JPS6158583A - Control of flow cultivation - Google Patents

Abstract

PURPOSE:To improve the production efficiency of metabolic product in the culture of microorganisms, by adding substrate to the culture system according to the change of oxygen consumption rate, etc. CONSTITUTION:A microorganism (e.g. streptomycin-producing bacteria) is cultured in a culture liquid containing a substrate (e.g. glucose) in a culture tank while introducing air through the line 7 and discharging generated gas through the line 13. In the above process, (i) the oxygen concentration of the inlet gas is determined by the gas flow meter 5 and the oxygen analyzer 6 attached to the line 7, (ii) the oxygen concentration of the outlet gas and the carbon dioxide gas concentration of the outlet gas are determined by the gas flow meter 10, the oxygen analyzer 11 and the carbon dioxide gas analyzer 12 attached to the line 13, and (iii) the rate of oxygen consumption and the rate of carbon dioxide gas generation in the culture liquid are calculated from the values determined by the above process. When the above rates are varied beyond preset ranges, the pump 8 is operated, and the substrate is supplied from the tank 3 through the line 9 to the culture liquid.

Description

【発明の詳細な説明】 〔発明の利用分野〕 本発明は微生物や動植物細胞などに抗生物質、アミノ酸
及び生理活性物質を生産させるに際し、目的とする物質
を効率よく生産させる流加培養制御方法に関するもので
おる。[Detailed Description of the Invention] [Field of Application of the Invention] The present invention relates to a fed-batch culture control method for efficiently producing target substances when producing antibiotics, amino acids, and physiologically active substances in microorganisms, animal and plant cells, etc. It's something.

〔発明の背景〕[Background of the invention]

微生物や動植物の細胞を培養する仁とにより、その代謝
物である抗生物質、アミノ酸またはホルモンなどの生理
活性物質などを生産することが行われている。この培養
方法は大部分が培養槽に培地と種菌を入れて培養するだ
けの回分培養で１゜まだ生産性は低い。BACKGROUND ART Metabolites such as antibiotics, amino acids, or physiologically active substances such as hormones are produced by culturing cells of microorganisms, animals and plants. This culture method is mostly a batch culture in which the culture medium and seed bacteria are placed in a culture tank and the productivity is still low.

そζで、生産性を高めるために培養途中で基質を添加す
る流加培養法が行われることがあるが、基質を培養中の
どの時点で添加するかが問題となっており、その明確な
指標はまだ知られていない。Therefore, a fed-batch culture method in which a substrate is added during the culture is sometimes used to increase productivity, but the problem is at what point during the culture the substrate should be added, and it is not clear how to do so. The indicators are not yet known.

本発明では流加培養制御方法を示す具体例として、　ｓ
ｔｒｅｐｔｏｍｙｃｅｉ＋　ｇｒｌｓｅｕａによるスト
レブトマイシン（ＳＭ）発酵の流加培養制御方法を示す
。In the present invention, as a specific example of the fed-batch culture control method, s
1 shows a fed-batch culture control method for streptomycin (SM) fermentation by treptomycei+ grlseua.

ＳＭ発酵においては主に回分培養がなされているが、Ｊ
ｎｏｕｅら（Ｊ　、　ｐｅｒｍｅｎｔ、　’ｌ’ｅｃｈ
ｎｏｌ、　６０　。Batch culture is mainly used in SM fermentation, but J
noue et al. (J, perment, 'l'ech
nol, 60.

Ｐ１０５〜１１０．１９８２）はアスパラギンを培地中
のグルコースが消費される前に添加することにより８Ｍ
生産量を向上させた。しかし、これにはグルコースをオ
ンラインで測定するのが難しいという問題がある。P105-110.1982) is 8M by adding asparagine before the glucose in the medium is consumed.
Improved production. However, the problem with this is that glucose is difficult to measure online.

