WO1992013959A1 - Fermentation process producing a monascus dye mixture - Google Patents

Fermentation process producing a monascus dye mixture Download PDF

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WO1992013959A1
WO1992013959A1 PCT/EP1991/002450 EP9102450W WO9213959A1 WO 1992013959 A1 WO1992013959 A1 WO 1992013959A1 EP 9102450 W EP9102450 W EP 9102450W WO 9213959 A1 WO9213959 A1 WO 9213959A1
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monascus
precursor substrate
nutrient medium
added
culture
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French (fr)
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Joachim Schindler
Michael Bahn
Holger Viehweg
Wolfgang Adams
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Henkel Kommanditgesellschaft Auf Aktien
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

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  • the invention relates to a fermentation process for the improved production of a red dye mixture which is produced by microorganisms of the genus Monascus.
  • Preparations made from mushrooms of the genus Monascus colored red or orange have been used as dyes for coloring food, particularly in Asian countries.
  • the dyes which are a mixture of defined chemical substances such as ankaflavin, monascin, monascorubrin, rubropunctain, monascorubramine or rubropunctamine, are considered to be tolerated by the body.
  • Various health benefits are also attributed to them.
  • the main use is the coloring of milk and meat products.
  • the fermentation of the mushroom is traditionally carried out on rice in the Emers culture, the preparations thus obtained being used directly after drying.
  • German patent 24 61 642 proposes to make Monascus pigments water-soluble with the help of chitosan.
  • US Pat. No. 4,927,760 suggests the use of poly (oxyethylene) sorbitan esters of palmitic acid and / or unsaturated triglycerides for the production of crystalline monacus pigments.
  • the invention thus relates to a process for producing a red dye mixture by culturing a microorganism of the genus Monascus in a nutrient medium under aerobic conditions with a pH between 4 and 8.5, characterized in that the nutrient medium is precursor substrate for the Adds dye formation.
  • Suitable precursor substrates are those which are easily broken down to C 2 building blocks in the course of the usual metabolic cycles or are already present as C 2 building blocks, in particular carboxylic acids having 2 to 4 C atoms and, if desired, an amino group. So C 4 compounds such as malic acid, fumaric acid or oxaloacetic acid are suitable, C 3 compounds such as Ala ⁇ in are also suitable. C 2 compounds such as glycine and in particular acetic acid are preferred. Glutamic acid is also suitable.
  • the compounds mentioned are preferably used in the form of their salts, in particular in the form of the sodium salts.
  • the addition of the acids in the form of their alkali salts not only leads to nutrient supplementation during cultivation, but also to a stabilization or shift of the pH environment in the medium into the alkaline range. Both effects together lead to a clear stimulation of the coloring.
  • the concentration of the precursor substrates is set between 0.1 and 7% by weight, based on the fermentation medium. According to a particularly preferred procedure, the precursor substrates are added in several portions in an amount of 0.1 to 0.5% by weight. It may be particularly preferred to add 0.1 to 0.5% by weight to the fermentation batches daily.
  • the process according to the invention allows, in particular, the yield
  • nutrient media are used in the method according to the invention, as are customary in the cultivation of fungi of the genus Monascus.
  • Emers cultures are preferably grown on rice.
  • suitable as C substrates are galactose, glucose, fructose, maltose, sucrose, starch, rice flour, corn flour, ethanol and the like.
  • Ammonium chloride, sodium nitrate, ammonium nitrate, sodium glutamate, peptone, yeast extract or other substrates commonly used in fermentation technology are used as N sources. Reference is also made here to the patents mentioned at the beginning and the fermentation media mentioned therein.
  • Particularly suitable C sources are glucose and starch, especially prehydrolyzed starch.
  • the optimal concentration is between 2 and 10% by weight, based on the fermentation medium, in particular between 5 and 7.5% by weight.
  • Good pigment production is often achieved when yeast extract is used as the main nitrogen source and the concentration is around 1% by weight, based on the fermentation medium.
  • the method according to the invention is suitable for numerous microorganisms of the genus Monascus, for example for a microorganism of the species Monascus anka, Monascus kaoliang, Monascus purpurescens, Monascus purpureus, Monascus ruber, Monascus rubiginosus, Monascus rubropunctatus or Monascus serorubescens.
  • Monascus purpureus DSM 1379 was inoculated on slant agar tubes (ME or YM agar), incubated at 10- ° C for about 10-14 days until optimal transport and then stored in the cold room at 5 ° C.
  • Ferenbach flasks (ME agar; incubated for 21 days at 30 ° C) were well washed away with 100 ml 0.1 m PO4 buffer (pH 6.5) and washed with 20 ml buffer. The spore suspension was then shaken for 5 minutes, filtered to remove mycelium fragments over sterile glass wool, sterile glycerol (final concentration 10%) and stored in portions at -25 ° C. The spore concentration was 6.3 ⁇ 10 5 / ml.
  • the strain was shaken in 500 ml Erlenmeyer flasks (with baffles, 100 ml culture solution) on the shaker (Infors type RC106; shaking frequency 140 rpm) (gross temperature: 30 ° C).
  • the flasks were inoculated with 0.1 to 05 ml spore suspension.
  • the pH of the culture solution should be kept between 5.8 and 6.8 in the growth phase and between 7.5 and 8.5 in the production phase.
  • Monascus purpureus stores the pigments formed predominantly in the mycelium and secrets some of them into the culture medium.
  • the extracellular pigment formation is measured as follows:
  • the mycelium is separated off by filtration.
  • the separated and washed mycelium was dried at 90 ° C. for 48 hours and then ground to a fine powder. 10 mg of crushed mycelium were mixed with 5 ml of methanol and extracted in screw-on test tubes for 30 minutes on the mini shaker. Insoluble components were then centrifuged off and the clear extract was measured at 420 and 490 nm against methanol.

