼
真希 木原
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関西化学機械製作株式会社
Ｂｉｏ－ｅｎｅｒｇｙ株式会社
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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

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  • the present invention relates to a method for producing lactic acid. More specifically, the present invention relates to a method for producing lactic acid by fermentation using microorganisms.
  • Lactic acid can be used as a raw material for biodegradable plastics such as polylactic acid and polyester polyol, or as a raw material for agricultural chemicals or pharmaceuticals.
  • Microorganisms that produce lactic acid from sugars such as glucose include lactic acid bacteria (for example, Lactobacillus (for example, Lactobacillus plantarum and Lactobacillus casei)) and molds (for example, Lactobacillus casei) Rhizopus is well known.
  • Non-Patent Document 1 Attempts have been made to produce lactic acid.
  • lactic acid bacteria can perform lactic acid fermentation with a high sugar yield, they are highly auxotrophic. For this reason, when an expensive material (for example, yeast extract etc.) is used for the fermentation medium of lactic acid bacteria, there exists a problem that the manufacturing cost of lactic acid becomes high.
  • corn steep liquor CSL: a concentrated soaking solution containing soluble components eluted from corn in the corn starch production process and components produced by lactic acid fermentation
  • fish protein hydrolysis products Attempts have been made to use it as an inexpensive nutrient source.
  • these nutrient sources other than yeast extract have poor nutritional value for lactic acid bacteria, so it is necessary to add them in large quantities, and the compounds contained in the nutrient sources cause impurities to adversely affect the lactic acid purification process.
  • the amount of nutrients to be added is limited in consideration of these factors, the lactic acid fermentation cell activity is low, so a sufficient lactic acid yield cannot be obtained, and the amount of sugar remaining in the fermentation broth increases. Adversely affects the purification process.
  • An object of the present invention is to provide a method for producing lactic acid, which can increase the amount of lactic acid produced and suppress the amount of residual sugar.
  • the present invention provides a method for producing lactic acid, the method comprising: (I) a step of preparing a culture solution having an initial pH, which is a pH suitable for growth of the lactic acid-producing microorganism, comprising a medium containing sugar and a lactic acid-producing microorganism, and culturing the microorganism; (Ii) culturing the lactic acid-producing microorganism under a pH lower than the initial pH, and (iii) culturing the lactic acid-producing microorganism at a pH suitable for the growth of the lactic acid-producing microorganism,
  • the pH of step (iii) is the same or different from the initial pH;
  • the step (ii) is before or after the step (i).
  • the step (i), the step (ii) and the step (iii) are included in this order, and the lactic acid-producing microorganism is not neutralized through the step (i) and the step (ii).
  • the lactic acid-producing microorganism is cultured while adjusting to a pH suitable for the growth of the lactic acid-producing microorganism.
  • the lactic acid-producing microorganism in the step (i), is cultured while adjusting the pH to the initial pH, and in the step (ii), the lactic acid-producing microorganism is cultured without being neutralized. In the step (iii), the lactic acid-producing microorganism is cultured while adjusting the pH to a pH suitable for the growth of the lactic acid-producing microorganism.
  • the lactic acid-producing microorganism is a lactic acid bacterium.
  • the pH suitable for the growth of the lactic acid-producing microorganism is from weakly acidic to near neutral, and pH lower than the initial pH is more acidic.
  • the culture solution further contains a saccharifying enzyme.
  • a method for producing lactic acid capable of increasing the amount of lactic acid produced and suppressing the amount of residual sugar.
  • the method for producing lactic acid according to the present invention can efficiently produce lactic acid and reduce the sugar remaining in the culture solution (fermentation solution) by a simple operation of controlling the pH of the culture solution (fermentation solution). it can.
  • Lactobacillus plantarum ldhL1 amyA strain
  • fermentation was started after re-adjusting the sugar concentration when neutralization was not performed until 18 hours from the start of culture and when neutralization was performed.
  • the graph which shows the time-dependent change of the lactic acid density
  • the pH in the culture solution is lowered by lactic acid produced by fermentation. For this reason, it is common to proceed the fermentation while neutralizing, but in the present invention, the pH is intentionally lowered during the production of lactic acid by fermentation.
  • the present invention provides a method for producing lactic acid.
  • This method includes the following steps (i) to (iii): (I) a step of preparing a culture solution containing a sugar-containing medium and a lactic acid-producing microorganism and having an initial pH that is suitable for growth of the lactic acid-producing microorganism, and culturing the microorganism; (Ii) culturing the lactic acid-producing microorganism under a pH lower than the initial pH, and (iii) culturing the lactic acid-producing microorganism at a pH suitable for the growth of the lactic acid-producing microorganism.
  • Step (ii) can be before or after step (i).
