WO2019027017A1 - Method for culturing lactic acid bacteria - Google Patents

Method for culturing lactic acid bacteria Download PDF

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WO2019027017A1
WO2019027017A1 PCT/JP2018/029124 JP2018029124W WO2019027017A1 WO 2019027017 A1 WO2019027017 A1 WO 2019027017A1 JP 2018029124 W JP2018029124 W JP 2018029124W WO 2019027017 A1 WO2019027017 A1 WO 2019027017A1
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lactic acid
culture
amount
acid bacteria
batch culture
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美桜 河合
思乃 山崎
啓雄 片倉
麻美 土屋
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株式会社明治
学校法人 関西大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

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  • the present invention relates to a method of culturing lactic acid bacteria.
  • Lactic acid bacteria acquire ATP by metabolizing sugars to pyruvate, and regenerate NAD + required in this process by converting pyruvate to lactate. For this reason, lactic acid produced along with the growth of lactic acid bacteria is accumulated to lower the pH of the culture medium, and the growth and substance production of lactic acid bacteria are inhibited.
  • the decrease in pH of the medium can be suppressed by adding an alkali.
  • alkali is added to the medium to maintain the pH, the accumulation of lactic acid causes growth inhibition of lactic acid bacteria by lactic acid itself, so high-density culture of lactic acid bacteria is considered to be difficult.
  • lactic acid produced from lactic acid bacteria affects not only growth but also the quality as a lactic acid bacteria material.
  • lactic acid bacteria have attracted attention as probiotics and are used for various foods, but since lactic acid or a salt thereof has a unique flavor, it is desirable that the amount of lactic acid in the culture be small.
  • the presence of lactic acid causes moisture absorption.
  • lactic acid produced during culture of lactic acid bacteria inhibits the growth of the lactic acid bacteria themselves, and reduces the quality of the lactic acid bacteria material.
  • JP 2001-211878 A Japanese Examined Patent Publication 6-69367
  • Methods of avoiding growth inhibition by lactic acid include a method of continuously culturing at a high dilution rate to suppress the generated lactic acid to a low concentration, and a method of removing lactic acid by filtration, electrodialysis or the like (for example, Patent Document 1 and 2).
  • Patent Document 1 and 2 a method of removing lactic acid by filtration, electrodialysis or the like.
  • NADH dehydrogenase of the respiratory chain As a method of suppressing lactic acid producing lactic acid bacteria, it activates NADH dehydrogenase of the respiratory chain by the addition of hemin to play the NAD + from NADH, although the conversion of pyruvate to lactate is inhibiting culture method, NADH Since it is necessary to supply the oxygen necessary for the oxidation of H. pylori, it can be used only for some oxygen-insensitive strains.
  • an object of the present invention is to provide a method for cultivating lactic acid bacteria, which suppresses lactic acid production of various lactic acid bacteria and enables high-density culture.
  • the present invention is a culture method of lactic acid bacteria for high-density culture, and characterized in that the lactic acid bacteria are cultured by fed-batch culture.
  • cultivation method of the lactic acid bacteria which suppresses the lactic acid production at the time of culture
  • the cell yield that is, the cell yield per used sugar can be increased.
  • FIG. 1 is a graph showing temporal changes in the amount of cells and the amount of lactic acid in Example 1.
  • FIG. 2 is a graph showing temporal changes in the amount of cells and the amount of lactic acid in Example 2.
  • FIG. 3 is a graph showing temporal changes in the amount of cells and the amount of lactic acid in Example 3.
  • FIG. 4 is a graph showing temporal changes in the amount of cells and the amount of lactic acid in Example 4.
  • FIG. 5 is a graph showing temporal changes in the amount of cells and the amount of lactic acid in Example 5.
  • the culture method of lactic acid bacteria according to the present invention is characterized in that the lactic acid bacteria are cultured by fed-batch culture.
  • Fed-batch culture is a culture method in which culture is performed while supplying a medium or a substrate in the medium during the culture.
  • lactic acid bacteria are cultured in a culture medium, the concentration of lactic acid produced along with the growth of lactic acid bacteria increases, and the growth inhibition of lactic acid bacteria by lactic acid itself occurs.
  • the inventor of the present application has found that by performing fed-batch culture, the production of lactic acid is significantly suppressed as compared with the case of performing batch culture.
  • Lactic acid bacteria obtain ATP by metabolizing sugars to pyruvate by glycolysis, and regenerate the NAD + required in this process by converting pyruvate to lactate by lactate dehydrogenase.
  • lactic acid bacteria have a pathway for regenerating NAD + in addition to lactate dehydrogenase, and also have a pathway for acquiring ATP besides glycolysis.
  • the inventors of the present invention have noticed that most of these pathways are suppressed when sugars, in particular glucose, are present at concentrations above a certain level. It is not necessary to supply the sugar necessary for culture at once at the start of culture, but by feeding small amounts by fed-batch culture to keep the concentration of glucose and other sugar in the medium low, these pathways are suppressed.
  • lactic acid bacteria in which these pathways do not have sufficient ability, it depends on the glycolytic system to acquire ATP and rely on lactate dehydrogenase for NAD + regeneration, but sugar is supplied in small amounts by fed-batch Not only can these pathways be suppressed, but also reduce the proportion of energy gain by glycolysis in energy production, and also reduce the contribution of lactate dehydrogenase in NAD + regeneration, As a result, it leads to suppression of lactic acid production. Since the inhibition of the growth of lactic acid bacteria can be reduced when the production of lactic acid is suppressed, it becomes possible to culture lactic acid bacteria at a high density (high concentration) as compared with the case of performing batch culture.
  • sugars that can be used as a sugar source include monosaccharides such as glucose, fructose, galactose and mannose, and disaccharides such as sucrose, lactose, maltose and trehalose.
  • monosaccharides such as glucose, fructose, galactose and mannose
  • disaccharides such as sucrose, lactose, maltose and trehalose.
  • glucose or sucrose is preferred, and sucrose is more preferred.
  • sucrose is used as a sugar source, the generation of lactic acid can be further suppressed as compared to the case where glucose is used.
  • the medium in addition to the sugar source, nutrients such as amino acids, vitamins, nucleic acids, metal salts, etc., and a buffer solution for suppressing pH fluctuation and the like are blended in accordance with a conventional method.
  • the strain of lactic acid bacteria that can be cultured by the culture method according to the present invention is not particularly limited, and various strains can be cultured at high density.
  • species and strains contained in Lactobacillus, Lactococcus, and Leuconostoc can be cultured at high density.
  • the strains that can be cultured are limited to some strains with low oxygen sensitivity, but the present invention relates to the present invention.
  • the culture method can be applied to the culture of various strains because it does not require oxygen supply.
  • the pH of the culture medium, the amount of dissolved oxygen, the culture conditions such as culture temperature, culture time, and the conditions generally applied to the culture of lactic acid bacteria may be used, and can be appropriately selected according to the type of strain. Moreover, it is preferable to perform preculture before culture
  • the pre-culture is a culture for preparing a seed to inoculate the main culture. By pre-culturing, it is possible to secure the amount of cells necessary as the inoculum of the main culture.
  • the culture method according to the present invention can be used to produce specific chemical substances (peptides, proteins, polysaccharides, etc.) produced by lactic acid bacteria.
  • the culture method according to the present invention is also applicable to the production of fermented milk containing lactic acid bacteria at a high concentration, and foods such as lactic acid bacteria beverages and cheese.
  • the culture method according to the present invention is applied to the production of a food, it becomes possible to produce a food containing a high concentration of a specific chemical substance having physiological activity and a lactic acid bacterium producing the same.
  • Example 1 Lactobacillus gasseri JCM1131 was used as a strain of lactic acid bacteria.
  • preculture was performed. 100 ⁇ L of the frozen stock of Lb. gasseri JCM 1131 was inoculated into 100 mL of MRS medium containing glucose or sucrose as a sugar source, and stationary cultured at 37 ° C. for 12 hours. Next, 100 mL of MRS medium with a sugar source concentration of 0 g / L was prepared. The OD 660 of the preculture fluid was measured, and the amount of culture fluid required to bring the main culture fluid to 200 OD units was calculated. The preculture liquid was centrifuged (8,000 ⁇ g, 4 ° C., 5 min), washed with physiological saline, and then resuspended in this culture liquid and inoculated.
