JP2002085083A - Method for producing metabolic product using microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new method that permits continuously producing a metabolic product such as L-lactic acid by microorganisms. SOLUTION: This method for producing a metabolic product is characterized by the step of biologically making a raw material supplied react biologically with a living microorganism having the maximal activity at a constant density in a reaction system by the microorganisms.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The invention of this application relates to L-lactic acid,
The present invention relates to a method for producing a useful substance such as ethanol and / or bacteriocin with high efficiency by a fermentation method using a microorganism.

[0002]

2. Description of the Related Art Anaerobic bacteria, unlike aerobic bacteria, use an electron transfer system to obtain energy for metabolism and growth. Therefore, accumulation of metabolites such as organic acids and alcohols as electron acceptors. It is known to involve. In addition, the generation of CO ₂ is limited to anaerobic decarboxylation, and is fundamentally different from the generation of CO ₂ due to the complete oxidation of organic substances found in aerobic bacteria. From this feature, a biochemical reaction using an anaerobic bacterium is advantageous as an eco-friendly bioprocess because it reduces the CO ₂ load on the environment and reduces the generation of heat of reaction.

On the other hand, synthetic plastics, which are representatives of the petrochemical industry, have problems due to their robustness, and there is a need for the development of environmentally friendly plastics having biodegradability. For example, polylactic acid is a promising product developed for that purpose. However, the raw materials used for the synthesis are limited to L-lactic acid due to the selectivity of optical isomers. Lactic acid produced in petrochemicals is naturally a DL-form, so it is not a raw material for polylactic acid. In order to industrialize polylactic acid, practical development of high-purity L-lactic acid by fermentation with optical isomer selectivity is expected.

[0004] From the above viewpoints, production by L-lactic acid fermentation is considered, and the most important factor in determining the fermentation process is how to increase the productivity. For that purpose, it is necessary to develop continuous fermentation technology. Continuous fermentation is an established technique for some aerobic bacteria.
In particular, continuous fermentation technology for the purpose of cell production is based on the dilution rate (di
It has been shown that tuning the lution rate = D) forms a steady state. Methods for detecting the timing of substrate feed are also well known, including methods using dissolved oxygen and DO in the culture medium as indicators, and methods for measuring the timing by changing pH. However, despite the development of such technology,
In continuous culture, even for aerobic bacteria, growth-linked
associated), and non-growth associated fermentation such as glutamate fermentation and antibiotic production still uses batch fermentation and fed-batch culture. I have.

Since anaerobic bacteria do not require oxygen for growth, methods using immobilized cells have been studied as a means of continuous fermentation. For the ethanol-producing bacteria Zymomonas sp. And Clostridium sp. Fermented acetone-butanol, continuous fermentation-type bioreactors by the entrapment immobilization method have been actively studied, but none of them has been evaluated to be practical. There are mainly two reasons. The first is that the cost of the photo-curing resin used for immobilization is high, and the second is that some of the immobilized bacteria are inactivated in the fixed bed, so that the fermentation rate is reduced and new bacteria are used. It is also difficult to replace. The resolution of these problems has yet to be determined. In addition, continuous fermentation with anaerobic bacteria has been studied. Particularly in lactic acid fermentation, chemostat with a high cell density has a very high productivity (up to 60 g / l at a cell concentration of 100 g / L or more). (In some cases, lactic acid productivity of 1h was obtained.)
Have been reported. However, these reports do not control the concentration of bacteria, maintain the activity represented by the specific lactic acid production rate, nor control the concentration of residual sugar, which is the most important in industrialization, at all.
It is far from practical use.

[0006] Under such circumstances, the inventor of the present application has developed a continuous culture technique using homolactic bacteria that selectively produce only the L-form isolated from the natural world. L-lactic acid fermentation reduces productivity for two reasons. 1
One is product inhibition that becomes more pronounced as the concentration of lactic acid produced and accumulated increases. For this reason, when the lactic acid concentration exceeds 30 g / L, the fermentation rate drops sharply, and the fermentation is practically stopped. The second is that the inventors generated sterile cells (steril
ecell formation). In general, not only lactic acid bacteria but also anaerobic bacteria such as genus Zymomonas and genus Clostridium generally form aerobic bacteria that lose their growth ability at a constant rate even in the initial stage of growth corresponding to the logarithmic growth phase. Therefore, cultivation of anaerobic bacteria reaches a plateau at a low bacterial concentration. This also constitutes an essential reason why the productivity of fermentation by anaerobic bacteria does not increase.

[0007] Therefore, when the electrodialysis culture for reducing the product inhibition by removing the generated lactic acid outside the culture system by sequential electrodialysis was performed, the effect was confirmed.
With this method, it was not possible to take measures against inactivation of the bacteria or control the sugar concentration, and the culture was unstable and was not practical.
On the other hand, we investigated the densification of a culture system that incorporated a cross flow UF (ultrafiltration) membrane and circulated the culture solution to increase the bacterial concentration. However, with this culture method, it was impossible to control the removal of the filtrate, and stable results were not obtained.

