KR20120061470A - Mutants of Lactobacillus paracasei with Improved Glucose Tolerance and Acid Tolerance and their Uses - Google Patents

Abstract

PURPOSE: A Lactobacillus paracasei mutant strain and a method for preparing the same are provided to improve sugar tolerance and acid resistance and to enhance lactic acid yield. CONSTITUTION: A Lactobacillus paracasei mutant strain has enhanced glucose tolerance and acid resistance comparing to wild type and grows in a medium containing 100-250 g/L of glucose and 20-200 g/L of lactic acid. The mutant stain is DSLA 6912(deposit number KCTC 11808BP) strain. A method for producing the mutant strain comprises: a step of culturing Lactobacillus paracasei strain in a medium; and a step of adding MNNG(N-methyl-N'-nitro-N-nitrosoguanidine) to the medium. A composition for producing lactic acid contains the mutant strain. A method for producing lactic acid comprises a step of culturing the mutant strain in a medium containing carbon source, energy source, and nitrogen source.

Description

Mutants of Lactobacillus paracasei with Improved Glucose Tolerance and Acid Tolerance and their Uses

The invention improves the feasibility and in the acid resistance and lactic acid production yield is increased by the Lactobacillus para casei (Lactobacillus paracasei ) The present invention relates to a mutant strain , a method of producing the same, and a method of producing lactic acid using the same.

Lactic acid (Lactic acid) is an organic acid that exists naturally in a wide range of naturally occurring enantiomeric D-type and L-type lactic acid, and is generally mixed with living organisms, and animal foods have a higher lactic acid content than vegetable foods. Lactic acid is also present and consumed in many foods, including fermented foods such as yogurt, butter, cheese, kimchi, soybean paste and soy sauce, or traditional pickles. In general, lactic acid is a raw material of polylactic acid (PLA), which is one of food preservatives, flavoring agents, acidifying agents, pH adjusting agents, hygroscopic agents, cleaning agents, dialysis solutions, solvents, anemia treatment agents, acne treatment agents and biodegradable plastics (bioplastics). It is widely used in various industries such as food, cosmetics, pharmaceuticals, chemicals, metals, electronics, textiles, dyeing, paint and leather industries for various uses, and the importance thereof is increasing.

Polylactic acid, one of biodegradable plastics, which has recently been in increasing demand, has higher melting point and higher strength when produced with optically pure D- and L-lactic acid. Due to these characteristics, optical purity is important for lactic acid used as a raw material of polylactic acid.

The production of lactic acid, a very industrially useful substance, includes petrochemical synthesis and biotechnological fermentation for sugar-based microbial fermentation. Petrochemical synthesis is a process in which acetaldehyde obtained by oxidation of ethylene derived from crude oil is made into lactonitrile through hydrogen cyanide addition, followed by distillation and purification to produce lactic acid by acid hydrolysis. Biotechnological fermentation is a process of producing lactic acid through a fermentation process using microorganisms based on carbohydrates such as glucose, starch and molasses. The former petrochemical synthesis method has the advantage of easily producing lactic acid compared to the latter biotechnological fermentation method, but it is a racemic mixture composed of 50% D-lactic acid and 50% L-lactic acid. Type lactic acid is produced, and polylactic acid is produced from this mixture as a raw material, which has a disadvantage in that it is a very low melting point amorphous polymer, which has many limitations in use. In comparison, the biotechnological fermentation method has the advantage of selectively producing D-type and L-type lactic acid according to microorganisms. For example, Lactobacillus Bulgari Syracuse, Ryu Kawano Stock microorganisms such mesen teroyi Death is mainly producing D-type lactic acid, Lactobacillus Para casei, Enterococcus Microorganisms such as Picalis are known to mainly produce L-lactic acid. In addition, biotech fermentation is the preferred method in which environmentally friendly efforts are being promoted globally to overcome international oil prices that fluctuate every year due to petrochemical energy depletion and global climate warming caused by environmental pollution. However, the reduction of productivity due to the inhibition of microbial growth by the substrate sugar and lactic acid production is a problem to be solved.

