KR960006578B1 - Separating method microorganisms from amino acid and nucleotide fermenting liquid - Google Patents

Separating method microorganisms from amino acid and nucleotide fermenting liquid Download PDF

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KR960006578B1
KR960006578B1 KR1019920023938A KR920023938A KR960006578B1 KR 960006578 B1 KR960006578 B1 KR 960006578B1 KR 1019920023938 A KR1019920023938 A KR 1019920023938A KR 920023938 A KR920023938 A KR 920023938A KR 960006578 B1 KR960006578 B1 KR 960006578B1
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fermentation broth
amino acid
cell
acid
sodium
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KR940014767A (en
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박한선
강기권
이계철
황이남
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주식회사미원
유영학
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
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    • C12P13/14—Glutamic acid; Glutamine
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/445—The saccharide radical is condensed with a heterocyclic radical, e.g. everninomycin, papulacandin

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Abstract

Amino acid or nucleic acid fermenting solution is treated to PH 3.0-5.0. Organic coagulant 50-300 ppm is added to solution and heated at temp. 50-80 deg.C for 0.5-2 hours. Coagulated microorganisms is settled and seperated from mother solution continuously by conical thickener. Sodium polyacrylate or sodium polyacrylamide is used as organic coagulant. This method has a better yield ratio of amino acid or nucleic economically than known methods.

Description

아미노산 및 헥산 발효액으로부터 균체를 분리하는 방법Method for separating cells from amino acid and hexane fermentation broth

제1도는 본원 발명의 공정도이다.1 is a process diagram of the present invention.

본 발명은 통상의 발효법에 의하여 제조한 아미노산 발효액 및 핵산 발효액으로부터 균체를 제거하는 방법 및 장치에 관한 것이며,보다 상세하게는 아미노산 발효액 및 핵산 발효액을 처리함에 있어서 발효액에유기계 응집제를 넣고 50∼80℃에서 30분∼2시간 가열한 후 균체가 응집된 액을 분리할때 씨크너(thickener)를 사용하여 연속적으로 균체를 포함하지 않는 상징액과 균체 포함액으로 분리함을 특징으로 하는 방법에 관한 것이다. 으로써 기존의 방법에 비해 고수율로 목적 화합물의 결정을 얻고, 정석 모액량 감소는 물론 기계적 처리량을 18-3l%로 감소시키는 균체 분리 방법에 관한 것이다.The present invention relates to a method and apparatus for removing cells from amino acid fermentation broth and nucleic acid fermentation broth prepared by a conventional fermentation method. More specifically, in the treatment of amino acid fermentation broth and nucleic acid fermentation broth, an organic coagulant is added to the fermentation broth at 50 to 80 ° C. After heating for 30 minutes to 2 hours at the time of separating the agglomerates agglomerates, the method is characterized in that the use of a thickener (thickener) to continuously separate the supernatant and the cell-containing liquid containing no cells. The present invention relates to a cell separation method of obtaining a crystal of a desired compound in a high yield compared to the conventional method, and reducing the amount of crystallization mother liquor as well as reducing the mechanical throughput to 18-3 l%.

아미노산 발효액은 콜로이드성 단백질, 지방 등의 유기 콜로이드 물질을 다량 함유하고 있는데 종래에는균체 분리시 균체 분리기나 울트라 필터 등 기계적 방법을 이용함으로써 미세 균체 및 현탁 물질의 제거가곤란하있다. 또한 규조토를 여과 조제로 다량 사용함으로써 여과 속도가 느리고 여액에 콜로이드성 단백질이 존재함으로 투명한 액을 얻는 것이 불가능하였다. 이러한 결점을 보완하기 위하여 몇가지 개선방안이 제안되었다.The amino acid fermentation broth contains a large amount of organic colloidal substances such as colloidal proteins and fats, but conventionally, it is difficult to remove microbial cells and suspension materials by using a mechanical method such as a cell separator or an ultra filter. In addition, by using a large amount of diatomaceous earth as a filter aid, the filtration rate was slow and the colloidal protein was present in the filtrate, making it impossible to obtain a transparent solution. Several improvements have been proposed to compensate for this drawback.

