JP2014050325A - Method for improving growth of lactic acid bacterium at low temperature - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a lactic acid bacterium of which growth at low temperature is improved by transformation.SOLUTION: Provided is a method for improving growth ability of lactic acid bacterium under a low temperature culture condition, i.e., 10°C or lower, by transforming a lactic acid bacterium with a plasmid enabling high expression of peroxiredoxin so that the bacterium can proliferate and produce peroxiredoxin at low temperature, where the plasmid is constructed by introducing a highly expressible peroxiredoxin gene of a specified base sequence. Also provided is a low temperature proliferating lactic acid bacterium that amply expresses peroxiredoxin as a result of the transformation.

Description

  The present invention relates to a method for improving the low temperature growth of lactic acid bacteria, a peroxiredoxin high expression gene used therein, a peroxyredoxin high expression plasmid, and a low temperature growth lactic acid bacterium introduced with this plasmid.

  In general, among lactic acid bacteria that are effectively used in foods, some lactic acid bacteria species have been proliferated rapidly even under appropriate low-temperature control conditions when attached to processed meat products, which deteriorated the quality of the product. Known from.

  Therefore, it is very important from the viewpoint of quality control to know the properties of this lactic acid bacterium species that significantly deteriorates the quality of the processed meat manufacturer, although there is no health hazard.

  In addition, recombinant plasmids have been constructed for the purpose of using products such as the low-temperature growth function of psychrophilic bacteria living in the ocean and psychrophilic enzymes (Patent Document 1).

  In addition, a method for obtaining a transformant by introducing a recombinant plasmid DNA linked with a drug resistance gene or the like into Lactobacillus debrueckii belonging to the genus Lactobacillus, which is a kind of lactic acid bacteria, by electroporation or the like is known (Patent Literature). 2).

JP 2001-238675 A JP-A-5-176777

  However, among the above-described conventional techniques, the invention described in Patent Document 1 efficiently uses heat-labile proteins, enzymes that exhibit activity under low-temperature conditions, and the like by genetic recombination techniques for low-temperature bacteria. It is intended for production, and does not indicate a technique for improving the cold resistance of the host bacterium itself.

  In addition, the invention described in Patent Document 2 discloses a technology for transforming a lactic acid bacterium belonging to the genus Lactobacillus by introducing a gene related to drug resistance (see paragraph 0011 of the same document). It was not a technique for improving cold resistance.

  Therefore, the object of the present invention is to solve the above-mentioned problems and to enhance the action of the in vivo mechanism related to the low temperature growth property of lactic acid bacteria, so that the lactic acid bacteria are related to the cold resistance related to the property of high growth ability at low temperature. By finding a predetermined gene and transforming with this gene, a lactic acid bacterium with improved low-temperature growth is obtained.

  As described above, the present invention improves the ability of lactic acid bacteria to proliferate at low temperatures by increasing the ability to produce a protein (peroxiredoxin) involved in the intracellular antioxidant mechanism as an in vivo mechanism related to the low temperature growth ability of lactic acid bacteria. Is based on having found that.

  And the inventors of the present application investigated the properties of Leuconostoc mesenteroides wild strain (NH04 strain) isolated from actually processed meat products that were actually degraded, and in low temperature environments, the fatty acid composition of the cell membrane is rich in branched and saturated fatty acids, In addition, the protein (peroxiredoxin) that acts as an antioxidant in cells is highly expressed compared to other strains, and the NH04 strain, which has a high temperature growth potential, may have developed an oxidative stress tolerance mechanism. Therefore, the low-temperature growth ability of lactic acid bacteria that deteriorate meat products is considered to correlate with the improvement of a predetermined antioxidant mechanism, and the present invention has been completed.

  That is, the means adopted to solve the above-mentioned problems is that the lactic acid bacteria preferably have a high peroxiredoxin concentration so that the lactic acid bacteria can have peroxiredoxin productivity and grow under low temperature culture conditions of 10 ° C. or less. It is to provide a method for improving the low-temperature growth of lactic acid bacteria, which comprises introducing and transforming an expression gene.

