JPH0724578B2 - Nisin-producing microorganism and method for producing nisin using the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing nisin and a nisin-producing microorganism. Nisin is a substance useful as a food preservative and for inhibiting the growth of Borrinus.

[Conventional technology]

Nisin is Streptococcus lactis (S
specific strains of treptococcus lactis (eg ATCC 114
54 strain) is a peptide antibacterial substance. It has been confirmed that this substance has the following primary structure (J. Amer. Chemi. Soci. Vol. 93, 4634 to 4635, 1971).

Abu ＝ α-aminobutyric acid Dha ＝ dehydroalanine Dhb ＝ dehydrobutyrine Nisin has antibacterial activity against a narrow range of Gram-positive bacteria such as Clostridium but is clinically Has not been realized. On the other hand, since lactic acid bacteria used for food production are produced, it is highly safe and is used in the United States for the purpose of preventing abnormal fermentation and decay of cheese. Further, JP-A-59-42322 discloses a carcinostatic agent containing nisin as an active ingredient.

Nisin, Streptococcus lactis ATCC11454
Nisin-producing strains such as strains are cultivated and purified and recovered from this culture solution. In order to prevent abnormal fermentation of cheese, these production strains are used as a starter.

Since the nisin producing strain often loses its productivity when it is cultured and passaged, it has been considered that a gene related to nisin production or a nisin producing gene is present on the plasmid. JP-A-60-66976 discloses a method for introducing nisin productivity into a target microorganism by conjugation from a nisin-producing microorganism, and WO / 90 0558 discloses a DNA encoding a nisin precursor. Is disclosed.

[Problems to be Solved by the Invention]

The present inventors have conducted research on microorganisms capable of producing nisin, and as a result, found that the production of nisin is increased under certain culture conditions. We also found a lactic acid bacterium that has a nisin-producing gene on the chromosome, which was previously thought to exist in a plasmid. The present invention was made based on these findings, and an object of the present invention is to provide a high-concentration production method of nisin.

[Means for Solving the Problems]

In the production of nisin, it is necessary to isolate a strain that has nisin-producing ability and is resistant to nisin produced by itself. For this purpose, a nisin sensitivity test and a nisin production test are performed.

As the nisin-producing microorganism, it is preferable to target a conventionally known lactic acid bacterium, but other microorganisms may be used.

The nisin sensitivity test is performed by a cup test using 10 ⁴ IU / ml of nisin, and the size is determined by the size of the production inhibition circle. After the strains to be screened are mixed, the test is performed by the stainless steel cup method.

Then, a nisin production test is carried out on the sensitivity test negative strain. Nisin production can be assayed by various methods, but Gass
on method (Gasson, MJFEMS microbiol.Lett.vol.21, p7
~ (1984)) is common. This is a nisin-sensitive strain Streptococcus cr
emoris ATCC 14365], the culture supernatant of a strain showing growth inhibition was further heat-treated under the condition of pH 8.0 and α-chymotrypsin treatment, and when the strain was inactivated by any of them, the strain was treated with nisin. It is determined to be positive and selected as a positive strain.

The nisin producing strain thus selected can be used as the nisin producing strain in the present invention. However, genes for nisin production and resistance are present on the plasmid, which is often shed by passage.
Therefore, it is preferable to select only those having a gene for nisin production on the chromosome from the selected production strains. For selection, screening using the nisin precursor gene disclosed in WO / 90 0558 can be employed. However, in the present invention, as a simpler method, a plasmid separation test was performed to select. According to this method, a plasmid in a microorganism is separated and detected, and a plasmid-free strain is determined to have a nisin-related gene in its chromosome.

The production enhancement of the nisin producing strain can be easily induced by adding an amino acid into the medium.

