KR19990080269A - Production method of ice nucleus active microbial preparation - Google Patents
Production method of ice nucleus active microbial preparation Download PDFInfo
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- KR19990080269A KR19990080269A KR1019980013374A KR19980013374A KR19990080269A KR 19990080269 A KR19990080269 A KR 19990080269A KR 1019980013374 A KR1019980013374 A KR 1019980013374A KR 19980013374 A KR19980013374 A KR 19980013374A KR 19990080269 A KR19990080269 A KR 19990080269A
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Abstract
본 발명은 빙핵 형성 활성을 갖는 미생물을 pH 6.6 ∼ 7.2 범위로 유지하면서 배양 온도 15∼20℃ 범위로 배양하고, 회수하는 것으로 이루어지는, 빙핵 형성 미생물을 생산하는 방법을 제시한다. 본 발명에 의해 배양 중 온도 변화 또는 배양 후 온도 변화와 같은 특별한 공정 없이도 최대 빙핵형성능을 갖는 빙핵미생물을 생산할수 있다.The present invention provides a method for producing ice-nucleated microorganisms comprising culturing and recovering microorganisms having ice-nucleating activity at a culture temperature of 15 to 20 ° C. while maintaining the pH at a range of 6.6 to 7.2. According to the present invention, ice nucleus microorganisms having maximum ice nucleation capacity can be produced without special processes such as temperature change during culture or temperature change after culture.
Description
본 발명은 빙핵활성 미생물의 제조방법에 관한 것으로 빙핵활성 미생물의 최대 빙핵 형성능을 갖는 미생물을 배양하는 방법에 관한 것이다.The present invention relates to a method for producing ice nucleus active microorganisms and to a method for culturing microorganisms having the maximum ice nucleation capacity of ice nucleus active microorganisms.
생물학적 원인에 의한 서리 및 냉해 현상과 관련하여, 지난 20여년 동안 빙핵의 생물 학적 근원에 대한 연구가 많은 과학자들에 의해 진행되어, 식물의 상해원인이 기후적인 영향에 기인한 단순한 현상이 아니라 미생물에 의해 촉진되는 생화학적 현상이라는 것이 밝혀지게 되었다. 상해의 원인이 미생물의 빙핵형성 단백질(ice nucleation protein)로부터 기인한다는 사실이 밝혀진 이후, 빙핵형성 단백질에 관한 연구가 본격적으로 시작되어, 이 단백질이 빙핵(ice-nuclei)으로 작용한다는 것을 알게 되었고, 이러한 빙핵 형성 단백질을 생산하는 미생물들은 식물잎의 표면에 서식하는 엽면 미생물(ephiphytic microorganisms)로 밝혀졌다. 일반적으로 빙핵 형성 단백질은 3가지 타입으로 분류한다. 즉,-2℃ ∼-5℃ 에서 빙핵을 형성시키는 단백질을 타입I, -6℃∼-8℃ 에서 빙핵을 형성시키는 단백질을 타입II, -8℃ ∼-10℃ 에서 빙핵을 형성시키는 단백질을 타입III로 분류한다.(Ruggles et al.1993 Journal of Bacterio1ogy 175(22):7216-7221)Regarding frost and freezing caused by biological causes, research on the biological origins of ice cores has been conducted by many scientists for the last two decades, suggesting that the causes of plant injury are not simply phenomena caused by climate effects, It turns out that it is a biochemical phenomenon that is promoted. Since the cause of the injury was found to be caused by the microbial ice nucleation protein, research on ice nucleation protein began in earnest, and it was found that the protein acts as an ice nuclei. The microbes that produce these nucleus-forming proteins have been identified as epiphytic microorganisms that inhabit the surface of plant leaves. Generally, nucleus nucleating proteins are classified into three types. That is, a protein that forms ice cores at -2 ° C to -5 ° C is a type I, and a protein that forms ice cores at -6 ° C to -8 ° C is a type II protein that forms ice cores at -8 ° C to -10 ° C. Class III (Ruggles et al. 1993 Journal of Bacterio1ogy 175 (22): 7216-7221).
