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EP4598360A2 - Compositions de colorant et procédés de production - Google Patents

Compositions de colorant et procédés de production

Info

Publication number
EP4598360A2
EP4598360A2 EP23875667.0A EP23875667A EP4598360A2 EP 4598360 A2 EP4598360 A2 EP 4598360A2 EP 23875667 A EP23875667 A EP 23875667A EP 4598360 A2 EP4598360 A2 EP 4598360A2
Authority
EP
European Patent Office
Prior art keywords
colorant composition
liquid media
acid
colorant
monascorubraminic
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
Application number
EP23875667.0A
Other languages
German (de)
English (en)
Inventor
Mauricio BRAIA
Sebastián Suárez MUÑOZ
Emilia MAZZA
Joaquín BERMEJO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Michroma Corp
Original Assignee
Michroma Corp
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Michroma Corp filed Critical Michroma Corp
Publication of EP4598360A2 publication Critical patent/EP4598360A2/fr
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources
    • 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
    • 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
    • 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
    • C12R2001/80—Penicillium

Definitions

  • the filamentous fungus Talaromyces atroroseus is known to produce red pigments.
  • Such pigments include compounds known as monascorubraminic acids, which are azaphilone compounds and include red, orange, and yellow pigments.
  • monascorubraminic acids which are azaphilone compounds and include red, orange, and yellow pigments.
  • Known production methods of monascorubraminic acids by culturing T. atroroseus have low efficiency and produce low yields of individual pigments which are not suitable for a broad range of uses in products such as food, cosmetics, and medicine.
  • New colorant compositions and methods for their production are needed in order to reduce input resources, improve colorant production output, and provide desirable color, stability, solubility, and other properties for use in a wide range of products.
  • aspects of the present disclosure provide methods of culturing pigment-producing fungus and production of colorant compositions.
  • the method comprises culturing a pigment-producing fungus in a liquid media that lacks a phosphate source.
  • the pigment-producing fungus is from the genus Talaromyces, Penicillium, or Monascus.
  • the liquid media comprises a mixed nitrogen source.
  • the mixed nitrogen source is peptone, tryptone, soy tryptone, yeast extract, one or more proteins, one or more peptides, or two or more amino acids.
  • the pH is decreased during the culturing. In some embodiments, the pH is decreased to about 4.5, about 4.0, about 3.5, about 3.0, or about 2.5 during the culturing.
  • the method further comprises inoculating the liquid media with mycelium of the pigment-producing fungus.
  • the fungus is selected from the group consisting of Talaromyces alroroseus, Talaromyces verruculosus, Talaromyces albobiverticillius, Talaromyces purpureogenus, Talaromyces amestolkiae, Talaromyces ruber, Talaromyces flavus, Penicillium purpurogenum, Penicillium oxalicum, Penicillium armenica, Penicillium marneffei, Penicillium atrovenetum, Penicillium rubrum, Monascus purpureus, Monascus ruber, and Monascus pilosus.
  • the first liquid media, the second liquid media, or both comprise a mixed nitrogen source.
  • the first liquid media comprises a first mixed nitrogen source.
  • the first mixed nitrogen source is selected from the group consisting of peptone, tryptone, soy tryptone, yeast extract, one or more proteins, one or more peptides, and two or more amino acids.
  • the second liquid media comprises a second mixed nitrogen source.
  • the second mixed nitrogen source is selected from the group consisting of peptone, tryptone, soy tryptone, yeast extract, one or more proteins, one or more peptides, and two or more amino acids.
  • the pH is decreased during the pigment production culturing step. In some embodiments, the pH is decreased to about 4.5, about 4.0, about 3.5, about 3.0, or about 2.5 during the pigment production culturing step.
  • the dissolved oxygen is maintained at about 30% to 35% in the mycelium production culturing step. In some embodiments, the dissolved oxygen is maintained at about 30% to 35% in the pigment production culturing step.
  • a second nitrogen source is provided during the pigment production culturing step.
  • the second nitrogen source is one or more amino acids.
  • the pigment production culturing step is performed in a fermenter.
  • the mycelium production culturing step continues for 24 to 36 hours.
  • the method further comprises diluting the inoculum culture before the inoculating of step (c).
  • the inoculum culture is diluted to 0.4x to 0.6x.
  • the second liquid media comprises starch.
  • the starch is depleted to below 0.1 g/L during the pigment production culturing step.
  • the second liquid media is stirred at a first stirring speed during the pigment production culturing step.
  • the method further comprises stirring the second liquid media at a second stirring speed after the starch is depleted to below 0.1 g/L during the pigment production culturing step, wherein the second stirring speed is higher than the first stirring speed.
  • the second stirring speed is 100 to 300 rpm higher than the first stirring speed.
  • the second liquid media comprises a sugar and a starch in a ratio of 1 :4 to 1 :2. In some embodiments, at the beginning of the pigment production culturing step the second liquid media comprises a sugar at 8 to 12 g/L, a starch at 25 to 35 g/L, and yeast extract at 0.8 to 1.2 g/L.
  • Also disclosed herein is a method for culturing a pigment-producing fungus, the method comprising the steps of: inoculating a first liquid media with conidia of the fungus; performing a mycelium production culturing step comprising culturing the conidia in the first liquid media to generate an inoculum culture comprising mycelium; inoculating a second liquid media with the inoculum culture or a portion thereof; and performing a pigment production culturing step comprising culturing the mycelium in the second liquid media to generate a mycelium culture; wherein the pigment-producing fungus is from the genus Talaromyces, Penicillium, or Monascus, and wherein the second liquid media comprises from 0 to 40 mg/L of phosphate during the second culturing step.