The technical development for producing lactic acid with high efficiency has been steadily for a long time. There technology relating to the production of lactic acid with high efficiency with improved naedang Enterococcus and Lactococcus acid Esp . Alkawai 1 Lactic acid production method using KCTC 8890P (Korean Patent Application Publication No. 2001-0008380), Lactobacillus isolated from Doenjang Esp . Lactic acid production method using Al- Kwai 2 KCTC 10353BP (Korean Patent No. 0453758), Lactobacillus isolated from Kimchi Para car lactic acid production method using the KCCM10542 J. (Republic of Korea Patent No. 0517065), isolated from nature, Lactobacillus Kazei Subspecs A method for producing lactic acid using Rhamnosus KCTC 0084BP (Korean Patent No. 0113816), a method for newly separating and industrially using an organism that produces lactic acid with high efficiency from pentose sugar and hexose obtained from nature (US Patent US7098009) has been presented. There is a bar. In addition, techniques for fermentation using inexpensive biomass to reduce the cost burden in lactic acid production include tapioca starch (Korean Patent Application Publication No. 2005-0010986), wheat bran (Korean Patent No. 0454597), rice bran (Korean patent). No. 0490953), wood-based saccharification (Korean Patent Application Publication No. 2003-0008187), and the like.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors research efforts result to the high concentration of L-lactic acid from carbohydrate by biotechnological and that the development of lactic acid producing strains for producing in a high yield, Lactobacillus Paracazei Mutants suitable for strains such as M-N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) can be treated to prepare mutants with improved glucose and acid resistance, and when using them, lactic acid can be produced in high yield. The present invention has been completed by discovering that there is.

An object of the present invention is to provide within the feasibility and the acid resistance are improved and the yield of lactic acid production is increased by the Lactobacillus casei para mutants.

Another object of the present invention is to improve lactobacillus and acid resistance and to increase the yield of lactic acid lactobacillus It is to provide a method for preparing paracay mutant strains.

Another object of the present invention to provide a method for producing lactic acid.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention exhibits improved growth in glucose tolerance and acid resistance compared to wild type, and shows sustained growth in a medium containing 100 g / L to 250 g / L of glucose and 20 g / L to 200 g / L of lactic acid. , Lactobacillus with lactic acid production yield of 92-99.5% It provides a parametric casei mutants.

The present inventors research efforts result to the high concentration of L-lactic acid from carbohydrate by biotechnological and that the development of lactic acid producing strains for producing in a high yield, Lactobacillus Paracazei Mutants suitable for strains such as M-N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) can be treated to prepare mutants with improved glucose and acid resistance, and when using them, lactic acid can be produced in high yield. It was found.

Lactobacillus of the invention Paracazei The mutant strains exhibited improved glucose and acid resistance compared to wild type. More specifically, it shows sustained growth at high sugar concentrations of glucose 100 g / L to 250 g / L, and growth at high acid concentrations of lactic acid 20 g / L to 200 g / L.

Lactobacillus of the invention Paracazei The term “sugar resistance” as used to refer to mutant strains, when cultured in a conventional shaking culture in the art (eg using a 50 mL culture flask), ranges from 100 g / L to 250 g / L of glucose, preferably from 110 g / L to glucose. By cultivation characteristic, which indicates sustained growth at a concentration of 150 g / L, more preferably at a concentration of 120 g / L of glucose. When incubated in conventional fermenters of the art (e.g., 5L fermenters with stirrers and automatic pH regulators), the term "sugar tolerance" refers to a concentration of glucose from 100 g / L to 250 g / L, preferably from 180 g / L to 230 g / By the characteristic of culture, which indicates sustained growth at a concentration of L, more preferably at a concentration of 200 g / L of glucose.

Lactobacillus of the invention Paracazei The term “acid resistance” as used to refer to mutant strains refers to 20 g / L to 200 g / L lactic acid, preferably 20 g lactic acid, contained in the initial culture medium when cultured in a conventional shake culture (eg using a 50 mL culture flask) in the art. / L to 100 g / L, more preferably from 20 g / L to 50 g / L of lactic acid, most preferably from 30 g / L of lactic acid. The term “acid resistance” when incubated in conventional fermenters of the art (eg, 5 L fermenters with stirrers and automatic pH regulators) refers to lactic acid 20 g / L to 200 g / L, preferably lactic acid 100 g / L to 200 g / L, more Preferably it refers to a culture characteristic showing sustained growth at a concentration of 180 g / L to 200 g / L, even more preferably 185 g / L to 200 g / L lactic acid.