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Abstract

Disclosed is a fermentation process for the production of a dye mixture from Monascus microorganisms cultivated in a nutrient medium in aerobic conditions at a pH value between 4 and 8.5. In order to increase the yield of dye, a precursor-substrate for the dye formation is added to the nutrient medium.

Description

Fermentationsverfahren zur Herstellung einer Monascus Farbstoffmischung Fermentation process for the production of a Monascus dye mixture
Die Erfindung betrifft ein Fermentationsverfahren zur verbesserten Herstellung einer roten Farbstoffmischung, die von Mikroorganismen der Gattung Monascus erzeugt wird. The invention relates to a fermentation process for the improved production of a red dye mixture which is produced by microorganisms of the genus Monascus.
Rot oder orange gefärbte Zubereitungen aus Pilzen der Gattung Monascus werden insbesondere in den asiatischen Ländern seit alters her als Farbstoffe zum Anfärben von Nahrungsmitteln eingesetzt. Die Farbstoffe, die eine Mischung definierter chemischer Substanzen wie Ankaflavin, Monascin, Monascorubrin, Rubropunctain, Monascorubramin oder Rubropunctamin darstellen, gelten als körperveträglich. Verschiedentlich wird ihnen auch eine gesundheitsförderliche Wirkung zugeschrieben. Schwerpunkt der Verwendung ist die Anfärbung von Milch- und Fleischprodukten. Preparations made from mushrooms of the genus Monascus colored red or orange have been used as dyes for coloring food, particularly in Asian countries. The dyes, which are a mixture of defined chemical substances such as ankaflavin, monascin, monascorubrin, rubropunctain, monascorubramine or rubropunctamine, are considered to be tolerated by the body. Various health benefits are also attributed to them. The main use is the coloring of milk and meat products.
Traditionell erfolgt die Fermentation des Pilzes in Emerskultur auf Reis, wobei die so erhaltenen Zubereitungen nach dem Trocknen direkt eingesetzt werden. The fermentation of the mushroom is traditionally carried out on rice in the Emers culture, the preparations thus obtained being used directly after drying.
In dem US-Patent 4,145,254 wird vorgeschlagen, Monascus-Pigmente in einem wäßrigen Fermentationsmedium unter aeroben Bedingungen herzustellen, wobei man die Wachstumsphase bei einem pH-Wert von 4 bis 7 und die Produktionsphase in einen anderen Medium bei einem pH-Wert von 2 bis 4 durchführt, um ausschließlich organgefarbene Produkte zu erhalten. In US Pat. No. 4,145,254 it is proposed to produce Monascus pigments in an aqueous fermentation medium under aerobic conditions, the growth phase at a pH of 4 to 7 and the production phase in another medium at a pH of 2 to 4 carried out in order to receive only orange-colored products.
In dem US-Patent 3,765,906 wird vorgeschlagen, ein wasserlösliches MonascusPigment dadurch herzustellen, daß man einen Mikroorganismus Monascus purpureus oder Monascus anka züchtet und das entstandene Pigment mit einem wasserlöslichen Protein, wasserlöslichen Peptiden oder Aminosäuren zur Erhöhung der Löslichkeit umsetzt. In US Pat. No. 3,765,906 it is proposed to produce a water-soluble Monascus pigment by growing a microorganism Monascus purpureus or Monascus anka and reacting the resulting pigment with a water-soluble protein, water-soluble peptides or amino acids to increase the solubility.
