KR19980015533A - Process for producing freeze-dried cells having excellent preservability - Google Patents
Process for producing freeze-dried cells having excellent preservability Download PDFInfo
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- KR19980015533A KR19980015533A KR1019960034898A KR19960034898A KR19980015533A KR 19980015533 A KR19980015533 A KR 19980015533A KR 1019960034898 A KR1019960034898 A KR 1019960034898A KR 19960034898 A KR19960034898 A KR 19960034898A KR 19980015533 A KR19980015533 A KR 19980015533A
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Abstract
동결건조 균체를 제조할 경우, 전동결시의 온도 강하 조건을 분당 5℃∼15℃의 속도로 하고, 또 동결건조 시료의 온도가 -5℃∼-15℃가 되었을 때 5분∼10분간 일정 온도를 유지시켜 제조한 동결건조 균체는 동결건조 균체의 보존시 균주의 안정성이 매우 높다.When lyophilized cells are prepared, the temperature drop condition of the electric decay is set at a rate of 5 ° C to 15 ° C per minute, and when the temperature of the lyophilized sample reaches -5 ° C to -15 ° C, , The stability of the strain is very high when the lyophilized cells are preserved.
Description
본 발명은 동결건조균체에 관한 것으로서, 더욱 상세하게는 동결건조 균체중 생균수를 월등이 향상시킬 수 있는 보존성이 우수한 동결건조균체의 제조방법에 관한 기술이다.The present invention relates to a lyophilized microbial cell. More particularly, the present invention relates to a method for producing a lyophilized microbial cell, which is superior in preservability to improve the number of viable cells in lyophilized microbial cells.
동결건조는 미생물을 장기 보존하거나, 효소 또는 기능성 단백질 등과 같이 수용액 상태에서 온도 등의 조건에 따라 상대적으로 불안정한 활성물질을 보존하거나, 식품의 향과 성상을 원형 그대로 보존하기 위하여 사용되어온 건조방법중의 하나이다. 특히 미생물을 장기 보존할 경우 동결건조방법으로 건조시켜 보관하는 것이 일반적인 기술로 알려져 있다.Freeze-drying is a method of preserving microorganisms for a long time, preserving relatively unstable active substances according to conditions such as temperature or the like in an aqueous solution state such as an enzyme or a functional protein, It is one. In particular, when microorganisms are stored for a long period of time, it is known as a general technique to dry and store by freeze-drying method.
종래의 미생물 균주를 동결건조하는 방법은, 스킴밀크, 당류 등의 성분 또는 이들의 혼합물로 이루어진 동결건조 보호제를 균체와 혼합후 급속히 냉각시키고 이를 동결건조장치에서 건조하는 방법에 의하여 행하여졌다. 그러나 상기한 종래의 동결건조 방법은 동결건조전에 급속히 냉각시키는 과정에서 균체가 동해를 입어 생균수가 감소되는 단점이 있었다.A conventional method for lyophilizing a microorganism strain is a method in which a lyophilized protective agent composed of components such as skim milk and saccharides or a mixture thereof is mixed with a microbial cell, rapidly cooled, and dried in a freeze-drying apparatus. However, the conventional freeze-drying method has a disadvantage in that the number of viable cells is reduced due to damage to the cells during rapid cooling before freeze-drying.
본 발명은 상기와 같은 종래 기술의 문제점을 해결하기 위하여 개발된 것으로서, 본 발명의 목적은 동결건조후 생균수가 많은 우수한 보존성을 동결건조균체의 제조방법을 제공하는 것이다.The present invention has been developed in order to solve the problems of the prior art as described above, and an object of the present invention is to provide a method for producing freeze-dried cells having high preservability,
도 1은 본 발명의 동결건조를 위한 시료 전동결시 시료의 온도 강하를 나타내는 그래프.BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the temperature drop of a sample during freeze drying for freeze-drying of the present invention. FIG.
상기와 같은 본 발명의 목적을 달성하기 위하여 본 발명은, 미생물을 동결건조함에 있어서, 상기 동결건조장치내의 온도를 분당 5℃∼15℃의 속도로 강하시키는 것을 특징으로 하는 동결건조균체의 제조방법을 제공한다.In order to accomplish the object of the present invention, the present invention provides a method for preparing a lyophilized microbial cell characterized by lowering the temperature in the freeze-drying apparatus at a rate of 5 ° C to 15 ° C per minute .
상기한 본 발명에 있어서, 상기 동결건조 시료의 온도가 -5℃∼-15℃가 되었을 때 5분∼10분간 일정 온도를 유지시키는 공정을 더욱 포함하는 것이 바람직하다. 그리고 상기 미생물은 사카로마이세스 세레비시아인 것이 바람직하며, 상기 미생물은 YPD 액체 배지에 배양한 후 농축하여 동결건조하는 것이 바람직하다.In the present invention, it is preferable that the method further comprises a step of maintaining a constant temperature for 5 minutes to 10 minutes when the temperature of the lyophilized sample reaches -5 ° C to -15 ° C. Preferably, the microorganism is Saccharomyces cerevisiae. The microorganism is preferably cultured in a YPD liquid medium, concentrated and lyophilized.