Furthermore, most microorganisms existing in a culture environment have lactic acid racemase or metabolize and produce racemic lactic acid. Therefore, fermentation using homo-L-lactic acid bacteria that produce only L-lactic acid is possible. Even so, if contamination with various bacteria is caused, the purity of L-lactic acid immediately decreases, and it becomes difficult to produce the desired high-purity L-lactic acid. In addition, if the culture system is operated for a long time in continuous fermentation, the danger of bacterial contamination is further increased.

The invention of this application has been made in view of the above-mentioned conventional technology and the study by the inventor of this application, and is intended to continuously produce a metabolic reaction product of a microorganism such as L-lactic acid. The task is to provide a new method that enables production.

Another object of the present invention is to provide a method for efficiently producing high-purity L-lactic acid while avoiding bacterial contamination.

[0011]

Means for Solving the Problems The present invention solves the above-mentioned problems, that is, to immobilize cells for the purpose of efficiently producing metabolites such as L-lactic acid. It is possible to remove bacteria that have lost growth ability and that have weak growth ability with reduced fermentation ability from the culture system, and keep fresh and highly active bacteria at all times to produce continuously. I do.

[0012] For this purpose, the invention of this application is to produce a metabolite by allowing a microorganism having the highest activity to survive at a certain concentration and reacting biologically with a supplied raw material in a reaction system using the microorganism. And a method for producing a metabolite using a microorganism.

One aspect of the method of the present invention is to maintain the maximum activity of a microorganism by controlling the pH value within a predetermined upper limit and lower limit, and controlling the turbidity constant in a reaction system. More specifically, the difference between the upper limit value and the lower limit value of pH is set to 0.1 or less.

Another embodiment of the method of the present invention is to elute inactive bacteria from the reaction system. In yet another embodiment of the method of the present invention, the microorganism is a homozygous L-lactic acid fermenter and the metabolite of the microorganism is L-lactic acid.

[0015] In still another embodiment of the method of the present invention, the microorganism is an alcohol-producing bacterium and the metabolite of the microorganism is an alcohol. In still another embodiment of the method of the present invention, wherein the microorganism is a bacteriocin-producing homozygous L-lactic acid-fermenting bacterium, and the metabolite of the microorganism is L-lactic acid and / or bacteriocin. In a preferred embodiment, the bacteriocin is specifically nisin A or nisin Z. When the bacteriocin is nisin A, the microorganism is Lactococcus lactis NCDO497, L
actococcus lactis NCDO2111, Lactococcus lactis NIZ
O R5, Lactococcus lactis INRA1, Lactococcus lactis
INRA2, Lactococcus lactis INRA3, Lactococcus lact
is INRA4, Lactococcus lactisINRA5, Lactococcus lac
tis INRA6, Lactococcus lactis NP4G, Lactococcus la
ctis NZI, Lactococcus lactis SI, or Lactococcus
Lactococcus lactis IO-1 (JCM7638), Lactobacco lactis IO-1 (JCM7638)
ctococcus lactis subsp.lactis A. Ishizaki Chizuka
(JCM 11180), Lactococcus lactis subsp.lactis A. I
shizaki Yasaka 5B (JCM 11181), Lactococcus lactis
subsp.lactis A. Ishizaki Yasaka 7B (JCM 11181), La
ctococcuslactis subsp.lactis A. Ishizaki Yasaka 8B
(JCM 11181), Lactococcus lactis subsp.lactis A. I
shizaki Yasaka 9B (JCM 11181), Lactococcus lactis
NIZO22186, Lactococcus lactis NIZO N9, Lactococcus
lactis ATCC 7962, Lactococcus lactis NCDO2597, La
ctococcus lactis NCDO2091, Lactococcus lactis NCK40
0, Lactococcus lactis LJN80, Lactococcus lactis IL
C11, Lactococcus lactis ILC19, Lactococcus lactis I
LC126, Lactococcus lactis ILCSL5, or Lactococcu
s lactis ILCSL20 is a preferred embodiment.

In other words, the inventor of the present application uses the above method, in particular, two-point control of the upper limit (upper limit for substrate feed) and the lower limit (lower limit for alkaline feed), and sets the set interval between the two points as much as possible. The inventors have found that a small control method prevents the inactivation of bacteria, activates the growth, and produces a great effect of promoting the rate of sugar consumption, thereby completing the present invention. Note that the same result may be obtained when the offset width is the same as the control width of the two points in the one-point control, and such a case is also included in the two-point control.

In fact, according to the invention of this application, DDC using a computer provided Lactococcus lactis IO
In the culture of -1 (JCM7638), the results show that no wash-out occurs even at a dilution rate D = 1.1 h-1 which is almost the same as the maximum specific growth rate μmax = 1.25 h-1 of this bacterium in a glucose medium. By culturing at such a high dilution rate, the concentration of lactic acid in the culture system decreases due to the dilution effect, and an effect not inferior to the inhibition reducing effect of electrodialysis culture is obtained. pH-de
In addition to the pendent method, in order to industrially control the residual sugar concentration, it is necessary to use a laser turbidity meter for pH dependent feed.
It is more effective to combine turbidostat with eed back control. This allows for high dilu
A high specific sugar consumption rate and a specific lactic acid production rate can be maintained while maintaining the
High efficiency continuous fermentation is possible, and the residual sugar concentration can be stably maintained at a low level.

