JPH05236944A - Method for culturing bacterium having activity against ice nucleus - Google Patents

Abstract

PURPOSE:To obtain a method for culturing a bacterium having activity against ice nucleus wherein a large amount of a substance sufficiently functioning as a substance having activity against ice nucleus is produced and a substance active against ice nucleus having high activity against ice nucleus is produced. CONSTITUTION:Erwinia herbicola (IFO 12,686), a substance having activity against ice nucleus is cultured in a medium containing 0.05-0.3% phospholipid (soybean lecithin or egg yolk lecithin). A culture solution containing a substance active against ice nucleus having high activity against ice nucleus is obtained.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing ice nucleation active substances. More specifically, it relates to a culture method for producing highly active ice nucleation active substances in ice nucleation active bacteria.

[0002]

2. Description of the Related Art Ice nucleation active substances are substances found because leaves of plants in which many specific bacteria are present (parasitic) are susceptible to frost damage. In the presence of ice nucleate material, it destroys normal water or aqueous solutions undercooled at higher temperatures,
Since it forms ice nuclei at -2.5 to -4.0 ° C, it is useful as a substance that causes freezing at lower energy than usual.

Therefore, application in the technical field related to freezing, such as artificial snowfall, climate control, food processing, frozen storage, and ground freezing, is expected.

[0004] The microorganisms (ice nucleation active bacteria) to produce ice nucleation active substance, the genus Pseudomonas, microorganisms of the genus Xanthomonas or Eruwinyia genus, for example, Pseudomonas syringe (Pseudomonas syringe), Pseudomonas fluorescens (Pseudomonas fluorescen
s ), Pseudomonas biridiflava ( Pseudomonas v
iridiflava ), Xanthomonas campestris ( Xant
homonas campestris), Eruwinyia-uredovora (Erwinia uredovora), Eruwinyia-Habikora (Erwinia herbicola), etc. have been known (Obata, Japan Refrigeration Association Papers, Vol. 5, No. 2, 1 over 10 pages).

[0005] Attempts have been made to produce ice nucleation active substances with high efficiency in these ice nucleation active bacteria, and a method of culturing by changing the medium composition, for example, changing the carbon source (JP-A-1-171477). Alternatively, a method of culturing by changing the nitrogen source (JP-A-1-171477, JP-A-2-76595) is disclosed.

However, these conditions are mainly intended to improve the cell yield of microorganisms.

On the other hand, as a result of academic research on the expression of the activity of the ice nucleation active substance, the expression of the ice nucleation activity is not limited to the ice nucleus protein which becomes the nucleus of ice nucleation, and some complex lipid (phospholipid, lipopolysaccharide). Are believed to be involved [Jo
urnal of Biological Chemistry, 263, 9333 (198
8)]. Nevertheless, no studies have been conducted to utilize such knowledge for improving the production of ice nucleation active substances by culturing ice nucleation active bacteria.

[0008]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned conventional problems, and an object thereof is to provide a method for culturing ice nucleus-activating bacteria which achieves improvement of ice nucleus activity.

[0009]

The method for culturing ice nuclei-activating bacteria of the present invention includes a step of culturing ice nuclei bacteria in a nutrient medium containing phospholipids, whereby the above object is achieved.

The ice-nucleating bacteria used in the present invention include:
Any bacterium can be used as long as it produces an ice nucleation active substance, but it is preferable to use one having a property of releasing the ice nucleation active substance to the outside of the bacterial cell such as Erwinia herbicola.

For culturing ice nucleation-active bacteria, a known nutrient medium to which phospholipids have been added is used. For example, in the nutrient medium, sugars such as glucose and saccharose, organic acids, alcohols and the like can be used as carbon sources. As the nitrogen source, in addition to inorganic nitrogen sources such as ammonia, yeast extracts, proteins such as trypticase and soy, and organic nitrogen sources such as amino acids can be used. In addition, vitamins and inorganic salts may be added. Each concentration can be determined depending on the microorganism.