일반적으로 물은 0℃에서 어는 것으로 알려져 있으나, 실제로 순수한 물은 -39℃ 정도의 과냉각 상태(super cooling condition)를 거쳐서 얼음이 된다. 그런데, 빙핵활성 단백질이 존재하면 물의 어는점을 -2。C내지 -4C 정도로 상승시켜 물의 결빙을 촉진시킬 수 있다. 즉, 결빙 과정에서 물은-39℃정도의 과냉각 상태가 필요하지만, 빙핵활성 단백질이 존재하면 -2℃내지 -4℃ 정도의 과냉각을 요구하므로 그만큼의 에너지를 절약할 수 있다. 이와 같은 에너지 절약 효과를 위하여 빙핵활성 단백질은 여러 산업 분야에 응용되고 있는데, 예를 들어 빙핵형성 미생물(ice nucleating microorganism)을 보다 높은 온도에서 인공적으로 눈을 제조하기 위한 빙핵으로 사용하는 것이 보고되었으며, 또 냉동식품의 동결촉진제로 이용하는 것이 보고되었다.Generally, water is known to freeze at 0 ° C., but in practice pure water becomes ice through super cooling conditions of about -39 ° C. By the way, the presence of ice nucleus activating protein can increase the freezing point of water to -2 ° C to -4 C to promote water freezing. That is, during the freezing process, water requires about -39 ° C of supercooled state, but if ice nucleating protein is present, it requires about -2 ° C to -4 ° C of supercooling, thereby saving energy. To achieve this energy saving effect, ice nucleating proteins have been applied in various industries, for example, the use of ice nucleating microorganisms as ice nuclei for artificially producing snow at higher temperatures. In addition, it has been reported to be used as a freezing accelerator of frozen food.
빙핵형성 미생물은 인공제설의 촉진제로 널리 이용되고 있으며, 인공강우 촉진제로 이용하면 한발에 대비하여 인공강우를 유도할 수 있어서 현재 현장시험을 진행하고 있다. 결론적으로, 동결과정에 관련된 전 산업분야에 응용될 수 있을 것으로 기대된다.Ice nucleation microorganisms are widely used as an accelerating agent of artificial snow removal, and when it is used as an artificial rainfall accelerator, it can induce artificial rainfall in preparation for a pair and is currently conducting field tests. In conclusion, it is expected to be applicable to all industries related to the freezing process.
한편, 이와 같은 산업적 이용을 위해서는 빙핵형성 미생물을, 원하는 빙핵활성을 보유하는 상태로 얻는 것이 필수적이다. 즉, 배양 후 얻어지는 빙핵형성 미생물 모두가 빙핵 형성능을 발휘하는 것은 아니며, 따라서 특별한 조건에서 배양하거나 또는 배양 후 별도의 처리를 통해 미생물의 빙핵형성능을 높이고자 하는 시도가 있었다.On the other hand, for such industrial use, it is essential to obtain ice nucleation microorganisms in a state having desired ice nucleation activity. That is, not all of the ice nucleation microorganisms obtained after the cultivation exhibit the ice nucleation ability, and therefore, there have been attempts to increase the ice nucleation capacity of the microorganisms by culturing under special conditions or by separate treatment.