As used herein, the term “sustained growth” refers to a cultivation characteristic in which microorganisms are not killed by high concentrations of glucose or lactic acid and continuous growth without interruption of growth. More specifically, the term refers to lactic acid bacteria at glucose concentrations (eg, glucose 150 g / L to 200 g / L) or lactate concentrations (eg, lactic acid 150 g / L to 200 g / L) in the conventional culture of lactic acid bacteria. In this growth curve, the glucose concentration or acid resistance glucose concentration and the time between the initial time of the stationary phase and the initial time of the death phase, that is, a period of time, and Mean incubation time at the lactate concentration is 50-100%, preferably 80-100%.

Lactic acid production yield in the present invention means the ratio of lactic acid production (weight / weight) to glucose used as a carbon source and energy source.

"Increased lactic acid production yield" in the present invention means higher lactic acid production yield than wild type, preferably 93% or more, more preferably 94% or more, even more preferably 96% or more, even more It means that the lactic acid yield is increased by more than 97%, most preferably 99.5% or more.

Lactobacillus strains used to prepare the mutant strains of the present invention Paracaze strains can be used without limitation, but Lactobacillus has a high L-lactic acid production rate. To use a para casei KCTC 13169, KCTC 3165 strains are preferred.

Lactobacillus with increased sugar resistance, acid resistance and lactic acid production yield including the following steps Paracazei Variation Notes

(a) Lactobacillus Paracazei Culturing the strain in the medium; And

(b) MNCG (N-methyl-N'-nitro-N-nitrosoguanidine) was added to the strain medium, followed by mutations, thereby reducing the Lactobacillus of step (a). Paracazei Preparing a para Lactobacillus casei mutants having an acid resistance and an increase in feasibility than the strain.

The present inventors have conducted research to develop lactic acid-producing strains that produce L-type lactic acid from carbohydrates at high concentrations and high yields biotechnologically, resulting in Lactobacillus. Paracazei The strain was treated with MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) to prepare mutants with increased sugar resistance, acid resistance and lactic acid production yield.

Lactic acid (Lactic acid bacteria) used in the present invention as a parent strain Lactobacillus para casei (Lactobacillus paracasei) strain of KCTC 13169 is subgingival of the first tooth plaque; isolated from (subgingival dental plaque sandwiched plaque in the furrow between the teeth and gums) KCTC 3165 is a strain isolated from saliva of the first child, and is a strain distributed from a Biological Resource Center.

By using a known method to produce a mutation in the parent strain (e.g. Lactobacillus casei para parent process MNNG mutagenesis cause the strain) Lactobacillus It can be prepared para casei mutants.

More preferably, the present invention Lactobacillus casei para mutant parent strain of Lactobacillus Para casei KCTC 13169, and processes the mutagenesis causes MNNG in KCTC 3165 strains, and improved within the feasibility and acid resistance compared to the wild type, medium containing glucose 100g / L to 250g / L and lactic acid 20g / L to 200g / L It indicates the growth continued at, and refers to a strain having a lactic acid production yield of 92% to 97%.

MNNG (N-methyl-N'-nitro-N-nitrosoguanidine), a substance for preparing a mutant strain used in the present invention, is a substance that causes carcinogenesis in human cells and other animal cells, as well as mutations by damaging DNA against bacteria. It is known to cause (Pleven, C. et. al , Glioblastomas and chemical mutagenesis in biology laboratories. Report of 3 deaths in the same institute (Fr.). Arch . Mal . Prof. , 44: 411-418 (1983); Martin, MS et al , Susceptibility of inbred rats to gastric and duodenal carcinomas induced by N-methyl-N'-nitro-N-nitrosoguanidine. J. natl Cancer Inst ., 53: 837-840 (1974); IARC Monographs, Suppl. 6: 394-398 (1987)

The chemical structure of the MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) is shown in the following formula (1).

Formula 1

According to another aspect of the present invention, there is provided a method for producing Lactobacillus paracasei, which has increased sugar resistance, acid resistance, and lactic acid production yield, comprising the following steps.

(a) Lactobacillus Paracazei Culturing the strain in the medium; And

(b) by adding MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) to the strain medium and mutating the Lactobacillus of (a) Lactobacillus having the feasibility and acid resistance in an increased para casei strain Preparing a para casei mutants.

