JPH0276595A - Production of ice nucleus active substance - Google Patents

Abstract

PURPOSE:To readily produce an ice nucleus substance at a low cost by culturing a specific ice nucleus active bacterium in a culture medium containing protein and/or amino acid and then subjecting the cultured product to membrane treatment to separate bacterium cell. CONSTITUTION:Erwinia uredovora (FERM P-10186) and Pseudomonas viridiflava (FERM P-10185) which are ice nucleus active bacteria are cultured in a culture medium containing protein and/or amino acid. The above-mentioned culture is preferably carried out at pH5-8 and 13-35 deg.C for 10-96hr. Then the cultured product is subjected to membrane treatment at temperature lower than that of culture temperature, preferably <=20 deg.C using a nitrocellulose membrane having 0.2-0.45mum pore size to separate bacterial cell and ice nucleus substance- containing liquid. The obtained ice nucleus substance-containing emulsion is directly preserved or freeze-dried and then preserved.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention This invention relates to a method for producing ice-nucleating substances from ice-nucleating bacteria.

An ice-nucleating bacterium that is expected to be used in applications such as artificial snowfall and food freezing preservation is Nyutomonas ngringae <Ps.
eudomonas syringae: l: Maki (I
・, Iku Maki) et al., 8pp1. M 1crobi
o1. Volume 28 (197=l), p. 456], Pseudomonas fluorescens
as Nuorescens) (Maki et al., supra),
P seu-dom
onas viridiflava) [Bolin (J
, P. Paulin), et al., Proc., 4 Lh
lnt, cont, PlanL Path,
Bact.

No. 725N (] 978)], Erwinia herbicola (No. 1 rwinia herbicola) [Gentian (S, E.

Lindow et al., Phytopathology,
Vol. 68 (1978), p. 523 1 and Erwinia uredov
ora Tei Kobuka et al., Proceedings of the 55th Autumn Annual Meeting of the Chemical Society of Japan 1■, No. 6+8 @ (1987)] are known.

However, it is difficult to put this kind of ice-nucleating active bacteria into practical use because it poses problems such as environmental pollution and frost damage to agricultural products when used as living cells in a single vehicle.

In order to solve these problems, a method has been proposed to sterilize ice-nucleating bacteria using radiation, and attempts have also been made to create artificial snow using the sterilized cells themselves as ice-crystal nuclei. One drawback is that complete sterilization significantly reduces ice nucleation activity.

This invention was made in order to provide an ice nucleating active material that does not have the problems and drawbacks mentioned in 1.

Means for solving the problem, namely, the present invention, comprises culturing ice-nucleating active bacteria selected from the group consisting of Erwinia urethophora and Neudomonas viridifrach in a protein- and/or amino acid-containing medium, and subjecting the culture to membrane treatment. The present invention relates to a method for producing an ice-nucleating active substance, which is characterized by separating bacterial cells.

The ice-nucleating active bacteria used in the present invention, Erwinia uredopora and Shuuthomonas biltifurach, were sent to the National Institute of Microbial Technology, Agency of Industrial Science and Technology, respectively.
．． No. 0186 and No. 10185.

The medium for inoculating the above-mentioned ice-nucleating bacteria contains protein and/or amino acids as an organic nitrogen source, such as yeast extract, meat extract, malt extract, peptone, dogwood powder, corn/powder, casein, serine, alanine, purine,
It contains loin, lysine, threonine, thyronine, glutamic acid, etc., and also contains hitamine B1, hitamine B7, folic acid, nicotinic acid, pantothenic acid, biotin, etc.
Furthermore, the usual cultivation l'I! ! Ingredients such as sugar, magnesium sulfate, potassium sulfate, potassium chloride, ammonium sulfate, potassium hydrogen phosphate, potassium dihydrogen phosphate, ammonium dihydrogen phosphate, citric acid, sodium citrate, glucose, fructose, malto- Contains starch, starch, etc. as appropriate.