  • the fungus is selected from the group consisting of Talaromyces air arose us.
  • Talaromyces verruculosus Talaromyces albobiverticillius, Talaromyces purpureogenus, Talaromyces amestolkiae, Talaromyces ruber, Talaromyces fla s, Penicillium purpurogenum, Penicillium oxalicum, Penicillium armenica, Penicillium marneffei, Penicillium atrovenetum, Penicillium rubrum, Monascus purpureus, Monascus ruber, and Monascus pilosus.
  • the second liquid media comprises phosphate.
  • the concentration of phosphate is reduced to below 1 mg/L during the pigment production culturing step.
  • the phosphate is completely depleted from the second liquid media during the pigment production culturing step.
  • pH is decreased during the pigment production culturing step. In some embodiments, the pH is decreased to about 4.5, about 4.0, about 3.5, about 3.0, or about 2.5 during the pigment production culturing step.
  • a second nitrogen source is provided during the pigment production culturing step.
  • the second nitrogen source is one or more amino acids.
  • the pigment production culturing step is performed in a fermenter.
  • the mycelium production culturing step continues for 24 to 36 hours.
  • the method further comprises diluting the inoculum culture before the inoculating of step (c).
  • the inoculum culture is diluted to 0.4x to 0.6x.
  • the second liquid media comprises starch.
  • the starch is depleted to below 0.1 g/L during the pigment production culturing step.
  • the second liquid media is stirred at a first stirring speed during the pigment production culturing step.
  • the method further comprises stirring the second liquid media at a second stirring speed after the starch is depleted to below 0.1 g/L during the pigment production culturing step, wherein the second stirring speed is higher than the first stirring speed.
  • the second stirring speed is 100 to 300 rpm higher than the first stirring speed.
  • the second liquid media comprises a sugar and a starch in a ratio of 1 :4 to 1 :2. In some embodiments, at the beginning of the pigment production culturing step the second liquid media comprises a sugar at 8 to 12 g/L, a starch at 25 to 35 g/L, and yeast extract at 0.8 to 1.2 g/L. [0027] In some embodiments, the method further comprises the step of extracting one or more pigments from the mycelium culture to obtain a colorant extract.
  • the one or more pigments are selected from the group consisting of N-glutaryl monascorubraminic acid, N-asparagyl monascorubraminic acid, N-aspartyl monascorubraminic acid, N-cysteinyl monascorubraminic acid, N-phenyl alanyl monascorubraminic acid, N-lysyl monascorubraminic acid, N-methionyl monascorubraminic acid, N-glutamyl monascorubraminic acid, and N-arginyl monascorubraminic acid.
  • the colorant extract is red or a reddish color.
  • a colorant extract made by a method described above.
  • the extract is a liquid.
  • the extract is dried or lyophilized.
  • a foodstuff comprising the colorant extract.
  • a cosmetic comprising the colorant extract.
  • colorant compositions comprising c/.s-N-glutaryl monascorubraminic acid and /ra/z.s-N-glutaryl monascorubraminic acid.
  • a colorant composition comprising: (a) about 60% to about 75% c/.s-N-glutaryl monascorubraminic acid; and (b) about 1% to about 7% Zraz/.s-N-glutaryl monascorubraminic acid.
  • the c/.s-N-glutaryl monascorubraminic acid is present in the colorant composition in an amount of about 65%. In some embodiments, the /ra/z.s-N-glutaryl monascorubraminic acid is present in the colorant composition in an amount of about 3%. [0031] In some embodiments, the colorant composition further comprises N-glutamyl monascorubraminic acid. In some embodiments, the colorant composition comprises N-glutamyl monascorubraminic acid in an amount of about 2% to about 6%. In some embodiments, the N- glutamyl monascorubraminic acid is present in an amount of about 3%.
  • the colorant composition further comprises N-glutaryl monascorubramine. In some embodiments, the colorant composition comprises N-glutaryl monascorubramine in an amount of about 1% to about 4%. In some embodiments, the colorant composition comprises N-glutaryl monascorubramine in an amount of about 2%. [0033] In some embodiments, the colorant composition further comprises ash. In some embodiments, the colorant composition comprises ash in an amount of about 3%.
  • the colorant composition comprises a co angle measured in the CIELAB color space in the range of about 30° to 40°. In some embodiments, the co angle is about 34°.
  • the colorant composition comprises a L* value of about 75 to about 85. In some embodiments, the L* value is about 78.
  • the colorant composition comprises an a* value of about 15 to about 25. In some embodiments, the a* value is about 22.
  • FIG. 1 Assessment of the biomass growth and color production in AMYG culture medium over a range of carbon source concentrations.
  • the productivity (Qp), specific productivity (qp), and biomass titer (X) were calculated at each concentration of starch.
  • FIG. 2 Effect of the inoculum composition on the color production.
  • the productivity (Qp) and specific productivity (qp) were calculated for systems containing AMYG culture medium inoculated with mycelium and conidia.
  • FIG. 4 Effect of inoculum size and age on color production in systems containing AYP culture medium.
  • the productivity (Qp) was calculated at each dilution of the inoculum for each incubation time.
  • FIGS. 6A-6B Assessment of color production in AMYG culture medium over a range of yeast extract concentrations.
  • the productivity (Qp) (FIG 6A) and specific productivity (qp) (FIG. 6B) were calculated at each concentration of yeast extract (YE).
  • FIGS. 7A-7B Assessment of color production in AMYG culture medium over a range of dipotassium phosphate concentrations.
  • the productivity (Qp) (FIG. 7A) and biomass yield (Yxs) (FIG. 7B) were calculated at each concentration of dipotassium phosphate.
  • FIG. 8 Effect of yeast extract on the color production in systems containing culture medium with and without dipotassium phosphate.
