WO2024083795A1

WO2024083795A1 - Leucoindigo salt mixture from non-synthetic sources for dyeing processes

Info

Publication number: WO2024083795A1
Application number: PCT/EP2023/078782
Authority: WO
Inventors: Erwin Lucic; Umberto DE VITA; Andreas Wickli
Original assignee: Archroma Ip Gmbh
Priority date: 2022-10-17
Filing date: 2023-10-17
Publication date: 2024-04-25

Abstract

Method for standardizing non-synthetic leucoindigo salt mixture in the textile industry containing no aniline or N-methylaniline, wherein the non-synthetic leucoindigo salt is obtained from non-synthetic sources such as microorganisms or plants, comprising the non-synthetic leucoindigo salt in the range of from 5 to 60 % by weight.

Description

LEUCOINDIGO SALT MIXTURE FROM NON-SYNTHETIC SOURCES FOR DYEING PROCESSES

FIELD OF THE INVENTION

[0001 ] The present invention relates to the field of aniline and N-methylaniline free nonsynthetic leucoindigo salt mixtures obtained from non-synthetic indigo obtained from naturaland/or microorganism sources and the manufacturing method.

BACKGROUND OF THE INVENTION

[0002] Indigo is a vat dye used for dyeing cellulose-containing textile materials.

[0003] For application on a textile material, indigo is subjected to reduction wherein a water-soluble leucoindigo salt is formed. This salt is then applied in aqueous solution to the textile material. Oxidation of the leucoindigo salt results in the formation of indigo, wherein the dyed textile material is obtained.

[0004] Synthetically produced indigo contains impurities based on aromatic amines entrapped therein due to the commonly used production processes, in particular aniline and/or N-methylaniline. For example, synthetically produced indigo may contain up to 15000 ppm aniline and up to 7000 ppm N-methylaniline. Aromatic amines such as aniline and N-methylaniline are not desired in textile applications. Further, the synthetic pathway has a high impact on the environment through massive wastewater production and energy consumption.

[0005] EP 3 441 430 B1 discloses a method to purify synthetically produced indigo from aniline and N-methylaniline. It is clearly shown in this application, how much effort is needed regarding technical equipment, time, and cost to remove these side products.

[0006] Alternative routes to produce indigo have been known for decades as well. These routes comprise the harvesting of plants which produce precursors of indigo as for example indican from Indigofera tinctoria. US 8 585 783 for example discloses such a method for dyeing fibers with the natural indigo obtained from an indigo plant by extracting a plant pigment precursor from said plant followed by natural oxidization with air oxygen. Further, US’783 describes an elaborate washing process to remove remaining residues from the natural sources as well as reaction additives required for the dyeing with leucoindigo stemming from the natural source as described.

[0007] However, the raising chemical industry led to the abandoning of the natural sources due to the higher reliability of the synthetically produced indigo regarding yield and purity of the compound. Additionally, the described disadvantages in the application of solutions from natural leucoindigo sources, as for example the described elaborate washing processes, were eliminated by the upcoming industrial production of indigo.

[0008] Another way to produce indigo in a natural context is by microbiologically modified microorganisms like e.g. disclosed in WO 2021/013371 A1. Said microorganisms are modified by transforming the respective plasmids into competent microorganism cells, wherein said plasmids contain the information used by the microorganisms to express proteins which are able to produce indigo or precursors thereof. Said expressed proteins are mostly enzymes which are required to catalyze the reactions leading to the desired product.

[0009] WO 2021/013371 A1 discloses a method for the synthesis of leucoindigo starting from an indole and/or an indole derivative which is converted to indoxyl using an oxidizing enzyme. In a second step, leucoindigo is synthesized by a reducing enzyme. This leucoindigo is used to dye textiles directly. The indole used in the first step can be prepared from tryptophan using a tryptophanase. The enzymes used herein were expressed by microorganisms, extracted and purified previous to the application in said method of the patent.

[0010] Both routes do not provide any solution as to the problem of varying quality.

[0011 ] The present invention offers a method to provide non-synthetic leucoindigo solutions having comparable application properties in dyeing processes like leucoindigo solutions from synthetic sources. OBJECT OF THE INVENTION

[0012] The problem to be solved by this invention is to provide a non-synthetic standardized leucoindigo salt mixture, as well as a manufacturing method thereof, having comparable application properties in dyeing processes as leucoindigo solutions from synthetic sources.

SUMMARY OF THE INVENTION

[0013] The object is solved by a method to reliably standardize a non-synthetic leucoindigo salt mixture regarding the contained amount of said non-synthetic leucoindigo salt and impurities stemming from the non-synthetic origin. This is advantageous, since the non- synthetic indigo does not contain any aniline or N-methylaniline and therefore these compounds do not need to be removed after or before hydrogenation as with synthetic indigo leading to less effluent and less hazardous wastewater compared to known synthetic routes. Thus, environmental impact is reduced as well as the cost for purification of the product and wastewater treatment. Further, due to the lack of unhealthy amine side products the risk for consumers is lowered. In addition, the standardized non-synthetic leucoindigo salt mixture according to the invention has comparable application properties in dyeing processes as synthetic leucoindigo solutions, i.e. leucoindigo solution wherein the leucoindigo is produced synthetically. This in turn means that that synthetic leucoindigo solutions can be easily substituted in dyeing processes by the non-synthetic leucoindigo salt mixtures according to the invention as manufactures in the method according to the invention.