  • the pH in step (iii) may be the same as the initial pH in step (i), or may be different as long as the lactic acid-producing microorganism used can grow.
  • lactic acid-producing microorganism refers to any microorganism that can produce lactic acid from a sugar such as glucose (in other words, has a lactic acid fermentation ability).
  • the lactic acid-producing microorganism may be either a naturally occurring microorganism or a genetically modified microorganism. Examples of microorganisms that exist in nature include lactic acid bacteria and molds (for example, the genus Rhizopus).
  • Lactic acid bacteria is a general term for bacteria that produce lactic acid from sugars by metabolism or fermentation. Lactic acid bacteria can be mainly classified into four types: bifidobacteria, enterococcus, lactobacilli, and streptococcus.
  • Lactic acid bacteria include Streptococcus, Lactobacillus, Bifidobacterium, Lactococcus, Pediococcus, or Leuconostoc Examples include the Streptococcus thermophilus (Streptococcus thermophilus), Streptococcus cremoris, Streptococcus faecalis, Streptococcus bulactic lus, Lactos Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus delbrueckii Arabinosus (Lactobacillus arabinosus), Lactobacillus caucasicus (Lactobacillus caucasicus), Lactobacillus lactis, Lactobacillus Leishmanni (Lactobacillus Leishmanni), Lactobacillus (musicus (Lactobacillus musicus) thermophilus, Lactobacillus plantarum, Bifidobacterium
  • Lactic acid bacteria also include sporic lactic acid bacteria.
  • the spore-forming lactic acid bacterium is a general term for the spore-forming lactic acid bacterium.
  • Examples of sporic lactic acid bacteria include bacteria belonging to the genus Bacillus.
  • the spore-forming lactic acid bacteria belonging to the genus Bacillus can have heat resistance (for example, capable of growing under high heat such as 45 ° C.), a high fermentation rate, and a wide sugar utilization.
  • bacteria belonging to the genus Bacillus include Bacillus coagulans (also known as “Sporolactic acid bacterium”) and Bacillus lincheniformis.
  • Examples of the mold include Rhizopus, and examples thereof include Rhizopus oryzae.
  • Examples of the genetically modified microorganism include a recombinant microorganism in which either L- or D-lactic acid synthase gene is incorporated or destroyed.
  • a genetically modified host a microorganism having a wealth of genome information and a sufficient track record is often selected.
  • yeast, Escherichia coli, lactic acid bacteria and the like can be used as genetically modified hosts.
  • Examples of genetically modified microorganisms include, for example, Lactobacillus plantarum ldhL1 :: amyA strain (Okano et al., Appl. Environ. Microbiol.
  • Lactobacillus plantarum ⁇ ldhL1 PxylAB-xpk1: : tkt- ⁇ xpk2 :: PxylAB strain (Yoshida et al., “Appl.” Microbiol. “Biotechnol.,” 2011, “Vol. 92,” 67-76).
  • the Lactobacillus plantarum ldhL1 :: amyA strain is a recombinant strain that secretes ⁇ -amylase and produces D-lactic acid from glucose.
  • Lactobacillus plantarum ⁇ ldhL1 :: PxylAB-xpk1 :: tkt- ⁇ xpk2 :: PxylAB strain Is a recombinant strain that produces D-lactic acid from both glucose and xylose.
  • a medium for culturing a lactic acid-producing microorganism for lactic acid fermentation contains a carbon source such as sugar as a main raw material, and is preferably a liquid medium.
  • Sugars include monosaccharides such as glucose, xylose, arabinose and fructose; disaccharides such as sucrose, maltose and trehalose; and polysaccharides such as starch, cellulose, hemicellulose and xylan; and molasses containing saccharides ( For example, sweet potato molasses).
  • Biomass a carbohydrate material derived from biological resources, can also be used as sugar. Examples of biomass include starch such as corn and rice, and molasses such as sugarcane molasses.
  • sugar raw materials that do not compete with food include lignocellulosic biomass composed of cellulose, hemicellulose, and lignin.
  • the biomass material may be subjected to a pretreatment (for example, a crushing treatment, a hydrothermal treatment, or a drying treatment) as necessary before being subjected to lactic acid fermentation culture.
  • the medium may further contain a nitrogen source.
  • Nitrogen sources include yeast extract, peptone, animal polypeptone, plant polypeptone, meat extract, corn steep liquor, fish protein hydrolysate, casamino acids, shochu, oil-like peptides or amino acids; ammonia, nitrates Inorganic nitrogen such as urea. Corn steep liquor, fish protein hydrolyzate, nitrate and the like are preferable because they can be used at low cost.
  • inorganic salts such as magnesium salts such as phosphate and magnesium sulfate, calcium salts, iron salts and manganese salts, which are usually added to the medium for lactic acid fermentation in the medium; vitamins; Fatty acids such as polysorbate may be added to the medium.