Further, in the production method of the present invention, as described in the above embodiment, fermentation of L-lactic acid while accumulating bacteriocin (antimicrobial peptide) using bacteriocin-producing homozygous L-lactic acid fermenting bacteria. Do. The accumulation of high concentrations of bacteriocin kills bacteria entering the culture system,
High-purity L-lactic acid can be produced without lowering the purity of L-lactic acid. In fact, Lactococcus l, a homo L-lactic acid bacterium that produces bacteriocin (Nisin Z),
In the culture system using actis IO-1 (JCM7638) or the like, I-Nisin Z is accumulated at a concentration 3 to 5 times that of the batch method, and no bacterial contamination occurs over a long period of time. -It is possible to produce lactic acid in large quantities. Furthermore, a culture system using such a bacteriocin-producing homozygous L-lactic acid fermentation bacterium is also effective for mass production of bacteriocin.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION In the invention of this application, the reaction of producing a metabolite by a microorganism may be various,
For example, if the metabolites are alcohols, L-lactic acid and / or
Alternatively, various substances such as bacteriocin may be used.

The production of L-lactic acid can be further explained as follows. Bacteria that produce L-lactic acid from glucose with homo L-lactic acid bacteria at a conversion rate of almost 100% shall be cultured. PH electrode, on-lin
e Set up a turbidity measurement device so that pH and OD can be accurately measured online. For example, a laser turbidity meter is the most excellent turbidity measuring device. In the pH control method, substrate feed is performed at the upper limit of pH and alkali feed is performed at the lower limit of pH.
Water for controlling turbidity and a substrate feed solution containing sugar as a main component for replenishing the substrate are used. In addition, a hollow fiber type ultrafiltration membrane of a fermenter external circulation type is installed to concentrate the cells. A pump for transporting these liquids and controlling the flow rate is required. The entire apparatus configuration can be exemplified as, for example, FIG. The reference numerals in the figure indicate the following.

[0021]

[Table 1]

For example, the following table

[0023]

[Table 2]

As can be seen in L-lactic acid continuous fermentation of Lactococcus lactis IO-1 (JMC7368) according to the method of the invention of this application, the bacterium was isolated between D = 0.5 h-1 and D = 1.1 h-1. Regeneration rate is about 5%, and despite operating at such a high dilution rate,
The actual specific growth rate μ of the bacteria shows a low value. The establishment of such a special culture system was first found and established in the world. From this, this culture system has a low bacterial regeneration rate despite the immobilization of bacteria,
This indicates that a culture process that is substantially the same as the immobilized cell method has been formed. That is, the method of the present invention is a continuous fermentation method in which cells are regenerated only by the amount of inactivation and cells with extremely high activity are permanently used without being immobilized. Has become the most rational process of anaerobic cultivation.

Thus, the invention of this application is provided as a new method for culturing anaerobic bacteria, which is an alternative to the globally immobilized immobilization method. For example, when producing L-lactic acid, the production method of the present invention can be carried out using a known strain belonging to the genus Lactococcus lactis. When producing alcohols, ethanol-producing bacteria Zymo
It can be carried out using known strains such as monas spp. and acetone / butanol fermentation bacteria Clostridium sp.

Furthermore, the method of the present invention can produce high-purity L-lactic acid for a long time by accumulating a high concentration of bacteriocin by using a bacteriocin-producing homozygous L-lactic acid fermenting bacterium. Make it possible.

The bacteriocin-producing homozygous L-lactic acid-fermenting bacteria are nisin A-producing bacteria belonging to Lactococcus lactis, NCDO497, NCDO2111, NIZO R5, INRA1, INRA2, INRA.
3, INRA4, INRA5, INRA6, NP4G, NZI, SI or ILC13 can be used. As bacteriocin Z-producing bacteria, Lactococcus lactis IO-1 (JCM7638), Lactococcu
slactis subsp.lactis A. Ishizaki Chizuka (JCM 1118
0), Lactococcus lactissubsp.lactis A. Ishizaki Yas
aka 5B (JCM 11181), Lactococcus lactis subsp.lacti
s A. Ishizaki Yasaka 7B (JCM 11181), Lactococcus l
actis subsp.lactis A. Ishizaki Yasaka 8B (JCM 1118
1), Lactococcus lactis subsp.lactis A. Ishizaki Ya
saka 9B (JCM 11181), and NIZO22186, NIZO N9, ATCC 7962, NCDO2597, NCDO2 belonging to Lactococcus lactis
091, NCK400, LJN80, ILC11, ILC19, ILC126, ILCSL5 or ILCSL20 can be used.

Among these strains, those with a JCM number in parentheses are strains independently isolated by the inventors of the present application, and are available in a separable state from the “RIKEN Microbial Strain Storage Facility”. Has been deposited. Also, Lactococcu
It is widely accepted in the art that s lactis NCDO497 is a nisin A producing bacterium, and there are many prior art documents. The other strains are nisin A and nisin Z-producing bacteria, respectively, according to the literature (J. Appl. Envi.
ron. Mictrobiol. 59 (1): 214, 1993). These known strains are available from the authors of the literature, respectively, and are also available from their respective depository institutions (NCDO and ATCC).
Etc.).

