Abstract:
Disclosed herein are a new mutant of Pseudomonads having ice nucleating activity and an improved method for making snow and ice using it. More particularly, Pseudomonas syringae SO754 of the present invention, which is derived from the parent strain, Pseudomonas syringae SO7 can retain its ice nucleating activity at room temperature. 
     Further, Pseudomonas syringae SO754 of the invention retains its high activity during the fermentation, recovery and drying steps and during the storage without freezing treatment.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the invention 
     The present invention relates to a new mutant having ice nucleating activity and an improved method for making snow and ice using it. More particularly, the present invention relates to a new mutant, Pseudomonas syringae whose ice nucleating activity is stable at room temperature and relates to an improved method for making snow and ice using it. 
     2. Description of the Prior Art 
     In U.S. Pat. No. 4,200,228, there is disclosed a method for the making of snow by suspending and then spraying microorganisms into the air. The microorganisms that are employed in this invention are of the type which are known to promote ice nucleation and a use of such microorganisms makes it possible to make snow at much higher temperatures than are ordinarily possible. Further, there is also disclosed a method for the recovery, in a dry form, of microorganisms that have ice nucleating activity from a medium containing the microorganisms that are grown in a conventional manner. 
     In U.S. Pat. No. 4,706,463, there is disclosed a method for recovering microorganisms that have ice nucleating activities. In this method, the temperature of the fermentation medium which contains Pseudomonas syringae is brought to a temperature of about 15° C. or less, a concentrate of said microorganism is formed and the concentrate is cooled to form frozen pellets, which is then frozen dried at a temperature below 25° C. 
     In U.S. Pat. No. 4,796,805, there is disclosed a method for making ice using ice nucleating microorganisms. The method comprises the steps of forming an aqueous suspension of ice nucleating microorganism, introducing the suspension into a water source to form an ice nucleated water source at a temperature below about 13° C. and distributing and freezing the ice nucleated water source. However, it was found that if the said suspension was allowed to stand at a temperature of 21° C. for about 24 hours, it completely lost the ice nucleating activity. 
     All of these prior arts are directed to prevent the employed ice nucleating microorganisms from losing their activities at room temperature. 
     Ice nucleating microorganisms that had been employed in these inventions mentioned above are for example Pseudomonads, particularly P. syringae. The preferred microorganism is P. syringae ATCC 53543 deposited on Sep. 23, 1986 with American Type Culture Collection. Another suitable examples of microorganisms that have ice nucleating activity are P. syringae KCTC 1832 deposited with Korean Collection for Type Cultures in Korea and P. syringae IFO 3310 deposited with Institute for Fermentation, Osaka in Japan. 
     Mr. L. R. Maki and his coworkers first discovered an ice nucleating microorganism in 1974(L. R. Maki et al., Applied Microbiology Vol. 28, p456, 1974), and thereafter many ice nucleating microorganisms have been extracted from leaves of various plants. 
     Examples of microorganisms having ice nucleating activity are Pseudomonas syringae, Pseudomonas fluorcens, Pseudomonas pici, Pseudomonas coronafaciens, Pseudomonas viridiflava, Erwinia herbicola, Erwinia uredovara, Erwinia ananas and Xanthomonas campestris. Pseudomonas syringae, among others, has higher ice nucleating activity and has been widely employed in the industry. 
