JPH09173792A - Treatment of fermented liquid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve the practical membrane permeation rate by subjecting a fermented liquid which is prepared by culturing microorganisms belonging to the Esherichia genus, to heat treatment at specified pH and temp. and thereafter, filtering the treated liquid with a membrane. SOLUTION: In this treatment, a fermented liquid prepared by curing microorganisms belonging to the Esherichia genus is subjected to heat treatment at a pH of 1 to 4.5 and at 110 to 200 deg.C. At that time, the microorganism cells themselves or impurities derived from the microorganism cells in the fermented liquid are aggregated or decomposed to effect the reduction in cake resistance at the time of filtering the treated liquid with a membrane or the prevention of formation of concn. polarization. Thereafter, the resulting liquid is subjected to membrane filtration to remove the microorganisms with the membrane at an improved permeation rate. Thus, this treatment can efficiently be performed without requiring any excessive auxiliary equipment.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for pretreating a membrane-occluding substance to remarkably improve the permeation rate at the time of sterilizing a membrane, shortening the treatment time, and minimizing the equipment.

[0002]

2. Description of the Related Art In recent years, fermentative production technology using genetically modified microorganisms has advanced, and Escherichia coli (hereinafter abbreviated as E. coli), which is easy to genetically manipulate, is often used for this purpose. It's being used. At the end of the fermentation, if the desired product is outside the cells, it is usually sterilized and then sterilized by membrane separation or centrifugation, and the obtained cell-free solution is subjected to the next treatment step. Also,
When the desired product is present in the cells, the cells are disrupted by freezing, using a homogenizer, a mill or the like, and then the cells or the disrupted cells are removed by membrane separation or centrifugation. A microfiltration membrane (hereinafter abbreviated as MF), an ultrafiltration membrane (hereinafter abbreviated as UF), and the like are generally used as the membrane used for cell separation. Membrane separation can completely remove the cells and a clearer sterilized liquid can be obtained compared to centrifugation. Therefore, culturing a microorganism belonging to the genus Brevibacterium or the genus Corynebacterium A membrane is used to separate the cells of the obtained fermentation broth. However, a fermentation broth obtained by culturing E. coli (hereinafter,
Abbreviated as E. coli fermentation broth. It was found that it is not practical to use a membrane for cell separation, because the membrane permeation rate becomes extremely low regardless of the presence or absence of cell crushing when (2) is subjected to membrane filtration by an ordinary method.

On the other hand, several methods are known for improving the permeation rate of the fermentation broth and pretreatment of the fermentation broth during membrane filtration. For example, Japanese Patent Laid-Open Publication No. Sho 57-91196
No. 5, inosine or guanosine fermentation broth at pH 5.5-9.0.
It is described that the heat treatment at 90 to 110 ° C. at 90 ° C. improves the membrane permeation rate when separating bacterial cells and polymer substances with an ultrafiltration membrane. In addition, Japanese Patent Laid-Open Publication No. 60-78588 describes that heat treatment of an amino acid fermentation liquid at 50 to 100 ° C. improves the ultrafiltration membrane permeation rate. In addition, Japanese Patent Publication
59-14795 describes that the rejection rate of impurities in the ultrafiltration treatment operation can be improved by adjusting the pH of the amino acid fermentation liquor using cane molasses as a fermentation raw material to pH 2-5.

However, as already mentioned, E. col
i When separating bacterial cells from the fermentation broth using a membrane, the membrane permeation rate becomes extremely low, which is not practical. Further, even if the method disclosed in the above patent publication is applied, it is not effective in improving the permeation rate during membrane filtration, and since the permeation rate is still low, there is a problem that the membrane filtration equipment becomes excessive. Has become.

[0005]

Therefore, it is desired to develop a practical method for improving the membrane permeation rate in membrane filtration of a fermentation broth obtained by culturing a microorganism belonging to the genus Escherichia.