In ähnlicher Weise schlägt das deutsche Patent 24 61 642 vor, MonascusPigmente mit Hilfe von Chitosan wasserlöslich zu machen. Das US-Patent 4,927,760 schließlich schlägt die Mitverwendung von Poly(oxyethylen)sorbitanestern der Palmitinsäure und/oder von ungesättigten Triglyceriden zur Herstellung kristalliner Monacus-Pigmente vor. Similarly, German patent 24 61 642 proposes to make Monascus pigments water-soluble with the help of chitosan. Finally, US Pat. No. 4,927,760 suggests the use of poly (oxyethylene) sorbitan esters of palmitic acid and / or unsaturated triglycerides for the production of crystalline monacus pigments.
Vor dem Hintergrund dieses Standes der Technik haben sich die Erfinder die Aufgabe gestellt, durch gezielte Beeinflussung des Nährmediums die Ausbeute an Farbstoffmischung zu erhöhen. Gegenstand der Erfindung ist somit ein Verfahren zur Herstellung einer roten Farbstoffmischung durch Züchten eines Mikroorganismus der Gattung Monascus in einem Nährstoffmedium unter aeroben Bedingungen mit einem pH-Wert zwischen 4 und 8,5, dadurch gekennzeichnet, daß man dem Nährstoffmedium ein Precursor-Substrat für die Farbstoffbildung zusetzt. Against the background of this prior art, the inventors have set themselves the task of increasing the yield of dye mixture by specifically influencing the nutrient medium. The invention thus relates to a process for producing a red dye mixture by culturing a microorganism of the genus Monascus in a nutrient medium under aerobic conditions with a pH between 4 and 8.5, characterized in that the nutrient medium is precursor substrate for the Adds dye formation.
Geeignete Precursor-Substrate sind solche, die im Rahmen der üblichen Stoffwechselzyklen leicht zu C2-Bausteinen abgebaut werden oder bereits als C2-Bausteine vorliegen, insbesondere Carbonsäuren mit 2 bis 4 C-Atomen und gewünschtenfalls einer Aminogruppe. So sind C4-Verbindungen wie Apfelsäure, Fumarsäure oder Oxalessigsäure geeignet, C3-Verbindungen wie Alaπin sind ebenfalls geeignet. Bevorzugst sind C2-Verbindungen wie Glycin und insbesondere die Essigsäure. Weiterhin geeignet ist auch die Glutaminsäure. Suitable precursor substrates are those which are easily broken down to C 2 building blocks in the course of the usual metabolic cycles or are already present as C 2 building blocks, in particular carboxylic acids having 2 to 4 C atoms and, if desired, an amino group. So C 4 compounds such as malic acid, fumaric acid or oxaloacetic acid are suitable, C 3 compounds such as Alaπin are also suitable. C 2 compounds such as glycine and in particular acetic acid are preferred. Glutamic acid is also suitable.
Die genannten Verbindungen werden, soweit sie als Säuren vorliegen, vorzugsweise in Form ihrer Salze eingesetzt, insbesondere in Form der Natriumsalze. Die Zugabe der Säuren in Form ihrer Alkalisalze führt während der Kultivierung nicht nur zu einer Nährstoffsupplementierung, sondern zugleich auch zu einer Stabilisierung bzw. Verschiebung des pH-Milieus im Medium in den alkalischen Bereich hinein. Beide Effekte gemeinsam führen zu einer deutlichen Stimulierung der Färbildüng. Die Konzentration der Precursor-Substrate wird zwischen 0,1 und 7 Gew.-%, bezogen auf das Fermentationsmedium, eingestellt. Nach einer besonders bevorzugten Vorgehensweise werden die Precursor-Substrate dazu in einer Menge von 0,1 bis 0,5 Gew.-% in mehreren Portionen zugegeben. So kann es besonders bevorzugt sein, 0,1 bis 0,5 Gew.-% täglich den Fermentationsansätzen beizufügen. Das erfindungsgemäße Verfahren erlaubt es, insbesondere die Ausbeute anInsofar as they are present as acids, the compounds mentioned are preferably used in the form of their salts, in particular in the form of the sodium salts. The addition of the acids in the form of their alkali salts not only leads to nutrient supplementation during cultivation, but also to a stabilization or shift of the pH environment in the medium into the alkaline range. Both effects together lead to a clear stimulation of the coloring. The concentration of the precursor substrates is set between 0.1 and 7% by weight, based on the fermentation medium. According to a particularly preferred procedure, the precursor substrates are added in several portions in an amount of 0.1 to 0.5% by weight. It may be particularly preferred to add 0.1 to 0.5% by weight to the fermentation batches daily. The process according to the invention allows, in particular, the yield
Farbstoff bei Fermentation von Pilzen der Gattung Monascus im Submersverfahren zu erhöhen. Dies ist in der Praxis besonders wichtig, da Monascus-Pilze im Submersverfahren nach bisherigem Stand der Technik bedeutend schlechtere Ausbeuten erbringen als in dem für eine technische Herstellung weniger geeigneten Emersverfahren. Gleichwohl kann die erfindungsgemäße Lehre jedoch auch auf Emerskulturen übertragen werden. Dabei ist zu beachten, daß bei Emerskulturen mit geringeren Flüssigkeitsmengen gearbeitet wird. Der Fachmann hat daher die Menge der PrecursorSubstrate umzurechnen, in dem er das Verhältnis nicht-wäßrige Bestandteile zu Precursor-Substrat von den Submerskulturen auf die Emerskulturen überträgt. Increase dye during fermentation of mushrooms of the genus Monascus in the submerged process. This is particularly important in practice, since Monascus mushrooms in the submerged process according to the prior art have yielded significantly poorer yields than in the Emers process, which is less suitable for industrial production. Nevertheless, the teaching according to the invention can also be applied to Emers cultures. It should be noted that lower amounts of liquid are used for emers cultures. The person skilled in the art must therefore convert the amount of precursor substrates by transferring the ratio of non-aqueous components to precursor substrate from the submerged cultures to the emersed cultures.
In dem erfindungsgemäßen Verfahren werden ansonsten Nährmedien verwendet, wie sie bei der Anzucht von Pilzen der Gattung Monascus üblich sind. So werden Emerskulturen vorzugsweise auf Reis angezogen. Für Submerskulturen eignen sich als C-Substrate Galaktose, Glukose, Fruktose, Maltose, Saccharose, Stärke, Reismehl, Maismehl, Ethanol und dergleichen. Als N-Quellen werden Ammoniumchlorid, Natriumnitrat, Ammoniumnitrat, Natriumglutamat, Pepton, Hefeextrakt oder andere in der Fermentationstechnik gebräuchliche Substrate eingesetzt. Verwiesen sei hier auch auf die eingangs erwähnten Patente und die darin genannten Fermentationsmedien. Otherwise, nutrient media are used in the method according to the invention, as are customary in the cultivation of fungi of the genus Monascus. For example, Emers cultures are preferably grown on rice. For submerged cultures, suitable as C substrates are galactose, glucose, fructose, maltose, sucrose, starch, rice flour, corn flour, ethanol and the like. Ammonium chloride, sodium nitrate, ammonium nitrate, sodium glutamate, peptone, yeast extract or other substrates commonly used in fermentation technology are used as N sources. Reference is also made here to the patents mentioned at the beginning and the fermentation media mentioned therein.