In the above-described method for improving low-temperature growth, since a predetermined peroxiredoxin high-expressing gene has been introduced into lactic acid bacteria, lactic acid bacteria preferably produce a large amount of peroxiredoxin even under low-temperature culture conditions of 10 ° C. or lower. To do.
At this time, the produced peroxiredoxin acts as a glutathione-metabolizing enzyme in the intracellular antioxidant mechanism, so that the antioxidant property against many reactive oxygen species is enhanced.

  Here, it is presumed that lactic acid bacteria in a low-temperature environment cause oxidative stress due to metabolism, thereby generating a lot of cytotoxic reactive oxygen species, thereby inhibiting the ability to grow at low temperature. A large supply of redoxin increases the ability to grow at low temperatures. From the experimental results to be described later, the transformed lactic acid bacteria have excellent low-temperature growth properties as expected under low-temperature culture conditions of 10 ° C. or lower.

As such an excellent method for improving the low-temperature growth of lactic acid bacteria, a peroxiredoxin highly expressing gene of lactic acid bacteria having the base sequence described in SEQ ID NO: 1 can be employed.
Specifically, it is preferable to transform lactic acid bacteria using a peroxiredoxin highly expressing plasmid constructed by incorporating the above-mentioned peroxiredoxin highly expressing gene of lactic acid bacteria, and thus obtained. A lactic acid bacterium with high cryogenic growth ability can be obtained by using a psychrotrophic lactic acid bacterium transformed by introducing a plasmid expressing high peroxiredoxin.

In this invention, since the lactic acid bacterium is transformed by introducing a peroxiredoxin high expression gene into the lactic acid bacterium so that the lactic acid bacterium has peroxiredoxin productivity and can be grown under low temperature culture conditions, There is an advantage that the in vivo mechanism related to the can be improved.
In addition, the present invention has an advantage that a lactic acid bacterium having an improved low temperature growth ability capable of efficiently growing at a low temperature can be obtained by transformation of a lactic acid bacterium using a plasmid incorporating a predetermined gene related to low temperature growth ability. .

Two-dimensional electrophoretic diagram showing the expression pattern of soluble protein by low temperature culture of cold tolerant strain (NH04) and standard strain (NBRC3832) Explanatory drawing showing the range of action of plasmids and restriction enzymes used in vectors Chart showing the relationship between the amount of growth and culture time in Example 1 (transformant: NBRC3832pGKahpC) and Comparative Example 1 (standard strain: NBRC3832pGK) SDS-PAGE electrophoretic diagram showing the expression of AhpC in Example 1

  The method for improving the low-temperature growth property of lactic acid bacteria according to an embodiment of the present invention is such that the peroxyl represented by SEQ ID NO: 1 with respect to the lactic acid bacteria so that the lactic acid bacteria have peroxiredoxin productivity and can grow under low-temperature culture conditions. A redoxin highly expressing gene is introduced and transformed.

  Lactic acid bacteria that are host cells capable of improving the low-temperature growth ability according to the present invention are not limited to specific species, and one or more types can be appropriately employed.For example, Lactobacillus, Bifidobacterium ), Enterococcus genus, Lactococcus genus, Pediococcus genus, and Leuconostoc genus industrially useful lactic acid bacteria.

  The peroxiredoxin (AhpC) high-expression gene of lactic acid bacteria having the base sequence of SEQ ID NO: 1 can be obtained, for example, by the following method, and a plasmid for overexpression of AhpC is constructed, The growth rate at low temperatures can be confirmed.

For example, as two types of lactic acid bacteria having different growth rates in a low temperature environment, L. mesenteroides (NH04 strain with excellent growth properties) and a standard strain (NH01 strain with standard growth properties) are cultured at 10 ° C., Dihydroethidium (DHE) is used to analyze the amount of superoxide anion accumulated in the microbial cells.
From the result, it was found that the NH04 strain having a high low temperature growth property accumulates a large amount of superoxide anion, for example, 1.7 times as much as the NH01 strain.