[0006]

Means for Solving the Problems As a pretreatment before carrying out membrane filtration, the present inventors have prepared a fermentation solution obtained by culturing a microorganism belonging to the genus Escherichia after adjusting it to a specific pH range, It was found that when heated at a high temperature, the cells themselves or impurities derived from the cells in the fermentation broth aggregate or decompose, leading to a reduction in cake resistance during membrane filtration or prevention of concentration polarization formation. Achieved an improvement in the permeation rate during bacteria. That is, the present invention is a method for membrane filtration of a fermentation broth obtainable by culturing a microorganism belonging to the genus Escherichia, in a method for separating bacterial cells, the fermentation broth having a pH of 1 to
It is a method for treating a fermented liquor, characterized by performing membrane filtration after heat treatment at 4.5 to 110 to 200 ° C.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The microorganism belonging to the genus Escherichia used in the present invention may be either a wild strain or a mutant strain, and a recombinant strain derived by a genetic technique such as cell fusion or genetic manipulation is also used. be able to. The target product of these microorganisms may be any substance that permeates the membrane used in the present invention and does not decompose even when heated at low pH. Examples of the desired product include amino acids such as lysine, glutamic acid, isoleucine, valine, leucine, and phenylalanine, nucleic acids such as inosine and guanosine, organic acids such as lactic acid and malic acid, and substances such as vitamins.

The fermentation broth used in the present invention can be obtained by culturing and growing the above-mentioned microorganism in an appropriate medium. There is no particular limitation on such a medium, and an ordinary medium containing an ordinary carbon source, a nitrogen source, an inorganic ion, and optionally an organic nutrient source may be used. The culturing may be carried out by a method described in WO 95/16042, EP Patent Application Publication 685,555, Japanese Patent Publication JP-A-08-047397, etc., under conditions suitable for the growth of the above-mentioned microorganism.

As the carbon source, any of the above microorganisms can be used as long as it is available. Specifically, sugars such as glucose, fructose, sucrose, maltose, trehalose, amylose, fumaric acid, citric acid, acetic acid, Organic acids such as propionic acid and salts thereof can be used.

As the nitrogen source, any of the above microorganisms can be used as long as it can be used. Specifically, ammonium salts of inorganic salts such as ammonium sulfate and ammonium chloride, ammonium salts of organic acids such as ammonium fumarate and ammonium citrate. It is possible to use salts, nitrates such as sodium nitrate and potassium nitrate, peptone, yeast extract, meat extract, organic nitrogen compounds such as corn steep liquor, or a mixture thereof.

In addition, inorganic salts, trace metal salts, vitamins, etc.
Nutrient sources used for ordinary cultivation can be appropriately mixed and used.

The heat treatment of the fermentation broth obtained by culturing the microorganism belonging to the genus Escherichia is pH 1 to 4.5, preferably pH 2 to 4 at 110 to 200 ° C., preferably
It is carried out at 110 to 150 ° C, particularly preferably 110 to 130 ° C. The heating time is not particularly limited, but is preferably such that the target product is not denatured, and usually 10 minutes or less. Mineral acids such as hydrochloric acid, sulfuric acid and phosphoric acid can be used to adjust the pH of the fermentation broth. In addition, the membrane filtration speed can be effectively improved by adding the polymer flocculant before or after the heat treatment of the fermentation liquid. Examples of the polymer flocculant include polyethyleneimine and sodium polyacrylate. The amount of polymer flocculant added is 0.0005 to 0.5% by weight relative to the fermentation broth.
Is suitable, and 0.001 to 0.05% by weight is more preferable.

Next, the fermented liquor thus heat-treated is subjected to membrane filtration. Both MF and UF membranes can be used in the present invention. As a membrane device, a flat membrane,
It can be used in any form such as hollow fiber, tubular membrane and spiral. The film material may be an organic film such as polysulfone, polyolefin, polyvinylidene difluoride, or Teflon, or an inorganic film such as ceramic. In the case of UF, molecular weight cutoff of 1,00
A membrane with a pore size of 0 to 500,000 is suitable, and in the case of MF, a membrane with a pore size of 0.05 to 1 μm is suitable.

During the membrane filtration, the heat-treated fermentation liquor
It is not necessary to adjust the pH, but it may be adjusted to an appropriate pH according to the material of the membrane used for filtration. Further, the fermentation liquid does not need to be particularly heated during the membrane treatment, but it can be used for membrane filtration by heating and maintaining the temperature at about 50 to 60 ° C. for the purpose of obtaining a higher permeation flux and preventing spoilage. .

In this way, the cells are separated from the fermentation broth obtained by culturing the microorganism belonging to the genus Escherichia, and a clear permeate containing the desired product can be obtained.

[0016]

EXAMPLES The method of the present invention will be described in detail below with reference to examples.

