KR101496503B1 - Method for High Cell Density Culturing of Lactic Acid Bacteria and Producing Their Metabolites By Using Bioreactor Equipped with Internal Filter System - Google Patents

Abstract

The present invention relates to a method for continuously producing lactic acid bacteria in a fermentation tank equipped with an internal filter to produce metabolites such as lactic acid, acetic acid and mannitol at a high yield. In the present invention, lactic acid bacteria can be produced at a high concentration by using a fermentation tank equipped with an internal filter, and lactic acid, acetic acid and mannitol, which are intermediate metabolites, can be continuously produced at a high yield. The lactic acid bacterium produced by the method of the present invention can be used as a starter for milk and fermented food, and mannitol can be used as a food additive, and lactic acid can be used as a raw material for biodegradable polymer, polylactate (PLA) .

Description

TECHNICAL FIELD The present invention relates to a method for producing lactic acid bacteria, and more particularly, to a method for producing lactic acid bacteria using a bioreactor equipped with an internal filter system and a method for producing a metabolic product,

The present invention relates to a method for continuously culturing a lactic acid bacterium at a high concentration using a bioreactor equipped with an internal filter system and its metabolites.

Lactobacillus can be used as a starter to produce milk and dairy products and a variety of fermented foods. In addition, mannitol, which is a metabolite produced through cultivation, can be used as an additive in food production, and lactic acid is increasingly utilized as a raw material for biodegradable polylactide (PLA). Of the lactic acid bacteria, Leuconostoc citreum HJ-P4 used for the cultivation in the present invention is a major lactic acid bacterium that proliferates at the early stages of fermentation of kimchi, and can be grown under both aerobic and anaerobic conditions. Various kinds of vegetables It is also found in the surface of fruit and other frozen meats and dairy products. Growth of this strain requires various nutrients, but it can grow by adaptation to various environments, and it has many sugars, protein metabolites, and vitamins, and it can grow well at low oxygen pressure. It is also found to be a beneficial microorganism used as a starter in the production of milk and dairy products and various fermented foods. Leuconostoc citreum The HJ-P4 strain is a nearly spherical cell but occasionally a very short bacillus. The size of these microorganisms is about 0.5 to 0.7 X 0.7 to 1.2 탆 and is arranged in the form of a pair or a chain. Growth in a nutrient-rich medium shows a short chain form, but under extreme conditions, the chain becomes longer. The optimum growth temperature is in the range of 20 to 30 캜, and the optimum pH range of the medium is 6 to 7. In addition, this strain is a lactic acid fermented lactic acid bacterium, which produces lactic acid, alcohol, and CO ₂ through the metabolism of hexose monophosphate (6-phosphate gluconate) and pentose phosphate pathway as a glycoprotein. The pH will drop to 4.4-5.0 due to the organic acid. In addition, when excess fructose is present as a carbon source, most fructose acts as an elctron acceptor by mannitol dehydrogenase and is reduced to mannitol. Studies on the use of starters of fermented products such as kimchi, sauerkraut, and kefir, and various applications of fermented products have been reported using these characteristics, and the future development of our traditional foods and biotechnology It is expected to play an important role in (2001), Novel Leuconostoc citreum starter culture system for fermentation of kimchi, a fermented cabbage product, Antonie (Kim, J., and Kim, van Leeuwenhoek., 15, 163-82). However, there has not yet been conducted an experiment on high concentration culture of Leuconostoc citreum HJ-P4 strain. In order to cultivate high-concentration cells, culturing should be performed while maintaining optimal conditions for cell growth. The biggest weakness of the conventional batch method is that the product concentration is low and the productivity is low. Unlike general chemical substances, microbial fermentation can not increase the concentration of reactants (such as media) to above a certain level, and it is difficult to keep the cell concentration high because the microorganism itself produces harmful substances. As a method to solve this problem, a continuous culture method using a filter has been attempted in a high concentration culture. (1993) Cell retention culture with an internal filter module: continuous ethanol fermentation., Biotechnol. Bioeng., 41, 677-681).