     Unfortunately, however, these microorganisms are not stable and easily lose their ice nucleating activities at room temperature. As a result, in order to retain their ice nucleating activities, these microorganisms should be cultured, recovered and dried only at low temperature. Further, the suspension of these microorganisms also needs to be stored at low temperature. 
     Besides, in case that these ice nucleating microorganisms are employed for the ice production of an industrial scale, a large amount of microorganism cells are needed and these microorganisms should be maintained at low temperature during culture, recovery and drying process, transport and storage. These restrictions increase the cost and make the processes complex. Accordingly, there has been a need to provide a new strain which has ice nucleating activity at a higher temperature, for example at room temperature. 
     The present inventors had made researches to provide a new strain which has nucleating activity even at room temperature and as a result thereof could provide Pseudomonas syringae SO754 retaining ice nucleating activity stably at room temperature. Pseudomonas syringae SO754 of the invention retains high activity during the fermentation, recovery and drying steps and during transport and the storage. 
     SUMMARY OF THE INVENTION 
     Therefore, an object of the present invention is to provide a new strain which has ice nucleating activity at a temperature below about 25° C. 
     And an other object of the present invention is to provide an improved method for making ice at room temperature using said strain. 
     DETAILED DESCRIPTION OF THE INVENTION 
     Ice nucleating microorganisms may be extracted from the leaves of the frosted plant. These microorganisms are subjected to mutations to select a mutant still having ice nucleating activity at room temperature. The mutation and selection procedures are as follows. 
     Isolation of ice nucleating microorganisms 
     The leaves of the frosted plant were washed with a physiological solution and then the obtained solution was cultured in the nutrient agar medium which contains 0.3% of meat extract, 0.5% of peptone, 2.5% of glyceral and 1.5% agar at a temperature of about 30° C. for 2 days. 
     Thereafter, for the first isolation, the fermentor was moved to the freezer of -4° C. and maintained there for about 10 minutes. And strain colonies which showed ice nucleation were isolated. 
     Each of 75 isolated colonies was cultured at a temperature of 30° C. And then strains showing the highest ice nucleating activity were secondly isolated. The second isolation was carried out by comparing ice nucleating activities of all the colonies. 
     The ice nucleating activity was determined as follows: The isolated strains were cultured in a nutrient liquid medium which contains 0.3% of meat extract, 0.5% of pepton and 2.5% of glycerol at 30° C. for 24 hours. The cultured strains were recovered and then suspended in 50 mM phosphate buffer solution(pH 7). Under this condition, ice nucleating activity could be determined by measuring the temperature at which ice nucleation was observed. 
     Ice nucleating activity was determined with the Freezing nucleus spectrometer(Mitsuwa model K-1, Japan) by the method of Mr. Vali (Vali, G.: J. Atmos. Sci., 28, pp402-409, 1971). 
     From 75 colonies, one colony which had the highest ice nucleating activity was isolated, which was used as a parent strain to obtain an improved mutant strain. 
     The identification of parent strain 
     The identification of parent strain was carried out by following the method of Mr. Krieg(Krieg, N. R. : Bergey&#39;s manual of systematic bacteriology, Vol 1, 1984) and morphological, fermentative and physiological characteristics are shown in Tables 1 and 2. 
     From the results of Tables 1 and 2, the parent strain was identified as Pseudomonas syringae, and designated as Pseudomonas syringae SO7. 
     