Lactic acid production yield in the present invention means the ratio of lactic acid production (weight / weight) to glucose used as a carbon source and energy source.

In the present invention, "lactic acid production yield is increased" means higher lactic acid production yield than wild type, preferably 93% or more, more preferably 94% or more, even more preferably 95% or more, even more It means that the lactic acid production yield is preferably increased at least 96%, most preferably at least 97%.

In the present invention, the medium in step (a) includes any medium used for culturing Lactobacillus strains, preferably, deMan Rogosa Sharpe (MRS) broth medium, APT (All Purpose with Tween) medium or BHI (Brain) Heart Infusion) medium, and most preferably MRS broth medium can be used.

In the present invention, step (b) is Lactobacillus by adding MNNG to the medium Paracaze strains. Specifically, in the present invention, the concentration of MNNG added to the medium is preferably 200 µg / ml to 600 µg / ml, more preferably 300 µg / ml to 500 µg / ml, and most preferably 350 µg / ml. Ml to 450 μg / ml.

According to a preferred embodiment of the present invention, the present invention further comprises the step of selecting a strain having a tolerance and acid resistance and increased lactic acid production yield from the strain medium treated with MNNG.

According to another aspect of the invention, the Lactobacillus It provides a method for producing lactic acid comprising the step of culturing the paracase mutants in a medium containing a carbon source, energy source and nitrogen source.

The carbon source and the energy source are glucose and the nitrogen source is any one component selected from the group consisting of yeast extract, yeast concentrate, beef extract, corn steep flour, corn steep liquor, tuna extract, peptone, ammonium citrate, ammonium phosphate and ammonia water Or a mixture of two or more thereof, but is not limited thereto.

More preferably, the nitrogen source may use corn immersion powder or corn immersion liquid or a mixture thereof in order to reduce manufacturing costs.

The features and advantages of the present invention are summarized as follows:

(Ⅰ) the present invention is to improve the lactic acid bacteria growth inhibition caused by the the product per the substrate lactic acid through the Lactobacillus para casei mutants with improved naedang and acid and lactic acid as compared to wild-type while using environmentally friendly biotechnological methods It provides a process for producing in yield.

(Iii) The present invention provides a method for producing lactic acid that can reduce costs by using inexpensive corn steep flour and corn steep liquor, which are wastes generated after treating corn instead of expensive yeast extract, as organic nitrogen sources.

Figure 1 shows the production of lactic acid (Lactic acid) from carbohydrate (Carbobydrate).
Figure 2 is a glucose 200g / L, yeast extract 15g / L, corn steep liquor of Lactobacillus para casei (Lactobacillus parent strain in a culture with a 40g / L containing medium paracasei ) KCTC 13169 and MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) were treated with 5L fermenter to incubate DSLA 6912, a lactobacillus paracasei strain, improved in glucose tolerance and acid resistance. According to the strain growth, glucose consumption, a graph showing the lactic acid production curve. Cell concentration and lactic acid in KCTC 13169 culture medium were indicated as ●, glucose as ○, and cell concentration and lactate in DSLA 6912 culture medium as ■ and glucose as □.
Figure 3 is glucose 200g / L, yeast extract 15g / L, corn steep liquor of Lactobacillus para casei (Lactobacillus parent strain in a culture with a 20g / L containing medium paracasei ) KCTC 13169 and MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) were treated with 5L fermenter to incubate DSLA 6912, a lactobacillus paracasei strain, improved in glucose tolerance and acid resistance. According to the strain growth, glucose consumption, a graph showing the lactic acid production curve. Cell concentration and lactic acid in KCTC 13169 culture medium were indicated as ●, glucose as ○, and cell concentration and lactate in DSLA 6912 culture medium as ■ and glucose as □.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are only for illustrating the present invention more specifically, it is apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. something to do.

Example

Throughout this specification, unless otherwise indicated, “%” used to indicate the concentration of a characteristic substance is (weight / weight)% solids / solid, (weight / volume)%, and Liquid / liquid is (volume / volume)%.

Example  One: Tolerance Acid resistance  Improved Isolation and Screening of Mutant strains

Fermentation process of the present invention was carried out in the same manner as a general batch fermentation. The medium of this process is shown in Table 1 below.