예를 들면, 단백질에 있는 아미노산을 분해하고 콜로이드성 물질을 분리하기 위하여 글루타민산 발효액에염산을 가하고 고온으로 가열한 후 계면활성제를 0,05∼0.2% 첨가하여 60℃에서 30분간 응집 침전 후 규조토 1%를 가하여 진공여과하는 방법(일본특허공고 소38-16460) 및 타침강성 불순물을 분리 제거 후 pH 3.2로 중화하는 방법(일본특허공고 소 40-12691)이 발표되었으나 이들 공지 방법에 따르면 응집제 사용량이많아 점성의 증가로 분리에 어려움이 있고 여과 속도가 느리거나 기계적 처리량이 많아 공업적으로 이용하기에는 곤란하였다.For example, hydrochloric acid is added to glutamic acid fermentation broth to decompose amino acids in proteins and to separate colloidal materials, and heated to high temperature, and then 0,05 to 0.2% of a surfactant is added. A method of vacuum filtration with the addition of% (Japanese Patent Publication No. 38-16460) and a method of separating and removing other precipitated impurity impurities to pH 3.2 (Japanese Patent Publication No. 40-12691) have been published. It is difficult to separate due to the increase in viscosity, and it is difficult to use industrially because of the slow filtration rate or high mechanical throughput.

또한 발효액을 이온 교환수지로 처리하여 글루타민산을 얻는 방법으로 발효액을 강산성 양이온 교환수지에 통액 후 글루타민산을 농축.정제하는 방법, 및 카르본산형 수지에서 발효액을 탈 양이온 시킨 후 강산성 양이온 교환수지에 글루타민산을 양이온 형태로 흡착시킨 후 용리하여 글루타민산을 회수하는 방법 또는음이온 교환수지에 글루타민산을 흡착시키는 방법 등이 발표 되었으나 수지 사용으로 인하여 용리제 및 재생제가 필요하여 폐수 발생량 증가는 물론 제조 단가가 증가하는 단점이 있고 특히 첫번째 방법의 경우 수지층 중에서의 글루타민산 농도가 높아 수지탑을 가온.보온해야 하는 단점이 있다.In addition, the fermentation broth is treated with an ion exchange resin to obtain glutamic acid, and the fermentation broth is passed through a strong acid cation exchange resin, followed by concentrating and purifying glutamic acid, and decarboxylation of the fermentation broth in a carboxylic acid resin. A method of recovering glutamic acid by eluting after adsorbing in cation form or adsorbing glutamic acid on an anion exchange resin has been announced.However, the use of resins requires an eluent and a regenerant to increase the wastewater generation and increase the manufacturing cost. In particular, the first method has a disadvantage of having to warm and insulate the resin tower due to the high concentration of glutamic acid in the resin layer.

본 발명자들은 종래 기술의 상술한 단점들을 보완하기 위하여 예의 연구한 결과, 아미노산 또는 핵산 발효액을 산을 사용하여 약산성으로 조절한 후 유기계 응집제를 첨가하여 가열한 후 씨크너를 사용항으로써상기 목적을 달성할 수 있음을 발견하고 본 발명을 완성하기에 이르렀다.The present inventors earnestly studied to compensate for the above-mentioned disadvantages of the prior art, and as a result, the amino acid or nucleic acid fermentation broth was adjusted to weak acid using acid, and then heated by adding organic flocculant to achieve the above object. It has been found that the invention can be completed.

즉,발명의 목적은 통상의 발효법으로 얻은 아미노산 발효액 또는 핵산 발효액으로부터 균체를 효율적으로 분리하는 방법을 제공하는 것이다.That is, an object of the present invention is to provide a method for efficiently separating cells from amino acid fermentation broth or nucleic acid fermentation broth obtained by conventional fermentation.