  • Biomass containing nutrients such as a nitrogen source can also be used as a sugar raw material. When biomass containing nutrients such as a nitrogen source is used as a sugar raw material, it is not necessary to add a nitrogen source separately. In this case, the pH is preferably adjusted to around neutral before fermentation.
  • a culture solution for lactic acid fermentation includes a medium containing sugar and a lactic acid-producing microorganism.
  • the culture solution for lactic acid fermentation can be prepared, for example, by adding microbial cells of lactic acid-producing microorganisms to the medium as described above.
  • the lactic acid-producing microorganism may be precultured in a medium suitable for growing the microorganism before being subjected to culture for lactic acid fermentation.
  • the culture solution for lactic acid fermentation may further contain a saccharifying enzyme.
  • the saccharifying enzyme depends on the type of sugar contained in the medium, but amylases such as ⁇ -amylase, ⁇ -amylase, glucoamylase, isoamylase and pullulanase; cellulases such as endoglucanase, cellobiohydrolase and ⁇ -glucanase; xylanase; A pullulanase etc. are mentioned, You may use combining these 1 or more.
  • the origin of the saccharifying enzyme is not particularly limited.
  • the culture step in the method of the present invention is not particularly limited, and a normal lactic acid fermentation method can be used.
  • the initial pH of the culture solution for lactic acid fermentation (the pH of the medium newly prepared for lactic acid fermentation) varies depending on the type of microorganism used, the type of medium, and the culture conditions, and can be appropriately determined as necessary.
  • the initial pH may be any pH value suitable for the growth of lactic acid-producing microorganisms.
  • the “pH value suitable for the growth of the lactic acid-producing microorganism” can depend on the type of the lactic acid-producing microorganism to be used, and can be, for example, from weakly acidic to near neutral.
  • the “near” pH can be, for example, in the range of pH 4.5-7, preferably 5.5-6.8. In the case of “near neutral”, for example, it may be in the range of 6 to 7, preferably 6 to 6.8.
  • the pH of the culture solution is lowered (acidified) by the lactic acid produced by the fermentation, so that the pH is controlled by neutralization when culturing microorganisms at a pH suitable for the growth of lactic acid-producing microorganisms.
  • the neutralizing agent used for pH control include alkali metal or alkaline earth metal hydroxides, alkali metal or alkaline earth metal carbonates, ammonium compounds, and mixtures thereof.
  • Examples thereof include sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, calcium carbonate, magnesium carbonate, aqueous ammonia, ammonium carbonate, and ammonium hydrogen carbonate.
  • Sodium hydroxide, potassium hydroxide and the like are preferable from the viewpoint of easy handling of the lactate obtained by neutralization.
  • the step of culturing a lactic acid-producing microorganism under an acidic pH that is, a lower pH
  • the culturing step under a more acidic pH may be either before being subjected to lactic acid fermentation or during the lactic acid fermentation process.
  • the lactic acid-producing microorganism is further cultured at a pH suitable for the growth of the lactic acid-producing microorganism (which is higher than the lowered pH).
  • the above steps (i), (ii) and (iii) are included in this order, and the steps (i) and (ii) (fermentation culture initial stage) Cultivating the lactic acid-producing microorganism through neutralization (to reduce the pH by lactic acid produced by fermentation) and neutralizing it through step (iii) (at a more acidic pH) Culturing the lactic acid-producing microorganism while adjusting the pH to a pH suitable for the growth of the lactic acid-producing microorganism (for example, from weakly acidic to near neutral).
  • step (i) in the above step (i) (early fermentation culture), while adjusting the pH to the initial pH (for example, from weakly acidic to near neutral), lactic acid Culturing the production microorganism, cultivating the lactic acid-producing microorganism without neutralization in the step (ii) (culture at a more acidic pH), and the step (iii) (pH suitable for the growth of the lactic acid-producing microorganism) Culturing the lactic acid-producing microorganism while adjusting the pH to a pH suitable for the growth of the lactic acid-producing microorganism (for example, from weakly acidic to near neutral).
  • Step (ii) can be before or after step (i).
  • step (iii) may be the same as the initial pH in step (i), or may be different as long as the lactic acid-producing microorganism used can grow.
  • step (ii) is before step (i)
  • step (i) and step (iii) can be combined into one step.
  • the means for lowering the pH of the culture solution may be reduced spontaneously by the action of lactic acid produced by the cells during fermentation (for example, the first embodiment and the second embodiment). Culture without neutralization) or may be forcibly reduced by some means (second embodiment). In step (ii) of the second embodiment, the pH may be lowered spontaneously or forcibly.
  • the timing of lowering the pH of the fermentation broth may be to lower the pH by stopping pH control during the fermentation culture or by adding acid, or by acclimatizing the cells with a low pH medium. May be.