By the way, superoxide anion is a free radical that is generated as a by-product of respiratory metabolism and can be converted to a more reactive hydroxy radical.
In the cells of NH04 strain, despite the presence of many reactive oxygen species exhibiting cytotoxicity, it exhibits higher growth than other strains, so this strain protects against cytotoxicity by reactive oxygen species. A mechanism is considered to exist.

  In addition, when this strain is cultured at 10 ° C., peroxiredoxin (AhpC), which is known as one of glutathione metabolizing enzymes, is produced at high temperature, and the NH04AhpC high-expressing strain has a lower growth rate at a lower temperature than the wild-type NH01 strain. Was big.

Therefore, it is considered that the intracellular antioxidant mechanism by NHp strain-derived AhpC is likely to be involved in the low temperature growth of food-degrading lactic acid bacteria, and the effect of NHp strain AhpC on the low temperature growth of lactic acid bacteria Is constructed, an NH04AhpC overexpression plasmid is constructed and introduced into cells by electroporation into NH01.
The method for improving the low temperature proliferation property of lactic acid bacteria according to the embodiment of the present invention will be described more specifically.

[AhpC gene]
The AhpC gene used in the present invention is derived from, for example, Leuconostoc mesenteroides (NH04 strain), which is a chilled lactic acid bacterium collected from a food production environment where lactic acid bacteria are used. The NH04 strain collected in such an environment is highly proliferative at low temperatures as compared to the commercially available Leuconostoc mesenteroides NBRC3832, and the protein expression patterns of both strains at 10 ° C. were examined in order to investigate the reason. When examined, AhpC protein was remarkably abundantly expressed (see FIG. 1).

A method for identifying a gene region involved in the expression of this protein is as follows.
The sequence of the AhpC gene of several types of lactic acid strains, including Leuconostoc mesenteroides (ATCC 8293 strain), whose gene sequence has already been deciphered and known, is compared, and using the highly common sequence portion, Primers assumed to be amplified also in the AhpC sequence are prepared, the sequence assumed to be the AhpC gene is amplified using the NH04 strain genome as a template, and the sequence of the amplified product is analyzed by a sequencer. In order to accurately determine the sequences of the 5 ′ and 3 ′ terminal portions of the AhpC sequence, an inverse PCR method is performed based on the previously analyzed sequence, and finally the total length of the AhpC sequence of the NH04 strain is determined.

[Construction of recombinant plasmid]
As shown in FIG. 2, in order to determine the ability of the above AhpC gene, this gene sequence is inserted into a plasmid and introduced into cells to synthesize AhpC protein. Commercially available pGK :: nucMCS (ATCC) or the like can be used as the plasmid for that purpose.

In order to incorporate such an AhpC gene into a vector (pGK :: nucMCS) and express the gene, it is incorporated with a promoter according to a standard method.
That is, an AhpC gene and a promoter sequence are introduced into the pGK :: nucMCS vector. First, a primer set for separately amplifying the AhpC gene sequence and the promoter sequence of the NH04 strain is prepared.

Incidentally, the same sequence as the AhpC gene is added at about 10 bases to the end of the reverse primer for amplifying the promoter sequence so that the promoter sequence and the AhpC gene can be directly linked (the promoter sequence is linked 5 ′ upstream of the AhpC gene). In addition, after linking the promoter sequence and the AhpC gene, when they are inserted between, for example, XhoI and SalI sites of pGK :: nucMCS, the end of the forward primer for amplifying the promoter sequence and the end of the reverse primer for amplifying the reporter sequence XhoI and SalI restriction enzyme recognition site sequences are added, respectively.
It should be noted that any restriction enzyme recognition site contained in this plasmid can be used, not limited to the restriction enzyme cleavage sites, XhoI and SalI.