본 발명의 또다른 목적은 통상의 발효법으로 얻은 아미노산 발효액 또한 핵산 발효액에 산을 첨가하여 그pH를 약산성으로 조절하고, 유기계 응집제를 50-300ppm의 양으로 넣고 가열한 후 씨크너를 사용하여 균체가 분리된 상징액과 균체 부피가 30.0% 정도의 균체 포함액을 얻는 것을 특징으로 하는 발효액으로부터균체를 분리하는 방법을 제공하는 것이다.Another object of the present invention is to add amino acid fermentation broth obtained by the conventional fermentation method to the acidic fermentation broth to adjust the pH to weakly acidic, the organic flocculant is added in an amount of 50-300ppm and heated, and the cells are separated using a Seekner It is to provide a method for separating the cells from the fermentation broth, characterized in that the obtained supernatant and the cell containing solution of the cell volume of about 30.0%.

이하, 본 발명을 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail.

본 발명에 따르면, 그로부터 균체를 제거하게 필 아미노산 발효액 또는 핵산 발효액은 통상의 방법을 얻을 수 있다. 이러한 발효법은 당업계에 주지되어 있으며 본 발명의 일부를 구성하지 않는다. 또한 아미노산과 핵산의 종류는 제한되지 않지 않다According to the present invention, the amino acid fermentation broth or nucleic acid fermentation broth to be removed from the cells can be obtained by the conventional method. Such fermentation methods are well known in the art and do not form part of the present invention. In addition, the types of amino acids and nucleic acids are not limited.

발효액의 pH를 약산성으로 조절하기 위해 사용되는 산은 무기산 또는 유기산일 수 있으며, 바람직하게는황산이 사용된다. 이들 산을 이용하여 아미노산 발효액 또는 핵산 발효액의 pH를 3.0-5.0으로 조절한다.The acid used to adjust the pH of the fermentation broth to weak acidity may be an inorganic acid or an organic acid, preferably sulfuric acid. These acids are used to adjust the pH of the amino acid fermentation broth or nucleic acid fermentation broth to 3.0-5.0.

이렇게 pH를 조절한 발효액을 가열조로 이송한 후 유기계 응집계를 첨가한다. 본 발명에서 사용가능한유기계 응집제는 당업계에서 통상적으로 사용되고 있는 것으로서, 특히 폴라아크릴레이토 나트륨(soldiump이yacrylate)계, 또는폴리아크릴아미드나트륨(sodiump이yacITlamide)계응집제이다·유기계응집제로는 시판되는 것, 예를 들면 일본순약(日本純藥) 제품인 아론비스-M(Aronvis-M) 또는 일본 부로스화학제품인 세파란(Separan) AP-30을 사용할 수 있다. 유기 응집제의 사용량은 10-500ppm, 바람직하게는50 - 300ppm 다.The pH-adjusted fermentation broth is transferred to a heating bath and then an organic flocculant is added. The organic flocculant usable in the present invention is commonly used in the art, and is particularly a polyacrylato sodium (polyacrylate) or polyacrylamide sodium (sodiump). For example, Aronvis-M (Japanese Pure Chemicals) or Separan AP-30 (Japanese Buros Chemical) can be used. The amount of the organic flocculant used is 10-500 ppm, preferably 50-300 ppm.

유기 응집제가 첨가된 발효액은 가일조에서 약 40-80℃로0.5-2시간동안 가일한 후 씨크너에 이송된 후일정한 속도로 제거된다 이때 pH가 조절된 발효액을 가일조로 이송시키는 것과 동시에 유기 응집제가 첨가된 발효액을 씨크너로 이송시킴으로써 두 과정을 동시에 연속적으로 행하는 겻이 바람직하다. 이때 두 발효액의 유속은 1-10 L/hr이 다. 이 공정에 의해 원 발효액은 발효액중 80% 이상이 균체 분피(ceelvolume)가 0.5% 이하인 균체가 제거된 상징액과 균체의 부피 함량이 약 30.0%인 균체 포함액으로 분리되게 된다.The fermentation broth to which the organic flocculant is added is removed at a constant rate after passing for 0.5-2 hours at about 40-80 ° C. in the sills, and then transferred to the Seeker, and at the same time as the organic flocculant is transferred to the sills. It is preferable to carry out two processes simultaneously simultaneously by transferring the fermentation broth to which is added to the Seeker. At this time, the flow rate of the two fermentation broths is 1-10 L / hr. By this process, more than 80% of the fermentation broth is separated into a supernatant from which the cells with a cell volume of 0.5% or less are removed, and a cell-containing solution having a volume content of about 30.0%.