  • the productivity (Qp) and specific productivity (qp) were calculated in the absence (AYP and AMY-P04 culture media) and presence of dipotassium phosphate (AMYG culture medium).
  • FIG. 9 Effect of pH on the color production in systems containing AYP culture medium.
  • the productivity (Qp) and biomass titer (X) were calculated at each pH.
  • FIG. 10 Effect of growth type on the color production in systems containing AYP culture medium (control) and diauxic culture medium.
  • the productivity (Qp) were calculated for each trial and summarized by system.
  • a two-sample Wilcoxon test was used to establish statistical significance in the difference among productive systems (p-value ⁇ 0.001).
  • Culture methods disclosed herein may begin with a phase of producing a seed culture suitable for inoculating a production culture from which a colorant composition may be prepared.
  • the seed culture phase may include inoculation of a liquid media with conidia of the pigment-producing fungus, followed by culturing to produce mycelium of the fungus.
  • the mycelium produced from the seed culture phase may then be used to inoculate liquid media for a production culture.
  • Production of sufficient mycelia suitable for inoculation of a production culture may include multiple passages. For example, liquid media may be inoculated with conidia and cultured until the culture reaches a certain density of mycelium.
  • That mycelium culture may then be used to inoculate additional fresh liquid media in order to increase the volume of mycelium culture available for inoculating a production culture.
  • the culturing steps that take place before inoculation of a production batch may be performed under conditions that optimize for mycelium proliferation rather than pigment production.
  • production cultures can be performed under conditions that optimize for production of pigment as opposed to maximizing mycelium proliferation.
  • the amount of conidia used to inoculate the liquid media in a seed culture process may be 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , or 10 8 conidia per ml of the liquid media, or between any two of these values.
  • the seed production process may continue for sufficient time to produce a mycelium culture suitable for inoculating the liquid media in the second step.
  • the first step may continue for 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours, or a range between any two of these values (e.g., 24 to 36, 24 to 30, or 18 to 36 hours).
  • the seed production culture may continue for 24 to 48 hours.
  • the culturing may continue until the culture reaches stationary phase, until a carbon source, nitrogen source, or phosphate source is depleted from the liquid media, or until productivity stops increasing.
  • more than one culture step is included in a process of culturing a pigment-producing fungus.
  • a process can include, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more passaging steps in which a culture of fungus is used to inoculate a culture in a next culturing step.
  • the conditions of the final culturing step i.e., a production culture — may be optimized for production of pigments such as by having a low amount or no source of phosphate in the liquid media used in the final culturing step.
  • one or more steps of the culturing of the pigment-producing fungus can be performed in a bioreactor or fermenter.
  • a fermenter may be used which includes a vessel in which culture conditions may be monitored and adjusted. Such culture conditions may include oxygen content; agitation; pH; concentrations of nutrient sources such as, for example, sugars, carbohydrates, nitrogen, phosphate; concentrations of metabolites and waste products; concentrations of pigments; and other components.
  • a fermenter may be configured to allow for exchange or addition of liquid media or components thereof during a culturing step.
  • one or more, or all, of the culturing steps are performed in a fermenter.
  • one or more, or all, of the culturing steps are performed in a continuous flow manner. In some embodiments, one or more, or all, of the culturing steps are performed in a batch mode, which may be performed in a fermenter or in a culture flask or vessel.
  • a pigment-producing fungus is cultured in a single culturing step.
  • the inoculant for the culture may be conidia of the fungus or mycelium of the fungus.
  • the single culturing step may be, for example, a batch culture or a continuous flow culture.
  • the inoculant may be, for example, frozen, lyophilized, or active.
  • the inoculant for the single-step culture process may be conidia or an active culture of mycelium.
  • the liquid media for the single culture step may have a low or limiting amount of phosphate or may be free of a source of phosphate.
  • the phosphate may be depleted or removed during the culturing step, as described in other embodiments herein.
  • the amount of time a culturing step continues is determined by the amount of biomass and/or pigment produced in the culture.
  • the amount of biomass and/or pigment in the culture may be monitored, and the culture step may be concluded when a desired amount of the biomass and/or pigment is present in the culture.
  • the biomass may then be harvested or the pigment may be extracted to produce a colorant composition.
  • Culturing methods disclosed herein can use a variety of liquid media suitable for culturing of pigment-producing fungi.
  • Culture media for methods disclosed herein include, among other ingredients, one or more carbon sources or nitrogen sources.
  • the culture media used for at least part of the culture process lacks a source of phosphate for at least a portion of the culturing time.
  • the liquid media in different culturing phases may be the same or different.
  • the carbon source or the nitrogen source may be different, or the concentrations of these and other components of the liquid media may be different.
  • the phosphate concentration is lower in the liquid media used in a production culture step than in a seed culture step or another culturing phase before the production phase.
  • Such depletion may occur over the course of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48, 60, 72, 84, 96, 108, 120, 132, 144, or 156 hours or more, or any range between these values.
  • Depletion of the phosphate source may be due to consumption of the phosphate source by the fungus or may be caused by an operator of the culturing process intervening to change the concentration of phosphate in the liquid media.
  • an operator may exchange or dilute the liquid media such that the amount of phosphate or other nutrient available to the cultured fungus is reduced to a low level, including to zero.
  • the initial concentration of the inorganic phosphate source in the liquid culture media is 1, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 mg/ml, or a range between any two of these values.
  • the culture may be rotated at a speed of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 rpm, or at a speed in a range between any two of these values.