In der Regel kommt durch ein Überangebot an C-Substrat und durch Limitierung der N-Quelle eine stärkere Pigmentproduktion zustande. As a rule, an oversupply of C-substrate and limitation of the N-source lead to stronger pigment production.
Besonders geeignete C-Quellen sind Glukose und Stärke, insbesondere vorhydrolysierte Stärke. Die optimale Konzentration liegt dabei zwischen 2 und 10 Gew.-%, bezogen auf Fermentationsmedium, insbesondere zwischen 5 und 7,5 Gew.-%. Eine gute Pigmentproduktion wird vielfach erreicht, wenn Hefeextrakt als Hauptstickstoffquelle eingesetzt wird und die Konzentration um 1 Gew.-%, bezogen auf Fermentationsmedium, beträgt. Particularly suitable C sources are glucose and starch, especially prehydrolyzed starch. The optimal concentration is between 2 and 10% by weight, based on the fermentation medium, in particular between 5 and 7.5% by weight. Good pigment production is often achieved when yeast extract is used as the main nitrogen source and the concentration is around 1% by weight, based on the fermentation medium.
Das erfindungsgemäße Verfahren ist für zahlreiche Mikroorganismen der Gattung Monascus geeignet, so beispielsweise für einen Mikroorganismus der Spezies Monascus anka, Monascus kaoliang, Monascus purpurescens, Monascus purpureus, Monascus ruber, Monascus rubiginosus, Monascus rubropunctatus oder Monascus serorubescens. The method according to the invention is suitable for numerous microorganisms of the genus Monascus, for example for a microorganism of the species Monascus anka, Monascus kaoliang, Monascus purpurescens, Monascus purpureus, Monascus ruber, Monascus rubiginosus, Monascus rubropunctatus or Monascus serorubescens.
B e i s p i e l e B e i s p i e l e
Stammkulturhaltung Root culture
Monascus purpureus DSM 1379 wurde auf Schrägagarröhrchen (ME- oder YM-Agar) überimpft, etwa 10 - 14 Tage bis zur optimalen Versporung bei 30°C inkubiert und anschließend im Kühlraum bei 5°C gelagert.  Monascus purpureus DSM 1379 was inoculated on slant agar tubes (ME or YM agar), incubated at 10- ° C for about 10-14 days until optimal transport and then stored in the cold room at 5 ° C.
Impfkulturen Vaccine cultures
Gut versporte Ferenbachkoleben (ME-Agar; 21 Tage bei 30°C inkubiert) mit je 100 ml 0,1 m Pθ4-Puffer (pH 6,5) abgeschwemmt und mit 20 ml Puffer nachgewaschen. Sporensuspension wurde anschließend 5 Min. geschüttelt, zur Abtrennung von Myzelfragmenten über sterile Glaswolle filtriert, mit sterilem Glycerin versetzt (Endkonzentration 10 %) und portionsweise bei -25°C gelagert. Die Sporenkonzentration betrug 6.3 × 105/ml. Ferenbach flasks (ME agar; incubated for 21 days at 30 ° C) were well washed away with 100 ml 0.1 m PO4 buffer (pH 6.5) and washed with 20 ml buffer. The spore suspension was then shaken for 5 minutes, filtered to remove mycelium fragments over sterile glass wool, sterile glycerol (final concentration 10%) and stored in portions at -25 ° C. The spore concentration was 6.3 × 10 5 / ml.
Emerskultur Emers culture
Zur Oberflächenkultur wurden flache Alu-Schalen (770 cm2-Fläche) verwendet, die zur Sterilhaltung mit Alufolie abgedeckt waren. Flat aluminum dishes (770 cm 2 area ) were used for the surface culture, which were covered with aluminum foil for sterile maintenance.
Submerskultur Submerged culture