[0014] A further particular advantage of the method according to the invention is that the standardized non-synthetic leucoindigo salt mixture can be varied in concentration individually for different requirements of the textile industry, e.g. in the range of from 5 to 60 % by weight, or 10 to 50 % by weight or 15 to 40 % by weight or 20 to 30% by weight based on the total weight of the mixture, which comply with the dyeing requirements in terms of middle and deep shades. [0015] Accordingly, the invention relates to the following items: According to the invention is a method for the preparation of a standardized non-synthetic leucoindigo salt mixture comprising no aniline or N-methylaniline according to ISO 14362-1 :2017, wherein the method comprises steps (A) to (C):

(A) mixing non-synthetic indigo from at least one natural indigo source or at least one microorganism indigo source or combinations thereof with an alkali metal source, and a dispersing agent and water to obtain a mixture;

(B) subjecting the mixture obtained in (A) to a catalytic hydrogenation step to obtain a non-synthetic leucoindigo salt mixture;

(C) standardizing the obtained mixture of step (B) to a concentration range of non- synthetic leucoindigo salt of from 5 to 60% by weight, based on the total weight of the mixture.

[0016] The method according to the invention can further encompass that the natural indigo source used in step (A) is obtained from plants capable of producing indigo or indigo precursors selected from the group comprising at least Indigofera tinctoria, /satis tinctoria, Amorpha fruticose, polygonum tinctorium, Persicaria tinctoria, Succisa pratensis, Baptisia australis, Wrightia tinctoria, Marsdenia tinctoria or new plants based on a further development of one of above or cross of 2 or more.

[0017] The method according to the invention can further encompass that the microorganism indigo source used in step (A) is obtained from microorganisms capable of producing indigo or indigo precursors selected from the group consisting of bacteria, yeast or fungi, preferably chosen from the group consisting of Streptomyces, Escherichia, Bacillus, Pseudomonas, Saccharomyces, Aspergillus.

[0018] The method according to the invention can further encompass that the concentration of the at least one natural indigo source, the at least one microorganism indigo source, or mixtures thereof used in step (A) is at least 5% to at most 60% by weight based on the total weight of the mixture.

[0019] The method according to the invention can further encompass that in step (A) and/or (C) an alkali metal source is used, wherein said alkali metal source is selected from the group consisting of lithium hydroxide, sodium hydroxide, potassium hydroxide or mixtures thereof. [0020] The method according to the invention can further encompass that the dispersing agent is selected from the group consisting of lignin sulfonate, turkey red oil, alkyl acryl sulphonates, alkyl sulphonates, soap powder, or mixtures thereof.

[0021 ] The method according to the invention can further encompass that after step (A) further steps (I) and/or (II) are conducted:

(I) pre-filtering the mixture obtained in step (A); and/or

(II) fine-filtering the mixture obtained in step (A) or step (I).

[0022] The method according to the invention can further encompass that the temperature in step (B) is at least 20 °C and at most 140 °C, and/or wherein the pressure in step (B) is at least 1 bar and at most 50 bar.

[0023] The method according to the invention can further encompass that the catalyst in step (B) is selected from the group consisting of Raney-Nickel, Palladium on carbon (Pd/C), tetrakis(triphenylphosphine) palladium(O), or a mixture thereof.

[0024] The method according to the invention can further encompass that after step (B) further steps (III) and/or (IV) and/or (V) are conducted:

(III) separating the catalyst used in step (B) out of the non-synthetic leucoindigo salt mixture; and/or

(IV) fine-filtering the mixture obtained from step (B) and/or step (III) to remove remaining contaminants; and/or

(V) analyzing the obtained mixture from step (B) and/or step (III) and/or step (IV) of the method according to the invention.

[0025] The method according to the invention can further encompass that the standardizing in step (C) is done by at least one of the following procedures: dilution with water, dilution with an alkali metal source, distillation of water.

[0026] Further according to the invention is an aqueous non-synthetic leucoindigo salt mixture obtained by the method according to the invention described above and also in the following in detail, wherein no aniline or N-methylaniline according to ISO 14362-1 :2017 is present.

[0027] The aqueous non-synthetic leucoindigo salt mixture according to the invention can further encompass that the concentration of the non-synthetic leucoindigo salt contained in the obtained mixture is adjusted to at least 5 % by weight, and at most 60 % by weight, based on the total weight of the non-synthetic leucoindigo salt mixture.

[0028] Further according to the inventions is an aqueous leucoindigo salt composition comprising or consisting of the aqueous non-synthetic leucoindigo salt mixture according to the invention and an aqueous synthetic leucoindigo salt mixture, wherein no aniline or N- methylaniline according to ISO 14362-1 :2017 is present.

[0029] Further according to the invention is a method of making indigo, comprising step (E): (E) oxidizing the aqueous leucoindigo salt composition according to the invention, or the aqueous non-synthetic leucoindigo salt mixture according to the invention, or as produced according to the method according to the invention.

[0030] The method of making indigo according to the invention can further comprise step (D) prior to step (E):

(D) treating a textile with the leucoindigo salt composition according to the invention, or the non-synthetic leucoindigo salt mixture according to the invention, or as produced according to the method according to the invention.