Lactic acid bacteria Fermenter Culture Badge composition division ingredient density(%) Equivalent (g / L) liquid measure ( mL , 2L standard) A glucose 10.0 ~ 20.0 100.0-200.0 1300-1400 MgSO ₄ ? 7H ₂ O 0.02 0.2 B Corn Soak 0.0-4.0 0.0-40.0 300 Yeast extract 1.5 ~ 2.5 15.0 ~ 25.0 Sodium Acetate
(Sodium-acetate) 0.1 1.0 C MnSO ₄ ? 5H ₂ O 0.006 0.06 100 FeSO ₄ ˜7H ₂ O 0.003 0.03 D K ₂ HPO ₄ 0.07 0.7 100 KH ₂ PO ₄ 0.05 0.5 E Cultivation 5.0-10.0 100-200

Medium components A, B, C, and D as shown in Table 1 were prepared in different containers, respectively, and used after autoclaving at 121 ° C. for 20 minutes.

In this embodiment, improved sugar resistance and acid resistanceLactobacillus Paracazei(Lactobacillus paracasei) In order to obtain a mutant strain, strains were prepared by using a medium containing glucose and lactic acid in the preparation of strain culture solution for MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) treatment and the culture of MNNG-treated strain Tolerance of sugar and acid resistance was maintained.

In order to prepare strain culture for MNNG treatment, 0.3 mL of glycerol cryopreserved strain Lactobacillus paracasei KCTC 13169 and KCTC 3165 were inoculated in 100 mL of MRS preculture medium and incubated at 37 ° C. and 200 rpm for 15 hours. The culture solution was inoculated into 2 L of fermentation culture medium of Table 1 containing 100-150 g / L of glucose and 0-80 g / L of lactic acid, which were added to a 5 L fermenter subjected to autoclaving by a flame-sterilization process, to proceed with fermentation. The temperature during fermentation was 37 ℃, the stirring speed was 200rpm, pH was maintained at 6.0 using ammonia water. Samples were taken every 8 hours during incubation to analyze the growth of the strain, glucose consumption, and lactic acid production. As a result, the culture medium of the fermentor to which the medium containing the highest concentration of lactic acid was added among the strains for which lactic acid production was maintained, that is, the culture medium of the fermentor containing 120 g / L glucose and 30 g / L lactic acid, was used as a strain culture medium for MNNG treatment. Ready.

After taking 10 mL of this culture, centrifugation was performed for 5 minutes at 3000 rpm using TM buffer (buffer, 50 mM Tris-HCl, 2.5 mM MgCl2; pH 7.0). Washed strains were suspended in the same volume of TM buffer. The suspension containing the strain was divided into 5mL and MNNG (N-methyl-N'-nitro-N-nitrosoguanidine; Fluka chemie AG, Switzerland) at 400μg / mL concentration was added, and then treated for 15 minutes at 37 ℃, 200rpm. Finally, after removing the remaining MNNG using saline, strains treated with MNNG in MRS plate medium containing 100 ~ 150g / L glucose, 0 ~ 80g / L lactic acid, a certain level, for example, 10-4 ~ The plate was diluted to 10-6. Plates with strains were incubated in an incubator at 37 ° C., and lactose centered on colonies grown first on MRS plates containing 120 g / L glucose and 30 g / L lactic acid on day 2 to 5 after incubation. Bacillus paracasei KCTC 13169 and KCTC 3165 mutant strains were selected 20 weeks each.

Example  2: lactic acid productivity confirmation culture of mutant strain on flask

The lactic acid production culture was carried out on OK MNNG (N-methyl-N'- nitro-N-nitrosoguanidine) , each one processes the selected note 20 Lactobacillus casei para KCTC 13169, KCTC 3165 flask, the mutant strain. The medium of this process is shown in Table 2 below.

Lactic acid bacteria flask culture Badge composition division ingredient density(%) Equivalent (g / L) A glucose 12.0 120.0 MgSO ₄ ? 7H ₂ O 0.02 0.2 B Yeast Concentrate 2.0 20.0 Sodium Acetate 0.1 1.0 K ₂ HPO ₄ 0.07 0.7 KH ₂ PO ₄ 0.05 0.5 C MnSO ₄ ? 5H ₂ O 0.006 0.06 FeSO ₄ ˜7H ₂ O 0.003 0.03 Adjust the pH of the medium to 6.5 with NaOH.