The following examples illustrate the method of the present invention in more detail and specifically, but the present invention is not limited to the following examples.

[0030]

Example 1 The bacterium used was Lactococcus lactis I, which was isolated by the inventor.
O-1 (JCM7638) was used. The inoculum used was a cryopreserved bacterium at −85 ° C. inoculated in a TGC medium, and the culture was allowed to stand. This is made up of glucose 3%, yeast extract 0.5%, polypeptone 0.5
% And NaCl 1% in 100 ml of medium, dispensed into Erlenmyer Flask, inoculated in autoclave sterilized at 120 ° C for 5 minutes,
After culturing for a time, the seed was used. The culture apparatus used for lactic acid fermentation is as shown in FIG. The main fermenter is a glass jar with a total volume of 1 liter. A magnetic stirrer stirrer is installed inside the jar, and the mixture is slowly stirred at about 400 rpm. The entire jar is immersed in a water bath, and the temperature is controlled by circulating hot water at 37 ° C. A composite glass electrode for pH measurement was attached to the jar, measured with a pH meter (manufactured by Toa Denpa), the measured value was transferred to a computer by RS232, and two points of upper limit and lower limit of pH were controlled by a self-made program. In continuous operation, the substrate field is performed at the upper pH limit, and the alkali (NaOH) field is performed at the lower pH limit. In addition, a process online turbidity meter probe was installed in the jar, and the output was input to a DDC controller (Model LA-300 ASR) to construct a turbidity control system for the jar.
Extract the culture from the jar and circulate the culture using a cross flow ultrafiltration membrane (MICROZA PSP103, Asahi Kasei)
While concentrating the microorganisms, the bacteria-free solution is extracted from the outside of the filtration membrane, and the lactic acid solution is extracted. On the other hand, as a turbidity control system, sterilized water is fed and a culture solution (bacterial solution) is extracted. At this time, the supply of water and the extraction of the culture solution were synchronized by a peristatic pump.

In the ligation culture, three kinds of liquids are supplied, and two kinds of liquids are extracted. That is, a feed solution containing a saccharide as a substrate is added at an upper pH limit, and an alkali solution (1 M-NaOH) for pH control is added at a lower pH limit. At this time, when both the feed solution and the alkaline solution are supplied to the jar, the disinfecting solution is withdrawn from the outside of the ultrafiltration membrane using a peristatic pump, and the amount of the jar solution is kept constant. As described above, water is supplied and the culture solution is extracted for turbidity control. A peristatic pump is also used to keep the volume of the jar constant.

The culture was performed with glucose 5%, yeast extract 1%,
The culture is started by inoculating 400 ml of a 1% polypeptone composition with 20 ml of a seed and feeding alkaline to maintain pH 6.0. Approximately 12 hours later, when the residual sugar concentration of the medium becomes almost zero, the continuous substrate feed (feed solution) with upper and lower two-point control (alkaline feed at the upper limit of pH 6.1 and substrate feed at the lower limit of pH 6.0) Is 35 g of glucose
/ L, yeast extract 1.0%, polypeptone 1.0%) at the same time as circulating the culture to increase the cell concentration.
When the cell concentration becomes constant by turbidity control, a steady state in which the residual sugar and lactic acid concentrations are kept constant is formed. FIG. 2 illustrates the results when the lactic acid concentration was kept constant at 25 g / L. Dilution rate at a cell concentration of 5 g / L
When a steady state is formed at 0.5 h ^-1 , lactic acid productivity is 13.5 g / 1
h, The effluent sugar concentration (residual sugar concentration) was 0.25 g / L. At this time, the cell extraction amount was 0.25 g / h at 5% of the total bacterial amount. The specific lactic acid production rate, which indicates the lactic acid fermentation activity of the bacteria, is 3.3
g / gh. The optical purity of the obtained L-lactic acid was 99.8%.

In the continuous fermentation, the culture solution is aseptically sampled every 12 hours, diluted with sterile water in several steps, and 100 μl of the diluted solution is added to a complete medium containing glucose (CMG).
Were developed and cultured at 37 ° C. for 24 hours, and the growing colonies were monitored. As a result, no bacterial contamination was monitored. Furthermore, the nisin Z concentration of the culture solution in the continuous fermentation state was 5 times as high as the nisin Z concentration of 3,500 AU / ml when the same bacteria were batch-cultured in the same medium. Example 2 The strain used was Lactococcus lactis IO-1 as in Example 1.
In (JCM7638), the refreshing of the stock and the preparation of the seed are as in Example 1. The culturing apparatus is the same as in Example 1 and is as shown in FIG.