  • the acclimatization of the microbial cells to the low pH medium is, for example, simply left without adjusting the pH in the lactic acid fermentation culture starting by adding the microbial cells to the medium of the initial pH from weakly acidic to neutral (that is, By allowing the pH of the culture solution to decrease as the lactic acid fermentation progresses, the cells are acclimated to a low pH medium), or before being subjected to lactic acid fermentation culture, the cells are pre-cultured in a lower pH medium. Can be made by Moreover, you may combine these methods.
  • the acid to be added examples include inorganic acids such as hydrochloric acid, sulfuric acid, carbonic acid, phosphoric acid, nitric acid, or any combination thereof. Hydrochloric acid is preferred.
  • the acidic pH (or lower pH) than the initial pH may depend on the type of microorganism used, but is a pH that is lower than the pH reduction that can be seen in culture under normal pH control as described below.
  • the pH is 0.7 or more lower than the initial pH, preferably 1 or more.
  • such a pH value can be in the range of pH 3 to 4.8, preferably pH 3.5 to 4.5.
  • the addition concentration of the acid depends on the kind of the acid, but may be any concentration suitable for causing neutralization, and may be, for example, in the range of 0.5M to 18M, preferably 1M to 6M.
  • the time for maintaining the culture solution (fermentation solution) at a more acidic pH may depend on the fermentation conditions and microorganisms used, but for example, 10 minutes to 12 minutes
  • the time may be 20 minutes to 6 hours, more preferably 30 minutes to 3 hours.
  • the pH is lowered spontaneously by culturing a lactic acid-producing microorganism without neutralization, it may depend on the fermentation conditions and microorganism used, but is preferably 6 to 24 hours in the fermentation culture medium.
  • a lactic acid-producing microorganism for example, the lactic acid bacterium Lactobacillus plantarum ldhL1 :: amyA strain of Example 1 below
  • a lactic acid-producing microorganism is cultured at an initial pH of 5.5 and cultured without neutralization for the above time (without pH control).
  • the above-mentioned more acidic pH for example, pH 3 to 4.8, preferably pH 3.5 to 4.5
  • the culture that has fallen below the initial pH is not necessarily limited to the above acidic pH value and time.
  • the pH of the culture solution can be lowered due to the produced lactic acid during the cultivation of lactic acid-producing microorganisms (even when pH is controlled).
  • the pH value during culture can vary.
  • the pH value at the time of cultivation includes a pH value that changes within a range that can occur other than a pH change by pH control. As an example, when the pH value during culture is set to 5.5, the pH value includes, for example, 5.4 to 5.6.
  • the culture may be batch culture, semi-batch culture, or continuous culture. Further, semi-batch culture or continuous culture in which only carbohydrates are added during culture may be used.
  • the culture time varies depending on the strain used, medium components, particularly the amount of carbohydrates, etc., but in the case of batch culture, for example, 1 day to 10 days, preferably 2 days to 8 days, more preferably 2 days to 7 days. Days.
  • the culture period is not limited thereto. It is desirable to lower the pH at a timing after the logarithmic growth phase of the cells. You may use a fermentation residue as a microbial bed. For example, after completion of one batch (batch) of fermentation, lactic acid-producing microorganisms contained in the fermentation residue may be used again for the next batch of fermentation.
  • the temperature of the fermentation culture can be set in consideration of the culture conditions such as the temperature at which the lactic acid-producing microorganism used grows and the action temperature of the added enzyme. For example, it can be set to 25 ° C. to 45 ° C., or 30 ° C. to 40 ° C., or 35 ° C. to 37 ° C., but may be higher or lower depending on the lactic acid-producing microorganism used.
  • the lactic acid-producing microorganism to be used is a thermostable microorganism such as a spore-forming lactic acid bacterium, it can be set to a higher temperature, for example, a temperature around 45 ° C. at which saccharifying enzymes generally work well.
  • Lactic acid can be recovered in the form of lactic acid or lactic acid alkali salt by removing the cells from the culture solution after culturing.
  • the method for recovering lactic acid from the culture solution is not particularly limited, and a known method can be used. For example, the method described in WO2007 / 114017 is mentioned. In addition, it is adsorbed on an ion exchange resin, washed and then eluted; a method in which an ester is formed by reacting with an alcohol (for example, methanol, ethanol, etc.) under addition of sulfuric acid; and insoluble lactic acid such as magnesium salt; Examples thereof include a method of recovering and purifying as a salt.
  • the lactic acid obtained by the present invention is useful as a raw material for biodegradable plastics such as polylactic acid and polyester polyol, and agricultural chemicals and pharmaceuticals.