Medium components A, B, and C as shown in Table 2 were prepared in different containers, respectively, and used after autoclaving at 121 ° C. for 20 minutes.

In this Example, 40 strains of strains were inoculated into 5 mL of MRS containing 30 g / L lactic acid to prepare precultures incubated at 37 ° C. and 200 rpm for 15 hours. 0.03 mL of the preculture was inoculated into a 50 mL flask to which 20 mL of the flask culture medium containing 30 g / L of lactic acid was added, followed by incubation at 37 ° C. and 200 rpm for 50 hours to compare the lactic acid productivity of the mutant strains.

The results of this example are shown in Table 3.

Tolerance Acid resistance  Productivity on improved strain flasks Strain ( Colony  number) pH OD (610 nm ) Lactic Acid (g / L) * Sugar (g / L) Yp / s (g / L) 12 4.95 8.4 3.57 6.19 93.70 13 4.90 8.0 3.63 6.15 94.29 16 4.89 7.8 3.60 6.05 91.14 KCTC  13169 4.88 8.2 3.33 6.05 84.30 19 4.85 7.9 3.93 6.02 98.74 23 4.82 7.3 3.89 6.05 98.48 28 4.91 7.6 3.79 6.05 96.01 KCTC  3156 4.87 8.3 3.53 6.05 89.35

* Lactic acid (g / L): Excluding lactic acid 30g / L contained in the flask culture medium

Through the example results of the embodiment, the para Lactobacillus casei KCTC 13169, KCTC 3165 mutants were screened six weeks of the 40 weeks. OD represents the optical density at 610 nm.

Example  3: lactic acid productivity confirmation culture of mutant strain on 5L fermenter

( Glucose 200g / L, Yeast Extract 15g / L, Corn Dipping Liquid 40g / L)

Fermentation process of the present invention was carried out in the same manner as a general batch fermentation. The medium of this process is shown in Table 1 and Table 4.

Lactic acid bacteria Cultivation Badge composition ingredient density(%) Equivalent (g / L) Yeast Concentrate 2.0 20.0 glucose 5.0 50.0 Sodium Acetate 0.3 3.0 MgSO ₄ ? 7H ₂ O 0.01 0.1 MnSO ₄ ? 5H ₂ O 0.01 0.1 K ₂ HPO ₄ 1.0 10.0 Adjust the pH of the medium to 6.5 with NaOH.

The media components as shown in Table 4 were prepared and used after autoclaving at 121 ° C. for 20 minutes.

MNNG (N-methyl-N'- nitro-N-nitrosoguanidine) treatment obtained by Lactobacillus casei para KCTC 13169, KCTC 3165 all the mutant strains 6 weeks showed a relatively high productivity of lactic acid in the flask over 40 weeks on the 5L fermenter the strain Inn Lactobacillus Para casei KCTC 13169, KCTC 3165 compared with the experiments were performed to confirm its superiority, as a result, Lactobacillus Para casei was selected the 12th colony of Lactobacillus casei para DSLA 6912 originated from KCTC 13169.

The waste in this embodiment for generating and processing the MNNG naedang and acid resistance that mutants of Lactobacillus para casei DSLA 6912 and the parental strain of Lactobacillus para casei KCTC 13169 improve after processing the corn in place of an expensive yeast extract Lactic acid productivity was compared using a medium in which cheap corn steep powder or corn steep liquor was replaced with an organic nitrogen source.

In this example, the glycerol cryopreserved strain was inoculated into 100 mL of the pre-culture medium of Table 4, and cultured at 37 ° C. and 200 rpm for 15 hours. The culture solution was inoculated into fermentation culture medium of Table 1 containing 200 g / L of glucose, 15 g / L of yeast extract, and 40 g / L of corn steep liquor added to a 5 L fermenter subjected to autoclaving by a flame-sterile process. . The temperature during fermentation was 37 ℃, the stirring speed was 200rpm, pH was maintained at 6.0 using ammonia water. Samples were taken every 8 hours during incubation to analyze the growth of the strain, glucose consumption, and lactic acid production. The results of comparing lactic acid productivity from fermentation broth containing glucose 200g / L, yeast extract 15g / L and corn steep liquor 40g / L are shown in Table 5 and FIG. In Tables 5 and 6, the yield refers to the ratio of the finally produced lactic acid content to the initial content of glucose used as a carbon and energy source.