                       TABLE 1______________________________________The morphological and fermentative characteristicsof the parent strain SO7Morphology          short rod______________________________________Size                0.7˜1.0 × 1.2˜1.6 μmGram-stain          negativeMobility            positiveColor of colony     light yellow-whiteOxygen requirement  aerobicOptimum growth temperature               28° C.Fluorescent pigment positiveGrowth at 41° C.               negativeLevan production    positiveArginine degradation               negativeOxy-Meige reaction  negativeDenitrification     negativeGelatin liquefaction               positiveStartch hydrolysis  negative______________________________________ 
    
     
                       TABLE 2______________________________________The physiological characteristics of the parent strain SO7Carbon source     Availability                 Carbon source                              Availability______________________________________Glucose   +           L-Alanine    +D-Ribose  ±        D-Alanine    -L-Arabinose     ±        L-Leucine    -D-Mannose ±        L-Histidine  -D-Fructose     ±        L-Tyrosine   -Raffinose +           L-Tryptophan -Fumarate  +           Putrescine   +Sucrose   ±        Sarcosine    +Saccharate     -           Linolenate   -Valerate  +           Ascorbate    +Pyruvate  +           Lecithin     -Sorbitol  ±        Asparagine   -______________________________________ 
    
     Preparation of mutant 
     The parent strain SO7 isolated as described above, had considerably high ice nucleation activity at 30° C. But unfortunately its activity was not stable against heat. Therefore, for the purpose of preparing new ice nucleating microorganism having the increased stability to heat, the parent strain SO7 was mutated and treated with heat. And then, mutant whose ice nucleating activity was very stable to heat was screened by measuring its ice nucleating activity. 
     Mutation was carried out by conventional technique using ultraviolet rays as a mutagen. 
     Pseudomonas syringae SO7 was cultured in a nutrient glycerol medium at 30° C. until an exponential growth phase. The recovered strains were suspended in a physiological solution, streaked onto a nutrient glycerol agar and then irradiated with a ultraviolet ray until extinction rate of germ became 95%. Immediately thereafter, the fermentor was moved to a dark room of 30° C., where culture was carried out for 24 hours. 
     Living strain colonies were replicated using a sterilized velvet and cultured at 30° C. for 24 hours. Replica was placed on the cooling bath of -4° C. Of them, a few colonies which showed ice nucleation earlier than the others were isolated. 
     The ice nucleating activity of the isolated colonies were measured by Freezing nucleus spectrometer. Among them, the strains whose ice nucleating activities were high and retained at room temperature were selected. These strains were cultured in a nutrient glycerol medium at 30° C. for 48 hours and centrifuged. The recovered strain pellets were washed with a physiological solution and freeze-dried. The freeze-dried strain pellets were suspended in 50 mM phosphate buffer solution(pH 7.0) and maintained in constant temperature bathes each of which temperature was 40° C., 35° C., 30° C., 25° C., 20° C., 10° C. and 4° C. respectively. After 24 hours, their activities were measured using Freezing nucleus spectrometer. 
     In these manner, a mutant whose activity was the highest and stable at room temperature was identified and designated as Pseudomonas syringae SO754. Pseudomonas syringae SO754 was deposited on July 19, 1993 in accordance with the Budapest Treaty with Korean Culture Culture Center of Microorganisms (KCCM), Department of Food Engineering, College of Engineering Yonsei University, Sodaemun-gu, Seoul 120-749, Korea and given an accession number KCCM-10039. 
     The mutant according to the present invention shows the same microbiological characteristics as those of the parent strain SO7 except the former strain retain its high ice nucleating activity at high temperature, and can be cultured in a fermentation medium which is commonly employed for the culture of Pseudomonas syringae and contains a suitable nitrogen and carbon source and inorganic salts under the controlled pH. These compositions of fermentation medium and culture conditions can be optimized without difficulty by any one skilled in the art. 
     Although the largest amount of solid ice nucleating microorganisms which had been used in the prior art can be collected at 28°˜30° C., these microorganisms lose their activities at this range of temperature. Therefore they should be cultured at a low temperature below about 21° C. 
     However, the mutant according to the present invention shows good ice nucleating activity at a temperature below 60° C., particularly at a temperature below 40° C. 
     PREFERRED EMBODIMENTS OF THE INVENTION 
     The present invention shall be illustrated in more detail by way of the following examples. The following examples are merely illustrative and it should be understood that the present invention is not limited to these Examples. 
    
    
     EXAMPLE 1. 
     Five samples(A, B, C, D and E) of Pseudomonas syringae SO754 (KCCM-10039) were cultured for 24 hours in a 3 liter fermentor containing nutrient glycerol medium [0.3% of meat extract, 0.5% of peptone and 0.5% of glycerol] at the aeration of 1.0 VVM and pH of about 6.0. The culture temperatures were 15° C. (sample A), 21° C. (sample B), 25° C. (sample C), 28° C. (sample D) and 30° C. (sample E), respectively. 
     After culture was completed, broths were centrifuged and pellets were collected. The collected pellets were suspended in 50 mM phosphate buffer solution(pH 7.0) and their ice nucleating activities were determined by Mr. Vail&#39;s technique(Vail, G. J. : Atomos. Sci., 28, pp402-409, 1971). Ice nucleating activity is determined by measuring the temperature(T50%) at which 50% of total amount of sample was frozen. 
     The activity of sample A(T50%) was -2.6° C.; sample B, -2.5° C.; sample C, -2.8° C.; sample D, -2.8° C.; and sample E, -3.0° C. 
     COMPARATIVE EXAMPLE 1. 
     In this Example, the procedure of Example 1 was repeated except that Pseudomonas syringae ATCC 53,543 was used. 
     The activity of sample A(T50%) was -2.7° C.; sample B, -2.8° C.; sample C, -3.5° C.; sample D, -4.2° C.; and sample E, -4.9° C. 
     The results in Example 1 and comparative Example 1 are shown in the following Table 3. 
     