[0031 ] Further according to the invention is a textile dyed with the aqueous leucoindigo salt composition according to the invention, or the aqueous non-synthetic leucoindigo salt mixture according to the invention, or as produced according to the method according to the invention

DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention relates to a method for the preparation of a non-synthetic leucoindigo salt mixture comprising no aniline or no N-methylaniline (= Oppm) according to ISO 14362-1 :2017, wherein the method comprises steps (A) to (C):

(A) Mixing non-synthetic indigo from at least one natural indigo source or at least one microorganism indigo source or mixtures thereof with water, an alkali metal source, and a dispersing agent to obtain a mixture;

(B) subjecting the mixture obtained in (A) to a catalytic hydrogenation step to obtain a non-synthetic leucoindigo salt mixture; (C) standardizing the obtained mixture of step (B) to a concentration range of nonsynthetic leucoindigo of from 5 to 60% by weight, based on the total weight of the mixture.

[0033] The term “non-synthetic” refers to indigo (and/or leucoindigo (salt), i.a. derived from said indigo) obtained from plants or microorganisms. This means that synthetic pathways are excluded. Due to the origin of the obtained indigo or leucoindigo (salt), natural residues like plant and cell debris, whole cells stemming from microorganisms or plants, proteins, clay, silicic acid, phosphoric acid, microorganism residues, sand, soil, indin, indirubin, isatine, anthranilic acid, indoxyl, indole, indolecarboxylic acid or mixtures thereof can be present in the indigo and/or leucoindigo (salt) derived therefrom. Further, the term “nonsynthetic” means that the produced indigo or leucoindigo salt products (e.g. powders or solutions) do not contain any aniline or N-methylaniline, i.e. , the aniline and N-methylaniline amount thereof is considered below the limit of detection by ISO 14362-1 :2017.

[0034] The term “synthetic pathways” refers to all methods for synthesizing indigo using chemical reactions, i.e. artificially instigated chemical reactions, e.g. starting from aniline, anthranilic acid, cinnamic acid or nitrobenzaldehyde.

[0035] In general, the term “purity” of non-synthetic indigo as used within the context of the present application, relates to the content of the indigo dye in the non-synthetic indigo harvested from either plants or microorganisms. Non-synthetic indigo is a composition that besides indigo contains a multitude of other components (also referred to in the present application as “contaminant”) like plant and cell debris, whole cells stemming from microorganisms or plants, proteins, clay, silicic acid, phosphoric acid, microorganism residues, sand, stones, soil, indin, indirubine, isatine, anthranilic acid, indoxyl, indole, indolecarboxylic acid, or mixtures thereof. As an example, 1 kg of non-synthetic indigo having a purity of 90 % by weight, contains 900 g of the actual indigo dye, and 100 g of impurites. The purity of non-synthetic indigo can be determined by a procedure that is described in detail in the example section.

[0036] The term “natural indigo source” refers to plants, capable of producing indigo or indigo precursors, of the group comprising but not limited to Indigofera tinctoria, Isatis tinctoria, Amorpha fruticose, polygonum tinctorium, Persicaria tinctoria, Succisa pratensis, Baptisia australis, Wrightia tinctoria, Marsdenia tinctoria or new plants based on a further development of one of above or cross of 2 or more. The plants thereby can be either wild forms or cultivated forms or genetically modified forms of the plants. The indigo can be obtained therefrom by fermenting obtained parts of the plant, oxidizing contained precursors and filtration of the obtained indigo.

[0037] The non-synthetic indigo stemming from natural indigo source can comprise at most 90 % by weight, or at most 70% by weight, or at most 60% by weight, or at most 50% by weight, or at most 40% by weight, or at most 30 % by weight, or at most 25 % by weight, or at most 20 % by weight, or at most 10 % by weight, or at most 5 % by weight, cell debris and/or other contaminants e.g. proteins, silicic acid, phosphoric acid, microorganism residues, sand, stones, soil, indin, indirubine, isatine, anthranilic acid, indoxyl, indole, indolecarboxylic acid, or mixtures thereof, like already defined above stemming from the natural indigo source, based on the total weight of the non-synthetic natural indigo. Or, in other words, the non-synthetic indigo harvested from the plant has a purity in the range of from 10% to 95% by weight, or from 30% to 90% by weight, or from 40% to 80% by weight, or from 50% to 75% by weight, or from 60% to 70% by weight based on the total weight of the indigo harvested from the plant. E.g. 1 kg of indigo harvested from the plant contains between 100 to 950 g of the indigo per se, whereas the remainder to 1 kg can be made up by other compounds (e.g. sand, stones, plant residues, water etc.).

[0038] The term “indigo” within the context of the present application refers to the following chemical compound

having the CAS number 482-89-3 and derivatives thereof, wherein the above shown chemical formula contains one or more substituents like sulfo groups (-SOs'lVT, wherein M⁺ is a suitable cation for balancing the anionic charge of the sulfo group like one or more of H⁺, Li⁺, Na⁺, K⁺ or substituted or unsubstituted ammonium cation), alkyl residues or halogen. A possible derivative of indigo may be indigotin, wherein the phenyl rings are substituted by sulfogroups. Indigo is used in the method according to the invention in step (A) as a powder or a water-containing slurry. As mentioned above, the indigo is stemming exclusively from natural, and/or microorganism indigo sources. This means that the indigo used in step (A) in the method according to the invention cannot contain any aniline or N- methylaniline (i.e. below limits of detection by method ISO 14362-1 :2017 of aniline and/or N-methylaniline); i.e. the aniline and/or N-methyl aniline content is 0 ppm. However, since the indigo stems from natural, and/or microorganism sources, the indigo used in step (A) in the method according to the invention can contain natural residues like plant and cell debris, whole cells stemming from microorganisms or plants, proteins, clay, silicic acid, phosphoric acid, microorganism residues, sand, stones, soil, indin, indirubine, isatine, anthranilic acid, indoxyl, indole, indolecarboxylic acid, or mixtures thereof.