  • the stirring speed is increased during a pigment production culturing step after a sugar, a starch, a nitrogen source, or a phosphate source is depleted below a certain amount such as, for example, 1, 0.5, 0.1, or 0.01 g/L. In some embodiments, the stirring speed is increased by 50, 100, 150, 200, 250, or 300 rpm, or a range between any two of these values. In some embodiments, the stirring speed is increased by 50, 100, 150, 200, 250, or 300 rpm after a starch in the liquid medium is depleted to below 1, 0.5, 0.1, or 0.01 g/L during a pigment production culturing step.
  • Liquid media used in embodiments described herein may have the following components in the indicated concentrations: glucose at 8 to 12 g/L, starch at 28 to 32 g/L, yeast extract at 0.8 to 1.2 g/L, Fe2SO4 at 0.3 to 0.5 g/L, KC1 at 0.4 to 0.6 g/L, and MgSCU at 4.0 to 6.0 g/L.
  • the indicated concentrations are the concentrations at the beginning of a culturing step, and the components are depleted over time.
  • components are maintained at the indicated concentrations (or within 1, 5, 10, or 15% of the indicated concentrations) during a culturing step by addition of components over time.
  • Methods of producing colorant compositions disclosed herein may produce at least about 0.5 g, 1.0 g, 1.5 g, 2.0 g, 2.5 g, 3.0 g, 3.5 g, 4.0 g, 4.5 g, 5.0 g, 5.5 g, 6.0 g, 6.5 g, 7.0 g, 7.5 g, 8.0 g, 8.5 g, 9.0 g, 9.5 g, or 10.0 g of recovered colorant composition per liter of liquid culture, or a range between any two of these values (e.g., 3.0 to 8.0, 4.0 to 8.0, 5.0 to 8.0, 6.0 to 8.0, 7.0 to 8.0, 4.0 to 7.5, 5.0 to 7.5, 6.0 to 7.5, etc.).
  • Another parameter that may be used to evaluate a culture is the ratio of grams of fungal biomass (dry weight) produced to grams of a carbon source initially provided in a liquid media (Yxs).
  • a culture of pigment-producing fungi has a ratio of biomass to grams of carbon source of at least 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6 grams of biomass (dry weight) produced per gram of carbon source, or a range between any two of these values.
  • liquid media described herein may be monitored and/or adjusted during or between culturing steps to achieve a desired outcome.
  • a concentration of a component present in the liquid media may be the same or different for liquid media used in different culturing steps.
  • Any aspect of a liquid media embodiment described herein may be combined with any other aspect.
  • Talaromyces verruculosus Talaromyces albobiverticillius, Talaromyces purpureogenus, Talaromyces amestolkiae, Talaromyces ruber, Talaromyces flavus, Penicillium purpurogenum, Penicillium oxalicum, Penicillium armenica, Penicillium marneffei, Penicillium atrovenetum, Penicillium rubrum, Monascus purpureas, Monascus ruber, or Monascus pilosus.
  • a person of ordinary skill in the art will recognize that the methods disclosed herein can be used for culturing additional pigment-producing fungi that have similar properties to the genera and species listed above.
  • Suitable strains of T. atroroseus for use in methods of producing colorant as described herein include, for example, strains 35816 and 1061 from the Agricultural Research Service Culture Collection (US), strain 9777 from the American Type Culture Collection (US), strains 257.37, 364.48, 234.60, 391.96, 113139, 113153, 113154, 124294, 133442, 133443, 133447, 133449, 133450 from the CBS-KNAW Culture Collection (the Netherlands).
  • Cultures of pigment-producing fungi may be used to make a colorant composition by a method that includes recovering pigments produced by the cultured fungi from a liquid culture. Pigments may be present in dissolved form in the liquid culture. Pigments may also be present within cells of the pigment-producing fungi.
  • An example of a method of recovering pigments from a culture of pigment-producing fungi to produce a colorant composition may include removing solids and biomass from the liquid culture, producing a pigment solution that is free of solids and biomass.
  • Removing solids and biomass can be done, for example, by filtration or centrifugation.
  • Removing the solids and biomass can be done with or without a step of extracting pigments from within the cultured cells.
  • Extraction can involve lysing the cells, thereby releasing pigments into solution. Extraction may also involve adding a solvent, such as ethanol, ethyl acetate, acetone, or hexane, to the liquid culture to solubilize pigment compounds.
  • the pigment solution may include the monascorubraminic acid pigments.
  • a method of producing a colorant composition may also include precipitating the pigments in the pigment solution by acidification of the pigment solution.
  • ethanol, ethyl acetate, acetone, or hexane extraction of the liquid culture or pigment solution is not performed before the acidification.
  • extraction including by, for example, ethanol, ethyl acetate, or other solvents, is not performed at all during the process of recovering pigments from a culture.
  • Acidification may be performed by adding an acid to the pigment solution.
  • the acid may be any suitable acid for precipitating pigments from the pigment solution.
  • the acid may be selected from, for example, sulfuric acid, hydrochloric acid, nitric acid, lactic acid, boric acid, carbonic acid, citric acid, oxalic acid, phosphoric acid, and acetic acid, or any combination thereof.
  • Acidification may be performed by adding sulfuric acid to the pigment solution.
  • the concentration of the sulfuric acid added to the pigment solution may be at least about 10%, 33.5%, 62.18%, 77.67%, or 98% w/v sulfuric acid.
  • the concentration of the sulfuric acid may be adjusted accordingly using water.
  • Acidification may be performed until the pigment solution or liquid culture is at a particular pH.
  • the acid e.g., 98% sulfuric acid
  • the pH may be adjusted to at most about 3.5, 3.0, 2.5, 2.0, or 1.5.
  • the pH may be adjusted from about 1.5 to 3.5, 2.0 to 3.0, 2.5 to 3.0, or 2.0 to 2.5.