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made efforts to develop a method for culturing lactic acid bacteria at a high concentration using a continuous culture method of lactic acid bacteria and obtaining a useful metabolite at a high yield. As a result, it is possible to continuously cultivate the lactic acid bacterium at a high concentration and to continuously produce the metabolite through the continuous cultivation of Leuconostoc citreum HJ-P4 strain using a bioreactor equipped with an internal filter Thereby completing the present invention.

Accordingly, it is an object of the present invention to provide a method of continuously culturing lactic acid bacteria at a high concentration using a bioreactor equipped with an internal filter and continuously producing metabolites.

The 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 provides a method of continuously producing lactic acid, acetic acid or mannitol by continuously culturing lactic acid bacteria comprising the steps of: (a) adding to a fermentation tank equipped with an internal filter an initial batch culture medium And then inoculating the lactic acid bacteria; (b) an initial batch culture of the inoculated lactic acid bacteria; And (c) culturing the lactic acid bacteria while continuously supplying the culture medium for continuous culture to the fermentation tank to produce lactic acid, acetic acid or mannitol.

Hereinafter, the present invention will be described in detail for each step.

(A) a step of putting an initial batch culture medium into a fermentation tank equipped with an internal filter, and inoculating the lactic acid bacteria therein

The present invention is based on the successful successive cultivation of lactic acid bacteria which had been difficult to continuously cultivate using a fermentation tank equipped with an internal filter.

Lactic acid bacteria that can be used in the continuous culturing of the present invention are not particularly limited and include, for example, lactic acid bacteria of the genus Lactococcus, lactobacillus of the genus Lactobacillus, lactobacillus of the genus Leuconostoc, Lactic acid bacteria belonging to Propionibacterium genus, Enterococcus genus lactobacillus, Bifidobacterium genus lactobacillus, Carnobacterium genus lactobacillus, Streptococcus genus lactobacillus, Oenococcus genus, Sporolactobacillus sp. Lactobacillus or Pediococcus sp. Lactic acid bacteria, preferably Leuconostoc sp. Lactic acid bacteria, and most preferably Leuconostoc citreum HJ- P4 strain.

The present invention is advantageous in that an intermediate metabolite of lactic acid bacteria such as lactic acid, acetic acid or mannitol can be obtained in high yield through continuous cultivation of lactic acid bacteria.

The internal filter provided in the fermenter of the present invention is for culturing a high concentration of lactic acid bacteria and includes the following components: (i) one or a plurality of cylindrical ceramic filtration membranes 60; (Ii) one or more silicon tubes (70) having one end connected to the cylindrical ceramic filtration membrane and the other end connected to a top cover; And (iii) a top cover (80) having a lower portion connected to the one or more silicon tubes and an upper portion connected to the outer tube.

The bioreactor of the present invention comprises a feed tank 10, a recovery tank 30, and a fermenter 20 for supplying a culture medium for continuous culture. An internal filter is installed in the fermentation tank 20 and a feed pump 40 is provided between the medium storage tank 10 and the fermentation tank 20 and a filtration pump 40 is provided between the fermentation tank 20 and the filtrate storage tank 30. [ 50 are provided so as to continuously supply, filter and discharge the medium by these pumps (see FIG. 1A).

The internal filter of the present invention comprises a cylindrical filtration membrane 60 made of a ceramic material, a silicon tube 70 connecting the cylindrical filtration membrane, and a top lid 80 serving to fix the entire filter Reference).

The cylindrical filtration membrane 60 in the filter of the present invention is made of a ceramic material and functions as a filter for filtering cells. The pore size of the ceramic filtration membrane 60 can be selected depending on the type of lactic acid bacteria and the culture conditions, and is preferably 0.05 to 0.2 탆, more preferably 0.07 to 0.15 탆, 0.1 mu m. The number of the cylindrical ceramic filtration membranes can be one or more, and the number of the ceramic filtration membranes and the pore size in the filtration membrane can be controlled according to the capacity of the fermentation tank.