이중 균체 포항액으로부터 균체 분리기를 이용하여 균체를 분리하고 나면 균체 포함액 부피의 68%에 해망하는 부피의 상징액을 얻게 된다. 이 상징액과 상기에서 얻은 상징액을 합하고 산을 이용하여 pH 3.0-3.5로 조정하여 정석하면 고순도의 아미노산 또는 헥산의 결정을 얻을 수 있다.After separating the cells from the cell suspension using a cell separator, a volume of supernatant solution of 68% of the cell containing solution volume is obtained. This supernatant and the supernatant obtained above are combined and adjusted to pH 3.0-3.5 using an acid to crystallize high purity amino acids or hexanes.

본 발명에서 사용되는 써크너는 원추형의 것이며, 내부에 응집제가 부상하는 것을 방지하기 위하여 원형분리판이 구비되어 있다.The circner used in the present invention is conical, and is provided with a circular separator to prevent flocculents from floating therein.

발명의 방법에 따르면, 통상의 방법과 비교할 때, 최종적으로 얻어지는 아미노산 또는 핵산의 결정 수율이 증가하고, 균체를 분리하기 위한 기계적 처리량이 18-31%로 줄어들기 때문에 생산 비율을 절감할 수있는 장정이 있다.According to the method of the invention, when compared with the conventional method, the yield of crystallization of the finally obtained amino acid or nucleic acid increases, and the mechanical throughput for separating the cells is reduced to 18-31%, which can reduce the production rate. There is this.

이하 실시예에 의해 본 발명을 보다 상세히 설명하지만 본 발명이 이들 실시예에 국한되는 것은 아니다.The present invention will be described in more detail with reference to the following Examples, but the present invention is not limited to these Examples.

(실시예 1)(Example 1)

통상의 발효법에 의해 얻은 농도가 105.0 g/L인 글루타민산 발효액에 황산을 사용하여 ph 5.0으로 조절한 후 pH 조절액 3L를 취하여 가열조에 넣고 교반시키면서 유기계 응집제의 일종인 아론비스(Aronvis)-M을 50ppm 첨가하고 50 ℃에서 30분간 가열하였다. 펌프를 사용하여 6.0L/hr의 유속을 pH 조절 발효액과가열 응집 발효액을 각각 가열조와 씨크너토 이송시키고 씨크너로부터 일정한 속도로 균체를 제거하였다.그 결과, 균체 부피가 0.6% 이하인 균체 제거 상징액 15L와 균체부피가 33.6% 인 균체 포함액 4.5L가 얻어졌다. 따라서, 균체를 제거하기 위한 기계적 조작량을 23% 이하로 줄일 수 있었다.Glutamic acid fermentation broth with a concentration of 105.0 g / L was adjusted to pH 5.0 using sulfuric acid, and then 3L of pH-adjusted solution was added to a heating bath and stirred, followed by stirring with aronvis-M, a type of organic flocculant. 50 ppm was added and it heated at 50 degreeC for 30 minutes. Using a pump, the flow rate of 6.0 L / hr was transferred to the pH-controlled fermentation broth and the heated coagulated fermentation broth, respectively, and the thinner was removed from the thinner at a constant rate. As a result, 15L of the cell removal supernatant having a cell volume of 0.6% or less was removed. 4.5 L of the cell-containing solution containing 33.6% of the cell volume was obtained. Therefore, the amount of mechanical operation for removing the cells could be reduced to 23% or less.