본 발명을 대표적인 실시예를 참고하여 보다 상세히 설명하면 다음과 같다.Hereinafter, the present invention will be described in more detail with reference to exemplary embodiments.
대표적인 실시예In a representative embodiment
사카로마이세스 세레비시아(Saccharomyces cerevisiae) 균을 글루코스 2중량%, 펩톤 1중량%, 효모엑기스 1중량%로 이루어진 YPD 액체 배지에서 배양하여 얻은 균체를 농축하고 ,스킴 밀크와 당으로 구성된 동결건조 보호제와 혼합한 후 유리 페트리 접시에 도말한다. 이를 동결건조장치의 동결건조상자에 배열한 후 동결건조상자의 온도를 -5℃∼-15℃가 될 때까지 분당 5℃∼15℃의 속도로 강하시키고 시료의 온도가 -5℃∼-15℃가 되었을 때 5분∼10분간 일정온도를 유지시킨다. 이후 다시 온도를 분당 5℃∼15℃의 속도로 강하시켜 -40℃∼-50℃가 되게 한다. 시료의 온도가 -40℃∼-50℃가 되었을 때 감압을하여 동결건조 한다.(도 1 참조) 동결건조시의 압력은 10-3∼10-5mmHg로 유지한다.Saccharomyces cerevisiae bacteria were cultured in a YPD liquid medium consisting of 2% by weight of glucose, 1% by weight of peptone and 1% by weight of yeast extract, and the resulting cells were concentrated and lyophilized After mixing with a preservative, the mixture is spread on a glass Petri dish. Dried at a rate of 5 ° C to 15 ° C per minute until the temperature of the freeze-dried box reaches -5 ° C to -15 ° C, and the temperature of the sample is decreased from -5 ° C to -15 ° C When the temperature is reached, maintain a constant temperature for 5 minutes to 10 minutes. Thereafter, the temperature is lowered at a rate of 5 ° C to 15 ° C per minute so as to become -40 ° C to -50 ° C. When the temperature of the sample reaches -40 ° C to -50 ° C, it is decompressed and lyophilized (see FIG. 1). The pressure during lyophilization is maintained at 10-3 to 10-5 mmHg.
동결건조 과정중 전동결과정시 상기의 방법에 의한 처리후 감압에 의한 건조를 하였을 때 종래의 방법과 같이 균체를 급속동결 한후 감압에 의한 건조를 하였을 때보다 동결건조된 균체중의 생균수를 현저히 높일 수 있었다.The result of the lyophilization process during the lyophilization process is as follows. When the cells are dried by decompression after the treatment by the above method, the number of living cells in the lyophilized cells is markedly increased as compared with the case where the cells are rapidly frozen and dried under reduced pressure I could.
이하 본 발명의 바람직한 실시예 및 비교예를 기재한다. 그러나 하기한 실시예는 본 발명의 이해를 돕기 위한 본 발명의 바람직한 일 실시예로서 본 발명이 하기한 실시예에 한정되는 것은 아니다.Hereinafter, preferred embodiments and comparative examples of the present invention will be described. However, it should be understood that the present invention is not limited to the following examples.
실시예Example
사카로마이세스 세레비시아 균을 글루코스 2중량%, 펩톤 1중량%, 효모엑기스 1중량%로 이루어진 YPD 액체 배지 60ml에서 24시간 배양후 YPD 액체 배지 3L 부피가 들어있는 발효조에 접종하여 임펠러 회전속도 400rpm, 분당 공기송입량 3Nl, 배양온도 30℃에서 24시간 배양하였다. 배양액을 7000rpm의 회전속도로 20분간 원심분리하여 얻은 균체를 최초 배지부피의 1/10정도로 다시 현탁하여 농축하였다. 농축액을 스킴밀크와 당으로 구성된 동결건조 보호제와 혼합한 후 유리 페트리접시에 도말하였다.Saccharomyces cerevisiae was cultivated in 60 ml of YPD liquid medium consisting of 2% by weight of glucose, 1% by weight of peptone and 1% by weight of yeast extract for 24 hours, and then inoculated into a fermenter containing 3 L of YPD liquid medium, 400 rpm, 3 Nl of air per minute, and incubation temperature of 30 캜 for 24 hours. The culture was centrifuged at a rotation speed of 7000 rpm for 20 minutes, and the resulting cells were suspended again at about 1/10 of the initial medium volume and concentrated. The concentrate was mixed with a freeze-dried preservative consisting of skim milk and sugar, and then plated on a glass Petri dish.