  • the pH may be about 2.5.
  • the method may also include recovering the pigments to form the colorant composition.
  • Recovering may include centrifuging the pigment solution after the acidification to form a pellet or allowing precipitated pigment to settle in the bottom of a vessel.
  • the acidified pigment solution may be centrifuged at a speed of at least about 1000 rotations per minute (rpm), 2000 rpm, 3000 rpm, 4000 rpm, 5000 rpm, or more.
  • the acidified extract solution may be centrifuged at at most about 5000 rpm, 4000 rpm, 3000 rpm, 2000 rpm, 1000 rpm, or less.
  • the acidified extract solution may be centrifuged from about 2000 rpm to 5000 rpm, 3000 rpm to 5000 rpm, 3000 rpm to 5000 rpm, 4000 rpm to 5000 rpm, 3000 rpm to 4000 rpm.
  • the pellet may include the pigments, including monascorubraminic acid pigments, and a supernatant.
  • the supernatant may be removed from the pellet.
  • the pellet may be separated from the supernatant by filtering.
  • the supernatant may be separated from the pellet by pouring the supernatant away from the pellet. Recovering precipitated pigments from the acidified pigment solution can also be performed by filtration.
  • the recovered precipitated pigment which may be in a centrifugation pellet or filter cake, may be washed with a wash solution.
  • the washing may involve resuspending the precipitated pigment composition in a wash solution and then centrifuging or filtering the precipitate. This process can be repeated multiple times.
  • the solution may completely dissolve the pellet.
  • the solution may partially dissolve the pellet.
  • the solution may be used to resuspend the pellet.
  • the wash solution may include a base dissolved in a protic solvent.
  • the base may be selected from, for example, potassium hydroxide, sodium hydroxide, barium hydroxide, cesium hydroxide, sodium carbonate, sodium bicarbonate, or calcium carbonate, or any other suitable base.
  • the base may be potassium hydroxide.
  • the protic solvent may be selected from, for example, water, ethanol, methanol, isopropanol, acetic acid, formic acid, or n-butanol.
  • the protic solvent may be water.
  • the wash solution may include a base (e.g., potassium hydroxide) having at least about 0.01% weight per volume (% w/v), 0.05% w/v, 0.1% w/v, 0.2% w/v, 0.3%, 0.4%, 0.5%, 0.6% w/v, or a range between any two of these values (e.g., 0.05 to 0.5% or 0.3% to 0.5%).
  • a base e.g., potassium hydroxide
  • the final volume of the extraction liquid may be a particular ratio of water portion to solvent portion (e.g., ethyl acetate).
  • the final volume ratio may be at least about 0.01, 0.05, 0.075, 0.1, 0.125, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 or more, or a range between any two of these values (e.g., 0.05 to 0.15).
  • the final volume ratio may be at most about 0.5, 0.4, 0.3, 0.2, 0.15, 0.125, 0.1, 0.075, 0.05, 0.01, or less.
  • a colorant composition produced by embodiments of methods disclosed herein may be a solid and may include, for example, one or more of the following pigments: N-glutaryl monascorubraminic acid, N-asparagyl monascorubraminic acid, N-aspartyl monascorubraminic acid, N-cysteinyl monascorubraminic acid, N-phenyl alanyl monascorubraminic acid, N-lysyl monascorubraminic acid, N-methionyl monascorubraminic acid, N-glutamyl monascorubraminic acid, and N-arginyl monascorubraminic acid.
  • the colorant compositions provided herein comprise cz -N- glutaryl monascorubraminic acid, which exhibits a red color.
  • cz -N- glutaryl monascorubraminic acid is present in the colorant compositions in an amount of about 60% to about 75%.
  • cv.s-N-glutaryl monascorubraminic acid is present in the colorant compositions in an amount of at least 55% (e.g., 57%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, or 76%).
  • cv.s-N-glutaryl monascorubraminic acid is present in the colorant compositions in an amount of at most 80%% (e.g., 78%, 76%, 74%, 72%, 70%, 68%, 66%, or 64%). In some embodiments, cv.s-N-glutaryl monascorubraminic acid is present in the colorant compositions in an amount of about 55% to about 78%, about 60% to about 76%, about 62% to about 70%, or about 62% to about 68%. In some embodiments, cz -N- glutaryl monascorubraminic acid is the principal pigment in the colorant compositions.
  • cv.s-N-glutaryl monascorubraminic acid is present in the colorant compositions in an amount of about 65%%. In some embodiments, cv'.s-N-glutaryl monascorubraminic acid is present in the colorant compositions in an amount of about 60% to about 75%. In some embodiments, cv.s-N-glutaryl monascorubraminic acid is present in the colorant compositions in an amount of about 60% to about 70%.
  • the colorant compositions provided herein comprise trans-N- glutaryl monascorubraminic acid, which exhibits a purple color.
  • trans-N- glutaryl monascorubraminic acid is present in the colorant compositions in an amount of at least 1% (e.g., 2%, 3%, 4%, 5%, 6%).
  • /ra/z.s-N-glutaryl monascorubraminic acid is present in the colorant compositions in an amount of at most 10% (e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%).
  • /ra/z.s-N-glutaryl monascorubraminic acid is present in the colorant compositions in an amount of about 1% to about 7%, about 2% to about 6%, about 2% to about 5%, or about 2% to about 4%. In some embodiments, /ra/z.s-N-glutaryl monascorubraminic acid is present in the colorant compositions in an amount of about 3%. In some embodiments, /ra/z.s-N-glutaryl monascorubraminic acid is present in the colorant compositions in an amount of about 1% to about 7%.
  • the cis- and trans- isomers of N-glutaryl monascaorubraminic acid can be identified by nuclear magnetic resonance, such as described in Example 11.