〔発明の目的〕[Purpose of the invention]

本発明の目的は微生物や動植物などの卸１胞を培贅し、
その代謝物を生産させるに際Ｌ、ｊＦｔ養中の細胞の酸
素消費速度−土たは炭酸ガス生成速度の変化を（１イ標
として基質を流加する流加培養制御方法を提供するもの
である。The purpose of the present invention is to cultivate whole cells of microorganisms, animals and plants,
In order to produce the metabolite, we provide a fed-batch culture control method in which the substrate is fed using the changes in the oxygen consumption rate - soil or carbon dioxide production rate of the cells during L, jFt feeding as one indicator. be.

〔発明の概要〕[Summary of the invention]

本発明者らはＳＭ発酵における基′！１１流加について
検討し、グルコースが有効であることを見い出した。つ
ぎにその流加時期について培養フラスコを用いて検討し
た。その結果が第１表に示しである。The present inventors have discovered the basis for SM fermentation! 11, and found that glucose was effective. Next, we investigated the feeding period using a culture flask. The results are shown in Table 1.

給　１　表 グルコースがｌ　Ｏｇ／を増加するよりに、培養１日目
、２日目、５日目に流力口したが、それ程大きな効果は
なかった。Feeding 1 In order to increase the glucose level in lOg/, water was given on the 1st, 2nd, and 5th days of culture, but there was no significant effect.

そこで、８Ｍ生産量が最大になった後にグルコースを流
加すれば良いのではないがと考え、培養２．５日目にグ
ルコースが５．１０及び２０ｇ／を増加するように流加
した。その結果を第２表に示す。Therefore, we thought that it would be a good idea to feed glucose after the 8M production reached the maximum, and fed the glucose so that it increased to 5.10 and 20 g/g on the 2.5th day of culture. The results are shown in Table 2.

第２表 これから培養２．５日目にグルコースが１０　ｇ／を増
加するように流加することにより、８Ｍ生産量を１．５
倍に向上できる仁とが分った。Table 2 From now on, on the 2.5th day of culture, glucose was fed in such a way that 10 g/g was added, increasing the 8M production to 1.5
I found out that I can improve myself twice as much.

つぎに、グルコースの添加時期を検討するため、５０を
培養槽を有する自動制御培養装置を用いて８Ｍ発酵を行
った結果が第２図（ａ）〜（Ｃ）及び第３図（ａ）〜（
ｄ）である。ここで、８Ｍ生産量がピークに達した培養
２日目に酸素消費速度及び炭酸ガス生成速度の急激な低
下がみられる。この時点の培養液中のグルコース濃度は
１０ｇ／ｌであシ、初期量の約５０優が残っていること
になる。それにもかかわらずこのような現象が生じるの
は、この時点で８Ｍ生産菌・の物置代謝が８Ｍ生産から
別の方向に変化したためであると考えられる。このよう
な酸素消費速度及び炭酸ガス生成速度の急激な変化は本
発明者らが初めて見い出したものでおる。そこで、この
変化を指標としてグルコースを流加することにより８Ｍ
生産量の向上を達成できたのである。Next, in order to examine the timing of glucose addition, 8M fermentation was performed using an automatically controlled culture device having a 50% culture tank. The results are shown in Figures 2 (a) to (C) and 3 (a) to (
d). Here, on the second day of culture when 8M production reached its peak, a rapid decrease in the oxygen consumption rate and carbon dioxide production rate was observed. The glucose concentration in the culture solution at this point is 10 g/l, which means that approximately 50% of the initial amount remains. Nevertheless, the reason why such a phenomenon occurs is considered to be that at this point, the storage metabolism of the 8M-producing bacteria changed from 8M production to a different direction. Such rapid changes in the oxygen consumption rate and carbon dioxide production rate were discovered for the first time by the present inventors. Therefore, by feeding glucose using this change as an indicator, 8M
They were able to achieve an increase in production.