The amino acids to be added include the amino acids contained in the structure of nisin, Ala, Leu, Ileu, Pro, Lys, Asn, Met, Val, Se.
Examples include r, His, amino acids that are precursors of these amino acids in metabolic pathways, amino acids that are essential for growth, or one or a mixture of two or more of these amino acids. In production, 0.1-10 mg / ml of amino acid to be added to ordinary medium is added per ml, and the strain may be cultured under optimal conditions for the target strain. As a culture method, any method can be adopted as long as it is a culture method suitable for a microorganism such as ordinary static culture and shaking culture. Further, the collection of nisin has been known conventionally. For example, chloroform extraction disclosed in JP-A-59-42322, U.S. Pat.
Any method such as acetone-methanol extraction disclosed in No. 3 can be adopted.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1 Selection of Nisin-Resistant Strains Nisin-resistant strains were selected from 78 Streptococcus lactis strains that the present inventors have stored.

Cultivated overnight in M17broth medium.
Dilute 10 times, and dilute 0.2 ml and 10 ml of Elliker agar medi
Um was poured into a flat plate. Tween 20 was added to this plate at a concentration of 1% to promote the diffusion of nisin. A stainless steel cup was placed on this detection plate, and a nisin solution (10 ⁴ IU / ml) dissolved in 0.02N hydrochloric acid was injected. Then, it was cultured overnight at 28 ° C., and the growth inhibition circle was measured.

Most strains are growth-inhibited by nisin,
A 20 mm stop circle was formed. The 16 strains shown in Table 1 did not form an inhibition circle and showed nisin resistance.

The M-17broth medium, Elliker agar medium has the following composition.

M-17broth medium [Appl.microbiol., 29, 807 (1975 ) refer to Fig polypeptone (BBL) 5.0g phytone peptone (BBL) 5.0g yeast extract (BBL) 2.5 g beef extract (Difco) 5.0g Lactose 5.0g L- ascorbic acid 0.5 g beta-glycerophosphate disodium _{19.0g 1M MgSO 4 · 7H 2 O} 1 ml distilled water 1 pH 7.15 sterilization conditions:. 121 ° C. 15 minutes Elliker agar medium [J.Dairy Sci, 39, 1611 (1956)] Tryptone 2% yeast extract 0.5% gelatin 0.25% dextrose 0.5% lactose 0.5% sucrose 0.5% dietary salt 0.4% sodium acetate 0.15% ascorbic acid 0.05% agar 1.5% pH6.8 Example 2. Nisin Selection of Production Strains Each of the 16 strains selected in Example 1 was cultured overnight in M-17 broth medium and then filtered with a 0.45 μm filter.
This culture supernatant was divided into the following three samples. A: (pH adjusted to 2.0 with hydrochloric acid), B (pH adjusted to 7.0 with NaOH),
C (pH adjusted to 8.0 with NaOH), A and C were heated at 100 ° C. for 30 minutes, and B was treated with 5 mg / ml trypsin or α-chymotrypsin at 37 ° C. for 1 hour. On the other hand, Streptococcus cremoris (St
reptococcus cremoris) ATCC14356 strain 1 in M-17 medium
After overnight culture, this culture solution was diluted 10-fold with physiological saline, and 0.2 ml of the diluted solution was mixed with 10 ml of Elliker agar medium (agar concentration 0.7%) to prepare Ellikr agar prepared in advance.
The er agar medium (agar concentration: 1.5%) was dispensed into a 20 ml Petri dish, and 4 ml was layered on a horizontally solidified plate to solidify. As in Example 1, in order to promote the diffusion of nisin, Tween 20 was added at a concentration of 1%. This plate was pre-cooled at 4 ° C. for 1 hour, placed on a stainless steel cup, and the above-mentioned culture supernatant, enzyme treatment solution, and heat treatment solution were injected. After culturing at 28 ° C. for 24 hours, the diameter of the growth inhibition circle was measured.

Similarly, ATCC 7962, ATCC 11454, NCDO, which are Nisin producers
The test was performed using the culture supernatant of 497 strain as a control.