한국특허공고공보 96-10903(미국특허 5,137,815)에는 빙핵핵활성을 갖는 미생물의 생산방법으로 발효배지의 pH를 약 pH 5.5-6.7, 특히 pH 6.6∼5.6 범위 내에서 조절하고,21℃에서 배양하는 방법이 기재되어 있다. 이 문헌에서는 빙핵 형성 미생물의 경우 배양 pH가 특허청구범위에 기재된 범위에서 벗어나는 경우, 예를 들어 pH 6.8과 같은 pH에서는 빙핵 형성능(INA,Ice nucleation protein)가 떨어진다고 보고하였다.Korean Patent Publication No. 96-10903 (U.S. Patent No. 5,137,815) discloses a method for producing microorganisms having ice nucleus activity, which adjusts the pH of fermentation broth to about pH 5.5-6.7, especially pH 6.6 to 5.6, and incubates at 21 ° C. The method is described. In this document, ice nucleation microorganisms report that if the culture pH is out of the range described in the claims, for example, pH 6.8, the ice nucleation protein (INA) falls.
미국특허 5,153,134에는 배지내의 질소함량을 세포중량으로 20g/L 되도록 성장시킬 수 있으며 성장말기에 빙핵형성을 저해하지 않는 양으로 한정하여 배양하면서 배양온도를 성장시기에 따라 29℃에서 24℃로 변환하면서 배양하는 생산방법이 기재되어 있다.US Pat. No. 5,153,134 shows that the nitrogen content in the medium can be grown to 20 g / L by cell weight, and is limited to an amount that does not inhibit ice nucleation at the end of the growth, while the culture temperature is changed from 29 ° C. to 24 ° C. depending on the growth period. A production method for culturing is described.
Journal of Bacteriology,1993 년 7 월호 p.4062-4070 에는 32℃ 에서 배양하여 얻은 배양액을 배양이 끝난 후 짧은 시간 내에 16℃로 온도를 변환시켜 빙핵형성능이 높은 빙핵단백질을 유도하는 방법이 기재되어 있다. 그러나 이와 같이 배양 후 빠른 시간 내에 배양액의 온도를 낮추는 방법은 대량 배양 방법에서는 별개의 공정이 더 추가되므로 공정을 번거롭게 할 뿐 아니라, 실제로 대량 배양에서 다량의 배양액의 온도를 빠른 시간에 큰 폭으로 떨어뜨리는 것은 용이하지 않다.Journal of Bacteriology, July, 1993, p.4062-4070, describes a method of inducing ice nucleus proteins with high ice nucleation capacity by converting the temperature of the culture broth obtained at 32 ° C. to 16 ° C. within a short time after incubation. have. However, the method of lowering the temperature of the culture solution in a short time after the cultivation not only makes the process cumbersome because more separate processes are added in the mass culture method, and in fact, the temperature of a large amount of the culture solution in the large culture is drastically reduced in a short time. It is not easy to shake.
본 발명의 목적은 빙핵형성능이 높은 미생물을 생산하는 방법을 제공하는 것이다.It is an object of the present invention to provide a method for producing microorganisms having high ice nucleation capacity.
본 발명의 또다른 목적은 배양 중의 온도 변화 없이 빙핵형성능이 높은 미생물을 대량생산할 수 있는 생산공정을 제공하는 것이다.Still another object of the present invention is to provide a production process capable of mass-producing microorganisms having high ice nucleation capacity without changing temperature during culture.
도 1는 배양온도에 따른 빙핵형성 미생물의 빙핵형성능을 나타낸 그래프이 다.1 is a graph showing the ice nucleation capacity of ice nucleation microorganisms according to the culture temperature.
도 2는 배양pH에 따른 빙핵형성 미생물의 빙핵형성능을 나타낸 그래프이 다.Figure 2 is a graph showing the ice nucleation capacity of ice nucleation microorganisms according to the culture pH.