  • the flow acceleration F (L / h) of the medium in fed-batch culture was calculated based on the following equation so as to keep the specific growth rate constant.
  • t is the culture time
  • the initial culture solution volume V 0 was 0.1 L.
  • the sugar concentration S F of the feed medium is 20 g / L
  • the cell yield Y X / S is 0.1 g-cell / g-sugar when using sucrose as a sugar source, and when using glucose as a sugar source , 0.2 g-cell / g-sugar.
  • the specific growth rate ⁇ * was set at 0.25 1 / h when using sucrose as a sugar source, and at 0.30 1 / h when using glucose as a sugar source.
  • condition setting is made the same between the case where sucrose is used as the sugar source and the case where glucose is used, a difference is generated in the specific growth rate of the measured values. Therefore, the case where sucrose is used as the sugar source and the case where glucose is used
  • the respective cell yield and specific growth rate were set in The flow acceleration was determined from the above equation using these set values.
  • stirring culture was performed using a 3 cm diameter magnet bar while controlling the pH of the culture solution to 6.5 with a 3 M NaOH aqueous solution.
  • batch culture was performed using MRS medium containing glucose or sucrose at a concentration of 20 g / L as a sugar source.
  • the amount of culture solution, the amount of inoculation and the culture conditions were the same as in the fed-batch culture.
  • the culture fluid was sampled from the sampling line at intervals of 30 minutes or at intervals of 1 hour using a 5 mL syringe. At the time of sampling, after discarding the culture solution remaining in the sampling line, 1 mL of culture solution was sampled.
  • the turbidity (OD 660 ) of the sampled culture solution is measured using a UV-visible spectrophotometer UV-1850 (manufactured by Shimadzu Corporation), and the amount of cells in the culture solution is calculated based on the measured OD 660 value. did. Further, the concentration of lactic acid in the sampled culture solution was measured using a biosensor BF-5 (manufactured by Oji Scientific Instruments) and an enzyme electrode (manufactured by Oji Scientific Instruments). The lactic acid concentration is the total concentration of D-lactic acid and L-lactic acid.
  • the specific production rate ⁇ was determined from the slope.
  • the amount of lactic acid Y L / X per amount of proliferating cells is defined by dividing ⁇ by ⁇ in the logarithmic growth period, and dividing the amount of cells by the amount of sugar consumed by the cell yield Calculated by
  • Table 1 shows the specific growth rate ⁇ , specific production rate ⁇ , and proliferating cell mass calculated from the amount of cells and lactic acid amount after a predetermined time (6 hours after fed-batch culture and 2 hours after batch culture) from the start of main culture. It shows the amount of lactic acid per unit ⁇ / ⁇ , and the cell yield. Further, FIG. 1 shows a graph of temporal changes in the amount of cells and the amount of lactic acid. In Tables 1 to 5 shown below, error ranges indicate 95% confidence intervals by regression analysis.
  • the amount of lactic acid per growing bacterial body was smaller in fed-batch culture than in batch culture, regardless of whether glucose or sucrose was used as a sugar source.
  • the amount of lactic acid per growing bacterial body was smaller when using a sucrose sugar source.
  • the cell yield (g-cell / g-sugar) was higher in fed-batch culture than in batch culture, regardless of whether glucose or sucrose was used as the sugar source.
  • the amount of cells when fed-batch culture exceeded the amount of cells when batch culture was performed 5 hours after the start of main culture.
  • the amount of lactic acid was compared 5 hours after the start of the main culture, it was suppressed by the fed-batch culture as compared to the batch culture.
  • Example 2 Lactobacillus plantarum subsp. Plantarum NBRC 15891 is used as a strain of lactic acid bacteria, batch culture using sucrose as a sugar source, fed-batch culture using sucrose as a sugar source, and flow using glucose as a sugar source And culture.
  • the culture method, the sampling method, and the method of calculating each evaluation value are the same as in Example 1.
  • Table 2 shows the specific growth rate ⁇ , specific production rate ⁇ , and proliferating cells calculated from the amount of cells and lactic acid amount after a predetermined time from the start of the main culture (6 hours after fed-batch culture and 4 hours after batch culture). It shows the amount of lactic acid per unit ⁇ / ⁇ , and the cell yield. Further, FIG. 2 shows a graph of temporal changes of the amount of cells and the amount of lactic acid.
  • the amount of lactic acid in the case of performing a fed-batch culture is the case of performing batch culture using sucrose as a sugar source. It was suppressed by comparison.
  • Example 3 Lactobacillus reuteri JCM1112 was used as a strain of lactic acid bacteria.
  • the culture method, the sampling method, and the method of calculating each evaluation value are the same as in Example 1.
  • Table 3 shows the specific growth rate ⁇ , specific production rate ⁇ ⁇ , and proliferating cell mass calculated from the amount of cells and lactic acid amount after a predetermined time (3 hours after fed-batch culture and 2 hours after batch culture) from the start of main culture. It shows the amount of lactic acid per unit ⁇ / ⁇ , and the cell yield. Further, FIG. 3 shows a graph of temporal changes of the amount of cells and the amount of lactic acid.
  • the amount of lactic acid per growing cell was smaller in fed-batch culture than in batch culture, regardless of whether glucose or sucrose was used as a sugar source. Moreover, when the fed-batch culture using a glucose sugar source was compared with the fed-batch culture using a sucrose sugar source, the amount of lactic acid per growth cell was smaller when using a sucrose sugar source. However, even in fed-batch culture using glucose as a sugar source, it is considered that the effect of glucose repression is sufficiently reduced. Also, the cell yield (g-cell / g-sugar) was higher in fed-batch culture than in batch culture, regardless of whether glucose or sucrose was used as the sugar source.
  • Example 3 when the batch culture using a glucose sugar source was compared with the batch culture using a sucrose sugar source, the amount of lactic acid per growth cell was smaller when using a sucrose sugar source.
  • Lb. reuteri has been reported to regenerate one molecule of NAD + by degrading sucrose to glucose and fructose and converting fructose to mannitol.
  • sucrose as a sugar source, NAD + is regenerated by mannitol production, and by performing a fed-batch culture with a sucrose sugar source, the flux of this pathway is further increased, which is considered to be linked to suppression of lactic acid production.
  • Example 4 Lactobacillus rhamnosus ATCC 53103 was used as a strain of lactic acid bacteria.
  • the culture method, the sampling method, and the method of calculating each evaluation value are the same as in Example 1.
  • Table 4 shows the specific growth rate ⁇ , specific production rate ⁇ , and proliferating cells calculated from the amount of cells and lactic acid amount after a predetermined time (5 hours after fed-batch culture and 2.5 hours after batch culture) after the start of main culture. It shows the amount of lactic acid per unit ⁇ / ⁇ , and the cell yield. Further, FIG. 4 shows a graph of temporal changes of the amount of cells and the amount of lactic acid.
  • the amount of lactic acid per growing cell was smaller in fed-batch culture than in batch culture, regardless of whether glucose or sucrose was used as a sugar source. Moreover, when the fed-batch culture using a glucose sugar source was compared with the fed-batch culture using a sucrose sugar source, the amount of lactic acid per growth cell was smaller when using a sucrose sugar source. However, even in fed-batch culture using glucose as a sugar source, it is considered that the effect of glucose repression is sufficiently reduced. Also, the cell yield (g-cell / g-sugar) was higher in fed-batch culture than in batch culture, regardless of whether glucose or sucrose was used as the sugar source.
  • Example 5 Lactobacillus plantarum NCIMB 8826 was used as a strain of lactic acid bacteria.
  • the culture method, the sampling method, and the method of calculating each evaluation value are the same as in Example 1.
  • Table 5 shows the specific growth rate ⁇ , specific production rate ⁇ , and proliferating cell mass calculated from the amount of cells and lactic acid amount after a predetermined time from the start of the main culture (5 hours after fed-batch culture and 4 hours after batch culture) It shows the amount of lactic acid per unit ⁇ / ⁇ , and the cell yield. Further, FIG. 5 shows a graph of temporal changes in the amount of cells and the amount of lactic acid.