Example 1 According to the method described in the example of WO95 / 16042, the B-399 / B-399 / E. Coli B-399 strain into which plasmid RSFD80 was introduced.
L-lysine was produced using RSFD80 strain. Culture using the following production medium, culture time 48 hours, temperature of 37 ° C.,
Agitation was performed at 114-116 rpm. (L- lysine production medium _{preparation) A: (NH 4) 2} SO 4 16g / L KH 2 PO 4 1g / L MgSO 4 · 7H 2 O 1g / L FeSO 4 · 7H 2 O 0.01g / L MnSO4 · 5H20 0.01g / L Yeast Ext. (Difco) 2g / L L-methionine 0.5g / L Adjusted to pH 7.0 with KOH, autoclave (16/20 volume) for 10 minutes at 115 ° C B: 20% Glucose (115 10 ° C autoclave) (4/20 volume) C: Japanese Pharmacopoeia CaCO ₃ (180 ° C for 2 days dry heat sterilization) (30g / L) A: B mixed at 4: 1 to 1L Then, 30 g of C is added and dissolved, and an antibiotic (streptomycin 100 μg / ml, kanamycin 5 μg / ml) is added.

The amount of L-lysine produced at the end of the culture was 9.2 g / L.
(Calculated as L-lysine hydrochloride). 98% sulfuric acid was added to 300 ml of this lysine fermentation liquid (pH 6.5), and each pH was adjusted to 4.
After adjusting to 0 and 2.0, heat treatment was performed at 120 ° C. An autoclave was used for heating, and the temperature was maintained for 10 minutes after reaching 120 ° C. Also, the same lysine fermentation liquid as a control
300 ml was used for membrane filtration experiments without heat treatment.

The membrane filtration experiment was carried out by Minitan manufactured by Millipore, Inc.
Two PTTK membrane plates manufactured by the same company (UF membrane, molecular weight cut off 30,000) are installed in the module, and the fermented solution after heat treatment is supplied to the UF sterilization facility at a rate of 1000 ml / min while being kept at 50 ° C. It was carried out at an average pressure of 0.9 kg-f / cm ² . The results shown in FIG. 1 were obtained as the change with time of the permeation rate until the permeation time of 30 minutes. As is clear from this figure, according to the method of the present invention, pH
By heating at 120 ° C. in 2 or 4, the permeation rate could be improved to nearly 3 times that in the case of no treatment.

Comparative Example 1 The E. coli fermentation broth obtained in Example 1 was treated in the same manner as in Example 1 with pH 4.0, 70 ° C. and pH 5.5, 90 ° C. and pH 6.5, 110 ° C. Heat treatment was performed for 10 minutes under each condition. FIG. 2 shows the change with time in the permeation rate when the fermented liquid after the treatment was subjected to membrane filtration under the same membrane filtration apparatus and filtration conditions as in Example 1. As is clear from this figure, when heat treatment was performed at pH 4.0 and 70 ° C,
In the case of heat treatment at H5.5, 90 ℃ or pH6.5, 110 ℃, the permeation rate after 30 minutes is about 20 L / m ² · hour (kg
-f / cm ² ), which is almost the same as in the case of no treatment. Therefore, in the E. coli fermented liquor, the conventional fermented liquor treatment method has almost no effect of improving the membrane permeation rate.

Example 2 A strain (FERM-P 527) belonging to the genus Escherichia, having a resistance to β-2-thienylalanine, and having an ability to produce L-leucine (FERM-P 527).
4), Example 1 of Japanese Patent Application Laid-Open No. 8-70879.
According to the method of 1., after performing a mutation treatment with N-methyl-N'-nitro-N-nitrosoguanidine, a strain having resistance to 4-azaleucine is isolated, and a strain having an improved L-leucine-producing ability is obtained. It was

The strain thus obtained was treated with glucose.
5 g / dL, (NH ₄ ) ₂ SO ₄ 2.5 g / dL, KH ₂ PO ₄ 0.2 g / dL, MgSO ₄・ 7H
₂ O 0.1g / dL, yeast extract 0.05g / dL, thiamin hydrochloride
_{1mg / L, FeSO 4 · 7H} 2 O 1mg / dL, MnSO 4 · 4H 2 O 1mg / dL, with a composition of calcium carbonate 2.5 g / dL, with an aqueous solution of medium pH 7.0, 72 hours stirring culture at 31 ° C. did. L- at the end of culture
The amount of leucine produced was 3.1 g / L.

After completion of the culture, the pH of the fermentation broth was adjusted to 4. with sulfuric acid.
The temperature was adjusted to 0 and heat treatment was performed at 120 ° C for 10 minutes by an autoclave. For comparison, the fermentation broth after the same pH adjustment was performed at 60 ° C for 30
A membrane permeation experiment was also performed on the liquid that had been heat-treated for a minute.