  • Example 1 Lactic acid fermentation from brown rice by Lactobacillus plantarum
  • 200 g of crushed brown rice and 10 mL of CSL manufactured by Nippon Starch Co., Ltd.
  • CSL manufactured by Nippon Starch Co., Ltd.
  • 1 L of this fermentation medium was heated at 60 ° C. for 3 hours in a 3 L fermenter (microbe fermenter: manufactured by EYELA), and finally kept at 37 ° C.
  • the lactic acid bacterium Lactobacillus plantarum ldhL1 :: amyA was statically cultured at 37 ° C. for 20 hours in a medium containing 2% (v / v) CSL and 0.5% (w / v) soluble starch in distilled water. I was allowed to.
  • the total sugar concentration in the fermentation supernatant is quantified by the phenol / sulfuric acid method, the glucose concentration is quantified using a biosensor BF-5 (manufactured by Oji Scientific Instruments), and the lactic acid concentration is quantified by high-performance liquid chromatography. did. Hereinafter, these are simply referred to as total sugar concentration, glucose concentration, and lactic acid concentration in the fermentation broth.
  • FIG. 1 shows the change over time in the pH of the fermentation broth at which pH control was started at different points in lactic acid fermentation from brown rice.
  • the horizontal axis indicates the fermentation elapsed time (h: time)
  • the vertical axis indicates the fermentation broth pH value.
  • X, white rhombus, white triangle, and white circle respectively perform pH control by neutralization at the time of 15 hours (15h), 8 hours (8h), 5 hours (5h), and 0 hours (0h) from the start of fermentation. Represents the result.
  • Table 1 shows that in lactic acid fermentation from brown rice whose pH control was started at different time points, lactic acid concentration (g / L) in the fermentation solution after 144 hours of fermentation, and 1 L of the initial fermentation solution in consideration of dilution by adding a neutralizing agent Lactic acid amount per gram (g), concentration of glucose remaining in the fermentation broth (g / L) and total sugar concentration (g / L), and yield of lactic acid against sugar calculated from the glucose content of rice (g / g-glucose) ).
  • FIG. 2 shows the change over time in the glucose concentration in the fermentation broth in which pH control was started at different points in lactic acid fermentation from brown rice.
  • the horizontal axis indicates the fermentation elapsed time (h: time)
  • the vertical axis indicates the glucose concentration (g / L) of the fermentation broth supernatant.
  • X, white rhombus, white triangle, and white circle represent the results of pH control at 15 hours (15h), 8 hours (8h), 5 hours (5h), and 0 hours (0h) from the start of fermentation, respectively. .
  • glucose produced by saccharification by the enzyme was present at a high concentration in the initial stage of fermentation (up to about 24 hours from the start of fermentation).
  • pH control is performed at 0 hours from the start of fermentation (control) and when pH control is started at 5 hours after the start of fermentation, the glucose consumption rate is low, and the concentration is high even at the end of fermentation (after fermentation 72 hours). Of glucose remained.
  • pH control was started 8 and 15 hours after the start of fermentation, glucose produced at the beginning of fermentation was consumed rapidly after the start of pH control, and the residual glucose concentration became extremely low after 72 hours of fermentation.
  • the pH of the fermentation liquor is reduced by lactic acid produced by fermentation, and then neutralization is started to set the pH suitable for lactic acid fermentation (for example, 5.5). It was considered that the lactic acid yield was increased and the yield of lactic acid was also increased.
  • the cause of the increase in residual sugar concentration when pH control was started after 5 hours was that the cell density at 5 hours from the start of fermentation was low, and the properties of the cells grown after the start of neutralization affected the fermentation results. It seems to be.
  • FIG. 3 shows changes over time in the concentrations of lactic acid and glucose in the fermentation broth maintained at pH 4.8 for 30 minutes or 3 hours after 17 hours from the start of fermentation.
  • the horizontal axis indicates the fermentation elapsed time (h: time)
  • the vertical axis indicates the concentration (g / L) of lactic acid or glucose in the fermentation broth.
  • the square represents the lactic acid concentration in the fermentation broth
  • the circle represents the glucose concentration in the fermentation broth
  • the white symbol is the result when maintained at pH 4.8 for 30 minutes
  • the black symbol is maintained at pH 4.8 for 3 hours. Is the result of the case.
  • Example 2 Lactic acid fermentation from a model sugar solution using various bacterial cells
  • Lactobacillus plantarum ldhL1 amyA strain
  • Lactobacillus plantarum ⁇ ldhL1 PxylAB-xpk1 :: tkt- ⁇ xpk2 :: PxylAB strain
  • Lactobacillus casei were examined.