  • the amount of lactic acid per growing cell was smaller in fed-batch culture than in batch culture, regardless of whether glucose or sucrose was used as a sugar source. Moreover, when the fed-batch culture using a glucose sugar source was compared with the fed-batch culture using a sucrose sugar source, the amount of lactic acid per growth cell was smaller when using a sucrose sugar source. However, even in fed-batch culture using glucose as a sugar source, it is considered that the effect of glucose repression is sufficiently reduced. Also, the cell yield (g-cell / g-sugar) was higher in fed-batch culture than in batch culture, regardless of whether glucose or sucrose was used as the sugar source.
  • Example 6 Lb. rhamnosus ATCC 53103 was used as a strain of lactic acid bacteria.
  • preculture 100 ⁇ L of the frozen stock of Lb. rhamnosus ATCC 53103 was inoculated into 100 mL of MRS medium containing 2% of glucose or galactose as a sugar source and statically cultured at 37 ° C. for 15 hours.
  • the OD 660 of the preculture fluid was measured, and the amount of culture fluid required to make the main culture fluid 100 OD units was calculated.
  • the preculture liquid was centrifuged (8,000 ⁇ g, 4 ° C., 5 min), washed with physiological saline, and then resuspended in this culture liquid and inoculated.
  • the flow acceleration F (L / h) of the medium in fed-batch culture was calculated based on the following equation so as to keep the specific growth rate constant.
  • the feed medium was fed to the medium at a calculated flow rate using a peristaltic pump.
  • t is culture time (h)
  • the initial culture solution volume V 0 was 0.1 L.
  • the sugar concentration S F of the feed medium was 10 g / L, and the cell yield Y X / S was 0.2 g-cell / g-sugar.
  • a temperature-controlled thermostatic bath was placed on a magnetic stirrer, and stirring culture was performed at 500 rpm.
  • batch culture was performed using MRS medium containing glucose or galactose at 2% as a sugar source.
  • the amount of culture solution, the amount of inoculation and the culture conditions were the same as in the fed-batch culture.
  • the culture fluid was sampled from the sampling line using a 5 mL syringe. At the time of sampling, after discarding the culture solution remaining in the sampling line, 1 mL of culture solution was sampled.
  • the turbidity (OD 660 ) of the sampled culture solution is measured using a UV-visible spectrophotometer UV-1850 (manufactured by Shimadzu Corporation), and the amount of cells in the culture solution is calculated based on the measured OD 660 value. did. Further, the concentration of lactic acid in the sampled culture solution was measured using a biosensor BF-5 (manufactured by Oji Scientific Instruments) and an enzyme electrode (manufactured by Oji Scientific Instruments). The lactic acid concentration is the total concentration of D-lactic acid and L-lactic acid. Based on the obtained amount of cells, as an evaluation value, the specific growth rate ⁇ , the specific production rate ⁇ , and the amount ⁇ / ⁇ of lactic acid per the amount of grown cells were calculated.
  • Table 6 shows the specific growth rate ⁇ , the specific production rate ⁇ , and the amount of lactic acid ⁇ / ⁇ per amount of proliferating bacterial cells calculated from the amount of lactic acid and the amount of lactic acid after 3 hours in both fed-batch culture and batch culture from the start of main culture. Show.
  • the error range indicates the 95% confidence interval by regression analysis.
  • Example 7 Lactococcus lactis MG1363 was used as a strain of lactic acid bacteria.
  • Lactis MG1363 was inoculated into 100 mL of MRS medium containing 2% of glucose or galactose as a sugar source, and static culture was carried out at 30 ° C. for 15 hours.
  • Table 7 shows the specific growth rate ⁇ , the specific production rate ⁇ ⁇ , and the proliferating cells calculated from the amount of cells and the amount of lactic acid after a predetermined time from the start of the main culture (3 hours after fed-batch culture and 2 hours after batch culture). The amount of lactic acid per unit ⁇ / ⁇ is shown.
  • the error range indicates the 95% confidence interval by regression analysis.
  • the present invention can be used for high-density culture of lactic acid bacteria, and in particular, can be used for the production of specific chemical substances produced by lactic acid bacteria, and for the production of foods containing lactic acid bacteria or their products at high concentrations. Since the culture obtained by the present invention has a small amount of lactic acid, the unique flavor of lactic acid is alleviated, and moisture absorption hardly occurs at the time of powder processing, so that it can be suitably used as a food or its material.

Abstract

Provided is a method for culturing lactic acid bacteria that inhibits lactic acid production in a variety of lactic acid bacteria and enables high density culture. Lactic acid bacteria are cultured by fed-batch culture. When lactic acid bacteria are cultured by fed-batch culture, lactic acid production is inhibited in comparison to culture by batch culture, and the inhibition of lactic acid bacterial growth caused by high lactic acid concentrations can then be reduced. The culture of lactic acid bacteria can thus be carried out at a higher density than in the case of the batch culture of lactic acid bacteria.

Description

乳酸菌の培養方法Cultivation method of lactic acid bacteria
　本発明は、乳酸菌の培養方法に関する。 The present invention relates to a method of culturing lactic acid bacteria.
　乳酸菌は糖をピルビン酸に代謝することによってＡＴＰを獲得し、この過程で必要となるNAD+をピルビン酸から乳酸への変換により再生する。このため、乳酸菌の増殖に伴って生成された乳酸が蓄積して培地のｐＨが低下し、乳酸菌の増殖や物質生産が阻害される。培地のｐＨの低下は、アルカリを添加することによって抑制することができる。しかしながら、培地にアルカリを添加してｐＨを維持しても、乳酸が蓄積すると乳酸自体による乳酸菌の増殖阻害が生じるため、乳酸菌の高密度培養は困難とされている。 Lactic acid bacteria acquire ATP by metabolizing sugars to pyruvate, and regenerate NAD + required in this process by converting pyruvate to lactate. For this reason, lactic acid produced along with the growth of lactic acid bacteria is accumulated to lower the pH of the culture medium, and the growth and substance production of lactic acid bacteria are inhibited. The decrease in pH of the medium can be suppressed by adding an alkali. However, even if alkali is added to the medium to maintain the pH, the accumulation of lactic acid causes growth inhibition of lactic acid bacteria by lactic acid itself, so high-density culture of lactic acid bacteria is considered to be difficult.
　さらに、乳酸菌から生成される乳酸は、増殖だけではなく、乳酸菌素材としての質にも影響を与える。近年、乳酸菌はプロバイオティクスとして注目を集めており、様々な食品に利用されているが、乳酸またはその塩は独特の風味を有するため、培養物中の乳酸量は少ないことが望ましい。また、乳酸菌を粉末素材に加工する際には、乳酸の存在は吸湿を引き起こす原因となる。このように、乳酸菌の培養時に生成される乳酸は、乳酸菌自身の増殖を阻害し、乳酸菌素材としての質を低下させる。 Furthermore, lactic acid produced from lactic acid bacteria affects not only growth but also the quality as a lactic acid bacteria material. In recent years, lactic acid bacteria have attracted attention as probiotics and are used for various foods, but since lactic acid or a salt thereof has a unique flavor, it is desirable that the amount of lactic acid in the culture be small. In addition, when processing lactic acid bacteria into powder materials, the presence of lactic acid causes moisture absorption. Thus, lactic acid produced during culture of lactic acid bacteria inhibits the growth of the lactic acid bacteria themselves, and reduces the quality of the lactic acid bacteria material.
特開2001-211878号公報JP 2001-211878 A 特公平6-69367号公報Japanese Examined Patent Publication 6-69367
　乳酸による増殖阻害を回避する方法として、生成された乳酸を低濃度に抑える高い希釈率で連続培養を行う方法や、濾過や電気透析等による乳酸を除去する方法がある（例えば、特許文献1及び2参照）。しかしながら、これらの方法には、排水処理及び設備にコストがかかるという問題がある。 Methods of avoiding growth inhibition by lactic acid include a method of continuously culturing at a high dilution rate to suppress the generated lactic acid to a low concentration, and a method of removing lactic acid by filtration, electrodialysis or the like (for example, Patent Document 1 and 2). However, these methods suffer from the cost of wastewater treatment and equipment.
　また、乳酸菌の乳酸生成を抑制する方法として、ヘミンの添加により呼吸鎖のNADHデヒドロゲナーゼを活性化してNADHからNAD+を再生し、ピルビン酸から乳酸への変換を抑制する培養法があるが、NADHの酸化に必要な酸素を供給する必要があるために酸素感受性の低い一部の菌株にしか利用できない。 As a method of suppressing lactic acid producing lactic acid bacteria, it activates NADH dehydrogenase of the respiratory chain by the addition of hemin to play the NAD + from NADH, although the conversion of pyruvate to lactate is inhibiting culture method, NADH Since it is necessary to supply the oxygen necessary for the oxidation of H. pylori, it can be used only for some oxygen-insensitive strains.