In the present invention, based on the above medium,
Used with phospholipids added. As phospholipids, ice nuclei produced by adding phospholipids such as phosphatidylinositol (PI), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) to a nutrient medium and culturing It is intended to provide a culture method for improving the ice nucleus activity of an active substance. Any phospholipid can be used. For example, if a commercially available product is used, soybean lecithin (PI: PC: PE) is used.
= 34: 37: 29), egg yolk lecithin (PI: PC: P)
E = 17: 82: 1) and the like can be used. It is preferable that these are contained in the medium in an amount of at least about 0.01 W / V%, and if the content is lower than this, they are merely metabolized as a nutrient source, and the ice nucleus activity improving effect cannot be obtained. Preferably,
0.05-0.3 W / V% (phospholipid content 95% [W / W]
As a degree is good.

When culturing is carried out in a natural medium containing an appropriate amount of phospholipids or a medium prepared from agricultural and livestock wastes, it is not necessary to add phospholipids, and when phospholipids are newly added to the synthetic nutrient medium. It is also possible to obtain an activity yield similar to.

The timing of addition of the phospholipid is not particularly limited, but it is preferably at the start of the culture, and if added from the early stage of the culture, a highly active ice nucleus active substance can be obtained.

Culturing of ice nucleating bacteria can be carried out under known culture conditions. The culture temperature is generally 10 ° C to 35 ° C, and the culture can be performed aerobically, and the culture can be performed by shaking or aeration and stirring.

The ice nucleation activity of the ice nucleation active substance contained in the culture medium can be determined by the ice nucleation spectrum, and the ice nucleation spectrum is determined by the method of G. Vali [Journal of Atmo.
Spherical Sciences, Volume 28 (1971), Volume 402
Page]] is obtained as follows. First, an aluminum wheel was brought into close contact with a copper plate of an ice nucleus spectrometer (manufactured by Mitsuwa Rikagaku Co., Ltd.) using vacuum grease, and 30 μl of 10 μl of the sample was dropped on the aluminum wheel, and the copper plate was heated at a rate of 1 ° C./min. Cool and count the number of frozen water drops at each temperature. Then, the number of ice nuclei per unit volume at each temperature is obtained by the Poisson distribution formula below and plotted against the temperature to obtain an ice nucleation spectrum.

N (T) =-(lnf) / V Here, N is the number of ice nuclei per 1 ml at the temperature T, and f is the ratio of unfrozen water droplets among all the water droplets (30 pieces) at the temperature T (that is, Frozen water drops / 30) and V represent the volume of one water drop (0.01 ml).

[0018]

INDUSTRIAL APPLICABILITY According to the present invention, when ice-nucleus-active bacteria are cultured in a medium containing phospholipid, a large amount of substances sufficiently functioning as ice-nucleus active substances are produced and the activity yield can be improved.

Phospholipids used for culture are available as inexpensive ones such as soybean lecithin and egg yolk lecithin which can be easily recovered from industrial by-products, and are suitable for mass-culturing ice-nucleus-active bacteria on an industrial level.

Hereinafter, description will be made based on examples.

[0021]

【Example】

(Example 1) In 1 liter of distilled water, 30 g of a commercially available yeast extract, 2.0 g of DL-serine, 2.0 g of DL-alanine, 10.0 g of saccharose and 8.6 of K ₂ SO ₄ were used.
and a liquid medium containing 1.4 g of MgSO ₄ .7H ₂ O at pH 7.
It was adjusted to 0, sterilized under high pressure at 120 ° C. for 20 minutes, and cooled. Next, Trypticase soy slant medium (casein 17.0) was added to 10 ml of the medium.
g, kidney bean powder 3.0 g, NaCl 15.0 g, K ₂
HPO ₄ 2.5 g and glucose 2.5 g, agar 15 g
1 platinum loop of Erwinia herbicola (IFO 12686) cultured in 1 liter of distilled water was prepared and precultured at 18 ° C. for 16 hours.
0.1 ml of the preculture liquid was added to 100 ml of the above liquid medium to which soybean lecithin or egg yolk lecithin (all manufactured by Nacalai Tesque) was added at a rate of 1 g / L.
Main culture was performed at 18 ° C. for 24 hours. After culturing, 7.0
The cells were centrifuged at 00 rpm for 30 minutes to remove the cells, and the culture solution was collected. The ice nucleus formation spectrum of the culture solution is shown in FIG.