[표 1] 글루타민산 발효액의 균체 제거 전.후 비교[Table 1] Comparison before and after cell removal of glutamic acid fermentation broth

한편, 본 발명에 따른 방법의 효과를 알아보기 위하여 대조로서 씨크너를 통과시키지 않은 발효액으로부터 균체를 제거하고 그 결과를 실시예 1의 본 발명의 결과와 비교하였다.On the other hand, in order to determine the effect of the method according to the present invention as a control, the cells were removed from the fermentation broth that did not pass through the Seekner and the results were compared with the results of the present invention of Example 1.

[표 2] 기존 방법과 본 발명의 비교Table 2 Comparison of Existing Methods and the Invention

(실시예 2)(Example 2)

동상의 발효법에 의해 얻은 L-페닐알라닌 농도 42g/L인 발효액에 황산을 사용 pH 4로 조절한 후 pH조절액 4L를 취하여 가열조에 넣고 교반시키면서 유기계 응집제인 세파란(Separan) AP-30(일본 부로스 화학(7。ロス化學) 제품)을 200ppm 첨가하여 50∼80℃에서 1∼2시간 가열한 후 펌프를 사용하여 2∼4L/hr의유속으로 pH 조절액(16L)과 균체 응집발효액을 각각 가열조와 씨크너로 이송시키면서 씨크너로부터 균체포함액을 제거하고, 균체 분리 전후의 각각의 발효액의 페닐알라닌 성분과 균체량을 측정하고 결과를 표 3에 나타내었다.After adjusting sulfuric acid to pH 4 in L-phenylalanine concentration 42g / L obtained by the frostbite fermentation method, 4L of pH-adjusting solution was taken into a heating bath and stirred while Separan AP-30 (organic coagulant) 200 ppm of Ross Chemical Co., Ltd. was added and heated at 50 to 80 ° C. for 1 to 2 hours, and then the pH adjusting solution (16 L) and the cell aggregate fermentation solution were respectively used at a flow rate of 2 to 4 L / hr using a pump. The cell containing liquid was removed from the thinner while being transferred to the heating bath and the thinner, and the phenylalanine component and the cell weight of each fermentation broth before and after cell separation were measured and the results are shown in Table 3.

[표 3] L-페닐알라닌 발효액의 균체 분리 전.후 비교[Table 3] Comparison of before and after cell separation of L-phenylalanine fermentation broth

상기 표 3의 결과에서 알 수 있듯이, 본 발명의 방법에 따라 균체를 제거한 결과 균체 부피가 0.6% 이하인 균체 분리 상징액 15L와 균체 부피가 33.6%인 균체 포함액을 4.5L 얻음으로써 기계적 조작량을 23%이하로 줄일 수 있다.As can be seen from the results of Table 3, the result of removing the cells in accordance with the method of the present invention by obtaining a cell separation supernatant 15L having a cell volume of 0.6% or less and 4.5L of a cell containing solution having a cell volume of 33.6% by 23% mechanical operation amount It can be reduced below.

(실시예 3)(Example 3)

통상의 발효법에 의해 얻은 L-라이신 농도 50.2g/L인 발효액에 황산을 사용하여 pH를 4.5로 조절한 후 세파란 AP-30 300ppm을 사용하여 상기 실시예 2와 동일한 방법으로 실시한 결과 표 4와 같은 결과를 얻었다.In the fermentation broth with L-lysine concentration of 50.2 g / L obtained by the conventional fermentation method, the pH was adjusted to 4.5 using sulfuric acid, and then, the same procedure as in Example 2 was performed using Separan AP-30 300ppm. The same result was obtained.

[표 4] L-라이신 발효액의 균체 분리 전,후 비교[Table 4] Comparison before and after cell separation of L-lysine fermentation broth

상기 표 4의 결과에서 알 수 있듯이, 본 발명의 방법에 따르면, 균체 부피가 0.7%인 균체 분리 상징액13.4L와 균체 부피가 37.2%인 균체 포함액을 6.2L 얻음으로써 균체 제거를 위한 기계적 조작량을 31% 이하로 줄일 수 있다.As can be seen from the results of Table 4, according to the method of the present invention, by obtaining 13.4L of the cell separation supernatant with a cell volume of 0.7% and 6.2L of the cell containing liquid with a cell volume of 37.2%, a mechanical manipulation amount for cell removal was obtained. It can be reduced below 31%.