　それ故に、本発明は、種々の乳酸菌の乳酸生成を抑制し、高密度培養を可能とする乳酸菌の培養方法を提供することを目的とする。 Therefore, an object of the present invention is to provide a method for cultivating lactic acid bacteria, which suppresses lactic acid production of various lactic acid bacteria and enables high-density culture.
　本発明は、高密度培養を行うための乳酸菌の培養方法であって、流加培養により乳酸菌を培養することを特徴とするものである。 The present invention is a culture method of lactic acid bacteria for high-density culture, and characterized in that the lactic acid bacteria are cultured by fed-batch culture.
　本発明によれば、種々の乳酸菌の培養時の乳酸生成を抑制し、高密度培養を可能とする乳酸菌の培養方法を提供できる。また、菌体収率、即ち、使用した糖あたりの菌体収量を高めることができる。 ADVANTAGE OF THE INVENTION According to this invention, the culture | cultivation method of the lactic acid bacteria which suppresses the lactic acid production at the time of culture | cultivation of various lactic acid bacteria and enables high-density culture can be provided. In addition, the cell yield, that is, the cell yield per used sugar can be increased.
図１は、実施例１における菌体量及び乳酸量の経時変化を示すグラフである。FIG. 1 is a graph showing temporal changes in the amount of cells and the amount of lactic acid in Example 1. 図２は、実施例２における菌体量及び乳酸量の経時変化を示すグラフである。FIG. 2 is a graph showing temporal changes in the amount of cells and the amount of lactic acid in Example 2. 図３は、実施例３における菌体量及び乳酸量の経時変化を示すグラフである。FIG. 3 is a graph showing temporal changes in the amount of cells and the amount of lactic acid in Example 3. 図４は、実施例４における菌体量及び乳酸量の経時変化を示すグラフである。FIG. 4 is a graph showing temporal changes in the amount of cells and the amount of lactic acid in Example 4. 図５は、実施例５における菌体量及び乳酸量の経時変化を示すグラフである。FIG. 5 is a graph showing temporal changes in the amount of cells and the amount of lactic acid in Example 5.
　本発明に係る乳酸菌の培養方法は、流加培養により乳酸菌を培養することを特徴とするものである。流加培養は、培養中に培地または培地中の基質を供給しながら培養を行う培養方法である。培地中で乳酸菌を培養すると、乳酸菌の増殖に伴って生成される乳酸の濃度が増加し、乳酸自体による乳酸菌の増殖阻害が生じる。本願の発明者は、流加培養を行うことによって、回分培養を行った場合と比べて有意に乳酸の生成が抑制されることを見出した。乳酸菌は糖を解糖系によってピルビン酸に代謝することによってATPを獲得し、この過程で必要となるNAD+を乳酸脱水素酵素によるピルビン酸から乳酸への変換により再生する。しかし、乳酸菌は乳酸脱水素酵素以外にもNAD+を再生する経路をもっており、また、解糖系以外にも、ATPを獲得する経路を持っている。本発明の発明者は、これらの経路のほとんどが糖、特に、グルコースがある程度以上の濃度で存在すると抑制されてしまうこと気づいた。これは、培養に必要な糖を、培養開始時に一気に与えるのではなく、流加培養によって少量ずつ与えて培地中のグルコースをはじめとする糖の濃度を低く保てば、これらの経路が抑制されるのを回避できることを意味している。また、これらの経路が十分な能力をもっていない乳酸菌の場合、ATPの獲得を解糖系に頼り、NAD+の再生を乳酸脱水素酵素に頼ることになるが、糖を流加によって少量ずつ供給すれば、これらの経路が抑制されることを回避できるだけでなく、エネルギー生産に占める解糖系によるエネルギー獲得の割合を下げ、また、NAD+再生に占める乳酸脱水素酵素の寄与を下げることができ、結果として、乳酸生産の抑制につながる。乳酸の生成が抑制されると、乳酸菌の増殖阻害を低減できるので、回分培養を行う場合と比べて、高密度（高濃度）で乳酸菌を培養することが可能となる。 The culture method of lactic acid bacteria according to the present invention is characterized in that the lactic acid bacteria are cultured by fed-batch culture. Fed-batch culture is a culture method in which culture is performed while supplying a medium or a substrate in the medium during the culture. When lactic acid bacteria are cultured in a culture medium, the concentration of lactic acid produced along with the growth of lactic acid bacteria increases, and the growth inhibition of lactic acid bacteria by lactic acid itself occurs. The inventor of the present application has found that by performing fed-batch culture, the production of lactic acid is significantly suppressed as compared with the case of performing batch culture. Lactic acid bacteria obtain ATP by metabolizing sugars to pyruvate by glycolysis, and regenerate the NAD + required in this process by converting pyruvate to lactate by lactate dehydrogenase. However, lactic acid bacteria have a pathway for regenerating NAD + in addition to lactate dehydrogenase, and also have a pathway for acquiring ATP besides glycolysis. The inventors of the present invention have noticed that most of these pathways are suppressed when sugars, in particular glucose, are present at concentrations above a certain level. It is not necessary to supply the sugar necessary for culture at once at the start of culture, but by feeding small amounts by fed-batch culture to keep the concentration of glucose and other sugar in the medium low, these pathways are suppressed. It means that you can avoid the problem. Moreover, in the case of lactic acid bacteria in which these pathways do not have sufficient ability, it depends on the glycolytic system to acquire ATP and rely on lactate dehydrogenase for NAD + regeneration, but sugar is supplied in small amounts by fed-batch Not only can these pathways be suppressed, but also reduce the proportion of energy gain by glycolysis in energy production, and also reduce the contribution of lactate dehydrogenase in NAD + regeneration, As a result, it leads to suppression of lactic acid production. Since the inhibition of the growth of lactic acid bacteria can be reduced when the production of lactic acid is suppressed, it becomes possible to culture lactic acid bacteria at a high density (high concentration) as compared with the case of performing batch culture.
　糖源として使用可能な糖としては、グルコース、フルクトース、ガラクトース、マンノース等の単糖類、スクロース、ラクトース、マルトース、トレハロース等の二糖類を例示できる。これらの糖の中でも、グルコースまたはスクロースが好ましく、スクロースがより好ましい。糖源としてスクロースを用いた場合、グルコースを用いた場合と比べて更に乳酸の生成を抑制することができる。 Examples of sugars that can be used as a sugar source include monosaccharides such as glucose, fructose, galactose and mannose, and disaccharides such as sucrose, lactose, maltose and trehalose. Among these sugars, glucose or sucrose is preferred, and sucrose is more preferred. When sucrose is used as a sugar source, the generation of lactic acid can be further suppressed as compared to the case where glucose is used.
　培地には、常法に従い、糖源のほかに、アミノ酸、ビタミン、核酸、金属塩等の栄養素や、ｐＨ変動を抑制するための緩衝液等を配合する。 In the medium, in addition to the sugar source, nutrients such as amino acids, vitamins, nucleic acids, metal salts, etc., and a buffer solution for suppressing pH fluctuation and the like are blended in accordance with a conventional method.
　本発明に係る培養方法で培養可能な乳酸菌の菌株は特に限定されず、種々の菌株を高密度培養することができる。例えば、本発明に係る培養方法では、Lactobacillus属、Lactococcus属、Leuconostoc属に含まれる種・株を高密度培養することができる。ヘミンの添加により乳酸の生成を抑制する方法では、上述したように、酸素の供給が必要となるため、培養できる菌株が酸素感受性の低い一部の菌株に限られてしまうが、本発明に係る培養方法は、酸素供給を必要としないため、種々の菌株の培養に適用することができる。 The strain of lactic acid bacteria that can be cultured by the culture method according to the present invention is not particularly limited, and various strains can be cultured at high density. For example, in the culture method according to the present invention, species and strains contained in Lactobacillus, Lactococcus, and Leuconostoc can be cultured at high density. In the method of suppressing the formation of lactic acid by the addition of hemin, as described above, since the supply of oxygen is required, the strains that can be cultured are limited to some strains with low oxygen sensitivity, but the present invention relates to the present invention. The culture method can be applied to the culture of various strains because it does not require oxygen supply.
