JP2779073B2 - Selective fermentation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a selective fermentation method by improving microorganisms.

[0002]

2. Description of the Related Art When two or more substrates are subject to metabolism in a certain microorganism, the phenomenon that a substrate which is easily used by the microorganism inhibits the induction of an enzyme which controls the catabolism of another substrate is caused by catabolite lyophilization. Compression (Catabolite Rep
ression). (Hartwell L.
Hartwell L. and B. Magasan
ik), Journal of Molecular Biology
(J. Mol. Biol), Vol. 7, pp. 401-420 (1963),
Monod J., Recherches-sur-la-Croissance-de-Courture-Bactaenne, Thees (Recherches sur la croissance des c)
ultures bacteriennes, thesis) and Arman (Harman)
, Paris, p. 145 (1942)] and bacteria belonging to the genus Pediococcus, for example, Pediococcus halophiluc
The above phenomenon also occurs when is cultured in a medium containing two or more types of substrates that can be used as an energy source by the bacterium.

[0003] The above catabolite repli- cations are sugars and amino acids which are substrates that serve as energy sources for bacteria.
It is generally known that the present invention extends not only to metabolic systems such as arginine and the like, but also to degradation systems for polymer substrates such as nucleic acids, polysaccharides and proteins, as well as metabolic systems such as organic acids. Therefore, in general, when an easily available substrate such as glucose or mannose is present, the enzyme biosynthesis of the metabolic system of the other substrate is suppressed, so that the other substrate is not available or the production of the desired enzyme is suppressed. There are many cases where this is not possible.

[0004] Catabolite repression is one of the very powerful mechanisms of metabolic regulation even in the genus Pediococcus, not only in the selective fermentation of preferential sub-substrates in a mixture of multiple substrates, but also in some fermentations. It is also a major problem in the production of enzymes and other secondary metabolites,
Normally, it was almost impossible to escape this suppression effect. Even if it can be partly done in special cases, it is necessary to cultivate for a very long time, or to specifically remove the substrate that interferes with the selective fermentation in the medium, which requires extra costs or is inexpensive. However, it is impossible to use a natural medium which is an excellent but complicated mixture, and a solution is strongly desired.

The present inventors have conducted various studies to release catabolite repression of microorganisms belonging to Pediococcus halophilus. As a result, the microorganisms include phosphoenol pileate-dependent microorganisms.
Sugar: Phosphotransferase system
(Phosphoenolpyruvate dependent Sugar: Phosphotrans
-ferase System) (hereinafter abbreviated as PTS), and found that catabolite repression can be released by deleting PTS.

[0006] PTS stands for enteric bacteria (ente bacteria).
ricbacteria), and is a sugar transport mechanism studied by Kundig W. et a
l.,), Proceedings National Academy
Of Science (Proc. Natl. Acad. Sci.), USA
(USA), Volume 52, pp. 1067-1074 (1964), PW Postma et al.,
Microbiol. Re
v.), Volume 49, pp. 232-269 (1985).

[0007] The present inventors have previously obtained a mutant having a reduced or defective PTS from a microorganism belonging to Pediococcus halophilus, and provided the mutant with a metabolic substrate that causes catabolite repression. A substance containing a metabolic substrate whose catabolism is inhibited by catabolite repression or a substance to which a nutrient is added if necessary is inoculated and cultured to selectively ferment the substrate whose catabolism is inhibited. And a method for removing it (see Japanese Patent Application Laid-Open No. 63-219368).

[0008]

However, in the above method, since galactose and the like are simultaneously fermented, the selective fermentability of xylose was not always satisfactory. Therefore, the present inventors have developed a triple-deficient novel strain in which PTS, phosphofructokinase (hereinafter abbreviated as PFK) and glucokinase (hereinafter abbreviated as GK) are deficient in microorganisms belonging to Pediococcus halophilus. Induction of the mutant strain, various studies to isolate, succeeded in obtaining the desired mutant strain, and further inoculate the thus obtained mutant strain in xylose-containing material, culture, and efficiently xylose The present inventors have found that fermentation can be selectively performed, and completed the present invention.

[0009]

That is, the present invention relates to a microorganism belonging to the genus Pediococcus, which is deficient in the phosphoenol pileate dependent sugar: phosphotransferase system, phosphoflactokinase and glucokinase. A selective fermentation method of pentose, characterized by inoculating and culturing pentose-containing substances and selectively fermenting pentose.