酸素消費速度は通気カスの酸素量（ｍｏｌ／ｈ）から排
ガスの酸素量（ｍｏｌ／ｈ　　）を差し引くことによシ
求められ、炭酸ガス生成速度は排ガスの炭酸ガス量（ｍ
ｏｌ／ｂ）から通気ガスの炭酸ガス量（ｍｏｌ／ｈ　）
を差し引くことによって求められる。The oxygen consumption rate is determined by subtracting the amount of oxygen in the exhaust gas (mol/h) from the amount of oxygen in the aeration scum (mol/h), and the rate of carbon dioxide production is calculated by subtracting the amount of carbon dioxide in the exhaust gas (mole/h).
ol/b) to the amount of carbon dioxide in the ventilation gas (mol/h)
It is found by subtracting

また、通気ガス量が一定の場合は、排ガス中の酸素濃度
及び炭酸ガス濃度の変化を指標とすることができる。Furthermore, when the amount of ventilation gas is constant, changes in the oxygen concentration and carbon dioxide concentration in the exhaust gas can be used as an index.

流加基質としてはグルコースが用いられるが、このほか
にフラクトースなどの糠中アミノ酸なども有効である。Glucose is used as the fed-batch substrate, but in addition to this, bran amino acids such as fructose are also effective.

実験を行い、その特定の条件の組合せにおいて酸素消費
速度及び／又は炭酸ガス生成速度の急激な変化点を求め
ておき、この予備実験に基づいて実際の流加時点を決め
るようにすることができる。It is possible to perform an experiment to determine the point of sudden change in the oxygen consumption rate and/or carbon dioxide production rate under that particular combination of conditions, and then determine the actual feeding point based on this preliminary experiment. .

本発明に用いられる分析計としては酸素に対しては隔膜
式の酸素センサーや磁気式ダンベル形分析計など、炭酸
ガスに対しては赤外線吸収式分析計などが用いられる。Analyzers used in the present invention include diaphragm-type oxygen sensors and magnetic dumbbell analyzers for oxygen, and infrared absorption analyzers for carbon dioxide.

本発明はＳＭ発酵を例として説明したが、他の抗生物質
発酵のみならずアミノ酸生産、動植物細胞により生理活
性物質生産など、代謝物生産を目的とした培養プロセス
に広く適用可能である。Although the present invention has been described using SM fermentation as an example, it is widely applicable to culture processes aimed at metabolite production, such as amino acid production, biologically active substance production using animal and plant cells, as well as other antibiotic fermentation.

つぎに本発明方法を実施するに必要な装置の一例を第１
図に示す。培養槽１内に培地と種菌を入れ、導管７によ
り培養槽１内に空気を吹込みつつ攪拌機２により培養液
を攪拌しながら８Ｍ生産菌を培養する。この時、導管７
に設置したガス量測定器５と酸素分析計６を用いて入口
ガス酸素量を、導管１３に設置したガス量測定器１０、
酸素分析計１１と炭酸ガス分析ｉ１′１２を用いて出口
ガス酸素量と炭酸ガス量を制御用電子計算機４を用いて
秀出し、これから酸素消費速度と炭酸ガス生成速度を求
める。これらの急激な低下が生じた時に、制御用電子計
算機４から基質流加ポンプ８に信号を出してポンプを動
かし、基質タンク３から導管９によりグルコースを培養
槽１に流加する。その後、一定時間培養し、培養液を回
収して生産されたＳＭを分離精製する。Next, an example of the equipment necessary to carry out the method of the present invention will be described in the first example.
As shown in the figure. A culture medium and seed bacteria are placed in a culture tank 1, and 8M production bacteria are cultured while blowing air into the culture tank 1 through a conduit 7 and stirring the culture solution with an agitator 2. At this time, conduit 7
The oxygen amount of the inlet gas is measured using the gas amount measuring device 5 and oxygen analyzer 6 installed in the gas amount measuring device 10 installed in the conduit 13,
Using the oxygen analyzer 11 and the carbon dioxide analyzer i1'12, the amount of oxygen and carbon dioxide in the outlet gas is determined using the control electronic computer 4, and from this the oxygen consumption rate and carbon dioxide production rate are determined. When these rapid decreases occur, a signal is sent from the control computer 4 to the substrate feeding pump 8 to operate the pump, and glucose is fed into the culture tank 1 from the substrate tank 3 through the conduit 9. Thereafter, the cells are cultured for a certain period of time, the culture solution is collected, and the produced SM is separated and purified.