　培地のpH、溶存酸素の量、培養温度、培養時間等の培養条件、通常乳酸菌の培養に適用される条件であれば良く、菌株の種類に応じて適宜選択することができる。また、培養（本培養）の前に、前培養を行うことが好ましい。前培養とは、本培養に接種するための種菌を調製するための培養である。前培養を行うことにより、本培養の種菌として必要な菌体量を確保することができる。 The pH of the culture medium, the amount of dissolved oxygen, the culture conditions such as culture temperature, culture time, and the conditions generally applied to the culture of lactic acid bacteria may be used, and can be appropriately selected according to the type of strain. Moreover, it is preferable to perform preculture before culture | cultivation (main culture). The pre-culture is a culture for preparing a seed to inoculate the main culture. By pre-culturing, it is possible to secure the amount of cells necessary as the inoculum of the main culture.
　また、本発明に係る培養方法は、乳酸菌が産生する特定の化学物質（ペプチド、タンパク質、多糖類等）を生産するために利用可能である。また、本発明に係る培養方法は、乳酸菌を高濃度で含有する発酵乳や、乳酸菌飲料、チーズ等の食品の製造にも利用可能である。本発明に係る培養方法を食品の製造に適用した場合、生理活性を有する特定の化学物質やこれを産生する乳酸菌を高濃度で含有する食品を製造することが可能となる。 In addition, the culture method according to the present invention can be used to produce specific chemical substances (peptides, proteins, polysaccharides, etc.) produced by lactic acid bacteria. The culture method according to the present invention is also applicable to the production of fermented milk containing lactic acid bacteria at a high concentration, and foods such as lactic acid bacteria beverages and cheese. When the culture method according to the present invention is applied to the production of a food, it becomes possible to produce a food containing a high concentration of a specific chemical substance having physiological activity and a lactic acid bacterium producing the same.
　以下、本発明の実施例を説明する。 Hereinafter, examples of the present invention will be described.
　（実施例1）
　実施例1では、乳酸菌の菌株として、Lactobacillus gasseriJCM1131を使用した。 Example 1
In Example 1, Lactobacillus gasseri JCM1131 was used as a strain of lactic acid bacteria.
　まず、前培養を行った。糖源として、グルコースまたはスクロースを配合したMRS培地100 mLに、Lb. gasseriJCM1131のフローズンストックから100 μLを植菌し、37℃で12時間静置培養した。次に、糖源濃度が0 g/LのMRS培地100 mLを調製した。前培養液のOD660を測定し、本培養液を200 OD unitsにするのに必要な培養液量を算出した。前培養液を遠心分離（8,000×g、4℃、5 min）して生理食塩水で洗浄した後、本培養液に再懸濁し植菌した。 First, preculture was performed. 100 μL of the frozen stock of Lb. gasseri JCM 1131 was inoculated into 100 mL of MRS medium containing glucose or sucrose as a sugar source, and stationary cultured at 37 ° C. for 12 hours. Next, 100 mL of MRS medium with a sugar source concentration of 0 g / L was prepared. The OD 660 of the preculture fluid was measured, and the amount of culture fluid required to bring the main culture fluid to 200 OD units was calculated. The preculture liquid was centrifuged (8,000 × g, 4 ° C., 5 min), washed with physiological saline, and then resuspended in this culture liquid and inoculated.
　流加培養における培地の流加速度Ｆ（L/h）は、比増殖速度を一定に保てるように下記式に基づいて計算した。
Figure JPOXMLDOC01-appb-M000001
ここで、ｔは培養時間であり、初期培養液量V0は0.1 Lとした。初期菌体濃度X０は、OD660＝1.0のときに0.25 g-cell/Lであるとして算出した。流加培地の糖濃度SFは20 g/Lとし、菌体収率YX/Sは、糖源としてスクロースを用いる場合、0.1 g-cell/g-sugarとし、糖源としてグルコースを用いる場合、0.2 g-cell/g-sugarとした。比増殖速度μ*は、糖源としてスクロースを用いる場合、0.25 1/hとし、糖源としてグルコースを用いる場合、0.30 1/hに設定した。尚、糖源にスクロースを用いる場合とグルコースを用いる場合とで条件設定を同じにすると、実測値の比増殖速度に差が生じてしまうため、糖源にスクロースを用いる場合とグルコースを用いる場合とでそれぞれの菌体収率及び比増殖速度を設定した。これらの設定値を用いて上記の式から流加速度を決定した。また、3 M NaOH水溶液で培養液のpHを6.5に制御しながら直径3 cmのマグネットバーを用いて攪拌培養を行った。 The flow acceleration F (L / h) of the medium in fed-batch culture was calculated based on the following equation so as to keep the specific growth rate constant.
Figure JPOXMLDOC01-appb-M000001
Here, t is the culture time, and the initial culture solution volume V 0 was 0.1 L. The initial cell concentration X 0 was calculated as 0.25 g-cell / L when OD 660 = 1.0. The sugar concentration S F of the feed medium is 20 g / L, and the cell yield Y X / S is 0.1 g-cell / g-sugar when using sucrose as a sugar source, and when using glucose as a sugar source , 0.2 g-cell / g-sugar. The specific growth rate μ * was set at 0.25 1 / h when using sucrose as a sugar source, and at 0.30 1 / h when using glucose as a sugar source. In addition, if condition setting is made the same between the case where sucrose is used as the sugar source and the case where glucose is used, a difference is generated in the specific growth rate of the measured values. Therefore, the case where sucrose is used as the sugar source and the case where glucose is used The respective cell yield and specific growth rate were set in The flow acceleration was determined from the above equation using these set values. In addition, stirring culture was performed using a 3 cm diameter magnet bar while controlling the pH of the culture solution to 6.5 with a 3 M NaOH aqueous solution.
　また、回分培養は、糖源としてグルコースまたはスクロースを20 g/Lの濃度で含有するMRS培地を用いて行った。培養液の量、植菌量及び培養条件は流加培養と同じとした。 In addition, batch culture was performed using MRS medium containing glucose or sucrose at a concentration of 20 g / L as a sugar source. The amount of culture solution, the amount of inoculation and the culture conditions were the same as in the fed-batch culture.
　5 mLのシリンジを用いて、30分間間隔または１時間間隔でサンプリングラインから培養液をサンプリングした。サンプリングに際して、サンプリングラインに残存した培養液を捨てた後、1 mLの培養液をサンプリングした。 The culture fluid was sampled from the sampling line at intervals of 30 minutes or at intervals of 1 hour using a 5 mL syringe. At the time of sampling, after discarding the culture solution remaining in the sampling line, 1 mL of culture solution was sampled.