The glucose used for the culture medium was a saccharified starch saccharified solution. 300 g of sago palm starch (obtained from Sarawak University, Malaysia) in terms of dry matter was suspended in 1 liter of water. After adjusting to pH 6.5, heat-resistant amylase derived from Bacillus subtilis (liquefied enzyme Klaistase T5 Daiwa Kasei,
(5,000 u / g) was added at 0.2% (v / v) and gelatinized by enzymatic reaction at 95 ° C. for 2 hours, followed by heat treatment at 130 ° C. for 10 minutes to inactivate the enzyme. 0.1% (v / v) of Klaistase T5 was added, and the mixture was again treated at 95 ° C. for 1 hour to complete the liquefaction reaction. After cooling, the pH was adjusted to 5.5, glucoamylase derived from Rhizopus delemar (saccharifying enzyme Glucozyme Amano Pharmaceutical, 4,200 U / g) was added at 8 U per 1 g of starch, and saccharification was carried out at 50 ° C. for 24 hours. After the saccharification reaction, the glucose obtained by a Glucose analyzer was determined. As a result, 1.05 g of glucose was obtained from 1 g of the dry starch.

In Example 2, the medium up to seed culture is exactly the same as in Example 1. The main fermentation medium is
The sago palm starch saccharified liquid 50 g was weighed in terms of glucose, and the soybean flake acid hydrolyzate 0.5% (v / v),
Natural rubber latex separated mother liquor dry powder
Serum powder: NRSP) was added to a concentration of 0.5% (v / v) to make 1 liter and used after autoclaving. The substrate feed solution was also adjusted so as to be 50 g / L in terms of glucose of sago palm starch saccharified solution. In the same manner as in Example 1, the seeds were added, and the culture was started by batch culture. When the residual sugar concentration became almost zero, the culture solution was recycled, the cell concentration was controlled by controlling turbidity, and the pH was controlled at two points (pH 6). Continuous feed with an alkaline feed at the upper limit of 0.1 and a substrate feed at the lower limit of pH 6.0 (sago palm starch saccharified liquid glucose 50 g / L, soybean flake acid hydrolyzate 0.5% (v / v), NRSP 0.5 %). Bacteria concentration 10 g
/ L, reached steady state at a dilution rate of 0.75h- ¹ , lactic acid productivity at that time was 31.5 g / Lh, and residual sugar concentration in the effluent was 0.3 g
/ L. Cell extraction volume is 0.35 g / h at 3% of the total bacterial mass
Met. The specific lactic acid production rate showing the lactic acid fermentation activity of the bacteria was 3.15 g / gh. The optical purity of the obtained L-lactic acid is 9
It was 9.8%.

FIG. 3 is a diagram illustrating the relationship between the dilution ratio of continuous production of L-lactic acid and the concentration of lactic acid in the reactor when the cell concentration is constant at 10 g / L. A germ contamination test was performed in the same manner as in Example 1, but no germ contamination was observed over the entire culture period. Furthermore, the nisin Z concentration of the culture solution in the continuous fermentation state was about 3.5 times that of the batch culture method. Example 3 The strain used was Lactococcus lactis IO-1 as in Example 1.
In (JCM7638), the refreshing of the stock, preparation of seed, and culturing apparatus (FIG. 1) were the same as those in Example 1.

The glucose used for the medium is corn starch (c
orn starch) An enzyme saccharified solution was used. 300 g of corn starch in terms of dry matter was suspended in 1 liter of water. After adjusting to pH 6.5, 0.1 g of thermostable amylase derived from Bacillus licheniformis (liquefaction enzyme Thermomyl Novo Nordisk, 120 KNU / g) was added.
% (V / v), enzymatically reacted at 90 ° C. for 2 hours to gelatinize, and then heat-treated at 130 ° C. for 10 minutes to inactivate the enzyme. After cooling p
Adjusted to 5.5, a mixed enzyme of glucoamylase from Aspergillusniger and pullulanase from Bacillus acidpullulyticum (Dextroyme Nove Nordisk, 225AGU
/ g) was added at 4 U per 1 g of starch, and saccharification was carried out at 50 ° C for 24 hours. After the saccharification reaction, glucose obtained by a Glucose analyzer was determined, and 1.05 g of glucose was obtained from 1 g of dry starch.

In the third embodiment, the medium up to seed culture is exactly the same as in the first embodiment. The main fermentation medium is
The above corn starch saccharified solution was weighed in an amount of 50 g in terms of glucose, and powdered CSL (corn steep liquor, manufactured by Shono Starch) was added to 1% to make 1 liter, and the mixture was sterilized by autoclave. The substrate feed solution was also adjusted so as to be 35 g / L in corn starch saccharified solution glucose equivalent. In the same manner as in Example 1, the seed was added, and the culture was started by batch culture. When the residual sugar concentration was almost zero, the culture solution was recycled, the cell concentration was controlled by controlling the turbidity, and the pH was controlled at two points (pH 2). Continuous substrate feed (glucose of corn starch saccharified solution) with alkaline feed at the upper limit of 6.1 and substrate feed at the lower limit of pH 6.0
50 g / L, CSL 1%). Bacteria concentration 12 g / L, dilution
A steady state is reached at a rate of 1.1 / h, and the lactic acid productivity at that time is
The residual sugar concentration in the effluent was 36 g / Lh, and the residual sugar concentration was 2.5 g / L. The cell extraction amount was 0.2 g / h at 2% of the total bacterial amount. The specific lactic acid production rate showing the lactic acid fermentation activity of the bacteria was 3.0 g / gh. The optical purity of the obtained L-lactic acid was 99.8%.