〔発明の実施例〕[Embodiments of the invention]

つぎに本発明の実施例について具体的に説明するが、本
発明はこれによりなんら限定されるものではない。Next, examples of the present invention will be specifically described, but the present invention is not limited thereto.

実施例１ 菌体：　Ｓｔｒｅｐｔｏｍｙｃｅｓ　ｇｒｉｓｅｕｓ　
ＨＵＴ　２２４７゜培地ニゲルコース２５　ｇ　、　　
（ＮＨ４）２８０４　２　ｇ。Example 1 Bacterial body: Streptomyces griseus
HUT 2247° medium Nigel course 25 g,
(NH4) 2804 2 g.

ＫＪｂＰＯｔ　０．４　ｇ　、ＮａｃｔＪＬ　Ｋ２８０
４６ＬＭｇＳＯ＜　、　７Ｆｈ　ＯＯ，２ｇ、　ＦｅＳ
Ｏ４，７ｌ−ｈｏｏ、　０１　ｇ　、　ＺｎＳＯ４、７
Ｈｚ００．０５ｇ＋Ｌ−ａｓｐａｒａｇｉｎｅ　７ｇ、
　ＣａＣ０ａ　　２Ｌ蒸留水ｍｔ、ＰＨ７，０゜ 培養条件＝５０を培養槽に培地を３０１仕込み、種菌を
６００　ｎ１１添加１〜だ。温度２８Ｃ，ｌ’Ｈ６，７
人口ガス１８ｔ／ｍｉｘに一定として培養。撹拌機回転
数を１４０〜２４５ｒｐｍ　に変えて溶 存酸素濃度を４．５ｍｇ／１以上に制御した。KJbPOt 0.4 g, NactJL K280
46LMgSO<, 7FhOO,2g, FeS
O4,7l-hoo, 01g, ZnSO4,7
Hz00.05g+L-asparagine 7g,
CaC0a 2L distilled water mt, PH7, 0°Culture conditions = 50, 301 medium was prepared in a culture tank, and 600 n11 seed bacteria were added. Temperature 28C, l'H6,7
Cultured with a constant artificial gas of 18t/mix. The stirrer rotation speed was changed from 140 to 245 rpm to control the dissolved oxygen concentration to 4.5 mg/1 or more.

結果：第４図（ａ）〜（Ｃ）及び第５図（ａ）〜（Ｃ）
に結果を示す。通気量一定条件下において、排ガス０２
＠度が最低値でかつ排ガスＣＯ２濃度が最高値に達した
時点、つまり酸素消費速度と炭酸カス生成速度が最高値
に達した時点から３０分後の５２時間目にａ　ｏ　ｏ　
ｇ／ｌ＠度のグルコースを１を流加した。この時点での
８Ｍ生産量は１５５ｍｇ／４であったが、グルコース流
加後の培養４日目には２６５ｍｇ／ｌの高い値になった
。対照実験として培養フラスコでＳＭ発酵゛を行った場
合では最大８Ｍ生産ｇ　＠　ｇｌｕｃｏｓｅ　、菌体収
率は０．２４ｇ−ｃｅｌｌ／　ｇ　。Results: Figures 4(a)-(C) and 5(a)-(C)
The results are shown in Under constant ventilation conditions, exhaust gas 02
At the 52nd hour, 30 minutes after the point when the temperature is the lowest and the exhaust gas CO2 concentration is the highest, that is, the oxygen consumption rate and carbon dioxide production rate are the highest.
1 g/l@degree glucose was fed. The 8M production amount at this point was 155 mg/4, but reached a high value of 265 mg/l on the fourth day of culture after glucose feeding. As a control experiment, when SM fermentation was performed in a culture flask, the maximum production was 8 Mg@glucose, and the bacterial cell yield was 0.24 g-cell/g.