Comparative Example 1 The same operation as in Example 1 was carried out except that a medium containing no phospholipid was used. The result is shown in FIG.

When soybean lecithin and egg yolk lecithin from Example 1 and Comparative Example 1 were added from the initial stage of culture,
It can be seen that the ice nucleus activity is enhanced as compared with the case where no ice is added.

(Example 2) Soybean lecithin (1 g / g) was added to a medium having the same composition as the slant medium described in Example 1 except that 0.1 ml of the preculture liquid obtained in Example 1 was not added to the agar. l) was added to 100 ml of the medium, and this was used to carry out main culture at 18 ° C. for 24 hours following preculture. The ice nucleation spectrum of the culture solution is shown in FIG.

Comparative Example 2 The same operation as in Example 2 was carried out except that a medium containing no phospholipid was used.

From the above-mentioned Example 2 and Comparative Example 2, it can be seen that even if the medium is changed, the one to which soybean lecithin is added from the early stage of culture has high ice nucleus activity.

Example 3 and Comparative Example 3 The culture solution obtained by adding and culturing the soybean lecithin of Example 1 was concentrated using an ultrafiltration membrane having a molecular weight cut off of 300,000, and a test sample was obtained. 10m so that the protein concentration is 20 μg / ml
It was adjusted with M Tris-HCl buffer (pH 7.0).

On the other hand, the culture solution of Comparative Example 1 was treated and diluted similarly to obtain Comparative Example 3.

FIG. 3 shows the ice nucleus formation spectra of the respective ice nucleus active substance-containing liquids.

Even if low-molecular substances such as phospholipids in the culture solution were removed by ultrafiltration, the ice nucleation activity did not change.

Reference Example 1 Both were not inoculated with bacteria,
The ice nucleation temperatures of the medium containing soybean lecithin used in Example 1 and the medium containing no soybean lecithin of Comparative Example 1 were measured according to the method of Example 1. The results are shown in Table 1.

[0032]

[Table 1]

From Table 1, it was found that the phospholipid itself does not affect ice nucleation.

Reference Example 2 50 μ of 0.1 M phosphate buffer (pH 7.0) containing 500 μg of soybean lecithin in 1 ml of the culture obtained by culturing under the same culture conditions as in Comparative Example 1.
After adding 1 and mixing, the mixture was left at 4 ° C. for 5 hours.

On the other hand, 1 ml of the culture solution obtained by the above-mentioned culture contains 0.1 M phosphate buffer solution (pH not containing soybean lecithin).
7.0) 50 μl was added and mixed, and the mixture was left at 4 ° C. for 5 hours.

FIG. 4 shows the ice nucleation spectrum of each ice nuclei-containing solution.

From FIG. 4, it was found that the addition of phospholipids to the culture medium after the completion of the culture did not show the activation effect.

[Brief description of drawings]

FIG. 1 is an ice nucleation spectrum when ice nucleating bacteria were cultured in a medium containing phospholipid and a medium not containing phospholipid.

FIG. 2 is an ice nucleation spectrum when ice nucleating bacteria are cultured in slant medium containing phospholipid and medium not containing phospholipid.

FIG. 3 shows a culture solution obtained by culturing ice nuclei-activating bacteria using a medium containing phospholipid and a medium not containing phospholipid, respectively.
It is an ice nucleation spectrum of each of which the molecular weight was fractionated and the protein concentration was adjusted.

FIG. 4 shows a culture solution obtained by culturing under the same culture conditions as in Comparative Example 1 with and without phospholipids added,
Each is an ice nucleus activity spectrum after standing at 4 ° C.

Claims (1)

[Claims] 1. A method for culturing ice nuclei active bacteria, which comprises the step of culturing ice nuclei active bacteria in a nutrient medium containing phospholipids.