[0039] The term “microorganism indigo source” refers to genetically modified procaryotic and/or eucaryotic host cells capable of producing indigo. Useful host cells may be bacteria, yeast or fungi as for example Streptomyces, Escherichia, Bacillus, Pseudomonas, Saccharomyces, Aspergillus, etc. The procaryotic host Escherichia coli represents one preferred host organism. The microorganisms are genetically modified to express e.g. at least one enzyme of the group of oxygenases.

[0040] The non-synthetic indigo stemming from microorganism source can comprise at most 99 % by weight, or at most 95% by weight, or at most 90% by weight, or at most 70% by weight, or at most 60% by weight, or at most 50 % by weight, or at most 40 % by weight, or at most 30 % by weight, or at most 25 % by weight, or at most 20 % by weight, or at most 10 % by weight, or at most 5% by weight, or at most 1 % by weight cell debris and/or other contaminants e.g. proteins, silicic acid, phosphoric acid, microorganism residues, indin, indirubine, isatine, anthranilic acid, indoxyl, indole, indolecarboxylic acid, or mixtures thereof like already defined above stemming from the microbiological synthesis based on the total weight of the non-synthetic indigo stemming from microorganism source. Or, in other words, the non-synthetic indigo harvested from microorganism indigo sources has a purity in the range of from 10% to 99% by weight, or from 20 to 95 % by weight, or from 30% to 90% by weight, or from 40% to 80% by weight, or from 50% to 75% by weight, or from 60% to 80% by weight, or from 60% to 70% by weight based on the total weight of the non-synthetic indigo harvested from the microorganism indigo source. [0041 ] The term “alkali metal source” as used within the context of the present application means lithium hydroxide, sodium hydroxide or potassium hydroxide or mixtures thereof. The respective cations of theses alkali hydroxides are used after step (B) to counter the anionic charge of the leucoindigo.

[0042] The used dispersing agent is selected from the group comprising lignin sulfonate, turkey red oil, alkyl aryl sulphonates, alkyl sulphonates, soap powder, or mixtures thereof.

[0043] The term “water” means tap water, demineralized water, deionized water, distilled water, or mixtures thereof.

[0044] The non-synthetic indigo from at least one natural indigo source or at least one microorganism indigo source or a mixture thereof is used in an amount of from 5% to 60% by weight, or from 15% to 50% by weight, or from 20% to 40% by weight, or from 25% to 35% by weight, based on the total weight of the mixture of step (A) of the method according to the invention.

[0045] The amount of the alkali metal source used in step (A) of the method according to the invention is in the range of from 1 % to 25% by weight, or from 2.5% to 15% by weight, or from 4% to 15% by weight, or from 5% to 10% by weight based on the total weight of the mixture of step (A) of the method according to the invention.

[0046] The amount of dispersing agent used is in the range of from 0.1 % to 20% by weight, or from 0.25% to 15% by weight, or from 0.3% to 10% by weight, or from 0.5 to 8 % by weight, based on the total weight of the mixture of step (A) of the method according to the invention.

[0047] The mixture of step (A) of the method according to the invention comprises or consists of 5% to 60% by weight of non-synthetic indigo from the at least one non-synthetic indigo source, from 1% to 25% by weight of an alkali metal source, and from 0.1 to 20% by weight of a dispersing agents, wherein the weight% are based on the total weight of the mixture. The remaining proportion to 100% by weight can be filled up with water and/or further additives.

[0048] Step (A) of the method according to the invention is a mixing step wherein said non- synthetic indigo, said alkali metal source, said dispersing agent and water are mixed to form a mixture. Said mixture can be a solution, a slurry, a dispersion, or a suspension. Said mixing step can be conducted in a separate homogenization reactor or directly in the hydrogenation reactor used in step (B) of the method according to the invention.

[0049] Said mixture obtained according to step (A) of the method according to the invention may comprise contaminants from the group comprising natural residues like plant and cell debris, whole cells stemming from microorganisms or plants, proteins, clay, silicic acid, phosphoric acid, microorganism residues, sand, stones, soil, indin, indirubine, isatine, anthranilic acid, indoxyl, indole, indolecarboxylic acid, or mixtures thereof, due to the nonsynthetic origin of the used indigo. The "contaminant content" of the mixture obtained in step (A) of the method according to the invention is defined as the weight percentage of contaminants stemming from the plants or microorganisms in the mixture. For example, if 100 kg of non-synthetic indigo having a purity of 50% is used for preparing 200 kg of the mixture, said mixture has a contaminant content of 25% (50 kg impurities in 200 kg mixture).

[0050] The contaminant content of the mixture obtained in step (A) of the method according to the invention can range within the wt% ranges as defined above with regard to the nonsynthetic indigo stemming from natural and/or microorganism sources.

[0051 ] The method according to the invention may optionally further comprise steps (I) and/or (II). Step (I) and/or step (II) can be conducted after step (A), i.e. between steps (A) and (B) of the method according to the invention:

(I) pre-filtering the mixture obtained in step (A) of the method according to the invention; and/or

(II) fine-filtering the mixture obtained in step (A) or step (I) of the method according to the invention.

[0052] The term “pre-filtering” refers to the use of filtration systems with a pore size of 1 mm to 5 mm, or 1 mm to 4 mm, or 1 mm to 3 mm. The pre-filtering can remove larger contaminants (e.g. sand or stones).