The pH of the medium is usually 5 to 8, and even if the pH is lower than this range, the growth of ice nucleating activity sterilization will be lowered and the ice nucleating activity of the ice nucleating substance will also be reduced.

The culture temperature and culture time for ice nucleation activation sterilization in the medium described in 1. are not particularly limited, but are usually 10 to 35°C, preferably 14 to 20°C for 10 to 96 hours, preferably 2
0 to 48 hours.

The culture is subjected to membrane treatment to separate the ice-nucleated active substance-containing liquid from the bacterial cells.

The pore size of the membrane is not particularly limited as long as it is large enough to separate the bacterial cells and the ice-nucleating active substance. The size of the gap is about 12 to 16 μm, and the size of the ice core active material is about 0.1 μm.
Therefore, a membrane with a pore diameter of 0°1 to 1.2 μm can be used.

For such a membrane, the pore size should be 0.2μm or 0.4μm.
A 5 μm nitrocellulose type membrane is exemplified.

The membrane treatment temperature is usually below the culture temperature, preferably below 20°C, and at high temperatures the ice nucleating activity of the ice nucleating substance decreases.

By subjecting the culture to centrifugation in advance before the membrane treatment described above, more efficient membrane treatment can be performed. Centrifugation is usually 5.000 to 20.00 Or
, p, preferably ]/1,000-] 6. OO
Do this with Or, p, and m.

If desired, the ice-nucleated active substance-containing liquid separated from the bacterial cells by membrane treatment may be further subjected to ultrafiltration treatment to remove low molecular weight components.

As the ultrafiltration membrane, a polysulfone type membrane having a molecular weight cut off of 30,000 is exemplified.

The ice-nucleating substance-containing emulsion (colloidal dispersion) thus prepared may be used or stored as it is, or may be lyophilized and stored if desired.

Whether it is known that a protein is involved in the ice nucleating active substance produced by the present invention, or the structure thereof is unknown and must wait for future research.

Hereinafter, the present invention will be explained by examples.

Example 1 Commercially available yeast extract 20g 1 sodium citrate 1.0g
, 2SO4o, 86g and M g SO4.7H
,OO,]4g]00m1! Dissolved in water at pH 7.
, ], autoclaved at 115°C for 15 minutes, and cooled, trypticase soy (Tryplicas
e 5oy) Culture solution (duck 421f, 0 bean powder 3.0
g, NaCl 5. Og, K, H P 0425g
1 mρ (3.0 x 108 cells/m) of a culture of Erwinia uretopo, or Eudomonas viridiflava presolvated with 25 g of glucose (dissolved in 1 liter of water) was added and incubated at 16°C for 12/1 h. The culture was cultured with shaking.The culture solution was centrifuged, and the upper layer was separated from the membrane (0.2μm
The living cells were further separated by ultrafiltration (molecular weight cutoff, 30,000) to obtain an emulsion (colloidal dispersion) of the ice-nucleating active substance.

Kushikushi carp-4 Commercially available Q? Meat extract 2. Og, glucose I L N
aCl 0.5g. , MgSO3・7H to 0
, 0 2 g-N" 41-1, PO4 0.Ig
and HPO40. Ig was dissolved in 100 mQ of water, adjusted to pI (7.I), autoclaved at 115°C for 15 minutes, and cooled. After germination, 16℃9
Culture was carried out with shaking for 24 hours. The culture solution was centrifuged, and the two layers were separated using a membrane (0, 2 μm or 0.45 μm) to further separate living cells and ultrafiltration (molecular weight cut off, 3
10,000) to obtain an emulsion (colloid-shaped dispersion) of the ice-nucleating active substance.