본 발명은 빙핵 형성 미생물을 배양함에 있어, 세포 생육과 세포의 빙핵 형성능을 동시에 만족시킬 수 있는 온도 범위에서 배양하는 것으로 이루어진다. 따라서 배양 공정 중에 배양 온도를 변화시킬 필요 없이, 높은 빙핵활성능을 갖는 빙핵미생물을 배양할 수 있다. 구체적으로, 본 발명은 빙핵 형성 미생물을 배양 중 pH를 pH 6.6 ∼ 7.2, 바람직하게는 pH 6.8 ∼ 7.0 으로 유지하고 배양 온도를 15℃ ∼ 20℃, 바람직하게는 17℃ ∼20℃로 유지하면서 배양하는 것으로 이루어지는, 빙핵 형성 활성을 가지는 미생물을 생산하는 방법에 관한 것이다.In the present invention, in culturing ice nucleus forming microorganisms, the cell growth and the ice nucleation forming ability of the cells are achieved by culturing in a temperature range that can satisfy simultaneously. Therefore, ice nuclei with high ice nucleation activity can be cultured without the need to change the culture temperature during the culture process. Specifically, the present invention cultures ice nucleus forming microorganisms while maintaining the pH at pH 6.6 to 7.2, preferably pH 6.8 to 7.0 and maintaining the culture temperature at 15 ° C to 20 ° C, preferably 17 ° C to 20 ° C. It relates to a method for producing a microorganism having ice nucleation activity consisting of.
본 발명의 방법에서 pH를 조절하는 방법은 미생물 발효 분야에서 통상적으로 사용되는 방법에 따른다. pH를 조절하기 위하여 통상적으로 사용되는 산 및 염기를 사용할 수 있으며, 예를 들어 황산, 수산화나트륨 등이 사용될 수 있다.The method of adjusting pH in the method of the present invention is according to the method commonly used in the field of microbial fermentation. Conventionally used acids and bases can be used to adjust the pH, for example sulfuric acid, sodium hydroxide and the like can be used.
본 발명은 빙핵활성능이 있다고 알려진 미생물, 예를 슈도모나스 시린제게(Psedomonas syringae)와 같은 슈도모나스속 미생물, 어위니아 허비콜라(Erwinia herbicola)와 같은 어위니아속 미생물 등의 배양에 적용될 수 있으며, 특히 본 발명의 방법을 적용할 수 있는 유용한 대표적인 미생물은 잔토모스나스(Xanthomons)속 미생물, 바람직하게는 잔토모나스 캄페스트리스 (Xanthomons campestris) 계열 미생물이다.The present invention can be applied to the culture of microorganisms known to have ice nucleating activity, for example, Pseudomonas microorganisms such as Psedomonas syringae, and Erwinia microorganisms such as Erwinia herbicola. Representative useful microorganisms to which the method of the invention can be applied are Xantomas genus microorganisms, preferably Xanthomons campestris family of microorganisms.
본 발명에서 빙핵 형성능은 발리의 동결 방울수 계측법으로 측정하였다(Schnell,R.Cand G.Vali,Nature,236:163-165(1972)). 즉, 배지에서 배양한 배양액을 pH 7.0인 10mM 인산완충용액에 현탁시켜 세포수를 600nm에서의 OD가 1이 되도록즉,3 × 109cell/ml이 되도록 현탁액을 만들고, pH 7.0인 10mM 인산완충액으로 연속 희석하여 일련의 10배 희석액을 제조하였다. 이 희석액들은 파라핀으로 코팅된 알루미늄 블럭에 5㎕ 씩 20방울을 분주하여 냉각 수조(Refrigerated batch circulater,JEIOTECH, 한국)에 올려 놓고, 0℃에서 -10℃까지 0.6℃/min의 일정한 속도로 냉각시키면서 과냉각 온도(Supercooling temperature)에 따른 동결 방울수를 계수하여 다음 식에 따라 계산한다. 특히 0℃에서 -5℃까지 냉각시키면서 생기는 동결 방울수를 계수하여 다음 식에 따라 계산하여 타입 I 빙핵수를 측정하였다.Ice nucleation capacity in the present invention was measured by the freezing droplet counting method of Bali (Schnell, R. Cand G. Vali, Nature, 236: 163-165 (1972)). In other words, the culture medium cultured in the medium was suspended in a 10 mM phosphate buffer solution at pH 7.0 to make a suspension so that the cell number becomes OD of 1 at 600 nm, that is, 3 × 10 9 cells / ml, and a 10 mM phosphate buffer pH 7.0. Serial dilutions were made to prepare a series of 10-fold dilutions. These dilutions were dispensed in 5 drops of 5 µl in a paraffin-coated aluminum block and placed in a refrigerated batch circulater (JEIOTECH, South Korea), cooling at a constant rate of 0.6 ° C / min from 0 ° C to -10 ° C. The number of frozen drops according to the supercooling temperature is counted and calculated according to the following equation. In particular, the type I ice core water was measured by counting the number of frozen drops generated while cooling from 0 ° C to -5 ° C according to the following equation.