  • Each lactic acid bacterium is treated with 2% (v / v) CSL and 0.5% (w / v) substrate (Lactobacillus plantarum ldhL1 :: amyA strain and Lactobacillus casei for glucose only; Bacillus plantarum ⁇ ldhL1 :: PxylAB-xpk1 :: tkt- ⁇ xpk2 :: PxylAB is mixed in an equal amount of glucose and xylose) in a distilled water and allowed to stand at 37 ° C. for 20 hours. A suspension was obtained.
  • 900 mL of a pH 5.5 medium containing 4% (v / v) CSL and 2% (w / v) substrate in distilled water was prepared, and 60 mL was prepared in a 3 L fermenter (microbe fermenter: manufactured by EYELA). After sterilizing at 3 ° C. for 3 hours, the temperature was kept at 37 ° C. 45 mL of the bacterial suspension was added thereto, and culture was performed while stirring at 37 ° C. and 200 rpm. The culture was continued for 18 hours without neutralization immediately after the start of the culture.
  • Lactobacillus casei was cultured in the same manner as described above except that the initial pH was 6.0 and the culture temperature was 30 ° C.
  • control experiment was performed in the same manner except that the pH during the culture was maintained at the initial pH value (5.5 or 6.0) by neutralizing with 5N NaOH immediately after the start of the culture. .
  • FIGS. 4 to 6 show the production during liquid culture after starting fermentation after readjusting the sugar concentration after 18 hours of culture for various lactic acid bacteria when neutralization is not performed until 18 hours from the start of culture and when neutralized.
  • FIG. 4 shows changes over time in the concentrations of lactic acid and glucose (FIG. 4, Lactobacillus plantarum ldhL1 :: amyA strain; FIG. 5, Lactobacillus plantarum ⁇ ldhL1 :: PxylAB-xpk1 :: tkt- ⁇ xpk2 :: PxylAB strain; and FIG. 6) , Lactobacillus casei).
  • the horizontal axis indicates the elapsed fermentation time (h: hour), and the vertical axis indicates the concentration (g / L) of the produced lactic acid or glucose in the fermentation broth.
  • the square represents the concentration of lactic acid produced in the fermentation broth
  • the circle represents the glucose concentration in the fermentation broth
  • the white symbol is the result of the control (pH adjusted immediately after cultivation)
  • the black symbol is the low pH period ( The result is that neutralization is not performed until 18 hours immediately after the culture).
  • the concentration of the produced lactic acid was expressed as a concentration value obtained by subtracting the lactic acid concentration immediately after the start of fermentation (at the time of readjusting the sugar concentration) from the measured lactic acid concentration in the fermentation broth.
  • Lactobacillus casei as shown in FIG. 6, the glucose consumption rate was faster and the lactic acid concentration was higher when the pH at the initial stage of the culture was lowered.
  • the glucose consumption rate was faster and the lactic acid concentration was higher when the pH at the initial stage of the culture was lowered.
  • Example 3 Effect of lactic acid fermentation by pH control without addition of nutrient sources
  • the lactic acid bacterium Lactobacillus plantarum ldhL1 :: amyA was statically cultured at 37 ° C. for 20 hours in a medium containing 2% (v / v) CSL and 0.5% (w / v) soluble starch in distilled water. Then, the cells were collected by centrifugation at 8,000 G for 5 minutes and suspended in 0.85% physiological saline.
  • FIG. 7 shows changes over time in the concentrations of lactic acid and glucose in the fermentation broth when pH control is started at 0 hours or 15 hours from the start of fermentation in lactic acid fermentation from brown rice without addition of a nutrient source.
  • the horizontal axis indicates the fermentation elapsed time (h: time), and the vertical axis indicates the concentration (g / L) of lactic acid or glucose in the fermentation broth.
  • the square represents the lactic acid concentration in the fermentation broth, the circle represents the glucose concentration in the fermentation broth, the white symbol indicates the pH control at the time of 15 hours (15 h) of fermentation start, and the black symbol indicates the time at 0 hour (0 h). Indicates.
  • Table 2 shows the results after 144 hours of fermentation when pH control was started at 0 hours or 15 hours from the start of fermentation in lactic acid fermentation from brown rice without addition of a nutrient source. From left to right, lactate concentration (g / L) in the fermentation liquor, the amount of lactic acid A LA of initial fermentation broth per 1L considering dilution by addition of a neutralizing agent (g), a pair of lactic acid was calculated from the glucose content of the rice Yield of sugar Y LA / Glc (g / g-glucose), concentration of glucose remaining in fermentation broth (g / L) and total sugar concentration (g / L), and optical purity of D-lactic acid (% ee) Show.
  • Example 4 Influence of lactic acid fermentation by pH control using repeated fermentation residues
  • the lactic acid bacterium Lactobacillus plantarum ldhL1 :: amyA was statically cultured at 37 ° C. for 20 hours in a medium containing 2% (v / v) CSL and 0.5% (w / v) soluble starch in distilled water. Then, the cells were collected by centrifugation at 8,000 G for 5 minutes and suspended in 0.85% (w / v) physiological saline.