(실시예 4)(Example 4)

통상의 발효법에 의해 얻은 5。-이노신산 나트륨 능도 41.8G/1인 5'-이노신산 나트륨 발효액의 pH를 황산으로 5.0으로 조절한 후 상시 실시예 3과 동일한 방법으로 실시한 결과 균체 부피가 0.7%인 균체 분리상징액 13.8L와 균체 부피가 37.6%인 균체 포함액을 5.8L 얻음으로서 기계적 조작량을 29% 이하로 줄일수 있었다. 결과를 표 5에 나타내었다.The pH of the 5'-inosinoate fermentation broth obtained by the conventional fermentation method with 51.8-sodium inosinonate ability of 41.8G / 1 was adjusted to 5.0 with sulfuric acid, and the same procedure as in Example 3 was carried out, and the cell volume was 0.7%. By obtaining 13.8L of the cell separation supernatant and 5.8L of the cell-containing solution containing 37.6% of the cell volume, the mechanical manipulation amount was reduced to 29% or less. The results are shown in Table 5.

[표 5] 5'-아노신산 나트륨 발효액의 균체 분리 전.후 비교[Table 5] Comparison of before and after cell separation of 5'-anosine fermentation broth

(실시예 5)(Example 5)

통상의 발효법에 의해 얻은 5。-구아닐산 나트륨 농도 45.2g/L인 5'-구아닐산 나트륨 발효액을 사용하여실시예 5와 동일한 방법으로 실시한 결과 표 6과 같은 결과를 얻었다.Using the 5'-sodium guanylate fermentation broth having a concentration of 55.2 g / L sodium guanylate obtained by a conventional fermentation method in the same manner as in Example 5, the results shown in Table 6 were obtained.

[표 6] 5'-구아닐산 나트륨 발효액의 균체 분리 전.후 비교[Table 6] Comparison of before and after cell separation of 5'-sodium guanylate fermentation broth

Claims (4)

통상의 발효법에 의해 얻은 아미노산 또는 핵산 발효액을 pH 3.0~5.0으로 조정한 후,여기에 유기응집제 50∼300ppm을 첨가하고,50∼80℃로 0.5∼2시간 동안 가열하면서 반응시킨 후, 균체를 응집시켜 원추형 씨크너(thickener)에 의해 연속적으로 균체와 상징액을 분리하는 방법.After adjusting the amino acid or nucleic acid fermentation broth obtained by a conventional fermentation method to pH 3.0-5.0, 50-300 ppm of an organic coagulant is added here, and it reacts, heating at 50-80 degreeC for 0.5 to 2 hours, and then, agglomerating a bacterial cell. To separate cells and supernatant successively by conical thickener. 제1항에 있어서, 유기계 응집제로 폴리아크릴레이트 나트륨(Sodium polyacrylate)계 또는 폴리아크릴아미드 나트륨(Sodium polyacryamide)계 응집제를 사용함을 특징으로 하는 방법.The method of claim 1, wherein the organic flocculant comprises sodium polyacrylate or sodium polyacryamide flocculant. 제1항에 있어서, 아미노산은 글루타민산, 페닐알라닌, 라이신 임을 특징으로 하는 방법.The method of claim 1 wherein the amino acid is glutamic acid, phenylalanine, lysine. 제1항에 있어서, 핵산은 5'-이노신산나트륩 및 5'-구아닐산나트륨 임을 특징으로 하는 방법.The method of claim 1, wherein the nucleic acid is 5′-nanosuccinate and 5′-sodium guanylate.
KR1019920023938A 1992-12-11 1992-12-11 Separating method microorganisms from amino acid and nucleotide fermenting liquid KR960006578B1 (en)

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