                       TABLE 3______________________________________Culture          Ice nucleating activity(T50%)Sample  Temperature  Example 1 C. Example 1______________________________________A       15° C.                -2.6° C.                          -2.7° C.B       21° C.                -2.5° C.                          -2.8° C.C       25° C.                -2.8° C.                          -3.5° C.D       28° C.                -2.8° C.                          -4.2° C.E       30° C.                -3.0° C.                          -4.9° C.______________________________________ 
    
     EXAMPLE 2. 
     Four samples(F, G, H and I) of Pseudomonas syringae SO754(KCCM-10039) cultured at 28° C. by the procedure of Example 1, were dried with a spraying dryer until the final temperatures of the dried sample were 60° C.(sample F), 50° C.(sample G), 40° C.(sample H) and 30° C.(sample I), respectively. 
     The activity of dried sample F(T50%) was -5.2° C.; sample G, -4.1° C.; sample H, -3.6° C.; and sample I, -3.1° C. 
     COMPARATIVE EXAMPLE 2. 
     In this Example, the procedure of Example 2 was repeated except that Pseudomonas syringae ATCC 53,543 was used. 
     The activity of dried sample F(T50%) was -8.3° C.; sample G, -6.7° C.; sample H, -5.7° C.; and sample I, -3.8° C. 
     The results in Example 2 and comparative Example 2 are shown in the following Table 4. 
     
                       TABLE 4______________________________________Final temperature            Ice nucleating activity(T50%)Sample of dried sample                Example 2  C. Example 2______________________________________F     60° C.  -5.2° C.                           -8.3° C.G     50° C.  -4.1° C.                           -6.7° C.H     40° C.  -3.6° C.                           -5.7° C.I     30° C.  -3.1° C.                           -3.8° C.______________________________________ 
    
     EXAMPLE 3. 
     The dried strain of sample H(final temperature: 40° C.) obtained in Example 2 was suspended into the distilled water to form a suspension. Several samples(Sample Nos. were a, b, c, d, e, f and g) of this suspension were placed for 12 hours in a constant temperature bath whose temperature was 4° C., 10° C., 20° C., 25° C., 30° C., 35° C. and 40° C., respectively. 
     Ice nucleating activity of the samples are expressed as a percentage of remaining activity to that of Example 2 and the results are shown in the following Table 5. 
     COMPARATIVE EXAMPLE 3. 
     In this Example; the procedure of Example 3 was repeated except that the dried strain of sample H(final temperature: 40° C.) obtained in Comparative Example 2 was used. 
     Ice nucleating activities of the samples are expressed as a percentage of remaining activity to that of C. Example 2 and the results are shown in the following Table 5. 
     
                       TABLE 5______________________________________            Total Ice nucleating activityTemperature      of remaining sample(%)Sample  of sample    Example 3  C. Example 3______________________________________a        4° C.                99.8% ± 5.4                           92.3% ± 2.5b       10° C.                91.2% ± 3.1                           89.6% ± 3.1c       20° C.                97.6% ± 2.3                           81.5% ± 3.4d       25° C.                95.4% ± 2.9                           75.3% ± 5.2e       30° C.                89.7% ± 3.4                           54.3% ± 6.7f       35° C.                82.3% ± 3.3                           25.9% ± 7.3g       40° C.                75.1% ± 5.1                           11.1% ± 6.4______________________________________ 
    
     As shown in Table 3˜5, the novel mutant SO754(KCCM-10039) of the present invention has higher activity than ATCC 53,543 over the wide range of temperature and more particularly it shows much higher ice nucleating activity at a temperature range of 25°˜40° C. 
     The invention has been described in detail with particular reference to preferred embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention.