JPS63112996A - Production of riboflavin - Google Patents
Production of riboflavinInfo
- Publication number
- JPS63112996A JPS63112996A JP26022086A JP26022086A JPS63112996A JP S63112996 A JPS63112996 A JP S63112996A JP 26022086 A JP26022086 A JP 26022086A JP 26022086 A JP26022086 A JP 26022086A JP S63112996 A JPS63112996 A JP S63112996A
- Authority
- JP
- Japan
- Prior art keywords
- culture
- source
- riboflavin
- yeast
- carbon source
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 title claims abstract description 54
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 title claims abstract description 25
- 235000019192 riboflavin Nutrition 0.000 title claims abstract description 25
- 229960002477 riboflavin Drugs 0.000 title claims abstract description 25
- 239000002151 riboflavin Substances 0.000 title claims abstract description 25
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 26
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 20
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 17
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 17
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 15
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 13
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 8
- 241000235070 Saccharomyces Species 0.000 claims abstract description 5
- 239000002609 medium Substances 0.000 claims description 13
- 238000012258 culturing Methods 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 claims description 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 abstract description 19
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 abstract description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 7
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 abstract description 7
- 229910052921 ammonium sulfate Inorganic materials 0.000 abstract description 7
- 235000011130 ammonium sulphate Nutrition 0.000 abstract description 7
- -1 etc. Chemical compound 0.000 abstract description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 abstract description 3
- 229910019142 PO4 Inorganic materials 0.000 abstract description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 abstract description 3
- 239000008103 glucose Substances 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 239000001888 Peptone Substances 0.000 abstract description 2
- 108010080698 Peptones Proteins 0.000 abstract description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 abstract description 2
- 239000004202 carbamide Substances 0.000 abstract description 2
- 235000013877 carbamide Nutrition 0.000 abstract description 2
- 235000019319 peptone Nutrition 0.000 abstract description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 abstract 2
- 239000010452 phosphate Substances 0.000 abstract 2
- 238000000855 fermentation Methods 0.000 description 7
- 230000004151 fermentation Effects 0.000 description 7
- 244000005700 microbiome Species 0.000 description 7
- VSGNNIFQASZAOI-UHFFFAOYSA-L calcium acetate Chemical compound [Ca+2].CC([O-])=O.CC([O-])=O VSGNNIFQASZAOI-UHFFFAOYSA-L 0.000 description 5
- 239000001639 calcium acetate Substances 0.000 description 5
- 235000011092 calcium acetate Nutrition 0.000 description 5
- 229960005147 calcium acetate Drugs 0.000 description 5
- 238000012136 culture method Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 241000193403 Clostridium Species 0.000 description 2
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 229910000160 potassium phosphate Inorganic materials 0.000 description 2
- 235000011009 potassium phosphates Nutrition 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 235000012501 ammonium carbonate Nutrition 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 229940099112 cornstarch Drugs 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 229940111685 dibasic potassium phosphate Drugs 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000002864 food coloring agent Nutrition 0.000 description 1
- 239000000576 food coloring agent Substances 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000011785 micronutrient Substances 0.000 description 1
- 235000013369 micronutrients Nutrition 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 229910017464 nitrogen compound Inorganic materials 0.000 description 1
- 150000002830 nitrogen compounds Chemical class 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- IBUIVNCCBFLEJL-UHFFFAOYSA-M sodium;phosphoric acid;chloride Chemical compound [Na+].[Cl-].OP(O)(O)=O IBUIVNCCBFLEJL-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明はサツカロミセス属の酵母を用いる酢酸からりボ
フラビンの製造方法に関するものである。DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method for producing acetic acid-induced boflavin using yeast of the genus Satucharomyces.