Zur Überprüfung von Wachstum und Pigmentfärbung unter Submersbedingungen wurde der Stamm in 500 ml Erlenmeyerkolben (mit Schikanen, 100 ml Kulturlösung) auf der Schüttelmaschine (Infors Typ RC106; Schüttelfrequenz 140 UpM) geschüttelt (Bruttemperatur: 30°C). Die Kolben wurden mit 0,1 bis 05 ml Sporensuspension beimpft.  To check growth and pigment coloration under submerged conditions, the strain was shaken in 500 ml Erlenmeyer flasks (with baffles, 100 ml culture solution) on the shaker (Infors type RC106; shaking frequency 140 rpm) (gross temperature: 30 ° C). The flasks were inoculated with 0.1 to 05 ml spore suspension.
Grundmedium (unter Zugrundelegung eigener Befunde und Literaturangaben) Basic medium (based on own findings and references)
0,10% NaH2PO4 0.10% NaH 2 PO 4
0,08% K2HPO4 0.08% K 2 HPO 4
0,05% MgSO4 · 7H2O 0.05% MgSO 4 .7H 2 O
0,02% MnCL2 · 4H2O 0.02% MnCL 2 .4H 2 O
0,10% (NH4)2SO4 0.10% (NH 4 ) 2 SO 4
0,80% Hefeextrakt  0.80% yeast extract
0,30% Pepton 7 , 50 % Maisstärke (vorhydrolysiert) 0.30% peptone 7, 50% corn starch (pre-hydrolyzed)
pH 6 ,2  pH 6.2
Der pH-Wert der Kulturlösung sollte in der Wachstumsphase zwischen 5,8 und 6,8 und in der Produktionsphase zwischen 7,5 und 8,5 gehalten werden. The pH of the culture solution should be kept between 5.8 and 6.8 in the growth phase and between 7.5 and 8.5 in the production phase.
Analytik a) Extrazelluläre Piαmentbildunq Analytics a) Extracellular Piαmentbildungunq
Monascus purpureus speichert in Submerskultur die gebildeten Pigmente überwiegend im Myzel und sezerniert jedoch einen Teil auch ins Kulturmedium. Die extrazelluläre Pigmentbildung wird wie folgt gemessen:  In submerged culture, Monascus purpureus stores the pigments formed predominantly in the mycelium and secrets some of them into the culture medium. The extracellular pigment formation is measured as follows:
1. Zunächst Abtrennung des Myzels durch Filtration. 1. First, the mycelium is separated off by filtration.
2. Anschließend Entfernung der Trübstoffe durch 2. Then remove the turbidity by
Zentrifugation.  Centrifugation.
3. Messung der klaren Kulturbrühe im Zeiss SpektraIphotometer PM2D bei 420 und 490 nm gegen dest. H2O als Blindwert (d = 1 cm). Bei Extinktionswerten über 1,0 entsprechende Verdünnung der Probe. 3. Measurement of the clear culture broth in the Zeiss spectrophotometer PM2D at 420 and 490 nm against dist. H 2 O as blank (d = 1 cm). If the absorbance is above 1.0, dilute the sample accordingly.
Die unbewachsene Kultur hat zwar anfangs eine Eigenextinktion (durch Hefeextraktanteil), da diese aber mit zunehmender Kulturdauer verschwindet, wurde als Blindwert dest. H2O benutzt. b) Intrazelluläre Piαmentbilduno The uncultivated culture initially has its own absorbance (due to yeast extract content), but since this disappears with increasing culture duration, dest. H 2 O used. b) Intracellular Piαmentbilduno
Das abgetrennte und gewaschene Myzel wurde 48 Std. bei 90°C getrocknet und anschließend zu feinem Pulver zermörsert. 10 mg zermörserten Myzels wurden mit 5 ml Methanol versetzt und in verschraubbaren Reagenzgläsern 30 Min. auf dem Minishaker extrahiert. Nichtlösliche Anteile wurden anschließend abzentrifugiert und der klare Extrakt bei 420 und 490 nm gegen Methanol gemessen.
Figure imgf000009_0001
The separated and washed mycelium was dried at 90 ° C. for 48 hours and then ground to a fine powder. 10 mg of crushed mycelium were mixed with 5 ml of methanol and extracted in screw-on test tubes for 30 minutes on the mini shaker. Insoluble components were then centrifuged off and the clear extract was measured at 420 and 490 nm against methanol.
Figure imgf000009_0001
Figure imgf000010_0001
Figure imgf000010_0001