CNI = - Ln ( 1 - F ) / ( V × D )CNI =-Ln (1-F) / (V × D)
CNI : 누적 빙핵수 f : 동결 방울의 분율CNI: cumulative ice core f: fraction of frozen drops
V : 방울의 부피 D : 희석 배율V: volume of droplet D: dilution factor
총 배양액의 빙핵활성능은 OD600nm:1(3 × 109cell/ml)의 타입1의 빙핵수에 배양액의 OD값을 곱하여 계산하였다The ice core activity of the total culture was calculated by multiplying the OD value of the culture by the type 1 ice core water of OD600nm: 1 (3 × 10 9 cells / ml).
이하, 실시예를 통하여 본 발명을 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 설명하기 위한 것으로, 이들 실시예에 의해 본 발명의 범위가 한정되지 않는다는 것은 본 발명이 속하는 분야에서 통상의 지식을 가진 자들에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.
실시예 1. 배양온도에 따른 빙핵 형성능Example 1. Ice core formation ability according to the culture temperature
대한민국 대전광역시 유성구 구성동에 위치한 생명공학연구소 유전자은행(KCTC)에 1996년 6월 21일에 기탁된 잔토모나스 캄페스트리스 (Xanthomons campestris) KCTC0251BP와 기존의 빙핵활성 미생물로 알려진 슈도모나스 시린제(Psedomonas syringae)KCTC1832 그리고 생명공학연구소 생물공정그룹에서 입수한 어위니아 허비콜라(Erwinia herbicola)를 표 1의 액체 배지 2L에서 배양온도를 각각 15℃,18℃,20℃,22℃,25℃,30℃로 달리하여 48시간 배양하였다. 배양 pH가 pH 6.8∼pH 7.0의 범위에서 유지되도록 3NNaOH와 3NH2SO4를 이용하여 조절하였고, 용존산소량은 20%이상을 유지하면서 48시간동안 회분배양하였다. 배양 발효조는 5리터 발효조(Korean Fermentor Co., Ltd. Korea)를 이용, 배양액을 2 리터로 하였다.Xanthomons campestris KCTC0251BP, deposited on June 21, 1996, at KCTC, Guseong-dong, Yuseong-gu, Daejeon, Korea, and Psedomonas syringae, known as the existing ice nucleus activating microorganism. Erwinia herbicola obtained from KCTC1832 and Biotechnology Research Group Bioprocessing Group was used in 2L of liquid medium in Table 1, and the incubation temperature was changed to 15 ° C, 18 ° C, 20 ° C, 22 ° C, 25 ° C and 30 ° C, respectively. Incubated for 48 hours. Culture pH was adjusted using 3NNaOH and 3NH 2 SO 4 to maintain the pH in the range of pH 6.8 ~ pH 7.0, dissolved oxygen was batch cultured for 48 hours while maintaining more than 20%. The culture fermenter used a 5 liter fermenter (Korean Fermentor Co., Ltd. Korea) to make the culture liquid 2 liters.
앞에 기재된 방법에 따라 타입 I 빙핵 형성능을 측정하였으며 그 결과를 표 2 및 도 1 에 나타낸다.Type I ice core formation ability was measured according to the method described above, and the results are shown in Table 2 and FIG. 1.