（従来の技術）
発酵法によるリボフラビンの製造方法についてはエレモ
セシウムＯアシビイ（Ｅｒｅｍｏｔｈａｃｉｕｍａｓｈ
ｂｙｉｉ）、アシビア・ゴッシビイ（Ａｓｈｂｙａｇｏ
ｓｓｙｐｉｉ）、キャンディダ・フラング４ （Ｃａｎ
ｄｉｄａ中
ｆｌａｒｅｒｉ）、クロストジウム・アセトブチリカム
（Ｃｌｏｓｔｒｉｄｉｕｍ ａｃｅｔｏｂｕｔｙｌｉｃ
ｕｍ ）等を塘質培地中で培養して培養液中にリボフラ
ビンを生成・蓄積せしめる方法が知られている（ｆログ
レス・インダストリアル・ミクロバイオロジー（Ｐｒｏ
ｇｒｅｓｓＩｎｄｕｓｔｒｉａｌ Ｍｉｃｒｏｂｉｏｌ
ｏｇｙ ）１巻１３９頁、１９５９）。(Prior art) Regarding the production method of riboflavin by fermentation method, Eremothesium O.
byii), Ashbyago
ssypii), Candida Frang 4 (Can
Clostridium acetobutylic (Clostridium acetobutylic)
um) etc. in a solid medium to produce and accumulate riboflavin in the culture solution (Flogres Industrial Microbiology (Pro
gressIndustrial Microbiol
ogy), Vol. 1, p. 139, 1959).
本発明者の一部は酢酸を炭素源とする発酵法によるリボ
フラビンの製造方法を報告して−る（アグリ力ルチアル
アンド バイオロジカル、ケミスト リ −（Ａｇｒ、
Ｂｉｏｌ 、Ｃｈｅｍ、）ｖｏｌ 、 ２８ ＋ ｐ
、５５９＋５６６＋７６５（１９６４））。Some of the inventors have reported a method for producing riboflavin by a fermentation method using acetic acid as a carbon source (Agr.
Biol, Chem, ) vol, 28 + p
, 559+566+765 (1964)).
また、変異株を用いる方法としては、本発明者らによる
サツカロミセス属に属するプリン要求性変異株を用いる
方法（特開昭６０−２４１８９５号）、サツカロミセス
属に属する３−アミノ−１，２ｔ４−）リアゾールに耐
性を有する変異株を用いる方法（特開昭６０−２４１８
９６号）、サツカロミセス属に属するプリン要求性復帰
変異株を用いる方法（特願昭６０−１２０１１９号）、
サツカロミセス属に属するアンモニウムイオンに耐性を
有する変異株を用いる方法（特願昭６１−２３４８６号
）か知られている。Further, as a method using a mutant strain, a method using a purine auxotrophic mutant strain belonging to the genus Satucharomyces by the present inventors (Japanese Patent Application Laid-open No. 60-241895), 3-amino-1,2t4-) belonging to the genus Satucharomyces Method using a mutant strain resistant to lyazole (Japanese Patent Application Laid-Open No. 60-2418
96), a method using a purine auxotrophic revertant strain belonging to the genus Satucharomyces (Japanese Patent Application No. 120119/1982),
A method using a mutant strain resistant to ammonium ions belonging to the genus Satucharomyces (Japanese Patent Application No. 61-23486) is known.
なお、上記文献において微生物の名称として、キャンデ
ィダ・ロブスタ（Ｃａｎｄｉｄａ ｒｏｂｕｓｔａ）が
用いられているが、その後キャンディダ・ロブスタの標
準様（タイゲストレイン）において胞子が見出されてい
るため、ロダー著「デ・イースト」１９７０年版におい
ては、キャンディダ・ロブスタはサツカロミセス・セレ
ビッシェ（Ｓａｃｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓ
ｉａｅ ）に再分類されている。In addition, although Candida robusta (Candida robusta) is used as the name of the microorganism in the above-mentioned literature, since spores were subsequently found in the standard form (tigest strain) of Candida robusta, In the 1970 edition of "De East", Candida robusta is referred to as Saccharomyces cerevisiae.
iae).
一般的な培養方法においては、適当な組成の培地に微生
物を接種し、そのまま培養を続ける回分法で培養を行う
ことが多い。これに対し近年、最初から多量に加えると
微生物の生育に阻害的に働くメタノール等の炭素源を培
養経過に応じて少量ずつ加える流加培養法が発展してき
た。パン酵母の製造に際しては、アルコールの副生を抑
えるために糖蜜を少量ずつ加える流加培養法が用いられ
ている。In general culture methods, microorganisms are often inoculated into a medium with an appropriate composition, and culture is continued in a batch manner. In contrast, in recent years, a fed-batch culture method has been developed in which a carbon source such as methanol, which inhibits the growth of microorganisms if added in large amounts from the beginning, is added little by little as the culture progresses. When producing baker's yeast, a fed-batch culture method is used in which molasses is added little by little to suppress the by-product of alcohol.
（発明が解決しようとする問題点）
培地中でサツカロミセス属の酵母を培養して、リボフラ
ビンを採取するりボフラビンの製造方法については、す
でに上記のような本発明者らによる変異株を用いる方法
等が知られているが、かかる発酵法によるＩＪ ｇフラ
ビンの製造を回分法で行うに当たっては培養中に蓄積さ
れるリボフラビ／の濃度をできるだけ高めることが工業
化に際しての大きな課題となる。また、回分法で行うに
当たって、培養の最初よシ高濃度の炭素源を仕込むと、
酵母の生育及びす？フラビンの生成が阻害されることが
多いという欠点がある。(Problems to be Solved by the Invention) A method for producing boflavin by cultivating yeast of the genus Satucharomyces in a medium and collecting riboflavin has already been proposed, such as a method using a mutant strain as described above by the present inventors. However, when producing IJ g flavin by such a fermentation method in a batch method, a major challenge for industrialization is to increase the concentration of riboflavin accumulated during culture as much as possible. In addition, when using the batch method, if a high concentration of carbon source is added at the beginning of culture,
Yeast growth and growth? The disadvantage is that flavin production is often inhibited.
（問題点を解決するための手段）
本発明者らは発酵法によるｖｅフラビンの製造方法を改
良すべく、上記のような炭素源ばかシでなく広く他の成
分について流加培養法を用いて鋭意検討した結果、サツ
カロミセス属の酵母を用いてリボフラビンを製造する方
法において、培養槽内の培地に炭素源及び窒素源を連続
的又は間欠的に加えて培養を行うことにより、リボフラ
ビンが高濃度蓄積されることを認め、本発明を完成する
に至った。(Means for Solving the Problems) In order to improve the method for producing veflavin by fermentation, the present inventors have used a fed-batch culture method for a wide range of other components, not just the carbon source as described above. As a result of extensive research, we found that in a method for producing riboflavin using yeast of the genus Satucharomyces, riboflavin accumulates at high concentrations by continuously or intermittently adding a carbon source and a nitrogen source to the culture medium in the culture tank. The present invention was completed based on this recognition.
即ち、本発明はサツカロミセス属の酵母を用いてりｚク
ラビンを製造する方法において、培養槽内の培地に炭素
源及び窒素源を連続的又は間欠的に加えて培養を行うこ
とを特徴とするりぜフラビンの製造方法である。That is, the present invention provides a method for producing z-clavine using yeast of the genus Satucharomyces, which is characterized in that the culture is carried out by continuously or intermittently adding a carbon source and a nitrogen source to a medium in a culture tank. This is a method for producing zeflavin.
（使用する微生物）
本発明に於て使用する微生物としては培地で培養するこ
とによシリボフラビンを生産する微生物であればいかな
るものでも良いが複雑な天然物などの使用量が少ない培
地で培養できりボフラビンを生産する微生物例えば酢酸
などを炭素源としてリボフラビンを生産するサツカロミ
セス（Ｓａｃｃｈａｒｏｍｙｃｅｓ）属の酵母などが好
ましい具体例トシてはサツカロミセス・センビッシェ（
Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ
） ＡＨＵ ３４０５から誘導された変異株であるサツ
カロミセス・セレビッン工ｐ−１５４（ＦＥゐｉ ＢＰ
−５６６） ＴＰｌ、０１０（ＦＥＲＭ ＢＰ−５６５
）、ＴＷ−５７３（ＦＥＲＭ ＢＰ−５６７）、ＴＲ−
２９（ＦＥＲＭ ＢＰ−７８２）、ＮＨ−２６８（ＦＥ
肢Ｉ ＢＰ−９６５）などが上げることが出来る。(Microorganisms used) The microorganisms used in the present invention may be any microorganisms as long as they can produce siliboflavin when cultured in a medium, but those that can be cultured in a medium with a small amount of use, such as complex natural products. Preferred examples include microorganisms that produce riboflavin, such as yeast of the genus Saccharomyces that produces riboflavin using acetic acid as a carbon source.
Saccharomyces cerevisiae
) AHU 3405-derived mutant strain Satucharomyces cerebinate p-154 (FEii BP
-566) TPl, 010 (FERM BP-565
), TW-573 (FERM BP-567), TR-
29 (FERM BP-782), NH-268 (FERM
Limb I BP-965) can be raised.
（培養方法）
本発明を実施するに当たって培養物中にリボフラビンを
蓄積させるために、炭素源、窒素源、リン酸源、その他
必要な栄養源を含む培地にサツカロミセス属に属するす
がフラビン生産能を有する酵母を接種し、培養を行う。(Culture method) In carrying out the present invention, in order to accumulate riboflavin in the culture, the flavin-producing ability of Saccharomyces spp. Inoculate the yeast containing the yeast and culture it.
炭素源としては酢酸、グルコン酸等の有ｔａ 酸、グル
コース、シュークロース、キシロース等のＲＴＪ 、エ
タノール、グリセリン等のアルコール類その他を使用で
きる。As carbon sources, talic acids such as acetic acid and gluconic acid, RTJs such as glucose, sucrose and xylose, alcohols such as ethanol and glycerin, and others can be used.
窒素源としては種々の形態の窒素化合物が使用でき、例
えば硫酸アンモニウム、塩化アンモニウム、炭酸アンモ
ニウム、硝酸アンモニウム、アンモニア水、尿素、アミ
ノ酸、ペプトン、コーンステイープリカー等を用いるこ
とができる。Various forms of nitrogen compounds can be used as the nitrogen source, such as ammonium sulfate, ammonium chloride, ammonium carbonate, ammonium nitrate, aqueous ammonia, urea, amino acids, peptone, and cornstarch liquor.
リン酸源としてはフリーのリン酸の他リン酸第−カリウ
ム、す／酸第二カリウム、す／酸第−ナトリウム等のリ
ン酸塩を用いることができる。As the phosphoric acid source, in addition to free phosphoric acid, phosphates such as potassium phosphate, dibasic potassium phosphate, and sodium chloride phosphate can be used.
また、炭素源、窒素源、リン酸源の他に硫酸マグネシウ
ム等の無機塩類を使用することが好ましい。また、必要
に応じてピオチン等のビタミン類、アミノ酸、核酸塩基
等の微量栄養素を添加すればリボフラビンの蓄積量を増
す場合が多い。本発明者が先に発明した亜鉛イオンを添
加してリボフラビン生産性を向上させ鉄イオンによる阻
害を防ぐ改良製法（％開昭６０−５８０８８号）を適用
することもできる。Moreover, it is preferable to use inorganic salts such as magnesium sulfate in addition to the carbon source, nitrogen source, and phosphoric acid source. Furthermore, if necessary, the amount of accumulated riboflavin can be increased in many cases by adding micronutrients such as vitamins such as piotin, amino acids, and nucleobases. It is also possible to apply an improved manufacturing method (% JP-A-60-58088) which was previously invented by the present inventor and which improves riboflavin productivity by adding zinc ions to prevent inhibition by iron ions.
培養には好気的条件が好ましい。培地の声は２乃至１０
とするが、６乃至９に調節すれば、最も好ましい結果が
得られる。温度は２０℃乃至３７℃の範囲のうち使用菌
株の生育及びリボフラビン生産性に適した温度を用いる
ことができる。Aerobic conditions are preferred for culturing. Medium voice is 2 to 10
However, if the value is adjusted to 6 to 9, the most preferable results will be obtained. A temperature suitable for the growth of the strain used and riboflavin productivity can be used within the range of 20°C to 37°C.
′本発明においては、培養の途中で消費された炭素源及
び窒素源を連続的又は間欠的に添加して補充していくこ
とが肝要である。これによシリボフラビンの蓄積量を著
しく高めることができる。また、リン酸源を添加するこ
とによシリボフラビ／の蓄積量を更に高めることができ
る。これら途中で添加する炭素源、窒素源、リン酸源は
、粉末状で加えてもよいし水溶液の形で添加してもよい
。'In the present invention, it is important to continuously or intermittently add and replenish the carbon and nitrogen sources consumed during the culture. This can significantly increase the amount of siliboflavin accumulated. In addition, by adding a phosphoric acid source, the amount of siliboflavin/ can be further increased. The carbon source, nitrogen source, and phosphoric acid source that are added during these steps may be added in powder form or in the form of an aqueous solution.
このようにして得られる培養液からのリボフラビンの採
取には公知の手法が適用できる。即ち培養液を６０℃〜
１２０℃に加熱しりボフラビンを溶解させたのち、遠心
分離によシ酵母菌体と濾液に分離し、濾液を必要に応じ
て濃縮した後ハ・「ドロサルファイド或いは三塩化チタ
ンによシ還元しり？２ラビンを沈降させる。このように
して得られたり？フラピンを空気中で酸化させた後、水
、酢酸水溶液等の溶媒を用いて再結晶を行い、精製する
ととが可能である。Known techniques can be applied to collect riboflavin from the culture fluid thus obtained. That is, the culture solution is heated to 60℃~
After heating to 120°C and dissolving the boflavin, it was separated into yeast cells and filtrate by centrifugation, and the filtrate was concentrated as necessary. 2 Rabin is precipitated.Furapine thus obtained can be oxidized in air and then recrystallized using a solvent such as water or an aqueous acetic acid solution to purify it.
更には、本発明者によるリボフラビンの取得法（特開昭
６１−２１０９６号）を用いて高純度のり？７ラピン結
晶を採取することが可能である。Furthermore, high-purity glue can be obtained using the method for obtaining riboflavin by the present inventor (Japanese Patent Application Laid-Open No. 61-21096). It is possible to collect 7 lapin crystals.
（実施例） 次に実施例を示す。(Example) Next, examples will be shown.
実施例１
サツカロミセスセレビッシェＮＨ−２６８（ＦＥＲＭ
ＢＰ−９６５）をグルコース２％、ポリ−２ニア°トン
０．５％、酵母エキス０．３Ｊ、麦芽エキス０．３チを
含む液体培地ＩＱＱｍ／に接種し、３０℃で４５時間振
盪培養する。との前培養液を第−表の発酵Ａ培地３００
０ｍｌに１５％の接種量で接種し、７Ｌ容ジャーファー
メンタ−を用いて通気量０．３３ ｖ、ｖ、ｍ、 、攪
拌翼の回転数６０ Ｏｒ、ｐ、ｍ、、３０℃で培養を開
始した。Example 1 Satsukaromyces cerevisiae NH-268 (FERM
BP-965) was inoculated into a liquid medium IQQm/ containing 2% glucose, 0.5% poly-2Niaton, 0.3J yeast extract, and 0.3J malt extract, and cultured with shaking at 30°C for 45 hours. . The pre-culture solution with Fermentation A medium 300 in Table 1.