The membrane permeation experiment was carried out by the same Millipor in the Minitan module.
Two VVLP plates (MF membrane, pore size 0.1 mm) manufactured by e company were installed and implemented. Liquid was supplied to the membrane device at a circulating flow rate of about 700 ml / min and operated at an average pressure of 0.9 kg / cm ² . The permeated liquid was operated while returning it to the stock solution, and the results shown in FIG. 3 were obtained as the change with time of the permeation flux up to the permeation time of 80 minutes. As is clear from this figure, even in the case of the L-leucine fermentation broth, the membrane permeation flux can be significantly improved by the heat treatment at 120 ° C., which is the method of the present invention, as compared with the conventional heat treatment. It was

Example 3 European Patent Publication No. 488424 or Japanese Patent Publication No.
Escherichia described in the example of 05-076352
E. coli AJ12604 strain was used as a medium for producing L-phenylalanine (glucose 20 g, disodium hydrogen phosphate 29.4 g, phosphoric acid 2
6g potassium hydrogen, 1g sodium chloride, ammonium chloride
2g, sodium citrate 10g, sodium glutamate
0.4 g, magnesium sulfate heptahydrate 3 g, calcium chloride 0.
23 g, thiamin hydrochloride 2 mg in 1 L of water)
After culturing at 24 ° C. for 24 hours, L-phenylalanine was produced.
The amount of L-phenylalanine accumulated at the end of the culture was 3.7 g / L.

This phenylalanine fermentation liquid (pH 6.5) 150
98% sulfuric acid was added to 0 ml to adjust the pH to 4.0, and then the mixture was heated at 120 ° C. for 10 minutes. A CS machine was used for heating, and treatment was performed so that the residence time from the heater to the cooler was 10 minutes. Also, as a control, the same phenylalanine fermentation liquid 15
After 98% sulfuric acid was added to 00 ml to adjust the pH to 4.0, it was sterilized at 60 ° C. for 30 minutes and used for the membrane filtration experiment.

The membrane filtration experiment was conducted by Toyo Roshi Kaisha, Ltd. MODEL UHP
-150, Toyo Roshi Kaisha (manufactured) PTFE membrane T100A142C (pore size 1.0μ
m) was used. In the membrane filtration experiment, the fermented broth after heat treatment at 120 ℃ and the fermented broth not treated at 120 ℃ were kept at 60 ℃, and coagulant sodium polyacrylate
(Product name “Panakayak”: manufactured by Nippon Kayaku Co., Ltd.) at 30 ppm (v
/ v) and added to the filtration device at a pressure of 0.7 kg-f / cm ² . The results shown in FIG. 4 were obtained as the change with time of the permeation rate with respect to the permeation time. As is clear from this figure, according to the method of the present invention, by heating at pH 4.0 at 120 ° C., the permeation rate could be improved 20 times or more as compared with the case of the conventional heat treatment.

[0028]

EFFECTS OF THE INVENTION As is clear from the text, when the method of the present invention is used, the membrane permeation flux of the fermentation broth is 1.
It can be greatly improved by 5 to 20 times. As a result, it is possible to obtain a practical membrane permeation flow rate, and it is possible to easily improve the membrane permeation flux of the fermentation broth without requiring excessive auxiliary equipment, and it is possible to improve the efficiency of the membrane filtration equipment. .

[Brief description of the drawings]

FIG. 1 is a view showing a change with time in permeation rate of a lysine fermentation broth obtained by culturing E. coli during membrane eradication.

FIG. 2 is a view showing a change with time in permeation rate of a lysine fermentation broth obtained by culturing E. coli during membrane eradication.

FIG. 3 is a view showing a change with time in permeation rate of a leucine fermentation broth obtained by culturing E. coli during membrane eradication.

FIG. 4 is a view showing a change with time in permeation rate of a phenylalanine fermentation broth obtained by culturing E. coli during membrane eradication.

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Claims (2)

[Claims] 1. A method for separating bacterial cells by membrane filtration of a fermentation broth obtained by culturing a microorganism belonging to the genus Escherichia, wherein the fermentation broth has a pH of 1 to 4.5, 110.
A method for treating a fermented liquor, which comprises performing membrane filtration after heat treatment at ˜200 ° C. 2. The method for treating a fermented liquor according to claim 1, wherein the membrane used for membrane filtration is an ultrafiltration membrane or a microfiltration membrane.