  • the lactic acid fermentation liquor is passed through a strainer, and the passed fermentation liquor is collected, centrifuged at 12,000 G for 10 minutes, and 40 to 78 g (wet weight) of precipitate (fermentation residue) Got.
  • This fermentation residue contained lactic acid bacteria.
  • This fermentation residue was added as a fungal bed to another fermentation medium heat-sterilized as described above.
  • saccharification enzyme Glucozyme # 20000 0.3 g, Spitase CP-40FG 0.39 g: both manufactured by Nagase ChemteX Corporation
  • was added was added, and fermentation of the next batch was started.
  • the fermentation was repeated up to 10 batches. In all batches, neutralization by dropwise addition of 5N NaOH (5M NaOH) was started in 15 hours from the start of fermentation.
  • FIG. 8 shows changes over time in lactic acid and glucose concentrations in the fermentation broth when pH 6.0 was controlled from 15 hours after the start of fermentation for each batch when 10 batches of fermentation were performed repeatedly using the residue as a fungus bed.
  • the horizontal axis indicates the fermentation elapsed time (h: time)
  • the vertical axis indicates the concentration (g / L) of lactic acid or glucose in the fermentation broth.
  • the black square represents the lactic acid concentration in the fermentation broth
  • the white circle represents the glucose concentration in the fermentation broth.
  • Example 5 Influence of lactic acid fermentation by pH control using fermentation residue
  • microbe fermenter: EYELA microbe fermenter: EYELA
  • the lactic acid bacterium Lactobacillus plantarum ldhL1 :: amyA was statically cultured at 37 ° C. for 20 hours in a medium containing 2% (v / v) CSL and 0.5% (w / v) soluble starch in distilled water. Then, the cells were collected by centrifugation at 8,000 G for 5 minutes and suspended in 0.85% (w / v) physiological saline.
  • the lactic acid fermentation liquor is passed through a strainer, and the passed fermentation liquor is collected, centrifuged at 12,000 G for 10 minutes, and 40 to 78 g (wet weight) of precipitate (fermentation residue) Got.
  • This fermentation residue contained lactic acid bacteria.
  • This fermentation residue was added as a fungal bed to another fermentation medium heat-sterilized as described above.
  • saccharification enzyme (glucoteam # 20000 0.3 g, spitase CP-40FG 0.39 g: both manufactured by Nagase ChemteX Corporation) was added, and fermentation of the next batch (second batch) was started.
  • neutralization by dropping 5N NaOH (5M NaOH) was started in 0 hours from the start of fermentation.
  • FIG. 9 shows the fermentation of the first batch when the pH is controlled from the time point of 15 hours from the start of fermentation and the second batch when the pH is controlled from the time point of 0 hours from the start of fermentation.
  • concentration in a fermented liquid at the time of performing fermentation is shown.
  • the horizontal axis indicates the fermentation elapsed time (h: time)
  • the vertical axis indicates the concentration (g / L) of lactic acid or glucose in the fermentation broth.
  • the black square represents the lactic acid concentration in the fermentation broth
  • the white circle represents the glucose concentration in the fermentation broth.
  • Example 6 Influence of lactic acid fermentation by pH control with cellulosic material
  • Hardwood pulp (powder) 15 g and Barex (manufactured by Sanwa Sake Co., Ltd.) 6 mL were dissolved in distilled water, adjusted to pH 5.5 and adjusted to 100 mL capacity to prepare a fermentation medium. 100 mL of this fermentation medium was transferred to a 200 mL fermenter (separable flask), sterilized in an autoclave at 121 ° C. for 15 minutes, and then finally kept at 37 ° C.
  • the lactic acid bacterium Lactobacillus plantarum ⁇ ldhL1 :: PxylAB-xpk1 :: tkt- ⁇ xpk2 :: PxylAB was transformed into 6% (v / v) Barex, 0.4% (w / v) glucose and 0.1% ( w / v) In a medium containing xylose in distilled water, static culture was performed at 37 ° C. to obtain a cell suspension.
  • FIG. 10 shows the changes over time of the lactic acid concentration and the total sugar concentration of glucose and xylose in the fermentation broth when the pH was controlled from the time point of 15 hours or 0 hours after the start of fermentation using cellulosic raw materials.
  • the horizontal axis indicates the fermentation elapsed time (h: time)
  • the vertical axis indicates the lactic acid concentration (g / L) in the fermentation broth or the total sugar concentration (g / L) of glucose and xylose.
  • the square represents the concentration of lactic acid in the fermentation broth
  • the circle represents the concentration of sugar (total sugar of glucose and xylose) in the fermentation broth
  • the white symbol is 15 hours (15 h) at the start of fermentation
  • the black symbol is 0 hour (0 h). The result of having performed pH control at the time is shown.