The silicon tube 70 connects the top cover 80 and one or more cylindrical ceramic filtration membranes 60. The silicone material of the silicone tube facilitates the bonding of the top cover and the cylindrical ceramic membrane after cleaning and cleaning, providing convenience.

The top lid 80 is preferably made of stainless steel tubing to prevent corrosion. Is connected to a filtration pump (50) outside the fermentation tank by one outer tube (90) of the top cover, and the culture filtrate is discharged to the recovery tank (30) by the connected pump.

Step (b): an initial batch culture of the inoculated lactic acid bacteria ;

In the present invention, the culture medium for early batch culture comprises a carbon source as an energy source and a cell constituent material source of lactic acid bacteria. Examples of the carbon source include glucose, dextrin, sucrose, galactose, And most preferably glucose. In the present invention, the continuous culture medium may further contain fructose (fructose) in addition to glucose as a carbon source when mannitol is produced.

The initial batch culture medium or continuous culture medium of the present invention includes a nitrogen source, and for example, a proteolytic substance or a yeast extract may be used as the nitrogen source, and a yeast extract is most preferably used.

The culture media for initial batch culture or continuous culture of the present invention may include other inorganic salts and vitamins, preferably K ₂ HPO ₄ , MgSO ₄ .7H ₂ O, MnSO ₄ .7H ₂ O, FeSO ₄ 7H ₂ O, CaCl ₂ .2H ₂ O, and NaCl. According to a specific embodiment of the invention, the medium of the present invention, glucose 50g per 1L, yeast extract 25g, 30g K ₂ HPO _4, MgSO ₄ and 7H ₂ O 0.2g, MnSO ₄ and 7H ₂ O 0.01g, FeSO ₄ and 7H include ₂ O 0.01g, CaCl ₂ and 2H ₂ O 0.02g, and 0.01g NaCl.

The initial batch culture temperature of the present invention is 20-36 캜, more preferably 22-34 캜, even more preferably 24-32 캜, and most preferably 26-30 캜.

The pH of the medium during the initial batch culture is 5-8, more preferably 6-7, most preferably 6.5. The stirring speed during culturing may vary depending on the culture conditions, but is preferably 100-500 rpm, more preferably 200-400 rpm, and most preferably 300 rpm.

Step (c): culturing the lactic acid bacteria while continuously supplying the culture medium for continuous culture to the fermentation tank to produce lactic acid, acetic acid or mannitol

In the present invention, the components of the medium for continuous culture are very similar to those of the medium for initial batch culture described above. That is, it contains a carbon source as an energy source of a lactic acid bacteria and a cell constituent material source, and most preferably contains glucose as a carbon source. In addition, yeast extract may be used as the nitrogen source, and other inorganic salts and vitamins may be contained. Preferably, K ₂ HPO ₄ , MgSO ₄ .7H ₂ O, MnSO ₄ .7H ₂ O, FeSO ₄ 7H ₂ O, CaCl ₂ .2H ₂ O, and NaCl.

In the present invention, fructose (fructose) is added to the medium for continuous culture in addition to glucose as a carbon source when producing mannitol.

In the present invention, the continuous culture is performed while the continuous culture medium is continuously supplied at the time when the carbon source is depleted by the initial batch culture. The temperature, the pH range, and the stirring speed in the continuous incubation of the present invention are performed in a manner similar to the temperature, the pH range and the stirring speed during the batch culture.

According to a preferred embodiment of the present invention, the continuous culture is performed so that the dilution ratio of the medium is in the range of 0.01 - 1 h ^-1 , more preferably 0.02 - 0.7 h ^-1 , still more preferably 0.03 - 0.6 h ^-1 , most preferably 0.07 - 0.2 h < ^{-1 &} gt ;. The higher the dilution rate, the higher the rate of the concentration increase of the lactic acid bacteria cells and the higher the production of the metabolites, but the disadvantage that the cost increases due to the increase of the consumption rate of the culture medium.