Strain
( L. paracasei ) Fermentation time
( hr ) Lactic acid (g / L) yield
(%) productivity
(g / L / h) KCTC  13169 56 178.0 89.0 3.2 DSLA  6912 50 190.0 95.0 3.8

Table 5 shows the productivity on mutant strain fermenters (glucose 200g / L, yeast extract 15g / L, corn steep liquor 40g / L) with improved sugar and acid resistance.

Example  4: Culture of Confirming Lactic Acid Product Production of Mutant in 5L Fermenter

( 200g / L glucose, 15g / L yeast extract, 20g / L corn steep liquor)

In this example, the fermentor culture medium of Table 1 containing glucose 200g / L, yeast extract 15g / L, corn steep liquor 20g / L was used, and other experimental methods, media, strains are the same as in Example 3. The results of comparing lactic acid productivity from fermentation broth containing glucose 200g / L, yeast extract 15g / L and corn steep liquor 20g / L are shown in Table 6 and FIG.

Strain
( L. paracasei ) Fermentation time
( hr ) Lactic acid (g / L) yield
(%) productivity
(g / L / h) KCTC  13169 90 176.1 88.1 2.0 DSLA  6912 68 188.7 94.4 2.8

Table 6 shows the productivity on mutant fermenters (glucose 200g / L, yeast extract 15g / L, corn steep liquor 20g / L) with improved sugar resistance and acid resistance.

Example  5: Culture of lactic acid production in mutant strains on 5 L fermenter

(150g / L glucose, 25g / L yeast extract)

In this example, the fermentor culture medium of Table 1 containing glucose 150g / L, yeast extract 25g / L was used, and other experimental methods, media, strains are the same as in Example 3. The results of comparing lactic acid productivity from fermentation broth containing 150 g / L glucose and 25 g / L yeast extract are shown in Table 7.

Strain
( L. paracasei ) Fermentation time
( hr ) Lactic acid (g / L) yield
(%) productivity
(g / L / h) KCTC  13169 30 144.5 96.3 4.8 DSLA  6912 26 149.3 99.5 5.7

Table 7 shows the productivity on mutant strain fermenters (glucose 150g / L, yeast extract 25g / L) with improved sugar and acid resistance.

Korea Biotechnology Research Institute KCTC11808 20101116

Claims (7)

Compared with wild type, improved glucose tolerance and acid resistance, sustained growth in medium containing glucose 100g / L to 250g / L and lactic acid 20g / L to 200g / L, yield of 92-99.5% lactic acid production Lactobacillus Para casei mutants.
The method of claim 1, wherein the Lactobacillus Para casei Lactobacillus variant is characterized in that the DSLA 6912 (accession number: KCTC 11808BP) strains Para casei mutants.
Lactobacillus with increased sugar resistance, acid resistance and lactic acid production yield including the following steps Paracazei Variation Notes
(a) Lactobacillus Paracazei Culturing the strain in the medium; And
(b) MNCG (N-methyl-N'-nitro-N-nitrosoguanidine) was added to the strain medium, followed by mutations, thereby reducing the Lactobacillus of step (a). Paracazei Preparing a para Lactobacillus casei mutants having an acid resistance and an increase in feasibility than the strain.
The method of claim 3, wherein the Lactobacillus Para casei mutants indicates a continued growth in the medium containing glucose 100g / L to 250g / L of lactic acid and 20g / L to 200g / L, characterized in that with a lactic acid yield of 92-99.5%.
Lactobacillus of claim 1 or 2 Para casei mutants for lactic acid producing composition containing a.
Lactobacillus of claim 1 or 2 Para car the second mutant comprising the step of culturing in a medium containing a carbon source, energy source and a nitrogen source, the carbon and energy source is glucose and the nitrogen source is yeast extract, yeast concentrate, beef extract, corn steep powder, corn steep liquor , Tuna extract, peptone, ammonium citrate, ammonium phosphate and at least one component selected from the group consisting of ammonia water.
7. The method of claim 6, wherein the nitrogen source is corn steep powder or corn steep liquor.

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