  • lactic acid can be efficiently produced by a simple process of controlling the pH of a culture solution (fermentation solution) in the process of lactic acid fermentation. Furthermore, since the sugar remaining in the culture solution (fermentation solution) can be reduced, the lactic acid purification step is facilitated, and the yield of lactic acid ester is also improved when lactic acid esters are obtained.
  • the production improvement of lactic acid and the reduction of residual sugar that can be achieved by the present invention lead to the improvement of the production efficiency and the cost reduction of polylactic acid and polyester polyol using lactic acid as raw materials, and agricultural chemicals or medicines.

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Abstract

　L'objectif de la présente invention est de fournir un procédé de production d'acide lactique qui permet d'augmenter la quantité d'acide lactique produite et de supprimer la teneur en sucre résiduelle. La présente invention concerne un procédé de production d'acide lactique, le procédé comprenant : (i) une étape de préparation d'un bouillon de culture contenant un milieu contenant du sucre et des microorganismes producteurs d'acide lactique, le bouillon de culture possédant un pH de départ qui est adapté à la croissance des microorganismes producteurs d'acide lactique, et de culture des microorganismes ; (ii) une étape de culture des microorganismes producteurs d'acide lactique à un pH inférieur au pH de départ ; et (iii) une étape de culture des microorganismes producteurs d'acide lactique à un pH adapté à la croissance des microorganismes producteurs d'acide lactique, avec une étape incluse, dans laquelle le pH de l'étape (iii) est identique ou différent du pH de départ et l'étape (ii) est réalisée avant ou après l'étape (i).
PCT/JP2014/079030 2013-11-05 2014-10-31 Procédé de production d'acide lactique WO2015068645A1 (fr)

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JPH11285397A (ja) * 1998-04-01 1999-10-19 Kankyo Technos Kk 有機廃棄物を原料とする乳酸の製造方法
JP2000236891A (ja) * 1999-02-23 2000-09-05 Kankyo Technos Kk 米焼酎蒸留粕からの乳酸の製造方法
JP2004065142A (ja) * 2002-08-08 2004-03-04 Kuraudo:Kk 乳酸菌による乳酸生成方法
JP2005073549A (ja) * 2003-08-29 2005-03-24 Oita Technology Licensing Organization Ltd 乳酸液の製造方法。
JP2007215427A (ja) * 2006-02-14 2007-08-30 Musashino Chemical Laboratory Ltd 乳酸の製造方法
WO2011049205A1 (fr) * 2009-10-23 2011-04-28 独立行政法人産業技術総合研究所 Procédé de fabrication d'acide lactique par fermentation non stérile

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JPS56148237A (en) * 1980-04-15 1981-11-17 Yoshihide Hagiwara Food or beverage made of lactic fermentation product from unpolished rice
JPS6236169A (ja) * 1985-08-07 1987-02-17 Masayoshi Takuma 玄米健康飲料水の製造方法
JPH06253871A (ja) * 1993-03-02 1994-09-13 Musashino Kagaku Kenkyusho:Kk 乳酸の製造方法
JPH08280341A (ja) * 1995-04-13 1996-10-29 Kano Shiyoujiyuan:Kk 乳酸発酵液の製造法
JPH11113513A (ja) * 1997-10-21 1999-04-27 Sooi:Kk 玄米発酵物
JP2010193846A (ja) * 2009-02-27 2010-09-09 Chube Univ 乳酸発酵方法
WO2012008589A1 (fr) * 2010-07-16 2012-01-19 国立大学法人九州大学 Procédé pour la production de l-lactose au moyen de lactobacille en présence de pentose et de cello-oligosaccharide

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0276592A (ja) * 1988-08-10 1990-03-15 Rhone Poulenc Chim 乳酸の製法
JPH11285397A (ja) * 1998-04-01 1999-10-19 Kankyo Technos Kk 有機廃棄物を原料とする乳酸の製造方法
JP2000236891A (ja) * 1999-02-23 2000-09-05 Kankyo Technos Kk 米焼酎蒸留粕からの乳酸の製造方法
JP2004065142A (ja) * 2002-08-08 2004-03-04 Kuraudo:Kk 乳酸菌による乳酸生成方法
JP2005073549A (ja) * 2003-08-29 2005-03-24 Oita Technology Licensing Organization Ltd 乳酸液の製造方法。
JP2007215427A (ja) * 2006-02-14 2007-08-30 Musashino Chemical Laboratory Ltd 乳酸の製造方法
WO2011049205A1 (fr) * 2009-10-23 2011-04-28 独立行政法人産業技術総合研究所 Procédé de fabrication d'acide lactique par fermentation non stérile

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