Lactic acid, acetic acid or mannitol, which are intermediate metabolites, can be obtained in a form contained in a continuously cultured medium. Separation and purification of these substances can be carried out through a method of isolating and purifying the compounds known in the art. For example, by centrifugation, filtration through a membrane filter, filtration through a silica gel or celite gel column, size exclusion chromatography using liquid column chromatography, ion exchange chromatography, partition chromatography, affinity chromatography or the like Chromatography, but are not limited thereto.

The present invention relates to a method for continuously producing lactic acid bacteria in a fermentation tank equipped with an internal filter to produce metabolites such as lactic acid, acetic acid and mannitol at a high yield. In the present invention, lactic acid bacteria can be produced at a high concentration by using a fermentation tank equipped with an internal filter, and lactic acid, acetic acid and mannitol, which are intermediate metabolites, can be continuously produced at a high yield. The lactic acid bacterium produced by the method of the present invention can be used as a starter of milk and fermented foods, and mannitol can be used as a food additive, and lactic acid can be used as a raw material of biodegradable polymer, polylactate (PLA) .

1A is a configuration diagram of a bioreactor of the present invention. The bioreactor of the present invention comprises a feed tank 10, a recovery tank 30, and a fermenter 20 for supplying a continuous culture medium. An internal filter is installed inside the fermentation tank 20 and a feed pump 40 is provided between the medium storage tank and the fermentation tank and a filtration pump 50 is provided between the fermentation tank and the filtrate storage tank, Continuous supply and discharge are performed. The inner filter is composed of a cylindrical filtration membrane 60 made of ceramic material, a silicone tube 70 connecting the cylindrical ceramic filtration membrane to the top cover 80, and a top cover 80 serving to fix the entire filter . The cylindrical ceramic filtration membrane 60 used in the embodiment of the present invention has a pore size of 0.1 탆 and has an inner diameter of 6 mm, an outer diameter of 8 mm, and a length of 65 mm, and 20 cylindrical filtration membranes are connected The surface area corresponds to 330 cm ² . The internal filter is designed to the effective volume 2.5 L of the fermenter, and the number of filtration membranes can be increased to increase the surface area. The top cover 80 connecting the twenty cylindrical filtration membranes 60 is made of a stainless steel pipe to prevent corrosion and has a cylindrical ceramic filtration membrane 60 and a top cover 80 for convenience of binding and cleaning of the filter. A silicon tube 70 was used. The twenty cylindrical filtration membranes were connected by a top cover so that only one tube 90 was connected to the pump outside the reactor to allow the filtrate to escape. In the present invention, the culture medium is supplied from a feed tank 10 to a fermenter 20 equipped with an internal filter through a feed pump 40, and the product produced in the fermentation tank is supplied to the upper part of the filtration pump 50 And is discharged to the recovery tank 30 through the pipe of the cover 80.
1B shows the structure of the internal filter installed inside the fermentation tank 20 of the present invention in more detail. The inner filter of the present invention comprises a cylindrical ceramic filtration membrane 60 and a silicone tube 70 and a top lid 80 connecting the cylindrical filtration membrane to the top lid 80. One pipe 90 is provided in the top lid, Is connected to the external filtrate storage tank (30).
FIG. 2 is a photograph showing the result of continuous culture of lactic acid bacteria by a bioreactor equipped with an internal filter of the present invention, which is a photograph of the lactic acid bacteria before culture and after culture. FIG.
FIG. 3 is a graph showing the results of measuring the cell growth rate and the production amount of metabolites while continuously culturing glucose as a carbon source by a bioreactor equipped with the internal filter of the present invention (dilution ratio 0.07 h ^-1 ).
FIG. 4 is a graph showing the results of measuring the cell growth rate and the production of metabolites while continuously culturing the cells using glucose and fructose as a carbon source by the bioreactor equipped with the internal filter of the present invention (dilution ratio 0.07 h ^-1 ).
FIG. 5 is a graph showing the results of measuring cell growth rate and metabolite production while continuously culturing glucose and fructose as a carbon source by a bioreactor equipped with an internal filter of the present invention (dilution ratio 0.13 h ^-1 ).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Methods