γ裡”-β 20 g of commercially available yeast extract, 0.5 g of glucose, 0.0 g of sodium citrate. 0 5g, (N+-14), S
0. OIg, K1-1, PO40.7g, l-
lPO40.7g and Mg5O, 71-1,0
Dissolve 0.02g in loOmQ water and obtain pI-(7.1
After sterilizing under high pressure at 115°C for 15 minutes and cooling, 1 ml of the culture solution of this strain precultured with trypticase soy culture solution was inoculated, followed by shaking culture at 16°C for 124 hours. The culture solution is centrifuged and the "-layer" is separated from the membrane (
The living cells were further separated by 0.062 μm or 0.45 μm) and subjected to ultrafiltration (molecular weight cut off, 30,000) to obtain an emulsion (colloid-like dispersion) of the ice-nucleating active substance.

Example-↓ Commercially available yeast extract 3. 0g, Serine 0 2g, Ara =
7-nin 0.2g,/yose sugar 1. gSK2SO4 0.86g
5MgSO4.7H, o0, 14g and KCI O71/Ig were dissolved in 100mρ water, adjusted to pI-(7.1, autoclaved at 115°C for 15 minutes, cooled,・After transplanting the culture solution of this strain pre-cultured with soybean culture solution]mQ, the culture solution was grown at 16℃.
It was cultured with shaking for 124 hours. Centrifuge the culture solution,
The upper layer was further separated from living cells using a membrane (0.2 μm or 0.45 μm), and ultrafiltration (molecular weight cut off, 3
10,000) to obtain an emulsion (colloidal dispersion) of the ice-nucleating active substance.

Examples 5-7 An emulsion (colloidal dispersion) of an ice-nucleating active substance was prepared in the same manner as in Example 1, except that peptone, meat extract, soybean powder, corn powder or malt extract was used instead of yeast extract. Obtained.

Example 8〉↓U Peptone, meat extract, soybean powder, 1 instead of yeast extract
- A milk/'lA liquid (colloidal dispersion) of an ice nucleating active substance was obtained in the same manner as in Example 1, except for using Umorokon powder or malt extract.

An emulsion (colloidal dispersion) of an ice-nucleating active substance was prepared in the same manner as in Example 3, except that peptone, meat extract, soybean powder, corn powder, or malt extract was used instead of yeast extract. liquid) was obtained.

Kaoru no L Juju]) - Peptone, meat extract, soybean powder, instead of Touko No. 1 extract
An emulsion (colloidal dispersion) of an ice nucleating active substance was obtained in the same manner as in Example 4 except that corn powder or malt extract was used.

The freezing temperature of the acid solution obtained by treating the culture cultured under the conditions of Example 4 using a membrane with a pore size of 02 μm or 0.457 zm was determined by G. Vali. How to CJ own
IO “8tmosphere sciences,
Vol. 28 (1971), p. 402], 1lt.
l+ was determined.

The measurement results are shown in Table-1.

■Example! ．． Example 1 except that a culture solution similar to that used in Example 4 was used instead of the culture cultured under the conditions of Example 4.
The freezing temperature of the filtrate was measured in the same manner as in Examples 7 and 18, and the results are shown in Table 1.

EXAMPLE The ice nucleation spectrum of the emulsion (colloidal dispersion) of the ice nucleating active substance prepared using Erwinia uretopora in Example 1 was lint (S.

E, Lindow) et al.'s method [P] ant P
Hysion, Volume 70 (1982), No. 108
Page 4].

The results are shown in (1) in FIG.

Comparative Example 2 The ice nucleation spectrum of living cells of Erwinia urea was measured in the same manner as in Example 19, and the results are shown in FIG.

Example 20 Emulsion (colloidal dispersion) of ice-nucleating active substance prepared using Shutomonas viridifrapa in Example 1
The ice nucleation spectrum of was measured in the same manner as in Example 19, and the results are shown as (4) in FIG.

1 Geigencho” The ice nucleation spectrum of living cells of Shutomonas bilitiflava was measured in the same manner as in Example 19, and the results were
Indicated by (5) in the figure.

Practical Example 7A The ice nucleation spectrum of silver iodide was measured in the same manner as in Example 19, and the results are shown in (3) in FIGS. 1 and 2.