  • Biomass growth and colorant production were assessed over a range of carbon source concentrations.
  • the mycelium suspension used in this experiment as inoculum was produced by inoculating 50 mL of MPPY medium in an Erlenmeyer flask with IxlO 7 conidia and incubating at a temperature of 28 °C, and an agitation speed of 200 rpm in an orbital shaker for 48 hrs.
  • Three culture systems were prepared in Erlenmeyer flasks containing 50 mL of AMYG medium with different concentrations of starch: 20, 40, and 60 g/L.
  • Conidia and mycelium inoculum (Talaromyces atroroseus) for liquid culture were compared.
  • Two culture systems were prepared in Erlenmeyer flasks containing 50 mL of AMYG medium.
  • One system was inoculated with 2xl0 8 conidia and the other with 10 %v/v of mycelium suspension.
  • the mycelium suspension used in this experiment as inoculum was produced by inoculating 50 mL of MPPY medium in an Erlenmeyer flask with 2xl0 8 conidia and incubating at a temperature of 28 °C, and an agitation speed of 200 rpm in an orbital shaker for 48 hrs.
  • a range of mycelium inoculant was tested for productivity. Two systems were prepared in Erlenmeyer flasks containing 50 mL of MPPY medium. One system was inoculated with 1 %v/v of mycelium suspension and the other with 10 %v/v of mycelium suspension. Both systems were incubated as described for the conidia/mycelium comparison above and then the final fungal biomass was measured as described in Example 1. Results are shown in FIG. 3. The higher inoculum level (10 %v/v) yielded 2.7 times more fungal biomass than 1 %v/v.
  • Mycelium suspensions were prepared by inoculating 50 mL of MPPY medium in an Erlenmeyer flask with IxlO 7 conidia and incubating at pH 5.0, at a temperature of 28 °C and agitated at 200 rpm in an orbital shaker for 48 hrs.
  • Four systems were prepared in Erlenmeyer flasks containing 50 mL of AMYG medium with different concentrations of glutamate: 4.0, 8.4, 15.0, and 20.0 g/L. Each system was inoculated with a 10 %v/v mycelium suspension and incubated under the conditions described in Example 2.
  • the titer of colorant and fungal biomass was measured as described in Example 1, with the exception that the wavelength at which the absorbance was measured varies with the concentration of glutamate; the 4 g/L of glutamate produced an orange colorant, with a maximum absorbance at 485 nm, the remaining concentrations of glutamate yielded a red color with a maximum absorbance at 500 and 505 nm (see FIG. 5B).
  • Qp and qp were calculated as described in the preceding examples. Results are shown in FIG. 5A.
  • Mycelium inoculum was prepared as described in Example 4.
  • Four culture systems were prepared in Erlenmeyer flasks containing 50 mL of AMYG medium with different concentrations of yeast extract (YE): 0, 0.5, 1.0 and 2.0 g/L. Each system was inoculated with a 10 %v/v mycelium suspension and incubated as described in the preceding examples. Titer of colorant, fungal biomass, and calculations of Qp and qp were performed as described in the preceding examples. Results are shown in FIG. 6A for Qp and FIG. 6B for qp. No significant difference in biomass or colorant titer was observed across the yeast extract concentrations tested.
  • Example 6 Evaluation of colorant production using different concentrations of phosphate
  • Mycelium inoculum was prepared as described in Example 4.
  • Three culture systems were prepared in Erlenmeyer flasks containing 50 mL of AMYG medium with different concentrations of dipotassium phosphate: 0, 0.5 and 1.0 g/L. Each system was inoculated with a 10 %v/v mycelium suspension and incubated as described in Example 2. Qp and Yxs were calculated as described in the preceding examples. Results are shown in FIG. 7A and FIG. 7B. The highest productivity was obtained in absence of phosphate. Although Yxs is the lowest in the absence of phosphate, the productivity was highest.
  • Example 7 Production of colorant using yeast extract in culture media without phosphate
  • Mycelium inoculum was prepared as described in Example 4.
  • Two systems were prepared in Erlenmeyer flasks containing 50 mL of AMYG medium without dipotassium phosphate (AMY-PO4) and AYP medium (without dipotassium phosphate and with yeast extract).
  • a system prepared with AMYG was used as a control.
  • Each system was inoculated with a 10 %v/v mycelium suspension and incubated at Ph 5.0, at a temperature of 28 °C and at an agitation rate of 200 rpm in an orbital shaker for 72 hrs.
  • Qp, qp, and biomass titer were calculated as described in the preceding examples.
  • Results are shown in Table 7 (biomass titer) and in FIG. 8.
  • the elimination of dipotassium phosphate from the culture medium negatively affected the production of biomass but resulted in a higher qp.
  • the addition of yeast extract to a culture medium lacking dipotassium phosphate (AYP) improved both the productivity and specific productivity of the colorant.
  • Mycelium inoculum was prepared as described in Example 4. Three systems were prepared in Erlenmeyer flasks containing 50 mL of AMYG medium and incubated at different Ph: 3.5, 5.0, and 6.5. Each system was inoculated with a 10 %v/v mycelium suspension and incubated at a temperature of 28 °C and agitated at 200 rpm in an orbital shaker for 48 hrs. Colorant production and biomass were measured as described in Example 1, and Qp and biomass titer (X) were calculated as described in the preceding examples. Results are shown in FIG. 9.
  • Results showed that colorant productivity is highest at pH 5.0 while fungal biomass is highest at pH 3.5.
  • the fermentation process takes 72 hrs and during that time, the absorbance and the biomass concentration (X) are measured to track the production of the colorant.