0 ml at a 15% inoculum volume, and cultured at 30°C using a 7 L jar fermenter with aeration volume of 0.33 v, v, m, rotation speed of stirring blades: 60 Or, p, m. It started.
培養開始７８時間目から酢酸カルシウムと硫酸アンモニ
ウムを流加し培養を継続した。全期間における酢酸カル
シウムと硫酸アンモニウムの総添加量は其々７６５ ｆ
ｌ 、 ２３．２ ｇであった。培養２６４時間でリボ
フラビン蓄積量が４．９１／／ｌになった。From 78 hours after the start of culture, calcium acetate and ammonium sulfate were added to continue the culture. The total amount of calcium acetate and ammonium sulfate added during the entire period was 765 f each.
1, 23.2 g. After 264 hours of culture, the amount of riboflavin accumulated was 4.91/l.
比較例１
実施例１と同様に培養を開始し、酢酸カルシウムと硫酸
アンモニウムの流加をおこなわず、通常の回分培養を行
った。培養１４０時間でリボフラビン蓄積量がＬ９ｆｌ
／ｌと最大になった。Comparative Example 1 Culture was started in the same manner as in Example 1, and normal batch culture was performed without feeding calcium acetate and ammonium sulfate. The amount of riboflavin accumulated after 140 hours of culture was L9fl.
It reached the maximum of /l.
実施例２
サツカロミセスセレビッシェＮＨ−２６８（ＦＥＲＭ
ＢＰ−９６５）ラブルコース２％、ポリペプトン０．５
係、酵母エキス０．３ ％ 、麦芽エキス０．３ ％を
含む液体培地１００ｍＪに接種し、３０℃で４５時間振
盪培養する。この前培養液を第二表の発酵Ｂ培地３００
０、ｗｌに３，３チの接種量で接種し、７Ｌ容ジャーフ
ァーメンタ−を用いて通気量０．５ ｖ、ｖ、ｍ、、攪
拌翼の回転数６００ ｒ、ｐ、ｍ、、３０℃で培養を開
始した。培養開始４８．５時間目と７４時間目にシュー
クロース１５０ｇを其々流加した。また、ＰＨ訳節を培
養開始直後よシーを７±０．５に維持するよう声コント
ローラーによ９２８％アンモニア水を流加し、窒素源流
加とした。全期間に１００．９の２８係アンモニア水を
流加した。培養１３９時間でリボフラビン蓄積量が７１
６１ｎｇ／ｌになった。Example 2 Satsukaromyces cerevisiae NH-268 (FERM
BP-965) Rubble course 2%, polypeptone 0.5
100 mJ of a liquid medium containing 0.3% yeast extract and 0.3% malt extract was inoculated and cultured with shaking at 30°C for 45 hours. This pre-culture solution was added to Fermentation B medium 300 in Table 2.
Inoculate 0, wl with an inoculum amount of 3.3 cm, and use a 7 L jar fermenter with aeration volume of 0.5 v, v, m, rotation speed of stirring blade 600 r, p, m,, 30 Culture was started at ℃. 150 g of sucrose was fed at 48.5 hours and 74 hours after the start of culture. Immediately after the start of culture, 928% ammonia water was added using a voice controller to maintain the pH value at 7±0.5, and a nitrogen source was added. During the entire period, 100.9% 28% ammonia water was fed. After 139 hours of culture, the amount of riboflavin accumulated was 71.
It became 61ng/l.
実施例３
サツ力ロミセスセレピッシェＮＨ−２６８（ＦＥＲ，’
ｌｉ’Ｉ ＢＰ−９６５）ラブルコース２％、ポリペプ
トン０．５ ％、酵母エキス０．３％、麦芽エキス０．
３チを含む液体培地１００ｍ／に接種し、３０℃で４０
時間振盪培養する。実施例１と同様に培養を開始した。Example 3 Satsuri Romyces Serepische NH-268 (FER,'
li'I BP-965) Rubble course 2%, polypeptone 0.5%, yeast extract 0.3%, malt extract 0.