  In consideration of the above, PCR amplification of the promoter sequence and the reporter sequence is performed separately, and then the respective PCR amplification fragments are mixed, and PCR is performed again using the forward primer for amplification of the promoter sequence and the reverse primer for amplification of the reporter sequence. To obtain a fragment in which a promoter and a reporter sequence are linked.

This fragment may be electrophoresed on a 1% agarose gel to cut out a band of a prescribed size, and then extracted and purified.
The resulting purified fragment and pGK :: nucMCS are each purified by digestion with a restriction enzyme.

  The recombinant plasmid obtained by the treatment can be introduced into E. coli, cultured, and extracted from the amplified E. coli to obtain a large amount of recombinant plasmid.

  In order to introduce the recombinant plasmid into the lactic acid bacterium, it may be appropriately performed according to a conventional method, for example, according to the well-known technique described in J Dairy Sci. 74: 1454-1460, 1991.

[Construction of overexpression plasmid]
The AhpC gene of SEQ ID NO: 1 was introduced into a commercially available vector (pGK :: nucMCS) together with a promoter sequence.
First, one set of primer sets for separately amplifying the NHp strain AhpC gene sequence and the promoter sequence is prepared, respectively, and the end of the forward primer for promoter sequence amplification and the end of the reverse primer for reporter sequence amplification are respectively XhoI, A SalI restriction enzyme recognition site sequence was added.

  Each of the promoter sequence and the reporter sequence is PCR amplified separately, and then each PCR amplified fragment is mixed, and PCR is performed again using the forward primer for amplifying the promoter sequence and the reverse primer for amplifying the reporter sequence. Ligated fragments were obtained.

  This fragment was electrophoresed on a 1% agarose gel to cut out a band of a prescribed size, and extracted and purified. The obtained purified fragment and pGK :: nucMCS were cleaved at 37 ° C. with restriction enzymes XhoI and SalI, respectively.

pGK :: nucMCS was subsequently treated with alkaline phosphatase, and the treated fragment and pGK :: nucMCS were treated with phenol / chloroform to remove the protein. Then, ethanol precipitation was performed and purified.
The purified fragment and a specified amount of pGK :: nucMCS were mixed, and ligated with “DNA Ligation Kit Ver. 2.1” manufactured by Takara.

The recombinant plasmid obtained by the treatment was subsequently introduced into Escherichia coli using “E. coli DH5α Competent Cells” manufactured by TaKaRa, and cultured at 37 ° C. on an LB plate supplemented with 500 μg / ml erythromycin. E. coli successfully introduced was obtained.
The Escherichia coli was fished, cultured with shaking in LB broth supplemented with 500 μg / ml erythromycin at 37 ° C., and the plasmid was extracted from the amplified Escherichia coli to obtain a large amount of recombinant plasmid.

[Introduction of recombinant plasmids into genus Leuconostoc and Lactobacillus]
Lactic acid bacteria for introducing the recombinant plasmid are inoculated with 50 ml of 1% glucose-added GAM bouillon (Nissui) according to the document J Dairy Sci. 74: 1454-1460, 1991, resulting in OD600 = 0.6 Cultured with shaking at 25 ° C. After centrifugation, the cells were washed twice with 75 ml of ice-cold sterilized water and suspended in 5 ml of ice-cold EPB (1 mM phosphate buffer (pH 7.4), 1 mM MgCl ₂ , 0.5 M sucrose). It became turbid and cooled on ice until use. This was used as a cell for introduction.

  Recombinant plasmid (1 μg / 1 to 4 μl) was mixed with 40 μl of the prepared bacterial solution, placed in a 0.2 cm electroporation cuvette, and cooled on ice for 5 minutes. Thereafter, electroporation was performed at 400Ω, 25 μF, and 8 kV / cm using a Gene Pulser Xcell PC system. Immediately thereafter, 1% glucose-added GAM broth with ice-cooling was added so that the total amount was 1 ml, and the mixture was statically cultured at 25 ° C for 2 hours. After culturing, the cells were smeared on a 1% glucose-added GAM plate supplemented with 5 μg / ml erythromycin and cultured at 25 ° C. for 72 hours. The obtained colonies were used as lactic acid strains into which the recombinant plasmid had been introduced, and were subjected to the following low-temperature growth test. .