Hereinafter, the present invention will be described in detail. First, belonging to the genus Pediococcus of the present invention, PTS, PFK and
Examples of the novel mutant deficient in GK include Pediococcus halophilus 3E4 (hereinafter sometimes referred to as the present mutant). The above Pediococcus halophilus 3E4 is a Pediococcus halophilus 13-2 strain
(FERM BP-702) is a novel mutant strain obtained by mutation treatment,
The mycological properties of Pediococcus halophilus 3E4 are:
As shown below, the mycological properties of Pediococcus halophilus 13-2 are described in JP-A-60-241885. Pediococcus halophilus 3E
4 Mycological properties The mycological properties of Pediococcus halophilus 3E4 are
Among the mycological properties of Pediococcus halophilus 13-2, "whether or not acid and gas are generated from sugars" and "PTS, PFK
And GK activity ”are as described below, and are exactly the same as the bacteriological properties of Pediococcus harophilus 13-2. (1) Generation of acid and gas from saccharides Acid generation Gas generation L-arabinose +-D-xylose +-D-glucose--D-mannose--D-fructose +-D-galactose--D-maltose- -Sucrose--lactose--trehalose--D-sorbitol--D-mannitol--inositol--glycerol--starch--(2) PTF, PFK and GK activity
GK activity is as shown in Table 1.

[0011]

[Table 1]

Measurement of PTS, PFK, and GK enzyme activities was performed by Abe et al.
(Abe K., and K. Uchida) [J. Bacteriol. 171:
1793-1800, (1989)]. The Pediococcus halophilus 3E4 has been deposited with the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology as Microbial Laboratories No. 12004 (FERM P-12004). As the mutation treatment, N-methyl-N′-nitro-N-nitrosoguanidine (hereinafter referred to as MNNG
Abbreviated. ), Mutagenic agents such as ethylmethanesulfonate and methylmethanesulfonate, ultraviolet irradiation, X
Methods include irradiation with radiation, irradiation treatment, spontaneous mutation, transformation with phage or plasmid, transduction, genetic recombination by conjugation, and the like.

In addition, aldolase (A
A similar effect can be expected with triple deficient strains of PTS, ALD and GK that have (LD) deficiency. As a medium used for culturing the present mutant strain, a medium used for culturing a strain belonging to general Pediococcus halophilus can be mentioned. The nitrogen source of the medium may be any available nitrogen compound or any containing it, for example,
One or more nitrogen sources such as yeast extract, peptone, meat extract, gelatin, corn steep liquor, amino acids, soybean or wheat koji leaching solution are used. One or more suitable inorganic salts such as manganese, phosphoric acid, potassium, magnesium and calcium are appropriately added to the nitrogen source, and carbon sources necessary for the growth of the bacterium, if necessary, for example, sugars, various organic substances, inorganic substances, vitamins And the like are preferably used as a medium, and a medium containing 2 to 17% of sodium chloride is preferable. In addition, moromi sap at the initial stage of preparation in a normal soy sauce production method is appropriately diluted and adjusted to about 15% salt.

The present mutant strain is preferably subjected to a liquid culture method, and is preferably cultured under static or anaerobic conditions. The culture temperature is 15 to 50 ° C, preferably 20 to 40 ° C. The cultivation time varies greatly depending on the salt concentration during cultivation.
-10 days, and the pH during the culturing was adjusted to pH 3 for both the synthetic medium (eg, MYP medium and YPG medium) and the moromi juice medium.
To 9 are preferred.

Next, any means may be used to collect the mutant cells from the culture thus obtained.
Separation is carried out according to a usual operation method such as centrifugation, filtration and the like, and washing is performed as necessary to obtain the present mutant strain. Then, in the present invention, the present mutant strain is inoculated into a pentose-containing substance and cultured,
Pentose, for example, xylose, is selectively fermented.

Examples of the pentose include arabinose, ribose and the like in addition to the above-mentioned xylose. Examples of the pentose-containing substance include soy sauce moromi, a wood hydrolyzate, and a cereal decomposition liquid. The inoculum amount of the present mutant strain is, for example, 10 ² −10 ⁹ cells / g, or
ml, preferably, 10 ⁴ -10 ⁶ cells / g, or number / ml.

The fermentation conditions may be any conditions as long as fermentation is achieved. For example, the fermentation may be performed in the same manner as the above-described culture conditions of the present mutant strain.

[0018]

As is evident from the above, if the present mutant strain is inoculated and cultured in a pentose-containing substance, pentose involved in the coloring substance in soy sauce moromi and the like can be efficiently and selectively fermented. The present invention is extremely useful industrially.

[0019]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples. However, these examples do not limit the technical scope of the present invention.