[0053] The term ’’fine-filtering” refers to the use of filtration systems with a pore size less than 1000 pm, or less than 500 pm, or less than 250 pm, or less than 100 pm, or less than 50 pm, or less than 10 pm, or less than 5 pm, or less than 1 pm.

[0054] Step (II) can be conducted after step (I). [0055] Each step (I) and/or step (II) can be conducted only once. Alternatively, each step (I) and/or step (II) can be conducted more then once, e.g. 2 times, 3 times, 4 times, six times, 10 times. In case each step (I) and/or step (II) is conducted more than once, the filtration system used for each repletion may be the same or may be different. E.g. in case step (I) is conducted twice, it is possible to use first a filtration system having a pore size of 5 mm, and in course of the repetition, a filtration system having the same pore size or a different pore size of e.g. 3 mm. The same applies for step (II).

[0056] During the pre-filtering and/or the fine-filtering step(s), the mixture subjected to filtration is filtered at least one time using one or more filters selected from the group consisting of candel filter, micro siew filter, magnetic filter, filter bags, membrane.

[0057] Step (B) of the method according to the invention comprises subjecting the mixture obtained in step (A) and/or step (I) and/or step (II) of the method according to the invention containing the non-synthetic indigo to a catalytic hydrogenation step to obtain a nonsynthetic leucoindigo salt mixture.

[0058] According to the method of the invention, the contaminant content of the nonsynthetic leucoindigo salt mixture obtained after the filtration step (B) is below 30 wt%, or below 20 wt%, or below 10 wt%, or below 5 wt%, or below 1 wt%, or below 0.5 wt%, or below 0.25 wt%, or below 0.1 wt%, wherein the wt% are based on the total weight of the non-synthetic leucoindigo salt mixture. In a preferred embodiment of the invention, the contaminant content is below 1wt%. The remaining contaminant content of the non- synthetic leucoindigo salt mixture obtained in step (B) of the method according to the invention can be e.g. calculated by weighing the impurities that have been filtered off in step (I) and/or step (II).

[0059] The catalytic hydrogenation step for making leucoindigo (salt) from indigo is well known in the literature.

[0060] The term “catalytic hydrogenation” refers to a redox reaction. During said redox reaction, the oxidation states of indigo changes, meaning the desired product leucoindigo is formed in a reduction process while an added reducing agent is oxidized. Any reducing agent known in the prior art can be used. Further, a catalyst is added to the reaction. Any catalyst known in the prior art can be used. [0061 ] The used reducing agent for said catalytic hydrogenation step can be molecular hydrogen.

[0062] The pressure of the molecular hydrogen can be adjusted individually to process requirements within wide ranges of from 1 bar to 50 bar.

[0063] The pressure of the molecular hydrogen during the reaction is adjusted to a range from 1 bar to 50 bar, or from 1 bar to 40 bar, or from 1 bar to 25 bar, or from 1 bar to 20 bar, or from 1 bar to 15 bar, or from 1 bar to 10 bar, or from 1 bar to 8 bar.

[0064] The used catalyst for said catalytic hydrogenation step can be Raney-Nickel, Palladium on carbon (Pd/C), tetrakis(triphenylphosphine) palladium(O), or a mixture thereof.

[0065] The used amount of the catalyst can be adjusted within wide ranges of from 0.1 to 20% by weight or from 0.25% to 15% by weight, or from 0.5% to 12.5% by weight, or from 0.75% to 10% by weight, or from 1 to 8 % by weight, based on the total weight of the complete mixture.

[0066] The temperature of step (B) of the method according to the invention according to the invention can be chosen within wide boundaries in the range of from 20 °C to 140 °C, or 40 °C to 120 °C, or 60 °C to 100 °C, or 70 °C to 90 °C, or is adjusted to 80 °C.

[0067] Step (B) of the method according to the invention is conducted for the time hydrogen adsorption is noticed or in a timeframe in the range of from 1 h to 48 h, or 3 h to 24 h, or 4.5 h to 18 h, or 6 h to 12 h, or 2 h to 6 h.

[0068] The mixture obtained after step (B) of the method according to the invention comprises a non-synthetic leucoindigo salt characterized in that said leucoindigo is encompassed as alkali metal salt and no aniline or N-methylaniline is contained (i.e. below limits of detection by method ISO 14362-1 :2017). The term “leucoindigo salt” within the context of the present application refers to the following chemical compound or derivatives thereof (like already defined above with respect to indigo):

The cations for balancing the anionic charge of the leucoindigo anion are selected from the group consisting of alkali metal cations Li⁺, Na⁺, K⁺ or hydrogen cation H⁺ or a mixture thereof. Preferred are Na⁺ and/or K⁺ as cations for balancing the anionic charge of the leucoindigo anion. The obtained mixture after step (B) of the method according to the invention does not contain any aniline or N-methylaniline (i.e. below limits of detection by method ISO 14362-1 :2017); i.e. the aniline and/or N-methyl aniline content is 0 ppm. However, the natural and/or microorganism origin means as well that the mixture obtained from step (B) of the method according to the invention can contain natural residues (also referred to herein as “contaminants”) like plant and cell debris, whole cells stemming from microorganisms or plants, proteins, clay, silicic acid, phosphoric acid, microorganism residues, sand, stones soil, indin, indirubine, isatine, anthranilic acid, indoxyl, indole, indolecarboxylic acid, or mixtures thereof.