Inoculate 100 m/liquid medium containing 3.
Incubate with shaking for an hour. Culture was started in the same manner as in Example 1.
培善開始６０時間目から酢酸カルシウムと硫酸アンモニ
ウムとリン酸第−カリウムを流加し培養を継続した。全
期間における酢酸カルシウムと硫酸アンモニウムとリン
酸第−カリウムの総添加量は其々３７０／＃１５．２ｇ
、６゜Ｏｙであった。培養２５０時間でリボフラビン蓄
積量が５．８ｇ／ｌとなった。From 60 hours after the start of culture, calcium acetate, ammonium sulfate, and potassium phosphate were added to continue the culture. The total amount of calcium acetate, ammonium sulfate, and potassium phosphate added during the entire period was 370/#15.2g each.
, 6°Oy. After 250 hours of culture, the amount of riboflavin accumulated was 5.8 g/l.
（発明の効果）
す、１．−フラビン発酵に於て、培養の途中で炭素源及
び窒素源を連続的又は間欠的に添加していくことによっ
てリボフラビン蓄積量を著しく高めることができた、リ
ボフラビンは医薬、飼料添加剤。(Effects of the invention) 1. - During flavin fermentation, the amount of riboflavin accumulated could be significantly increased by continuously or intermittently adding a carbon source and a nitrogen source during the culture. Riboflavin is used as a medicine and feed additive.
食品用の着色剤などとして非常に有用な物質であり、本
発明により、工業的規模での効率的なりボフラビンの製
造が可能となった。Boflavin is a very useful substance as a food coloring agent, and the present invention has made it possible to efficiently produce boflavin on an industrial scale.
Claims (2)
製造する方法において、培養槽内の培地に炭素源、窒素
源を連続的又は間欠的に加えて培養を行うことを特徴と
するリボフラビンの製造方法。(1) A method for producing riboflavin using yeast of the genus Saccharomyces, which comprises culturing by continuously or intermittently adding a carbon source and a nitrogen source to a medium in a culture tank.
源を連続的又は間欠的に加えて培養を行うことを特徴と
する特許請求第１項記載の方法。(2) The method according to claim 1, characterized in that the culture is carried out by continuously or intermittently adding a phosphoric acid source in addition to a carbon source and a nitrogen source to the culture medium in the culture tank.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP26022086A JPS63112996A (en) | 1986-10-31 | 1986-10-31 | Production of riboflavin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP26022086A JPS63112996A (en) | 1986-10-31 | 1986-10-31 | Production of riboflavin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS63112996A true JPS63112996A (en) | 1988-05-18 |
Family
ID=17345018
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP26022086A Pending JPS63112996A (en) | 1986-10-31 | 1986-10-31 | Production of riboflavin |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63112996A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5589355A (en) * | 1992-12-07 | 1996-12-31 | Kyowa Hakko Kogyo Co., Ltd. | Process for producing riboflavin |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS524628A (en) * | 1975-06-30 | 1977-01-13 | Kawasaki Heavy Ind Ltd | Tunnel excavator |
| JPS5926275A (en) * | 1982-07-08 | 1984-02-10 | マンネスマン・タリ−・ゲゼルシヤフト・ミツト・ベシユレンクテル・ハフツング | Pendulum mechanism for reciprocating machine section |
-
1986
- 1986-10-31 JP JP26022086A patent/JPS63112996A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS524628A (en) * | 1975-06-30 | 1977-01-13 | Kawasaki Heavy Ind Ltd | Tunnel excavator |
| JPS5926275A (en) * | 1982-07-08 | 1984-02-10 | マンネスマン・タリ−・ゲゼルシヤフト・ミツト・ベシユレンクテル・ハフツング | Pendulum mechanism for reciprocating machine section |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5589355A (en) * | 1992-12-07 | 1996-12-31 | Kyowa Hakko Kogyo Co., Ltd. | Process for producing riboflavin |
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