1. Strain, medium and culture conditions

The lactic acid bacteria strain used for the culture of this experiment is Leuconostoc citreum HJ-P4. The growth medium for inoculation was Lactobacilli MRS. The growth medium in the initial batch culture was prepared by adding 20 g of glucose, 10 g of yeast extract, 20 g of K ₂ HPO ₄ , 0.2 g of MgSO ₄ .7H ₂ O, MnSO ₄ .7H ₂ O 0.01 _{g, FeSO 4 · 7H 2 O} 0.01 g, CaCl 2 · was added to prepare _{2H 2 O 0.02 g, NaCl 0.01} g. In a continuous culture using a fermentation tank equipped with an internal filter, a feeding solution was prepared by adding 50 g of glucose, 25 g of yeast extract, 30 g of K ₂ HPO ₄ , MgSO ₄ .7H ₂ a _{O 0.2 g, MnSO 4 · 7H} 2 O 0.01 g, FeSO 4 · 7H 2 O 0.01 g, CaCl 2 · 2H 2 O 0.02 g, NaCl 0.01 g were used. For the production of mannitol, 100 g of fructose was further added to the feeding solution. The culture was carried out while maintaining the culture volume at 1.5 L, the temperature at 28 ° C, the pH at 6.5, and the stirring speed at 300 rpm.

2. Continuous culture process

A continuous culture process was carried out for mass culture of the strain and production of metabolites. In the continuous culture process, 1.5 L of an initial batch culture medium was put into a 2.5 L incubator to start the fermentation of Leuconostoc citreum HJ-P4, and from 6 hours after the fermentation, Was supplied into the incubator and the internal filter was operated. The culture medium in the incubator was maintained at a constant volume of 1.5 L by continuously supplying new medium as much as the medium decreased in the culture medium due to filtration of the culture medium.

3. Analysis method

The following method was used to measure the concentration of leuconostoc citreum HJ-P4 strain and the metabolites in the medium under the above conditions. The concentration of the cells was measured at 660 nm using a UV-spectrophotometer (Shimadzu 1650-PC, Japan). The analysis of fructose, mannitol, lactic acid and acetic acid was carried out by centrifuging the culture at 15,000 rpm for 10 minutes After separation, the supernatant was filtered through a membrane filter (pore size 0.45 μm) and analyzed using HPLC (YL 9100, Younglin, Inc., Korea). Fructose and mannitol were analyzed with a RI detector at Zorbax Carbohydrate Analysis column, mobile phase acetonitrile and water (75:25, v / v), flow rate 1.4 mL / min, and column temperature 35 ° C. Lactic acid and acetic acid were analyzed by a UV detector at a wavelength of 210 nm on a Zorbax SB-Aq column, mobile phase acetonitrile (1%) and 20 mM NaHPO4 (99%, pH 2) at a flow rate of 1.0 mL / min and a column temperature of 35 ° C. Glucose concentration was measured using Glucose-E kits (YD Diagnostics, Korea) using glucose oxidase-peroxidase method.

4. Change in dilution rate

Continuous culture during a step, dilution to determine the change of cell growth and metabolites in accordance with the dilution rate 0.07 h ^-1, and 0.13 h ^- were studied by changing to ^1.