  • Absorbance at 505 nm was used to calculate the titer of colorant (grams of colorant produced per liter of broth) using the following equation (0.0346*absorbance)-0.0000087.
  • Fungal biomass was measured as dry weight by filtering the whole broth with cellulose paper with a pore size of 30-40 pm and drying the filter plus the solids at 120 °C for 5 minutes.
  • the final titer of colorant together with the final biomass dry weight was used to calculate the productivity of the fermentation process (Qp, grams of colorant produced per liter of broth in one hour) and specific productivity (qp, grams of colorant produced by a gram of biomass in one hour), respectively, as shown in Table 9.
  • Results showed that the best stirring strategy is to start at 600 rpm and the switch to 800 rpm when starch depletes.
  • Example 10 Optimization of the culture medium composition.
  • the model was used to find the highest predicted Qp among the conditions set for this experiment.
  • the selected values for the factors under study are shown in Table 11. Its predicted Qp was 0.1221 g/Lh, while its observed Qp was 0.1248 g/Lh.
  • HPLC indicates a 77% yield of c/.s-N-glutaryl monascorubraminic acid.
  • the crude reaction mixture is purified by column chromatography.
  • the HPLC chromatogram and absorbance spectrum of cis- N-glutaryl monascorubraminic acid can be seen in FIG. 13A-B.
  • the cis-N-glutaryl monascorubraminic acid compound can be converted to the trans- N-glutaryl monascorubraminic acid by application of UV light for 3 hours.
  • the HPLC chromatogram and absorbance spectrum of /ra/z.s-N-glutaryl monascorubraminic acid can be seen in FIG. 14A-B.
  • the following protocol describes the methodology for identification, assay and purity evaluation of the red colorant.
  • the cv.s-N-glutaryl monascorubraminic acid and compounds /ra//.s-N-glutaryl monascorubraminic acid, N-glutaryl monascorubramine and N-glutamyl monascorubraminic acid are identified by comparison of the retention times of the peaks in the chromatogram at 520 nm in the Sample Preparation and in the System Suitability Mix.
  • the assay of cv.s-N-glutaryl monascorubraminic is determined by employing a calibration curve.
  • the purity is determined by means of internal normalization (IN).
  • Instrumental conditions include: Flow: 0.35 mL/min; Detection: 520 nm; Column temperature: 50 °C; Autosampler temperature: 15 °C; Injection volume: 5 pL (loop volume 20 pL); Run time: 33 minutes; Needle wash solvent: MeOELEEO (20:80). The gradient in Table 12 was used.
  • the mobile phase and solvent solution consist of: Mobile Phase A: 0.1% formic acid, filter; Mobile Phase B: 0.1% formic acid in acetonitrile, filter; Solvent solution (SS): MeOELEEO (9: 1). An HPLC chromatogram of the solvent solution can be seen in FIG. 16.
  • Samples are prepared by adding about 10.0 mg of sample to a 10 mL volumetric flask and dissolving in Solvent Solution. An ultrasonic bath is used where necessary. The solution was diluted to a final concentration of 1 mg/mL.
  • the system suitability mix is prepared with the c/.s-N-glutaryl monascorubraminic acid, /ra/z.s-N-glutaryl monascorubraminic acid, N-glutaryl monascorubramine, and N-glutamyl monascorubraminic acidpurified compounds.
  • An HPLC chromatogram of the system suitability mix can be seen in FIG. 17.
  • Table 15 shows the relative areas and retention times for several components of the colorant composition as prepared by methods described herein.
  • HPLC Thermo ScientificUltimate 3000 RSLC
  • HPLC system consists of an autosampler organizer, column manager and heater, quaternary pump, TSQ Quantum Access Max triple quadrupole mass analyzer and VWD-3400RS detectors.
  • Enterprise Chromeleon 7.3 software is used for data acquisition and system control.
  • the column used is Poroshell 120 Phenyl-Hexyl 150 mm x 2.1 mm, 2.7 pm.
  • An analytical balance capable of reading to at least 0.1 mg is used and a membrane filter with 0.22 pm pore size is used.
  • FIG. 13E The spectrum (FIG. 13E) shows the presence of the cv.s-N-glutaryl monascorubraminic acid, water, and an unknown impurity responsible for a signal at 1.93 ppm (singlet). All samples were prepared by dissolving 9 mg of solid in 0.6 mL of D2O. The J- couplings of about 12 Hz are indicative of the cis-configuration of the double bond.
  • both the 2D WH-COSY spectrum (FIG. 13F) and the 2D HSQC spectrum (FIG. 13G) of cv.s-N-glutaryl monascorubraminic acid reveal the methine hydrogen of the glutamic fragment (alpha hydrogen, 2'-H), the signal of which overlaps with that of water at approximately 4.93 ppm in the 'H NMR spectrum. According to the HSQC spectrum, this hydrogen atom is bonded to a methine carbon with a carbon-13 chemical shift of approximately 69 ppm, consistent with the structure of cv.s-N-glutaryl monascorubraminic acid.
  • Colorant compositions were made by methods differing from those described herein.
  • colorant compositions were prepared using methods described in Rasmussen, K. B. (2015). Taloromyces atroroseus: Genome sequencing, Monascus pigments and azaphilone gene cluster evolution.
  • the colorant properties e.g., CIELAB
  • CIELAB CIELAB
  • the fermentation conditions were 30°C, 800 RPM, dissolved oxygen over 30%, and pH according to Table 18.
  • the fermentation process takes 72 hours. Absorbance was measured through all the fermentation and used to calculate the titer of colorant (grams of colorant produced per liter of broth) using the next equation (0,0346*absorbance)-0, 0000087.