[0069] The term “alkali metal ion" encompasses the respective cations of lithium, sodium and potassium and combinations of two or three thereof.

[0070] Accordingly, in one embodiment, the alkali metal ion may be lithium or sodium or potassium.

[0071 ] In another embodiment, the alkali metal ion may be lithium and sodium, or lithium and potassium, or sodium and potassium.

[0072] In another embodiment, the alkali metal ion is lithium, sodium and potassium.

[0073] After hydrogenation is completed, the hydrogen atmosphere is exchanged against an inert gas atmosphere.

[0074] The term “inert gas atmosphere” refers to the atmosphere under which the respective step was done or the mixture is stored. In said atmosphere no oxygen is contained. Beside volatile components of the mixtures comprised said atmosphere, the main components of said atmosphere are molecular nitrogen and/or argon.

[0075] . The method according to the invention may further comprise steps (III) and/or (IV) and/or (V). Step (III) and/or step (IV) and/or step (V) can be conducted after step B, i.e. between the steps (B) and (C) of the method according to the invention:

(III) separating the catalyst used in step (B) according to the method of the invention out of the obtained non-synthetic leucoindigo salt mixture; and/or (IV) fine-filtering the mixture obtained from step (B) and/or step (III) to remove remaining contaminants; and/or

[0076] The term “separating” refers to any separation method known in the prior art. The catalyst thereby can be separated by one or more means selected from filtration, decanting, centrifuging.

[0077] The term “analyzing” means any physical or chemical method known in the prior art to systematically investigate the parts of mixtures, e.g measuring the concentration or purity of the obtained non-synthetic leucoindigo salt mixture, e.g. via UV/Vis analytical methods.

[0078] The term “fine-filtering” according to step (IV) has the same definition as provided above for step (II). Step (IV) can also be conducted once or more times using the same or different filtration systems.

[0079] After step (B) of the method according to the invention, a non-synthetic leucoindigo salt mixture is obtained.

[0080] The non-synthetic leucoindigo salt mixture obtained in step (B) and/or step (III) and/or step (IV) and/or step (V) optionally conducted after step (B) and prior to step (C), can be designed as solution, suspension, slurry or dispersion, preferably as solution. The concentration of the non-synthetic leucoindigo salt in the mixture obtained in step (B) can be in the range of from 5% to 60% by weight, or from 10% to 50% by weight, or from 15% to 40% by weight, or from 20% to 30% by weight, based on the total weight of the leucoindigo salt mixture. Further, the proportion of the remaining contaminants in said mixture obtained after step (B) and/or step (III) and/or step (IV) and/or step (V) of the method according to the invention compared to the mixture obtained in step (A) and/or step (I) and/or step (II) is lower. The mixture obtained after step (B) and/or step (III) and/or step (IV) and/or step (V) of the method according to the invention comprises at most 20wt%, or at most 10 wt%, or at most 5 wt% or below 1 wt% of contaminants stemming from the natural and/or the microorganism indigo source based on the total weight of the non- synthetic leucoindigo salt mixture. [0081 ] In one embodiment the non-synthetic leucoindigo salt mixture obtained in step (B) and/or step (III) and/or step (IV) and/or step (V) optionally conducted after step (B) and prior to step (C) is a solution.

[0082] The non-synthetic leucoindigo salt within the non-synthteic leucoindigo salt mixture obtained in step (B) and/or step (III) and/or step (IV) and/or step (V) optionally conducted after step (B) and prior to step (C) has a purity in the range of from 80% to 99% by weight, or from 85% to 99% by weight, or from 90% to 99% by weight, or from 95% to 99% by weight. The definition of the term “purity” disclosed above applies here as well. The concentration of the non-synthetic leucoindigo salt obtained in step (B) and/or step (III) and/or step (IV) and/or step (V) optionally conducted after step (B) and prior to step (C) is within the range of from 1 % to 90% by weight, or from 2 to 80% by weight, or from 5% to 60% by weight, or from 10% to 50% by weight, or from 15% to 40% by weight, or from 20% to 30% by weight, based on the total weight of the leucoindigo salt mixture.

[0083] The term “concentration” as used within the context of the present application means the amount of the non-synthetic leucoindigo salt per se present within the complete non-synthetic leucoindigo salt mixture. E.g. if the weight of the complete non-synthetic leucoindigo salt mixture (containing the non-synthetic leucoindigo salt, water, and further components) is 10 kg and among these 10 kg, 1 kg is made up by the non-synthetic leucoindigo salt, then the concentration of the non-synthetic leucoindigo salt mixture is 10%, i.e. 10% of the complete non-synthetic leucoindigo salt mixture is made up by the non-synthetic leucoindigo salt.

[0084] For a better understanding of the meaning of the terms “concentration” and “purity as used herein throughout the present application another example is given in the following: 200 kg of a 50% concentrated non-synthetic leucoindigo salt mixture is obtained in step (B) and/or step (III) and/or step (IV) and/or step (V) optionally conducted after step (B) and prior to step (C). This means that 100kg non-synthetic leucoindigo salt is present in the mixture, wherein the remained is due to e.g. solvent (like water) or other components which were added in course of the method according to the invention (e.g. stabilizing agents, catalyst or the like). The 100 kg non-synthetic leucoindigo salt present in the non- synthetic leucoindigo salt mixture obtained in step (B) and/or step (III) and/or step (IV) and/or step (V) optionally conducted after step (B) and prior to step (C) can have a purity of 99%by weight, which then means that among the 100 kg of non-synthetic leucoindigo salt, 99 kg are leucoindigo salt and 1 kg are contaminants stemming from the natural and/or microorganism source.