Experiment result

Example 1: Culture using glucose as a carbon source

Batch culture of Leuconostoc citreum HJ-P4 strain was started in the initial culture condition, and feeding solution containing glucose as a carbon source was supplied from a time point of 6 hours after the carbon source depleted culture , The internal filter was started to operate and the new medium was continuously fed at a dilution rate of 0.07 h ^-1 as the medium was filtered and the medium was reduced in the incubator so that the culture volume in the incubator was maintained at 1.5 L for 105 hours . As a result of culturing, it was confirmed that the cell concentration was increased up to 95 hours after the continuous culture. The dissolved oxygen (DO) concentration remained almost zero after 5 hours. The maximum density (OD) of the cell was increased up to 75 and the lactic acid was constantly about 20 g / L.

Example 2: Culture using glucose and fructose as a carbon source

In the same manner as in Example 1, continuous feeding was carried out at a dilution rate of 0.07 h < ^-1 > for 105 hours by feeding a feeding solution using glucose and fructose as a complex carbon source at 6 hours after the culture with depleted carbon source. As a result, the cell concentration was increased up to 95 hours after the continuous substrate feed. The cell concentration was increased to a maximum OD value of 75, and lactic acid was constantly produced at about 40 g / L and acetic acid at about 20 g / L. Mannitol was also produced by adding fructose as a carbon source. The mannitol production increased rapidly at the early stage of culture, and the production rate decreased after 40 hours of incubation, but was constantly about 80 g / L after 60 hours.

Example 3: Change in dilution rate

The culture was continuously performed at a dilution rate of 0.13 h ^-1 for 75 hours by supplying a feeding solution using glucose and fructose as a complex carbon source at 6 hours after the culture with the carbon source depleted in the same manner as in Example 2 . As a result, the concentration of mannitol increased rapidly at the initial rapid rate, and was constantly about 75 g / L after 25 hours of incubation. The cell concentration was increased more rapidly than the dilution rate of 0.07 h ^-1 The OD value was 70 at the time. By changing the dilution rate, the production time and productivity of the cells and metabolites could be controlled, and the dilution rate available was 0.01 - 1 h ^{- 1} .

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

A method for mass production of lactic acid, acetic acid and mannitol by continuously culturing Leuconostoc citreum HJ-P4 comprising the following steps:
(a) Inoculating an initial batch culture medium into a fermentation tank equipped with an internal filter, and inoculating Leuconostoc citreum HJ-P4 thereto,
(I) one or more cylindrical ceramic filtration membranes (60); (Ii) one or a plurality of silicon tubes 70 having one end connected to the cylindrical ceramic filtration membrane 60 and the other end connected to the top cover 80; And (iii) a top cover (80) having a lower portion connected to the one or more silicon tubes (70) and an upper portion connected to the outer tube (90)
The initial batch culture medium was glucose, yeast extract, K ₂ HPO ₄ , MgSO ₄ .7H ₂ O, MnSO ₄ .7H ₂ O, FeSO ₄ .7H ₂ O, CaCl ₂ .2H ₂ O, and NaCl The method comprising the steps of:
(b) an initial batch culture of the inoculated leuconostoc citreum HJ-P4; And
(c) the fermentation tank contains glucose and fructose as carbon sources after the carbon source in the initial batch culture medium has been exhausted, and yeast extract, K ₂ HPO ₄ , MgSO ₄ .7H ₂ O, MnSO ₄ .7H ₂ O, FeSO ₄ and 7H ₂ O, CaCl ₂ and 2H ₂ O, and with a feed of for continuous culture medium containing NaCl continuously flow Pocono stock sheet reum by culturing the (Leuconostoc citreum) HJ-P4 to produce lactic acid, acetic acid and mannitol step.
delete delete delete delete delete The method according to claim 1, wherein the initial batch culture is performed at a temperature of 20-36 캜, a pH of 6-7, and a stirring speed of 200-400 rpm.
According to claim 1, wherein said continuous culture is 0.01h ^-1 - characterized in that for performing a dilution rate of 1h ^-1.

Images