　サンプリングした培養液の濁度（OD660）を紫外可視分光光度計UV-1850（島津製作所社製）を用いて測定し、測定したOD660の値に基づいて培養液中の菌体量を算出した。また、サンプリングした培養液中の乳酸濃度は、バイオセンサーBF-5（王子計測機器株式会社製）及び酵素電極（王子計測機器株式会社製）を用いて測定した。乳酸濃度は、D-乳酸及びL-乳酸の合計濃度である。得られた菌体量に基づいて、菌体量の自然対数を培養時間に対してプロットしたグラフの傾きから実際の比増殖速度μを、積算菌体量に対して乳酸量をプロットしたグラフの傾きから比生産速度ρを求めた。増殖菌体量当たりの乳酸量YL/Xは、対数増殖している区間において、ρをμで除すことによって、菌体収率は消費した糖量で増加した菌体量を除すことによって算出した。 The turbidity (OD 660 ) of the sampled culture solution is measured using a UV-visible spectrophotometer UV-1850 (manufactured by Shimadzu Corporation), and the amount of cells in the culture solution is calculated based on the measured OD 660 value. did. Further, the concentration of lactic acid in the sampled culture solution was measured using a biosensor BF-5 (manufactured by Oji Scientific Instruments) and an enzyme electrode (manufactured by Oji Scientific Instruments). The lactic acid concentration is the total concentration of D-lactic acid and L-lactic acid. The graph which plots the amount of lactic acid against the integrated amount of cells from the inclination of the graph which plotted the natural logarithm of the amount of cells against the culture time based on the amount of cells obtained and the slope of the graph The specific production rate ρ was determined from the slope. The amount of lactic acid Y L / X per amount of proliferating cells is defined by dividing ρ by μ in the logarithmic growth period, and dividing the amount of cells by the amount of sugar consumed by the cell yield Calculated by
　表１に、本培養開始から所定時間後（流加培養では６時間後、回分培養では２時間後）の菌体量および乳酸量から算出した比増殖速度μ、比生産速度ρ、増殖菌体量当たりの乳酸量ρ/μ、菌体収率を示す。また、図1に、菌体量及び乳酸量の経時変化のグラフを示す。尚、以下に示す表1～5において、誤差範囲は回帰分析による95%信頼区間を示している。 Table 1 shows the specific growth rate μ, specific production rate ρ, and proliferating cell mass calculated from the amount of cells and lactic acid amount after a predetermined time (6 hours after fed-batch culture and 2 hours after batch culture) from the start of main culture. It shows the amount of lactic acid per unit ρ / μ, and the cell yield. Further, FIG. 1 shows a graph of temporal changes in the amount of cells and the amount of lactic acid. In Tables 1 to 5 shown below, error ranges indicate 95% confidence intervals by regression analysis.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　表1に示すように、増殖菌体当たりの乳酸量は、糖源としてグルコース及びスクロースのいずれを用いた場合においても、回分培養よりも流加培養で少なくなった。また、グルコース糖源を用いた回分培養と、スクロース糖源を用いた回分培養とを比べると、増殖菌体当たりの乳酸量は、スクロース糖源を用いた場合により少なかった。ただし、グルコースを糖源とした流加培養でもグルコースリプレッションによる影響が十分に軽減されたものと考えられる。また、菌体収率（g-cell/g-sugar）は、糖源としてグルコース及びスクロースのいずれを用いた場合においても、回分培養よりも流加培養で高くなった。 As shown in Table 1, the amount of lactic acid per growing bacterial body was smaller in fed-batch culture than in batch culture, regardless of whether glucose or sucrose was used as a sugar source. In addition, when batch culture using a glucose sugar source was compared with batch culture using a sucrose sugar source, the amount of lactic acid per growing bacterial body was smaller when using a sucrose sugar source. However, even in fed-batch culture using glucose as a sugar source, it is considered that the effect of glucose repression is sufficiently reduced. Also, the cell yield (g-cell / g-sugar) was higher in fed-batch culture than in batch culture, regardless of whether glucose or sucrose was used as the sugar source.
　また、図1に示すように、流加培養を行った場合の菌体量は、本培養開始から5時間で回分培養を行った場合の菌体量を上回った。また、本培養開始から5時間における乳酸量を比較しても、回分培養に比べて流加培養で抑制されていた。 In addition, as shown in FIG. 1, the amount of cells when fed-batch culture exceeded the amount of cells when batch culture was performed 5 hours after the start of main culture. In addition, even if the amount of lactic acid was compared 5 hours after the start of the main culture, it was suppressed by the fed-batch culture as compared to the batch culture.
　（実施例2）
　実施例2では、乳酸菌の菌株として、Lactobacillus plantarum subsp. plantarumNBRC15891を使用し、スクロースを糖源に用いた回分培養と、スクロースを糖源に用いた流加培養と、グルコースを糖源に用いた流加培養とを行った。培養方法、サンプリング方法、各評価値の算出方法は実施例1と同じである。 (Example 2)
In Example 2, Lactobacillus plantarum subsp. Plantarum NBRC 15891 is used as a strain of lactic acid bacteria, batch culture using sucrose as a sugar source, fed-batch culture using sucrose as a sugar source, and flow using glucose as a sugar source And culture. The culture method, the sampling method, and the method of calculating each evaluation value are the same as in Example 1.
　表2に、本培養開始から所定時間後（流加培養では6時間後、回分培養では4時間後）の菌体量および乳酸量から算出した比増殖速度μ、比生産速度ρ、増殖菌体量当たりの乳酸量ρ/μ、菌体収率を示す。また、図2に、菌体量及び乳酸量の経時変化のグラフを示す。 Table 2 shows the specific growth rate μ, specific production rate ρ, and proliferating cells calculated from the amount of cells and lactic acid amount after a predetermined time from the start of the main culture (6 hours after fed-batch culture and 4 hours after batch culture). It shows the amount of lactic acid per unit ρ / μ, and the cell yield. Further, FIG. 2 shows a graph of temporal changes of the amount of cells and the amount of lactic acid.
Figure JPOXMLDOC01-appb-T000003
 　表2に示すように、糖源としてスクロースを用いた場合における増殖菌体当たりの乳酸量は、回分培養よりも流加培養で少なくなった。また、グルコース糖源を用いた回分培養と、スクロース糖源を用いた回分培養とを比べると、増殖菌体当たりの乳酸量は、スクロース糖源を用いた場合により少なかった。また、菌体収率（g-cell/g-sugar）は、糖源としてグルコース及びスクロースのいずれを用いた場合においても、回分培養よりも流加培養で高くなった。
Figure JPOXMLDOC01-appb-T000003
 As shown in Table 2, when sucrose was used as a sugar source, the amount of lactic acid per grown cell was smaller in fed-batch culture than in batch culture. In addition, when batch culture using a glucose sugar source was compared with batch culture using a sucrose sugar source, the amount of lactic acid per growing bacterial body was smaller when using a sucrose sugar source. Also, the cell yield (g-cell / g-sugar) was higher in fed-batch culture than in batch culture, regardless of whether glucose or sucrose was used as the sugar source.
　また、図2に示すように、糖源としてグルコース及びスクロースのいずれを用いた場合においても、流加培養を行った場合の乳酸量は、糖源としてスクロースを用いた回分培養を行った場合と比べて抑制されていた。 Further, as shown in FIG. 2, in the case of using either glucose or sucrose as a sugar source, the amount of lactic acid in the case of performing a fed-batch culture is the case of performing batch culture using sucrose as a sugar source. It was suppressed by comparison.
　（実施例3）
　実施例3では、乳酸菌の菌株として、Lactobacillus reuteriJCM1112を使用した。培養方法、サンプリング方法、各評価値の算出方法は実施例1と同じである。 (Example 3)
In Example 3, Lactobacillus reuteri JCM1112 was used as a strain of lactic acid bacteria. The culture method, the sampling method, and the method of calculating each evaluation value are the same as in Example 1.
　表3に、本培養開始から所定時間後（流加培養では3時間後、回分培養では2時間後）の菌体量および乳酸量から算出した比増殖速度μ、比生産速度ρ、増殖菌体量当たりの乳酸量ρ/μ、菌体収率を示す。また、図３に、菌体量及び乳酸量の経時変化のグラフを示す。 Table 3 shows the specific growth rate μ, specific production rate 増 殖, and proliferating cell mass calculated from the amount of cells and lactic acid amount after a predetermined time (3 hours after fed-batch culture and 2 hours after batch culture) from the start of main culture. It shows the amount of lactic acid per unit ρ / μ, and the cell yield. Further, FIG. 3 shows a graph of temporal changes of the amount of cells and the amount of lactic acid.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　表3に示すように、増殖菌体当たりの乳酸量は、糖源としてグルコース及びスクロースのいずれを用いた場合においても、回分培養よりも流加培養で少なくなった。また、グルコース糖源を用いた流加培養と、スクロース糖源を用いた流加培養とを比べると、増殖菌体当たりの乳酸量は、スクロース糖源を用いた場合により少なかった。ただし、グルコースを糖源とした流加培養でもグルコースリプレッションによる影響が十分に軽減されたものと考えられる。また、菌体収率（g-cell/g-sugar）は、糖源としてグルコース及びスクロースのいずれを用いた場合においても、回分培養よりも流加培養で高くなった。更に、実施例3では、グルコース糖源を用いた回分培養と、スクロース糖源を用いた回分培養とを比べると、増殖菌体当たりの乳酸量は、スクロース糖源を用いた場合により少なかった。Lb. reuteriは、スクロースをグルコースとフルクトースに分解し、フルクトースをマンニトールに変換することで、1分子のNAD+を再生することが報告されている。糖源にスクロースを用いることで、マンニトール生成によりNAD+を再生し、スクロース糖源で流加培養を行うことでさらにこの経路のフラックスが大きくなり、乳酸生成抑制に繋がったと考えられる。 As shown in Table 3, the amount of lactic acid per growing cell was smaller in fed-batch culture than in batch culture, regardless of whether glucose or sucrose was used as a sugar source. Moreover, when the fed-batch culture using a glucose sugar source was compared with the fed-batch culture using a sucrose sugar source, the amount of lactic acid per growth cell was smaller when using a sucrose sugar source. However, even in fed-batch culture using glucose as a sugar source, it is considered that the effect of glucose repression is sufficiently reduced. Also, the cell yield (g-cell / g-sugar) was higher in fed-batch culture than in batch culture, regardless of whether glucose or sucrose was used as the sugar source. Furthermore, in Example 3, when the batch culture using a glucose sugar source was compared with the batch culture using a sucrose sugar source, the amount of lactic acid per growth cell was smaller when using a sucrose sugar source. Lb. reuteri has been reported to regenerate one molecule of NAD + by degrading sucrose to glucose and fructose and converting fructose to mannitol. By using sucrose as a sugar source, NAD + is regenerated by mannitol production, and by performing a fed-batch culture with a sucrose sugar source, the flux of this pathway is further increased, which is considered to be linked to suppression of lactic acid production.