[0085] In step (C) of the method according to the invention, the in step (B) obtained nonsynthetic leucoindigo mixture is standardized under an inert gas atmosphere and thus a standardized mixture is obtained.

[0086] The term “standardized mixture” herein refers to a non-synthetic leucoindigo salt mixture which has a reliable purity as well as a reliable concentration of non-synthetic leucoindigo salt adjusted for the further application in industrial processes. The standardization of the non-synthetic leucoindigo salt mixture can be conducted by diluting the non-synthetic leucoindigo salt mixture with water and optionally an alkali metal source as defined above, or by distillation. It is a matter of course that upon dilution the content of contaminants, e.g cell debris or other contaminants from the natural/microorganism sources, are further decreased.

[0087] The concentration of non-synthetic leucoindigo (salt) of the standardized non- synthetic leucoindigo salt mixture obtained after step (C) of the method according to the invention is in the range of from 5 to 60 % by weight, or 10 to 50 % by weight, or 15 to 40 % by weight, or 20 to 30 % by weight, wherein the percentages are based on the total weight of the non-synthetic leucoindigo salt mixture. After step (C) of the method according to the invention, a non-synthetic leucoindigo salt mixture is obtained. Said mixture contains no aniline or N-methylaniline (=0ppm) (i.e. below limits of detection by method ISO 14362- 1 :2017). The purity of the non-synthetic, standardized leucoindigo salt mixture obtained is or is up to 100% by weight, or is or is up to 99.9 % by weight, or is or is up to 99.75 % by weight, or is or is up to 99.5 % by weight, or is or is up to 99% by weight, or is or is up to 95% by weight, or is or is up to 90% by weight, or is or is up to 80% by weight, or is or is up to 70% by weight, or is or is at least 60% by weight. This in turn means that contaminants from natural/microorganism sources can be contained in said mixture such as natural residues like plant and cell debris, whole cells stemming from microorganisms or plants, proteins, clay, silicic acid, phosphoric acid, microorganism residues, sand, soil, indin, indirubine, isatine, anthranilic acid, indoxyl, indole, indolecarboxylic acid, or mixtures thereof and may be comprised in an amount of at most 40% by weight, or at most 30% or at most 20% by weight, or at most 10% by weight, or at most 5 % by weight, or at most 1 % by weight, or at most 0.5% by weight, or at most 0.25% by weight, or at most 0.1 % by weight and/or at least 0.1 % by weight, or at least 0.25% by weight, or at least 0.5% by weight, or at least 1 % by weight based on the total weight of the non-synthetic, standardized leucoindigo salt mixture.

[0088] In case the impurities are no longer detectable in the non-synthetic leucoindigo salt mixture obtained from step (C) of the method according to the invention, said mixture may be termed as pure non-synthetic leucoindigo salt mixture. The non-synthetic leucoindigo salt mixture obtained in step (C) can be designed as solution, suspension, slurry or dispersion. According to the invention, it is preferred that the non-synthetic leucoindigo salt mixture obtained in step (C) is designed as solution, preferably as alkali metal leucoindigo salt solution.

[0089] The amount of alkali metal cation in the non-synthetic leucoindigo salt solution essentially corresponds to an amount which stoichiometrically corresponds to the amount which is necessary for the complete formation of the leucoindigo salt. Preferably, the salt and/or the solution contains from 1.5 to 2.5 mole alkali per mole leucoindigo, more preferably of from 2.0 to 2.5 mole alkali, still more preferred of from 2.1 to 2.5 mole alkali [0090] In one embodiment, the leucoindigo salt is a sodium salt.

[0091 ] In one embodiment, the salt is in the form of a mixed alkali metal salt such as a mixed sodium and potassium salt.

[0092] In one embodiment, sodium and potassium are present in a molar ratio from 7 : 1 to 1 : 3.

[0093] In another embodiment, sodium and potassium are present in a molar ratio of from 3 : 1 to 1 : 3.

[0094] In another embodiment, sodium and potassium are present in a molar ratio of from above 1 : 10 to 10 : 1 such as 1 : 7 to 1 : 5, or 1 : 8 to 1 : 4, or 2 : 1 to 1 : 2, or 4 : 1 to 8 : 1 or 5 : 1 to 7 : 1 , or 7 : 1 to 1 : 3.

[0095] The person skilled in the art is capable of selecting suitable concentration ranges in which the respective purified non-synthetic leucoindigo salt solutions made from the nonpurified, non-synthetic leucoindigo salt solutions are stable depending on the used molar ratios. [0096] Thus, within the addressed molar ranges of sodium to potassium, stable and concentrated solutions may be prepared.

[0097] In a preferred embodiment, the non-synthetic leucoindigo salt is provided in the form of a mixed alkali metal salt, preferably a mixed sodium and potassium salt, further preferably in a molar ratio of sodium to potassium in the range of from 3 : 1 to 6 : 1 . .

[0098] It is possible to provide the required amount of alkali metal hydroxide at once prior to step (B) of the method according to the invention or in portions during step (B) of the method according to the invention, or prior and during step (B) of the method according to the invention. If necessary, additional alkali metal ion sources may also be added after step (B) or in step (C) of the method according to the invention.

[0099] The mixture obtained after the standardization in step (C) of the method according to the invention can be stored afterwards in dedicated tanks.