[0020]

[Example 1] sugar: Mutation induction of PTS, PFK and GK triple deficient strains Pediococcus halophilus 3E4 Table 2 Yeast extract 0.3 g / 100 ml polypeptone 0.5 dipotassium hydrogen phosphate 0.5 sodium thioglycolate 0.1 NaCl 5.0 xylose 1.0 Table 2 medium (hereinafter abbreviated as YPX-1-5) in 5 ml of Pediococcus halophilus 13-2 strain (FERM BP-702)
The cells were inoculated and allowed to stand still at a temperature of 30 ° C for 4 days to obtain a culture.
100 μl of the culture was added to YPX-1-5 at 150 mM 2-deoxygluc
5 ml of medium (YPDGX-1-5) supplemented with ose (2DG)
C. for 4 days. The 2DG-resistant bacteria thus grown were diluted and inoculated on a medium shown in Table 3 (MYPX-1-5CaCO ₃ plate) so that about 100 colonies were formed. MYPX
Colonies grown on -1-5CaCO ₃ plate were transferred to YPX-1-5
A medium (YPGX-1-5) supplemented with 0 mM glucose was inoculated and allowed to stand still at a temperature of 30 ° C. for 4 days to select a bacterium with little decrease in glucose and significant decrease in xylose. Table 3 Meat extract 0.5 g / 100 ml Yeast extract 0.5 Polypeptone 0.5 Sodium thioglycolate 0.1 Calcium carbonate 0.1 Agar 1.5 NaCl 5.0 Xylose 1.0 For the determination of glucose and xylose, see Abe et al. (Abe K., an
d K. Uchida) method (J. Bacteriol. 171: 1793-1800)
(1989)).

About strains selected on YPGX-1-5 medium PT
The S, PFK and GK activities were measured and the novel mutant Pediococcus halophilus 3E4 (F
ERM P-12004) was obtained.

[0022]

Example 2 This novel mutant strain Pediococcus halophilus 3E4 (FERM P-12004) and Pediococcus halophilus I-1 having a reduced PTS activity described in JP-A-63-21936 ( FERM BP-1303) was used in Example 1.
One loopful of platinum loop was inoculated into 5 ml of YPX-1-5 medium, and cultured at 30 ° C. for 4 days to obtain a culture.

Next, the culture was subjected to 18,000 r.
The cells were centrifuged at pm for 10 minutes to obtain cells, and the cells were
After washing with 5 ml of an aqueous solution of Cl and drying by a conventional method, 0.750 mg and 0.70 mg of dry cells of Pediococcus halophilus 3E4 and Pediococcus halophilus I-1, respectively.
80 mg were obtained.

[0024]

Example 3 Table 4 Yeast extract 0.3 g / 100 ml polypeptone 0.5 dipotassium hydrogen phosphate 0.5 sodium thioglycolate 0.1 NaCl 5.0 glucose 5.0 mannose 0.5 galactose 0.5 arabinose 0.5 xylose 1.0 parent strain Pediococcus halophilus 13-2 and this mutation The strain Pediococcus halophilus 3E4 was cultured in the medium described in Table 4.
(YPM-1-5) was inoculated into 5 ml, cultivated at 30 ° C. for 4 days, and the amount of sugar remaining in the culture was compared. Table 5 Residual amount (g / 100 ml) Strain name Galactose arabinose xylose glucose mannose 13-2 0.45 0 0.42 4.65 0.41 3E4 0.46 0 0.10 4.74 0.45

[0025]

Example 4 100 kg of defatted soybeans were steam-denatured and 105 kg of wheat were crushed by roasting. The seed koji was inoculated into the mixture, followed by 42 hours of ventilation koji making to obtain soy sauce koji. Add 360L of salt water cooled to 15 ℃ containing 90kg of salt to 600L
The charge was carried out in a closed charging tank.

Next, the present mutant strain Pediococcus halophilus 3E4 (FERM P-12004) and Pediococcus halophilus I-1 (FERM BP-13) obtained in Example 2 were obtained.
03), parent strain Pediococcus halophilus 13-2 (FERM
BP-702) and wild strain I (FERM BP-1302) were obtained from the soy sauce moromi three weeks after preparation by aseptic filtration using a membrane filter with a pore size of 0.22 μm (manufactured by Nippon Millipore). One platinum loop was inoculated into 100 ml (4 categories) of the obtained moromi sap, and cultured at 30 ° C. for 7 days to obtain respective cultures.

Table 6 shows the results of comparison of the amount of residual sugar in the obtained culture. The residual sugar content was determined by the method of Sinner M.Puls J. [Journal of Chromatography (J.
ogr.), vol. 156, p. 197 (1978)].

[0028]

As evident from Table 6, arabinose and xylose were selectively and completely fermented with a high concentration of glucose remaining. High selection was also shown when compared to the PTS-deficient strain and the parent strain 13-2.

Claims (1)

(57) [Claims] A microorganism belonging to the genus Pediococcus and lacking the phosphoenol pileate dependent sugar: phosphotransferase system, phosphofructokinase and glucokinase is inoculated into a pentose-containing substance and cultured. A selective fermentation method for pentose, comprising selectively fermenting pentose.