Claims

Patentansprüche Claims
1. Verfahren zur Herstellung einer roten Farbstoffmischung durch Züchten eines Mikroorganismus der Gattung Monascus in einem Nährstoffmedium unter aeroben Bedingungen bei einem pH-Wert zwischen 4 und 8,5, dadurch gekennzeichnet, daß man dem Nährstoffmedium ein Precursor-Substrat für die Farbstoffbildung zusetzt. 1. A process for the preparation of a red dye mixture by growing a microorganism of the genus Monascus in a nutrient medium under aerobic conditions at a pH between 4 and 8.5, characterized in that a precursor substrate for dye formation is added to the nutrient medium.
2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, daß man als Precursor-Substrat eine Carbonsäure mit 2 bis 4 C-Atomen und gewünschtenfalls eine Aminogruppe und/oder insbesondere ein wasserlösliches Salz einer solchen Carbonsäure zusetzt. 2. The method according to claim 1, characterized in that a carboxylic acid having 2 to 4 carbon atoms and, if desired, an amino group and / or in particular a water-soluble salt of such a carboxylic acid is added as the precursor substrate.
3. Verfahren nach einem der Ansprüche 1 und 2, dadurch gekennzeichnet, daß man die Konzentration des Precursor-Substrats zwischen 0,1 und 7 Gew.-%, bezogen auf Fermentationsmedium, einstellt. 3. The method according to any one of claims 1 and 2, characterized in that adjusting the concentration of the precursor substrate between 0.1 and 7 wt .-%, based on the fermentation medium.
4. Verfahren nach einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, daß man das Precursor-Substrat in einer Menge von 0,1 bis 0,5 Gew.-%, bezogen auf Fermentationsmedium pro Tag, zudosiert. 4. The method according to any one of claims 1 to 3, characterized in that the precursor substrate in an amount of 0.1 to 0.5 wt .-%, based on the fermentation medium per day.
5. Verfahren nach einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, daß man als Precursor-Substrat Essigsäure, Glycin, Alanin und/oder Glutaminsäure zusetzt und/oder ein wasserlösliches Salz einer der genannten Säuren. 5. The method according to any one of claims 1 to 4, characterized in that acetic acid, glycine, alanine and / or glutamic acid is added as a precursor substrate and / or a water-soluble salt of one of the acids mentioned.
6. Verfahren nach einem der Ansprüche 1 bis 5, dadurch gekennzeichnet, daß man in Submerskultur arbeitet. 6. The method according to any one of claims 1 to 5, characterized in that one works in submerged culture.
7. Verfahren nach einem der Ansprüche 1 bis 6, dadurch gekennzeichnet, daß man einen Mikroorganismus der Spezies Monascus anka, Monascus kaoliang, Monascus purpurescens, Monascus purpureus, Monascus ruber, Monascus rubiginosus, Monascus rubropunctatus oder Monascus serorubescens einsetzt. 7. The method according to any one of claims 1 to 6, characterized in that a microorganism of the species Monascus anka, Monascus kaoliang, Monascus purpurescens, Monascus purpureus, Monascus ruber, Monascus rubiginosus, Monascus rubropunctatus or Monascus serorubescens is used.
8. Verfahren nach einem der Ansprüche 1 bis 7, dadurch gekennzeichnet, daß man als C-Quelle des Nährstoffmedium Glukose und/oder Stärke in Mengen von 2 bis 10 Gew.-%, vorzugsweise von 5 bis 7,5 Gew.-%, einsetzt. 8. The method according to any one of claims 1 to 7, characterized in that as the C source of the nutrient medium glucose and / or starch in amounts of 2 to 10 wt .-%, preferably from 5 to 7.5 wt .-%, starts.
9. Verfahren nach einem der Ansprüche 1 bis 5 und 7, dadurch gekennzeichnet, daß man in Emerskultur arbeitet. 9. The method according to any one of claims 1 to 5 and 7, characterized in that one works in Emers culture.
PCT/EP1991/002450 1991-01-31 1991-12-19 Fermentation process producing a monascus dye mixture WO1992013959A1 (en)

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DE4102906A DE4102906A1 (en) 1991-01-31 1991-01-31 FERMENTATION PROCEDURE
DEP4102906.2 1991-01-31

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995014786A1 (en) * 1993-11-26 1995-06-01 Anna Kujumdzieva Method for obtaining food colouring product

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2505856A1 (en) * 1981-05-16 1982-11-19 Hayashibara Biochem Lab PIGMENT PRODUCTION OF MONASCUS
GB2204879A (en) * 1987-03-13 1988-11-23 Shell Int Research Increasing monascus pigment production

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2505856A1 (en) * 1981-05-16 1982-11-19 Hayashibara Biochem Lab PIGMENT PRODUCTION OF MONASCUS
GB2204879A (en) * 1987-03-13 1988-11-23 Shell Int Research Increasing monascus pigment production

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
AGRICULTURAL AND BIOLOGICAL CHEMISTRY, Vol. 39, No. 9, September 1975, pages 1789-1795, MINORU YOSHIMURA et al., "Production of Monascus-Pigment in a Submerged Culture". *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995014786A1 (en) * 1993-11-26 1995-06-01 Anna Kujumdzieva Method for obtaining food colouring product

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