  • the mycelium suspension used in this experiment as inoculum was produced by inoculating 200 mL of MPPY medium in an Erlenmeyer flask with 4xl0 6 conidia and incubating at 28 C and 200 rpm in an orbital shaker for 48 hs. Table 19
  • a sample of a Talaromyces atroroseus conidia suspension was added to 200 mL of sterilized MPPY (Table 20) in a 1 L flask to a final concentration of 2xl0 5 conidia/mL. The solution was incubated for 24 hours at 30°C and 130 rpm in an orbital shaker until fungal biomass reaches 2 g/L.
  • the 200 mL inoculum was transferred to the bioreactor containing 1.8 L of DOP culture media (Table 21, 10% v/v).
  • the initial fermentation conditions are 30°C, pH 5.0, 600 rpm, dissolved oxygen 100%, and aeration 1 vvm.
  • the fermentation step is divided into two stages. The first stage consists of the beginning of the fermentation to starch depletion (approximately 45 hours). During this stage, there is no control of the pH so it decreases from 5.0 to approximately 3.4 and dissolved oxygen decreases to 40%.
  • stage 2 from 45 hours to the end of the fermentation process (approximately 72 hours), pH is increased back to 5.0 using 2 N NaOH, aeration is lowered to 0.1 vvm, and stirring is set to 800 rpm. Samples are taken two times per day, and the fungal biomass and red colorant composition concentration are measured as dry weight and absorbance, respectively.
  • the dry weight of fungal biomass was measured by filtering the whole broth with cellulose paper with a pore size of 30-40 pm and drying the filter plus the solids at 120°C for 5 minutes.
  • the absorbance of the fermentation broth was measured by centrifuging the broth at 3000 RPM (1000g) for 5 minutes and measuring the absorbance at 500 nm of the supernatant.
  • the titer is calculated using the equation (0.0346*absorbance)-0.0000087 which relates absorbance with g/L of red colorant composition.
  • the final titer of the fermentation process is 3.6 g of colorant composition per liter of fermentation broth calculated using the absorbance at 500 nm of the centrifuged fermentation broth.
  • the fungal biomass is removed at 9400 rpm in a continuous stack centrifuge. 300 g of ethanol is added at 96°C to 125 g of supernatant broth containing the red colorant composition in a 500 mL bottle. The mixture is incubated at 4°C for at least 24 hours.
  • the mixture is filtered to separate the precipitate which may be in part composed of exopolysaccharides (EPS) from the supernatant containing the red colorant composition.
  • the supernatant is concentrated by evaporation using a rotary evaporator in a 1 L glass ball at 55°C, 200 rpm, -0.09 MPa or -27.5 inHg vacuum, for 30 minutes.
  • Sulfuric acid (98%) was added to the concentrated supernatant to lower the pH to 2.5 and boost the precipitation of the red colorant composition, this solution was incubated overnight at 4°C.
  • the supernatant was separated by centrifugation at 4000 rpm for 10 minutes and the absorbance was measured to calculate the acid precipitation efficiency.
  • the precipitated red colorant composition was re-suspended in 15 mL of potassium hydroxide (0.1 M) and the volume and absorbance was measured. The red colorant composition was then dried in the oven at 70°C overnight or sprayed at these conditions with a nozzle air rate of 1.6 bar, air flow of 70 m 3 /h, inlet temperature of 185°C, outlet temperature of 74°C, and feeding rate of 1200 g/h.
  • composition of the colorant composition was determined by the methods as described in Examples 12 and 13 to be comprised of ash, succinate, N-glutamyl monascorubraminic acid, c/.s-N-glutaryl monascorubraminic acid, /ra/z.s-N-glutaryl monascorubraminic acid, N-glutaryl monascorubramine, and other compounds (see Table 22)
  • the fungal biomass was removed at 9,400 rpm in a continuous stack centrifuge.
  • the supernatant was incubated at 70°C for 30 minutes to denature and precipitate proteins and exopolysaccharides.
  • the supernatant was filtered through a 40 pm paper filter and the pH was lowered to 2.5 with sulfuric acid (98%).
  • 25 mL of ethyl acetate was added for every 125 g of filtered supernatant and mixed thoroughly at room temperature, resulting in phase separation.
  • the upper ethyl acetate phase containing the red colorant composition was separated from the aqueous phase suing a separatory funnel, the absorbance of the aqueous phase was measured.
  • the red colorant composition was extracted from the 25 mL ethyl acetate phase with 15 mL of a 0.1 M potassium hydroxide solution.
  • the lower water phase from the liquid extraction was recovered and the exact volume and absorbance was measured.
  • the ethyl acetate phase was dried in the oven at 70°C overnight or spray dried to isolate the dried colorant composition.

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Abstract

La présente invention concerne des procédés de culture d'un champignon produisant des pigments, comprenant les étapes suivantes : inoculation d'un premier milieu liquide avec des conidies du champignon ; réalisation d'une étape de culture de production de mycélium comprenant la culture des conidies dans le premier milieu liquide pour générer une culture d'inoculum comprenant du mycélium ; inoculation d'un deuxième milieu liquide avec la culture d'inoculum ou une partie de cette culture ; et réalisation d'une étape de culture de production de pigments comprenant la culture du mycélium dans le deuxième milieu liquide pour générer une culture de mycélium produisant des pigments ; le champignon produisant des pigments étant du genre Talaromyces, Penicillium, ou Monascus, et le premier milieu liquide, le deuxième milieu liquide, ou les deux, étant dépourvus de source de phosphate. L'invention concerne également des compositions produites selon les procédés de culture de l'invention.
EP23875667.0A 2022-10-03 2023-10-02 Compositions de colorant et procédés de production Pending EP4598360A2 (fr)

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