Since nisin is an antibacterial substance for peptide purification, its thermal stability changes depending on pH. It also has the property of being hydrolyzed by some proteases and inactivated. The culture supernatants of these known nisin-producing strains are stable when heat-treated at pH 2.0, but deactivate at pH 8.0. It is stable with trypsin but inactivated with α-chymotrypsin. Utilizing this property, the above (A), (B) and (C) of the culture supernatants of 16 strains isolated in Example 1 were compared with the culture supernatant of the standard strain, and Table 1 shows (+). The four strains indicated as "A" were determined to be positive.

In addition, the nisin-producing gene is said to exist in close proximity to the sucrose fermentation gene by Gasson et al. (Gasson
FEMS microbiol. Lett. Vol. 21, 7 (1984)].

Therefore, the sucrose fermentability of 16 strains was searched and Table 1
The results shown in are obtained.

From the above results, 4 out of 16 strains SBT1212, 1213, 1227, S2001 were determined to be nisin-producing strains.

Example 3. Plasmid search of nisin-producing strain As described above, the nisin-producing resistance gene of the nisin-producing strain is presumed to be present on the plasmid according to the reports so far. For this reason, it is considered that the production ability is lost due to the subculture. Therefore, the search for strains in which the produced gene was integrated into the chromosome was carried out by modifying the method of Anderon, Mckay [see Appl. Envirom. Microbiol. 46 , 549 (1983)].

Four strains determined to be positive in Example 2, SBT1212,1213,1227, S2
001, a strain that has been confirmed to be a nisin-producing bacterium, but the involvement of a plasmid has not been confirmed, NCDO 497, and ATCC 11454 strain that has been confirmed to have a nisin-producing gene on the plasmid, ATCC 7962 A total of 7 strains were searched for the presence of the plasmid.

Each strain was cultured in M-17 broth medium at 32 ° C. for 4 hours, and cells were collected by centrifugation.

Suspend the cells in 1 ml of 10 mM tris-NaCl (pH8.0),
Centrifuge at m for 5 minutes, discard the supernatant, and collect the cells for 6.7% sucrose-50 mM.
The cells were resuspended in 380 μm of Tris · Cl (pH8.0) and left at 37 ° C. for 5 minutes. Next, add 97 μ of lysozyme dissolved in Tris · HCl at 10 mg / ml, and leave at 37 ° C. for 10 to 15 minutes. Then add 0.25M EDTA-50mM Tris.HCl (pH8.0) 48μ, 200m
50mM Tris ・ HCl-20mM EDTA (p
28μ of SDS dissolved in H8.0) was added and mixed, and 3.0N Na
Add 28μ of OH and mix, then 2.0M Tris ・ HCl (pH 7.
Add 50μ of 0) and mix gently. 5.0M NaCl 72μ
Is added, and the mixture is ice-cooled for 1 hour and then centrifuged at 15000 rpm for 30 minutes to obtain a supernatant. To this supernatant, 500 μl TE buffer saturated phenol is added and mixed, and the mixture is centrifuged at room temperature at 15000 rpm for 5 minutes. The obtained upper layer solution is collected, and 500 μl of a chloroform-isoaluminum alcohol (24: 1) mixed solution is added and mixed. The upper layer liquid is collected, 1 ml of ethanol cooled to -20 ° C is added and mixed, and then cooled in ice water for 30 minutes.
Centrifuge at 15000 rpm for 30 minutes at 0 ° C to remove ethanol, add 20μ of TE buffer, and add RNase solution (10mg / ml, 10mM
Tris.HCl, 15mM NaCl, pH7.5) was added at 1μ and the temperature was 30 ℃ at 37 ℃.
Reaction for minutes to obtain purified DNA.

The DNA obtained as described above was electrophoresed with agarose NA (Pharmacia). As a comparative example, HindIII cleaved λ phage was treated in the same manner. The results of electrophoresis are shown in FIG. No chromosomal DNA was detected in SBT 1212 and SBT 1227.

From this result, it was confirmed that these two types of nisin-related genes exist on the chromosome. This SBT 1212, SBT 122
7 has been deposited with the Institute of Microtechnology [Deposit No. Microorganism Institute No. 11674, Microorganism Institute No. 11675
issue〕. These two strains are useful as nisin-producing strains.