[Low temperature growth test of transformed lactic acid bacteria]
The test method for culturing Leuconostoc mesenteroides NH04 strain, NBRC3832 strain, and other transformed strains at low temperature was as follows. GAM bouillon with 1% glucose added to freeze stock and colonies formed on plates (5 μg / ml erythromycin in the case of transformed strains) 50 μl or 1 colony is inoculated into 5 ml, and after subculture for 2 passages at 25 ° C., the cell concentration is OD600 in a dedicated L-shaped test tube (the same medium) for Biophoto Recorder (Biorad) = 0.01, diluted and inoculated, cultured at 10 ° C. and 70 rpm, and measured by monitoring an increase in turbidity due to an increase in bacterial cells, and the results are shown in FIG.

  As a result of the evaluation test, it was confirmed that the time required for growth to a constant concentration was shortened for the NBRC3832 strain when cultured under a low temperature condition of 10 ° C.

[AhpC expression level analysis test]
For the NBRC3832 strain into which the recombinant plasmid was introduced and the NBRC3832 strain into which pGK :: nucMCS was introduced as a control, the number was increased with a biophoto recorder in the same manner as described above, and the cells were collected, washed, sonicated, and soluble protein recovered. Concentration measurement was performed, and 10 μg of protein was used for SDS-PAGE for confirming the difference in the expression level of AhpC. SDS-PAGE was carried out at 15 mA for 90 minutes using ePAGE (12.5% acrylamide concentration) manufactured by ATTO. Staining and detection were performed in the same manner as in two-dimensional electrophoresis, and an electrophoretogram showing the protein expression pattern is shown in FIG.

  Bacteria grown up to the stationary phase at 10 ° C by the above method are collected by centrifugation in 5 ml, washed twice with 100 mM Tris-HCl Buffer (pH 7.0), suspended in this buffer again, and the cells. Was sonicated.

  After crushing, the mixture was centrifuged at 15,000 rpm for 10 minutes to obtain a soluble protein fraction in the supernatant. The concentration of this protein solution was measured by the Bradford method, and 150 μg of the protein was used for two-dimensional electrophoresis, Ready Strip IPG strips 17 cm pH 4-7 (BioRad). The first dimension electrophoresis was performed with PROTEAN IEF Cell (Bio-Rad), and the second dimension was subjected to SDS-PAGE corresponding to the strip size. After the electrophoresis, the cells were stained with SYPRO Ruby (Invitrogen) and detected with Typhoon 9400 (GE Healthcare).

  3 and 4, the transformed NBRC3832 strain (referred to as NBRC3832_pGKahpC in the figure) and the NBRC3832 strain into which pGK :: nucMCS was introduced as a control (NBRC3832_pGK in the figure). The transformed NBRC3832 strain expressed the AhpC protein and improved its growth ability when cultured under a low temperature condition of 10 ° C.

Claims (5)

  A method for improving the low-temperature growth ability of lactic acid bacteria, comprising transforming the lactic acid bacteria by introducing a peroxiredoxin high-expressing gene so that the lactic acid bacteria have peroxiredoxin productivity and can grow at low temperatures.   The method for improving low-temperature growth of lactic acid bacteria according to claim 1, wherein the low temperature is a low temperature of 10 ° C or lower.   A peroxiredoxin highly expressing gene of a lactic acid bacterium having the base sequence of SEQ ID NO: 1, which is used in the method for improving the low temperature proliferation property of lactic acid bacterium according to claim 1 or 2.   A peroxiredoxin highly expressing plasmid constructed by incorporating the gene according to claim 3.   A psychrotrophic lactic acid bacterium transformed by introducing the peroxiredoxin highly expressing plasmid according to claim 4.