ｇｌｕｃｏｓｅ、生産性は３　Ｂ−９ｍｇ”　ＳＭ／ｇ
−ｃｅ目であった。Glucose, productivity is 3B-9mg” SM/g
- It was the ce order.

これより、グルコース添加前では８Ｍ生産量は１５５１
１１ｇ／を程度であるが、流加により８Ｍ生産−鼠を１
．７倍に向上できた。From this, before adding glucose, the production amount of 8M is 1551
It is about 11g/, but 8M production by fed-batch - 1 mouse
．． I was able to improve it by 7 times.

【図面の簡単な説明】[Brief explanation of the drawing]

第１図は本発明の喧養方法を示す概略図、第２図及び第
３図は５０１自動制御培養槽でのＳＭ発酵実験結果を表
わすグラフ、第４図及び第５図は５０を自動制御培養槽
で基質流加を行ったＳＭ発酵実験結果を表わすグラフで
ある。 １・・・培養槽、２・・・攪拌機、３・・・基質タンク
、４・・・制御用電子計算機、５・・・ガス量測定器、
６・・・酸素分析岨、７・・・導管、８・・・基質流加
ポンプ、９・・・導管、ｌＯ・・・ガス鼠測定器、１１
・・・酸素分析＃１．１２・・・炭酸ガス分析ｉ［，１
３・・・導管。Fig. 1 is a schematic diagram showing the cultivation method of the present invention, Figs. 2 and 3 are graphs showing the results of SM fermentation experiments in the 501 automatically controlled culture tank, and Figs. 4 and 5 are graphs showing the automatically controlled culture tank 50. It is a graph showing the results of an SM fermentation experiment in which substrate feeding was performed in a culture tank. DESCRIPTION OF SYMBOLS 1...Culture tank, 2...Agitator, 3...Substrate tank, 4...Control electronic computer, 5...Gas amount measuring device,
6... Oxygen analysis cap, 7... Conduit, 8... Substrate fed-batch pump, 9... Conduit, lO... Gas meter measuring device, 11
... Oxygen analysis #1.12 ... Carbon dioxide analysis i[,1
3... Conduit.

Claims (1)

【特許請求の範囲】 １、微生物または動植物の細胞を好気的に培養し、その
代謝物を生産させるに際し、培養中の細胞の酸素消費速
度及びまたは炭酸ガス生成速度の急激な低下を指標とし
て、基質を流加することを特徴とする流加培養制御方法
。 ２、培養槽への通気ガス量が一定の場合は、排ガス中の
酸素濃度または炭酸ガス濃度の変化を指標とする特許請
求の範囲第１項記載の流加培養制御方法。 ３、細胞が微生物細胞であることを特徴とする特許請求
の範囲第１または２項記載の流加培養制御方法。 ４、代謝物が抗生物質であることを特徴とする特許請求
の範囲第１項、第２項及び第３項のいずれかに記載の流
加培養制御方法。 ５、基質がグルコースであることを特徴とする特許請求
の範囲第１項、第２項、第３項及び第４項のいずれかに
記載の流加培養制御方法。[Claims] 1. When microorganisms or animal or plant cells are cultured aerobically and their metabolites are produced, a rapid decrease in the oxygen consumption rate and/or carbon dioxide production rate of the cells during culture is used as an indicator. , a fed-batch culture control method characterized by feeding a substrate. 2. The fed-batch culture control method according to claim 1, wherein when the amount of aeration gas to the culture tank is constant, a change in the oxygen concentration or carbon dioxide concentration in the exhaust gas is used as an indicator. 3. The fed-batch culture control method according to claim 1 or 2, wherein the cells are microbial cells. 4. The fed-batch culture control method according to any one of claims 1, 2, and 3, wherein the metabolite is an antibiotic. 5. The fed-batch culture control method according to any one of claims 1, 2, 3, and 4, wherein the substrate is glucose.