도1에서 확인되는 바와 같이, 볼 수 있듯이, 배양온도 15∼20℃ 에서 타입I(-5。C 이상에서 빙핵 형성)의 빙핵 형성능을 갖는 빙핵 미생물을 다량 생산할 수있었다.As can be seen from Figure 1, it was able to produce a large amount of ice core microorganisms having ice core formation ability of Type I (ice nucleation at -5 ° C or more) at a culture temperature of 15 to 20 ℃.
[표 2]TABLE 2
실시예 2 배양 pH 에 따른 빙핵 형성능Example 2 ice core formation ability according to the culture pH
배양온도를 18℃로 하고 배양pH를 각각 pH 5.5,6.0,6.5,7.0,7.5,8.0 까지 달리하는 것을 제외하고는 실시예 1과 같이 잔토모나스 캄페스트리스 (Xanthomons campestris)KCTC0251BP, 슈도모나스 시린제(Psedomonas syringae)KCTC1832 와 어위니아 허비콜라(Erwinia herbicola)를 회분식배양하였다. 앞에 기재된 방법에 따라 각온도별 타입 I 빙핵 형성능을 측정하였으며 그 결과를 표 3 과 도 2에 나타냈다. 도 2에서 확인되는 바와 같이,pH7.0에서 가장 높은 역가를 보였다.The culture temperature was 18 ° C. and the culture pH was changed to pH 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0, respectively, as in Example 1 Xantomas campestris KCTC0251BP, Pseudomonas syringe ( Psedomonas syringae) KCTC1832 and Erwinia herbicola were batch cultured. Type I ice core formation ability was measured at each temperature according to the method described above, and the results are shown in Table 3 and FIG. As confirmed in FIG. 2, the highest titers were at pH 7.0.
[표 3]TABLE 3
실시예 3 대량배양Example 3 Mass Culture
빙핵활성미생물을 대량생산하는 경우에도 빙핵 형성능이 높은 미생물을 생산할 수 있음을 확인하기 위하여, 표 1의 배지조건으로 배양온도를 18。C, 배양pH를 pH6.8 ∼ 7.0으로, 용존산소량을 20% 이상으로 유지하면서 450리터 발효조(Korean Fermentor Co., Ltd. Korea)와 (주)동아바이오텍사의 25m3발효조(Komatsugawa, Co.,ltd,Japan)를 이용하여 대량 배양하였으며 결과를 표 4에 나타낸다. 발효조 크기에 별다른 영향없이 5L 발효조와 유사한 결과를 얻을 수 있었다.In order to confirm that even in the case of mass production of ice nucleus active microorganisms, it is possible to produce microorganisms having high ice nucleation ability, and the culture temperature is 18 ° C, the culture pH is pH6.8 to 7.0, and the dissolved oxygen amount is 20 450 l fermentation vessel while maintaining a% or more (Korean fermentor Co., Ltd. Korea) and Co., was mass cultured using a 25m 3 East Biotech Inc. fermenter (Komatsugawa, Co., ltd, Japan ) the results are shown in Table 4 . Similar results were obtained with 5 L fermenters without significant effect on fermenter size.
[표 4]TABLE 4
본 발명에 의해 배양 중 온도 변화 또는 배양 후 온도 변화와 같은 특별한 공정 없이도 최대 빙핵형성능을 갖는 빙핵미생물을 생산할 수 있으며, 본 발명을 이용하여 종래의 빙핵활성 미생물보다 빙핵활성이 뛰어난 빙핵활성 미생물 제제를 생산할 수 있다.According to the present invention, ice nucleus microorganisms having a maximum ice nucleation capacity can be produced without a special process such as temperature change during culture or temperature change after culture, and ice nucleus active microbial preparations having superior ice nucleus activity than conventional ice nucleus microorganisms by using the present invention Can produce
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