　（実施例4）
　実施例4では、乳酸菌の菌株として、Lactobacillus rhamnosusATCC53103を使用した。培養方法、サンプリング方法、各評価値の算出方法は実施例1と同じである。 (Example 4)
In Example 4, Lactobacillus rhamnosus ATCC 53103 was used as a strain of lactic acid bacteria. The culture method, the sampling method, and the method of calculating each evaluation value are the same as in Example 1.
　表4に、本培養開始から所定時間後（流加培養では5時間後、回分培養では2.5時間後）の菌体量および乳酸量から算出した比増殖速度μ、比生産速度ρ、増殖菌体量当たりの乳酸量ρ/μ、菌体収率を示す。また、図4に、菌体量及び乳酸量の経時変化のグラフを示す。 Table 4 shows the specific growth rate μ, specific production rate ρ, and proliferating cells calculated from the amount of cells and lactic acid amount after a predetermined time (5 hours after fed-batch culture and 2.5 hours after batch culture) after the start of main culture. It shows the amount of lactic acid per unit ρ / μ, and the cell yield. Further, FIG. 4 shows a graph of temporal changes of the amount of cells and the amount of lactic acid.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　表4に示すように、増殖菌体当たりの乳酸量は、糖源としてグルコース及びスクロースのいずれを用いた場合においても、回分培養よりも流加培養で少なくなった。また、グルコース糖源を用いた流加培養と、スクロース糖源を用いた流加培養とを比べると、増殖菌体当たりの乳酸量は、スクロース糖源を用いた場合により少なかった。ただし、グルコースを糖源とした流加培養でもグルコースリプレッションによる影響が十分に軽減されたものと考えられる。また、菌体収率（g-cell/g-sugar）は、糖源としてグルコース及びスクロースのいずれを用いた場合においても、回分培養よりも流加培養で高くなった。 As shown in Table 4, the amount of lactic acid per growing cell was smaller in fed-batch culture than in batch culture, regardless of whether glucose or sucrose was used as a sugar source. Moreover, when the fed-batch culture using a glucose sugar source was compared with the fed-batch culture using a sucrose sugar source, the amount of lactic acid per growth cell was smaller when using a sucrose sugar source. However, even in fed-batch culture using glucose as a sugar source, it is considered that the effect of glucose repression is sufficiently reduced. Also, the cell yield (g-cell / g-sugar) was higher in fed-batch culture than in batch culture, regardless of whether glucose or sucrose was used as the sugar source.
　（実施例5）
　実施例5では、乳酸菌の菌株として、Lactobacillus plantarumNCIMB8826を使用した。培養方法、サンプリング方法、各評価値の算出方法は実施例1と同じである。 (Example 5)
In Example 5, Lactobacillus plantarum NCIMB 8826 was used as a strain of lactic acid bacteria. The culture method, the sampling method, and the method of calculating each evaluation value are the same as in Example 1.
　表5に、本培養開始から所定時間後（流加培養では5時間後、回分培養では4時間後）の菌体量および乳酸量から算出した比増殖速度μ、比生産速度ρ、増殖菌体量当たりの乳酸量ρ/μ、菌体収率を示す。また、図5に、菌体量及び乳酸量の経時変化のグラフを示す。 Table 5 shows the specific growth rate μ, specific production rate ρ, and proliferating cell mass calculated from the amount of cells and lactic acid amount after a predetermined time from the start of the main culture (5 hours after fed-batch culture and 4 hours after batch culture) It shows the amount of lactic acid per unit ρ / μ, and the cell yield. Further, FIG. 5 shows a graph of temporal changes in the amount of cells and the amount of lactic acid.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　表5に示すように、増殖菌体当たりの乳酸量は、糖源としてグルコース及びスクロースのいずれを用いた場合においても、回分培養よりも流加培養で少なくなった。また、グルコース糖源を用いた流加培養と、スクロース糖源を用いた流加培養とを比べると、増殖菌体当たりの乳酸量は、スクロース糖源を用いた場合により少なかった。ただし、グルコースを糖源とした流加培養でもグルコースリプレッションによる影響が十分に軽減されたものと考えられる。また、菌体収率（g-cell/g-sugar）は、糖源としてグルコース及びスクロースのいずれを用いた場合においても、回分培養よりも流加培養で高くなった。 As shown in Table 5, the amount of lactic acid per growing cell was smaller in fed-batch culture than in batch culture, regardless of whether glucose or sucrose was used as a sugar source. Moreover, when the fed-batch culture using a glucose sugar source was compared with the fed-batch culture using a sucrose sugar source, the amount of lactic acid per growth cell was smaller when using a sucrose sugar source. However, even in fed-batch culture using glucose as a sugar source, it is considered that the effect of glucose repression is sufficiently reduced. Also, the cell yield (g-cell / g-sugar) was higher in fed-batch culture than in batch culture, regardless of whether glucose or sucrose was used as the sugar source.
　（実施例6）
　実施例6では、乳酸菌の菌株として、Lb. rhamnosusATCC53103を使用した。 (Example 6)
In Example 6, Lb. rhamnosus ATCC 53103 was used as a strain of lactic acid bacteria.
　まず、前培養を行った。糖源として、グルコースまたはガラクトースを2%で含有するMRS培地100 mLに、Lb. rhamnosusATCC53103のフローズンストックから100 μLを植菌し、37℃で15時間静置培養した。 First, preculture was performed. 100 μL of the frozen stock of Lb. rhamnosus ATCC 53103 was inoculated into 100 mL of MRS medium containing 2% of glucose or galactose as a sugar source and statically cultured at 37 ° C. for 15 hours.
　次に、糖を含有しないMRS培地100 mLを調製した。前培養液のOD660を測定し、本培養液を100 OD unitsにするために必要な培養液量を算出した。前培養液を遠心分離（8,000×g、4℃、5 min）して生理食塩水で洗浄した後、本培養液に再懸濁し植菌した。流加培養における培地の流加速度F（L/h）は、比増殖速度を一定に保てるように下記式に基づいて計算した。ペリスタルティックポンプを用いて、算出した流速で流加培地を本培地に流加した。 Next, 100 mL of MRS medium containing no sugar was prepared. The OD 660 of the preculture fluid was measured, and the amount of culture fluid required to make the main culture fluid 100 OD units was calculated. The preculture liquid was centrifuged (8,000 × g, 4 ° C., 5 min), washed with physiological saline, and then resuspended in this culture liquid and inoculated. The flow acceleration F (L / h) of the medium in fed-batch culture was calculated based on the following equation so as to keep the specific growth rate constant. The feed medium was fed to the medium at a calculated flow rate using a peristaltic pump.
Figure JPOXMLDOC01-appb-M000007
ここで、tは培養時間（h）であり、初期培養液量V0は0.1 Lとした。初期菌体濃度X0は、OD660＝1のときに0.25 g-cell/Lであるとして算出した。流加培地の糖濃度SFは10 g/Lとし、菌体収率YX/Sは、0.2 g-cell/g-sugarとした。マグネットスターラーの上に温度設定した恒温槽を置き、500 rpmでの撹拌培養を行った。
Figure JPOXMLDOC01-appb-M000007
Here, t is culture time (h), and the initial culture solution volume V 0 was 0.1 L. Initial cell concentration X0 was calculated as 0.25 g-cell / L when OD 660 = 1. The sugar concentration S F of the feed medium was 10 g / L, and the cell yield Y X / S was 0.2 g-cell / g-sugar. A temperature-controlled thermostatic bath was placed on a magnetic stirrer, and stirring culture was performed at 500 rpm.
　また、回分培養は、糖源としてグルコースまたはガラクトースを2％で含有するMRS培地を用いて行った。培養液の量、植菌量及び培養条件は流加培養と同じとした。 In addition, batch culture was performed using MRS medium containing glucose or galactose at 2% as a sugar source. The amount of culture solution, the amount of inoculation and the culture conditions were the same as in the fed-batch culture.
　5 mLのシリンジを用いて、サンプリングラインから培養液をサンプリングした。サンプリングに際して、サンプリングラインに残存した培養液を捨てた後、1 mLの培養液をサンプリングした。 The culture fluid was sampled from the sampling line using a 5 mL syringe. At the time of sampling, after discarding the culture solution remaining in the sampling line, 1 mL of culture solution was sampled.
　サンプリングした培養液の濁度（OD660）を紫外可視分光光度計UV-1850（島津製作所社製）を用いて測定し、測定したOD660の値に基づいて培養液中の菌体量を算出した。また、サンプリングした培養液中の乳酸濃度は、バイオセンサーBF-5（王子計測機器株式会社製）及び酵素電極（王子計測機器株式会社製）を用いて測定した。
乳酸濃度は、D-乳酸及びL-乳酸の合計濃度である。得られた菌体量に基づいて、評価値として、比増殖速度μ、比生産速度ρ、増殖菌体量当たりの乳酸量ρ/μを算出した。 The turbidity (OD 660 ) of the sampled culture solution is measured using a UV-visible spectrophotometer UV-1850 (manufactured by Shimadzu Corporation), and the amount of cells in the culture solution is calculated based on the measured OD 660 value. did. Further, the concentration of lactic acid in the sampled culture solution was measured using a biosensor BF-5 (manufactured by Oji Scientific Instruments) and an enzyme electrode (manufactured by Oji Scientific Instruments).
The lactic acid concentration is the total concentration of D-lactic acid and L-lactic acid. Based on the obtained amount of cells, as an evaluation value, the specific growth rate μ, the specific production rate 乳酸, and the amount ρ / μ of lactic acid per the amount of grown cells were calculated.
　表6に、本培養開始から流加培養と回分培養ともに3時間後の菌体量および乳酸量から算出した比増殖速度μ、比生産速度ρ、増殖菌体量当たりの乳酸量ρ/μを示す。尚、誤差範囲は回帰分析による95%信頼区間を示している。 Table 6 shows the specific growth rate μ, the specific production rate ρ, and the amount of lactic acid ρ / μ per amount of proliferating bacterial cells calculated from the amount of lactic acid and the amount of lactic acid after 3 hours in both fed-batch culture and batch culture from the start of main culture. Show. The error range indicates the 95% confidence interval by regression analysis.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　表6に示すように、Lb. rhamnosusATCC53103をガラクトースを用いて流加培養した場合、増殖菌体量当たりの乳酸量は2.56 g-lactate/g-cellとなり、最も抑制された。グルコースの回分培養と比較すると36%にまで抑制された。グルコースの流加培養、ガラクトースの回分培養と比較するとそれぞれ40%、49%にまで抑制されることが分かった。 As shown in Table 6, when Lb. rhamnosus ATCC 53103 was fed-batch cultured using galactose, the amount of lactic acid per amount of grown cells was 2.56 g-lactate / g-cell, which was most suppressed. It was suppressed to 36% as compared to glucose batch culture. It was found that the concentration was suppressed to 40% and 49%, respectively, as compared to the fed-batch culture of glucose and the batch culture of galactose.
　（実施例7）
　実施例7では、乳酸菌の菌株として、Lactococcus lactis MG1363を使用した。 (Example 7)
In Example 7, Lactococcus lactis MG1363 was used as a strain of lactic acid bacteria.
　まず、前培養を行った。糖源として、グルコースまたはガラクトースを2%で含有するMRS培地100 mLに、Lc. lactis MG1363のフローズンストックから100 μLを植菌し、30℃で15時間静置培養した。 First, preculture was performed. 100 μL of the frozen stock of Lc. Lactis MG1363 was inoculated into 100 mL of MRS medium containing 2% of glucose or galactose as a sugar source, and static culture was carried out at 30 ° C. for 15 hours.
　表7に、本培養開始から所定時間後（流加培養では3時間後、回分培養では2時間後）の菌体量および乳酸量から算出した比増殖速度μ、比生産速度ρ、増殖菌体量当たりの乳酸量ρ/μを示す。尚、誤差範囲は回帰分析による95%信頼区間を示している。 Table 7 shows the specific growth rate μ, the specific production rate 増 殖, and the proliferating cells calculated from the amount of cells and the amount of lactic acid after a predetermined time from the start of the main culture (3 hours after fed-batch culture and 2 hours after batch culture). The amount of lactic acid per unit ρ / μ is shown. The error range indicates the 95% confidence interval by regression analysis.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
　表7に示すように、Lc. lactisMG1363をガラクトースを用いて流加培養した場合、増殖菌体量当たりの乳酸量は1.09 g-lactate/g-cellとなり、最も抑制された。グルコースの回分培養と比較すると11%にまで大幅に抑制され、ガラクトースの回分培養と比較すると、28%にまで抑制されることが分かった。 As shown in Table 7, when Lc. Lactis MG1363 was fed-batch cultured using galactose, the amount of lactic acid per mass of grown cells was 1.09 g-lactate / g-cell, which was most suppressed. It was found to be significantly suppressed to 11% compared to glucose batch culture and to 28% compared to galactose batch culture.
　以上のように、流加培養により乳酸菌を培養することにより、菌体量当たりの乳酸生成量が抑制されることが確認された。また、流加培養による乳酸生成量の抑制効果は、種々の乳酸菌の菌株において発現することが確認された。 As described above, it was confirmed that the amount of lactic acid production per cell mass is suppressed by culturing the lactic acid bacteria by fed-batch culture. Moreover, it was confirmed that the suppression effect of the lactic acid production amount by fed-batch culture is expressed in the strain of various lactic acid bacteria.
　本発明は、乳酸菌の高密度培養に利用でき、特に、乳酸菌が産生する特定の化学物質の生産や、乳酸菌またはその産生物を高濃度で含有する食品の製造に利用できる。本発明により得られた培養物は、乳酸量が少ないため、乳酸の有する独特の風味が緩和されており、粉末加工時には吸湿が起こりにくいことから、食品やその素材として好適に利用できる。 INDUSTRIAL APPLICABILITY The present invention can be used for high-density culture of lactic acid bacteria, and in particular, can be used for the production of specific chemical substances produced by lactic acid bacteria, and for the production of foods containing lactic acid bacteria or their products at high concentrations. Since the culture obtained by the present invention has a small amount of lactic acid, the unique flavor of lactic acid is alleviated, and moisture absorption hardly occurs at the time of powder processing, so that it can be suitably used as a food or its material.

Claims (3)

  1. 　高密度培養を行うための乳酸菌の培養方法であって、
    　流加培養により乳酸菌を培養することを特徴とする、乳酸菌の培養方法。     A culture method of lactic acid bacteria for high density culture, which comprises:
    A method for cultivating lactic acid bacteria, which comprises culturing the lactic acid bacteria by fed-batch culture.
  2. 　糖源として、スクロースまたはガラクトースを用いることを特徴とする、請求項１に記載の乳酸菌の培養方法。 The method for cultivating lactic acid bacteria according to claim 1, wherein sucrose or galactose is used as a sugar source.
  3. 　乳酸菌が、Lactobacillus gasseri、Lactobacillus plantarum、Lactobacillus reuteri、Lactobacillus rhamnosus及びLactococcus lactisのいずれかである、請求項１または２に記載の乳酸菌の培養方法。 The method for cultivating lactic acid bacteria according to claim 1 or 2, wherein the lactic acid bacteria are any of Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus and Lactococcus lactis.
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JP2019030292A (en) * 2017-08-04 2019-02-28 株式会社明治 Culture method of lactic acid bacteria
JP7444370B2 (en) 2017-08-04 2024-03-06 株式会社明治 Cultivation method of lactic acid bacteria

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