50% of a droplet of 10 μρ freezes jm + degree (1)) When using a membrane with a pore size of 0.2 μm (c) When a membrane with a pore size of 0.2 μm is used. /15μ
According to the present invention, when a membrane of M is used, it not only has no inferiority in terms of ice nucleus activity to that of a living cell (111), but also has no effect on environmental pollution or agricultural products. Ice nucleating active substances that do not cause problems such as frost damage can be easily produced at a low cost.

Therefore, the ice core active substance obtained by the present invention can be used in a wide range of applications such as artificial snow, artificial rain, ice heat storage agents, freezing agents, artificial ground freezing agents, food freezing agents, etc. ]! It can be used in f.

[Brief explanation of the drawing]

Figures 1 and 2 are ice nucleation spectra. (1) is an ice-nucleating active substance prepared using Erwinia urethora, (2) is a living cell of Erwinia urethora, (3) is silver iodide, and (4) is prepared using Shutomonas viridifrach. Ice-nucleated active substance and (5
) shows the ice nucleation spectra when living cells of Shutomonas virideifrach were used. Patent applicant: Hitoshi Obata and two other attorneys; one patent attorney, Haka Aoyama Figure 1: Written by ε Miyako. (''C) Procedural amendment written by the Commissioner of the Patent Office ☆ January 25, 1999 2 Title of the invention Process for producing ice-nucleated active substance 3 Relationship to the case of the person making the amendment Patent applicant address 6 Mukonosho, Amagasaki City, Hyogo Prefecture Chome 3-5 Name: Hitoshi Kobaku (and 2 others) 7. Contents of amendment (1) The "Claims" column of the specification will be amended as shown in the attached sheet. (2) In the specification, page 3, lines 12 to 14, "Erwinia 9. Ice-nucleating bacteria" is corrected to "Erwinia uretopora, ice-nucleating bacteria." (3) Ibid., page 3, line 19 to page 4, line 2, ``and deposited'' was replaced with ``deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology, No. 10186 It was deposited as ``.'' (4) Ibid., p. 7, lines 6 to 7, ``or...biriteifrachj'' is deleted. (5) Ibid., p. 10, line 12, ``and 18'' is deleted. Delete. (6) ``Ibid., page 11, line 2, and 18'' are deleted. (7) Same, page 11, line 4, ``Example 19'' is corrected to ``Example 18 Snow.'' (8) Same, page 11, line 14, replace ``19'' with ``
18,” he corrected. (9) Same, page 11, line 16 to page 12, line 5, [
Example 20 is shown below. ” will be deleted. (10) Same, page 12, line 6, [Comparative Example 4] is corrected to "Comparative Example 3." (11) Same, page 12, line 7, replace "19" with "
18,” he corrected. (12) Ibid., page 12, line 8, the words ``and Figure 2'' are deleted. (13) Same, page 12, Table 1 is corrected as follows (a
) Emulsion (colloidal dispersion) of ice-nucleated active substance 10μρ
The temperature at which 50% of the droplets freeze (b) When using a membrane with a pore size of 0.2 μF (c) When using a membrane with a pore size of 0.45 μF” (14) Ibid., p. 13, line 10 , ``and Figure 2'' are deleted. (15) Ibid., p. 13, lines 14 to 17, ", (3
) is silver iodide49. ``living cells'' is replaced with ``and (3)
) is corrected to "silver iodide." (16) Figure 2 attached to the specification will be deleted. ”- =4=

Claims (1)

[Claims] 1. Erwinia uredovora and Pseudomonas
A method for producing an ice-nucleating substance, which comprises culturing ice-nucleating bacteria selected from the group consisting of Viridiflava in a protein- and/or amino acid-containing medium, and subjecting the culture to membrane treatment to separate the bacterial cells. . 2. The method according to claim 1, wherein the membrane has a pore diameter of 0.1 to 1.2 μm. 3. Culture at 10-35°C and pH 5-8.
The method according to claim 1, which is carried out for 96 hours. 4. An ice nucleating active substance obtained by the production method according to claim 1.