Example 4. The amino acid requirement of the two strains obtained in Example 3 was examined.

Kozak and Dobrzanski medium (Kozak, W., Dobr−zanski, W.
T.; Acta Microbiologica Polonica, 26 , 361 (1977))
The medium supplemented with 10 mM sodium pyruvate was used as a completely synthetic medium. The test strain was cultured overnight in M17broth medium, washed twice with physiological saline, and then 5% in complete synthetic medium.
I inoculated. The amino acid requirement was judged by the presence or absence of growth of the strain after 28 hours at 28 ° C.

The results are shown in Table 2. Both strains showed diverse amino acid requirements.

Example 5 Enhancement of Nisin Production by Addition of Amino Acid Of the two nisin producing strains selected in Example 4, SBT 1212
The strain was used to test the enhancement of nisin production by the addition of amino acids.

SBT 1212 strain was cultured in M-17 medium. Amino acids shown in Table 3 were added to M-17 medium at 0.67 mg / ml.
After culturing at 24 ° C. for 24 hours, the nisin activity in the culture supernatant was determined in Example 2
It was measured by the cup assay method using the S. cremoris ATCC 14365 strain described in 1. As a nisin standard, Apli
It was determined from a calibration curve prepared in advance using nisin (10 ⁶ IU / g) manufactured by n & Bar-rett.

As shown in Table 3, the three amino acids Ser, Phe, and Hyp enhanced the production of nisin, and Ser and Phe were particularly remarkable. Further, as a control, only growth-requiring amino acids were added and cultured.

Ser is a constituent amino acid of nisin, and it is considered that the addition thereof promoted the synthesis of nisin. It was also considered that the addition of phenylalanine was related to glucose metabolism and the metabolism was inclined to the synthesis of serine.

Example 6. Whole milk 30 was sterilized and then cooled, and 20.1 g of serine was added to this milk, dissolved, and cultivated in advance as a starter SB
The T 1212 strain was added, and the mixture was cultured at 28 ° C for 24 hours. The nisin activity of this culture was 10 ³ unit / ml. Add hydrochloric acid to adjust the pH to 2.
As 0, the curd and whey were separated by centrifugation. The pH of the whey fraction is adjusted to 4.2 to 4.5, and this is emulsified with 5% chloroform and 0.1% secondary octyl alcohol. Then, the mixture is allowed to stand to obtain a formed chloroform gel, and a small amount of alcohol is added to this chloroform gel to obtain a precipitate of the nisin active substance. This precipitate is dissolved in dilute hydrochloric acid, adjusted to pH 2.5-3.0, and then an equal amount of alcohol is added to remove the non-precipitate as a precipitate, which is then concentrated under reduced pressure to adjust the pH to 4 and recover the precipitated precipitate did. This precipitate was dissolved in 0.01N hydrochloric acid and freeze-dried. About 11.9 g of nisin was recovered.

Effect of the Invention According to the method of the present invention, the production efficiency of nisin can be enhanced by organisms. Further, the microorganism of the present invention has a nisin-producing gene in its chromosome and can be used as a stable strain for nisin production.

[Brief description of drawings]

FIG. 1 shows agarose electrophoresis of purified DNA of lactic acid bacteria.

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Claims (3)

[Claims] 1. A nisin-producing Streptococcus lactis having the following characteristics. When DNA is extracted from the cells and tested for the presence of the plasmid, the DNA corresponding to the plasmid is not detected. Nisin production is enhanced by the addition of serine or phenylalanine to the culture. The nisin producing gene is present in the chromosome. 2. A nisin-producing Streptococcus lactis strain SBT 1212 (Microtechnology Research Institute No. 11674) or SBT 122.
Streptococcus lactis according to claim (1), which is 7 (Microbiology Research Institute No. 11675). 3. Streptococcus according to claim 1.
A method for producing nisin, which comprises culturing lactis and producing nisin, in which 0.1 to 10 mg / ml of serine or phenylalanine is added to the culture solution and culturing to enhance nisin production.