In addition, the same bacterial contamination test as in Example 1 was conducted, but no bacterial contamination was observed over the entire culture period. Furthermore, the nisin Z concentration of the culture solution in the continuous fermentation state was about 3.5 times that of the batch culture method. Example 4 The used bacterium was Zymomonas mobilis NRRLB-14023.
The inoculum used was a cryopreserved bacterium at −85 ° C. inoculated in a YM medium, and the culture was allowed to stand. 100 g of glucose, yeast extract 1
0 g, KH ₂ PO ₄ 1 g, (NH ₄ ) ₂ SO ₄ 1 g, Mg (SO ₄ ) ・ 7H ₂ O 0.
100 ml of a medium prepared by dissolving 5 g in 1 L of deionized water
The seeds were dispensed into Flasks, inoculated into autoclave sterilized at 120 ° C for 5 minutes, and cultured for about 8 hours. The culture device used for ethanol fermentation is the same as that shown in FIG. That is, the main fermenter is a glass jar having a total volume of about 1 liter, and is equipped with a stirrer with an internal magnetic stirrer and is slowly stirred at a rotation speed of about 400 rpm. The entire jar is immersed in a water bath, and the temperature is controlled by circulating hot water at 37 ° C. Attach a composite glass electrode for pH measurement to the jar and measure with a pH meter (manufactured by Toa Denpa).
The data was transferred to the computer in step 2, and two-point control of the upper limit and lower limit of pH was performed by a self-made program. When the continuous operation is started, the substrate is fed at the upper pH limit and the alkali (NaOH) feed is performed at the lower pH limit. Also, a process online turbidity probe is installed in the jar, and the output is DDC cont.
A system to control the turbidity of the jar by inputting to the roller (Model LA-300 ASR) was constructed. Extract the culture solution from the jar and use a cross flow type ultrafiltration membrane (MICROZA PSP1
03, Asahi Kasei), circulate the culture solution, concentrate the microorganisms, extract the disinfecting solution from the outside of the filtration membrane, and remove the lactic acid solution. On the other hand, as a turbidity control system, acidified water is fed, and a culture solution (bacteria-containing solution) is extracted. At this time, the supply of water and the extraction of the culture solution were synchronized by a peristatic pump.

In the ligation culture, three kinds of liquids are supplied, and two kinds of liquids are extracted. That is, a feed solution containing a saccharide as a substrate is added at an upper pH limit, and an alkali solution (1 M-NaOH) for pH control is added at a lower pH limit. At this time, when both the feed solution and the alkaline solution are supplied to the jar, the disinfecting solution is withdrawn from the outside of the ultrafiltration membrane using a peristatic pump, and the amount of the jar solution is kept constant. As described above, water is supplied and the culture solution is extracted for turbidity control. A peristatic pump is also used to keep the volume of the jar constant.

The main fermentation culture has the same composition as the seed, ie
Glucose 10%, Yeast extract 1%, KH_TwoPO_Four 0.1%, (N
H_Four)_TwoSO_Four 0.1%, Mg (SO_Four) ・ 7H_TwoO in 400 ml of 0.05 g medium
Inoculate 20 ml of seed to maintain pH 5.5
Start with a feed culture. After about 8 hours,
Two-point upper and lower limit control when the residual sugar concentration becomes almost 0
(Alkaline feed at pH 5.55 upper limit, pH 5.50 lower limit
Continuous substrate feed (feed solution)
Is glucose 35 g, yeast extract 10 g, KH_TwoPO _Four 1
g, (NH_Four)_TwoSO_Four 1g, Mg (SO_Four) ・ 7H_TwoO 0.05 g)
At the same time, the culture is circulated to increase the cell concentration.
When the cell concentration becomes constant by turbidity control, the residual sugar and lactic acid
A steady state is formed that will be maintained constant.

Cell concentration 7.5 g / L, dilution rate 0.7h ^-1
At a steady state with an ethanol production rate of 26.25 g / hL,
The effluent sugar concentration (residual sugar concentration) was 0.25 g / L. At this time, the cell extraction amount was 0.26 g / h at 5% of the total bacterial amount. Example 5 The strain used was a homo-L-lactic acid fermentation bacterium and nisin
Lactococcus lactis NCDO497, which is an A-producing bacterium. The stock is refreshed and the seed is prepared as in Example 1. The culturing apparatus is the same as in Example 1 and is as shown in FIG.