상기 시료가 도말된 페트리접시를 동결건조 장치의 동결건조 상자에 배열한 후 동결건조 상자의 온도를 -5℃∼-15℃가 될 때까지 분당 5℃∼15℃의 속도로 강하시키고 시료의 온도가 -5℃∼-15℃가 되었을 때 5분-10분간 일정온도를 유지시켰다. 이후 다시 온도를 분당 5℃∼15℃의 속도로 강하시켜 -40℃∼-50℃가 되게 하였다. 시료의 온도가 -40℃∼-50℃가 되었을 때 감압을 하여 동결건조하였다.(도 1 참조) 동결건조시의 감압의 조건은 10-3∼10-5mmHg로 유지하도록 하였다.The Petri dish on which the sample was smoothed was arranged in a freeze-drying box of the freeze-drying device, and the temperature of the freeze-drying box was dropped at a rate of 5 ° C to 15 ° C per minute until the temperature reached -5 ° C to -15 ° C, Was kept at -5 ° C to -15 ° C for 5 minutes to 10 minutes. Thereafter, the temperature was lowered at a rate of 5 ° C to 15 ° C per minute so as to become -40 ° C to -50 ° C. When the temperature of the sample reached -40 ° C to -50 ° C, it was decompressed and lyophilized (see FIG. 1). The condition of reduced pressure during lyophilization was maintained at 10-3 to 10-5 mmHg.
상기의 온도와 감압조건에서 12시간∼36시간 동안 건조한 후의 시료를 사용하여 생균수를 측정하였다.The number of viable cells was measured using a sample after drying for 12 to 36 hours under the above-described temperature and reduced pressure conditions.
비교예Comparative Example
대조군으로는 배양, 농축 및 전동결 후 건조조건 등은 상기한 실시예와 동일하지만 전동결시 시료의 온도가 -40℃∼-50℃가 되도록 급속히 동결을 시킨 시료를 사용하였다.As a control, the samples after the incubation, concentration and freeze-drying conditions were the same as those in the above-mentioned examples, but rapidly frozen so that the temperature of the sample became -40 ° C to -50 ° C at the time of freezing.
상기한 실시예 및 비교예에서 제조된 동결건조균체의 생균수 측정은 적정량을 멸균 생리 식염수로 적정 희석 배수로 희석후 YPD 한천 고체배지에 균의 콜로니가 고체 배지 페트리 접시당 30∼300개정도 나타나게 도말한 후 30℃ 항온기에서 약 48시간을 배양하여 균수를 측정하였다.The viable cell count of the lyophilized cells prepared in the above-mentioned Examples and Comparative Examples was determined by diluting an appropriate amount of the cells with sterile physiological saline at a suitable dilution ratio and then culturing the cells in a YPD agar solid medium at a concentration of 30-300 cells / After incubation, the cells were incubated at 30 ° C for 48 hours.
상기의 전동결조건을 사용하여 동결건조시킨 동결건조균체에서의 생균수는 동결건조균체 그람당 1.5×1011 생균으로 대조군의 전동결조건으로 동결건조시킨 동결건조균체에서의 생균수 동결건조균체 그람당 1.2×1010생균에 비하여 10배이상 높은 생균을 포함하고 있는 것을 나타났다.The number of viable cells in the lyophilized cells lyophilized using the total freezing conditions was 1.5 x 10 < 11 > viable cells per lyophilized bacterium. The lyophilized cells in the lyophilized cells lyophilized under the freezing conditions of the control group, And contained 10 times more live cells than 1.2 × 1010 live cells.
이상에서 살펴본 바와 같이 본 발명에 따른 동결건조균체의 제조방법을 사용할 경우 동결건조균체내에 존재하는 생균의 수가 매우 높았다. 즉 본 발명의 전동결건조를 행할 경우 동결건조 후 높은 생균수를 포함하는 동결건조균체를 제조할 수 있다. 이와 같은 본 발명의 동결건조균체의 제조방법을, 정장제로 상품화되고 있는 사카로마이세스 세레비시아 균체의 동결건조 방법에 사용할 경우, 종래의 전동결단계에서 생기는 동해로 인하여 생균수가 감소하는 문제를 해결할 수 있다.As described above, the number of viable cells in the lyophilized cells was very high when the method of the present invention was used. That is, in the case of performing the pre-freeze drying of the present invention, it is possible to produce a lyophilized microbial cell containing a high viable cell count after lyophilization. When such a method for producing a lyophilized microbial cell of the present invention is used in the freeze-drying method for microbial cells of Saccharomyces cerevisiae which is commercialized as a formulating agent, there is a problem that the viable cell count is reduced due to the frost- Can be solved.
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