The glucose used for the medium was a saccharified sago palm starch saccharified solution. 300 g of sago palm starch (obtained from Sarawak University, Malaysia) in terms of dry matter was suspended in 1 liter of water. After adjusting to pH 6.5, heat-resistant amylase derived from Bacillus subtilis (liquefied enzyme Klaistase T5 Daiwa Kasei,
(5,000 u / g) was added at 0.2% (v / v) and gelatinized by enzymatic reaction at 95 ° C. for 2 hours, followed by heat treatment at 130 ° C. for 10 minutes to inactivate the enzyme. 0.1% (v / v) of Klaistase T5 was added, and the mixture was again treated at 95 ° C. for 1 hour to complete the liquefaction reaction. After cooling, the pH was adjusted to 5.5, glucoamylase derived from Rhizopus delemar (saccharifying enzyme Glucozyme Amano Pharmaceutical, 4,200 U / g) was added at 8 U per 1 g of starch, and saccharification was carried out at 50 ° C. for 24 hours. After the saccharification reaction, the glucose obtained by a Glucose analyzer was determined. As a result, 1.05 g of glucose was obtained from 1 g of the dry starch.

In this Example 5, the medium up to seed culture is exactly the same as in Example 1. The main fermentation medium is
The sago palm starch saccharified liquid 50 g was weighed in terms of glucose, and the soybean flake acid hydrolyzate 0.5% (v / v),
Natural rubber latex separated mother liquor dry powder
Serum powder: NRSP) was added to a concentration of 0.5% (v / v) to make 1 liter and used after autoclaving. The substrate feed solution was also adjusted to be 53 g / L in terms of glucose of sago palm starch saccharified solution. In the same manner as in Example 1, the seeds were added, and the culture was started by batch culture. When the residual sugar concentration became almost zero, the culture solution was recycled, the cell concentration was controlled by controlling turbidity, and the pH was controlled at two points (pH 6). Substrate feed (substrate feed solution composition: sago palm starch saccharified solution glucose 53 g / L, with alkaline feed at the upper limit of .1 and substrate feed at the lower limit of pH 6.0)
Soybean flake acid hydrolyzate 5 ml / L, NRSP 7 ml / L). Steady state was reached at a bacterial concentration of 7 g / L and a dilution rate of 0.8 L / h, and continuous fermentation could be continued stably for 215 hours thereafter. The obtained L-lactic acid was 4.13 kg, and the optical purity was 99.9.
%Met.

In addition, a germ contamination test was conducted in the same manner as in Example 1, but no germ contamination was observed over the entire culture period. Furthermore, the nisin A concentration of the culture solution in the continuous fermentation state was determined by the nisin A concentration when this bacterium was batch-cultured in the same medium.
It had about four times the activity of the concentration. Example 6 The strain used was Lact, which was independently isolated by the inventors of this application.
ococcus lactis subsp.lactis A. Ishizaki Chizuka
(JCM11180). This strain is a homo-L-lactic acid fermentation bacterium and a Nishi Z-producing bacterium. Refresh of the stock, preparation of seed, and culture apparatus (FIG. 1) were the same as in Example 1.

The glucose used for the medium is corn starch (c
orn starch) An enzyme saccharified solution was used. 300 g of corn starch in terms of dry matter was suspended in 1 liter of water. After adjusting to pH 6.5, 0.1 g of thermostable amylase derived from Bacillus licheniformis (liquefaction enzyme Thermomyl Novo Nordisk, 120 KNU / g) was added.
% (V / v), enzymatically reacted at 90 ° C. for 2 hours to gelatinize, and then heat-treated at 130 ° C. for 10 minutes to inactivate the enzyme. After cooling p
Adjusted to 5.5, a mixed enzyme of glucoamylase from Aspergillusniger and pullulanase from Bacillus acidpullulyticum (Dextroyme Nove Nordisk, 225AGU
/ g) was added at 4 U per 1 g of starch, and saccharification was carried out at 50 ° C for 24 hours. After the saccharification reaction, glucose obtained by a Glucose analyzer was determined, and 1.05 g of glucose was obtained from 1 g of dry starch.

In this Example 6, the medium up to seed culture is exactly the same as in Example 1. The main fermentation medium is
The above corn starch saccharified solution was weighed in an amount of 50 g in terms of glucose, and powdered CSL (corn steep liquor, manufactured by Shono Starch) was added to a concentration of 1% to make 1 liter, and the mixture was subjected to autoclave sterilization. The substrate feed solution was also adjusted so as to be 35 g / L in terms of corn starch saccharified solution glucose. In the same manner as in Example 1, the seed was added, and the culture was started by batch culture. When the residual sugar concentration was almost zero, the culture solution was recycled, the cell concentration was controlled by controlling the turbidity, and the pH was controlled at two points (pH 2). Continuous substrate feed (glucose of corn starch saccharified solution) with alkaline feed at the upper limit of 6.1 and substrate feed at the lower limit of pH 6.0
53 g / L, CSL 1 g / L). Bacteria concentration 6 g / L, dilution
A steady state was reached at a rate of 0.7 L / h, and continuous culture was continued for 150 hours. The obtained L-lactic acid was 2.15 kg, and the optical purity was 99.8%
Met.

In addition, a germ contamination test was carried out in the same manner as in Example 1, but no germ contamination was observed over the entire culture period. Furthermore, the nisin Z concentration of the culture solution in the continuous fermentation state reached about three times that of the batch culture method.

[0049]

According to the method of the present invention, microorganisms that do not immobilize cells can be used permanently, and continuous production with a (surprising) high dilution rate of 1.0 h ^-1 or more can be achieved. As a result, lactic acid in the reaction system becomes low and no inhibition by lactic acid occurs, so that high productivity can be maintained. In addition, since the bacterium has a high activity, the sugar in the reaction system is well pumped, and the residual sugar concentration is kept low, so that waste of raw materials can be reduced. As a result, high productivity of 30 g / Lh or more of L-lactic acid can be obtained. This is 100 Kl
This indicates that it is possible to produce 20,000 tons of L-lactic acid annually in the above reaction tank, and the productivity is about 20 times that of L-glutamic acid fermentation, which is a representative of industrial fermentation boasting high productivity.
If alcohol-producing bacteria are cultured, ethanol and the like can be produced continuously and in large quantities.

Furthermore, by using bacteriocin-producing L-lactic acid bacteria such as nisin A and Z in the culture system of the present invention, it is possible to accumulate bacteriocin in a concentration 3 to 5 times that of batch culture. Not only large-scale cultivation is possible, but also high-purity L-lactic acid can be produced while avoiding various bacterial contamination.

[Brief description of the drawings]

FIG. 1 is a configuration diagram illustrating an apparatus for the present invention.

FIG. 2 is a diagram showing a relationship between a dilution ratio and L-lactic acid productivity when L-lactic acid is continuously produced in a reactor having a constant lactic acid concentration.

FIG. 3 is a graph showing the relationship between the dilution ratio of continuous production of L-lactic acid and the concentration of lactic acid in a reactor at a cell concentration of 10 g / L.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) // (C12N 1/20 (C12N 1/20 A C12R 1:01) C12R 1:01) F term (Reference) 4B064 AC03 AD33 AG01 CA02 CC01 CC06 CC07 CC09 CC10 CC15 CC30 CD09 CD20 CD22 DA01 DA10 4B065 AA01X AC15 BB15 BB24 BB27 BB29 BC02 BC03 BC09 BC11 BC13 BC17 BC50 CA06 CA10 CA24 CA34 CA41 CA43 CA44 CA60

Claims (11)

[Claims] In a reaction system using microorganisms, a microorganism characterized by producing a metabolite by causing a microorganism having the highest activity to survive at a certain concentration and biologically reacting with a supplied raw material. Method of producing metabolites. 2. The method according to claim 1, wherein in the reaction system, the maximum activity of the microorganism is maintained by controlling the pH value within a range between a predetermined upper limit and a lower limit, and controlling the turbidity constant. . 3. The method according to claim 2, wherein the difference between the upper limit value and the lower limit value of the pH is 0.1 or less. 4. The production method according to claim 1, wherein inactive bacteria are discharged from the reaction system. 5. The method according to claim 1, wherein the microorganism is a homozygous L-lactic acid fermentation bacterium, and the metabolite of the microorganism is L-lactic acid. 6. The microorganism according to claim 1, wherein the microorganism is an alcohol-producing bacterium and the metabolite of the microorganism is an alcohol.
Manufacturing method. 7. The microorganism is a bacteriocin-producing homozygous L-lactic acid-fermenting bacterium, and the metabolite of the microorganism is L-lactic acid and / or L-lactic acid.
5. The method according to claim 1, wherein the method is bacteriocin. 8. The method according to claim 7, wherein the bacteriocin is nisin A. 9. The method according to claim 7, wherein the bacteriocin is nisin Z. 10. The bacteriocin-producing homologous L-lactic acid fermenting bacterium may be Lactococcus lactis NCDO497, Lactococcus lacti.
s NCDO2111, Lactococcus lactis NIZO R5, Lactococcu
s lactis INRA1, Lactococcus lactis INRA2, Lactococ
cus lactisINRA3, Lactococcus lactis INRA4, Lactoco
ccus lactis INRA5, Lactococcus lactis INRA6, Lactoc
occus lactis NP4G, Lactococcus lactis NZI, Lactoco
9. The method according to claim 8, which is ccus lactis SI or Lactococcus lactis ILC13. 11. A bacteriocin-producing homologous L-lactic acid-fermenting bacterium, comprising Lactococcus lactis IO-1 (JCM7638), Lactococc
us lactis subsp.lactis A. Ishizaki Chizuka (JCM 11
180), Lactococcus lactis subsp.lactis A. Ishizaki
Yasaka 5B (JCM 11181), Lactococcus lactis subsp.la
ctis A. Ishizaki Yasaka 7B (JCM11181), Lactococcus
lactis subsp.lactis A. Ishizaki Yasaka 8B (JCM 11
181), Lactococcus lactis subsp.lactis A. Ishizaki
Yasaka 9B (JCM 11181), Lactococcus lactis NIZO2218
6, Lactococcus lactis NIZO N9, Lactococcus lactis
ATCC 7962, Lactococcus lactis NCDO2597, Lactococcu
s lactis NCDO2091, Lactococcus lactis NCK400, Lact
ococcus lactis LJN80, Lactococcus lactis ILC11, Lac
tococcus lactis ILC19, Lactococcus lactis ILC126,
Lactococcus lactis ILCSL5 or Lactococcus lactis
The production method according to claim 9, which is ILCSL20.