[00100] To increase storage time in said dedicated tanks, oxygen is excluded from said dedicated tanks by applying an inert gas atmosphere.

[00101] To increase storage time in said dedicated tanks, the temperature in said dedicated tanks is kept over -10 °C.

[00102] Further, an aqueous non-synthetic leucoindigo salt mixture according to the invention is claimed characterized in that no (=0 ppm) aniline or N-methylaniline is comprised (i.e. below limits of detection by method ISO 14362-1 :2017), wherein the aqueous non-synthetic leucoindigo salt mixture is obtained by the method according to the invention. The aqueous non-synthetic leucoindigo salt mixture has a reliable purity as well as a reliable concentration of non-synthetic leucoindigo salt. The aqueous non-synthetic leucoindigo salt mixture according to the invention has comparable application properties in dyeing processes as leucoindigo solutions from synthetic sources.

[00103] The concentration of non-synthetic leucoindigo (salt) in the standardized non- synthetic leucoindigo salt mixture according to the invention is in the range of from 5 to 60 % by weight, or 10 to 50 % by weight, or 15 to 40 % by weight, or 20 to 30 % by weight, wherein the percentages are based on the total weight of the non-synthetic leucoindigo salt mixture. The purity, determined according to the method described in detail in the Example section, of the non-synthetic standardized leucoindigo salt mixture according to the invention is or is up to 100% by weight, or is or is up to 99.9 % by weight, or is or is up to 99.75 % by weight, or is or is up to 99.5 % by weight, or is or is up to 99% by weight, or is or is up to 95% by weight, or is or is up to 90% by weight, or is or is up to 80% by weight, or is or is up to 70% by weight, or is or is at least 60% by weight. This in turn means that the contaminants from natural/microorganism sources can be contained such as natural residues like plant and cell debris, whole cells stemming from microorganisms or plants, proteins, clay, silicic acid, phosphoric acid, microorganism residues, sand, soil, indin, indirubine, isatine, anthranilic acid, indoxyl, indole, indolecarboxylic acid, or mixtures thereof, and may be comprised in an amount of at most 40% by weight, or at most 30% or at most 20% by weight, or at most 10% by weight, or at most 5 % by weight, or at most 1 % by weight, or at most 0.5% by weight, or at most 0.25% by weight, or at most 0.1 % by weight and/or at least 0.1 % by weight, or at least 0.25% by weight, or at least 0.5% by weight, or at least 1 % by weight based on the total weight of the non-synthetic leucoindigo salt mixture.

[00104] The non-synthetic leucoindigo salt mixture according to the invention contains leucoindigo salt, wherein the leucoindigo salt is derived only from (a) natural indigo source(s) or only from microorganism indigo source(s) or is derived from a combination thereof, i.e. contains leucoindigo salt derived from a natural indigo source(s) and leucoindigo salt derived from microorganism indigo source(s).

[00105] The term “aqueous” means that the non-synthetic leucoindigo salt mixture comprises water, e.g. as the only solvent or as a part of a solvent mixture. The solvents comprised in mixtures can be all water miscible solvents known in the prior art as for example short-chain alcohols.

[00106] The terms “non-synthetic”, “water” and “leucoindigo” are defined as before. The aqueous non-synthetic leucoindigo salt mixture according to the invention is preferably designed as solution.

[00107] Said aqueous non-synthetic leucoindigo salt mixture may be converted to indigo or may be used in a vat dyeing process for dyeing a textile.

[00108] The term “vat dyeing” refers to dyeing methods characterized in that waterinsoluble pigments are used to dye a textile. Said water-insoluble pigments are reduced to a water-soluble form previous to the application on said textiles. [00109] The present invention may further comprise a method of making nonsynthetic indigo, comprising step (E):

(E) oxidizing the aqueous non-synthetic, standardized leucoindigo salt mixture according to the invention, or as produced in steps (A) to (C) of the method according to the invention or the leucoindigo salt composition according to the invention defined in the following.

[00110] The terms “non-synthetic”, “indigo”, “aqueous” and “leucoindigo” are defined as before.

[00111] Said non-synthetic indigo is characterized in that no aniline or N-methylaniline is comprised, and further that it has a purity as defined above with regard to the non- synthetic leucoindigo salt; in particular obtained after step (C).

[00112] The present invention may further comprise a method (D) prior to (E):

(D) treating a textile with the aqueous non-synthetic, standardized leucoindigo salt solution according to the invention, or as produced in steps (A) to (C) of the method according to the invention, or with the leucoindigo salt composition according to the invention as defined in the following.

[00113] The terms “aqueous”, “non-synthetic” and “leucoindigo” are defined as before.

[00114] The term “treating a textile” refers to any method known in the prior art for dyeing.

[00115] In a preferred embodiment, said textile treated with the aqueous non- synthetic leucoindigo salt solution according to the invention or the leucoindigo salt composition according to the invention possesses comparable application properties as textiles dyed with synthetic leucoindigo solutions.

[00116] Further claimed is a “leucoindigo salt composition” comprising or consisting of the non-synthetic leucoindigo salt mixture according to the invention (wherein the leucoindigo salt is derived only from natural indigo source(s) or only from microorganism indigo source(s) or is derived from a combination thereof, i.e. contains leucoindigo salt derived from a natural indigo source(s) and leucoindigo salt derived from microorganism indigo source(s)) and a synthetic leucoindigo salt mixture, wherein the amount of aniline or N-methylaniline present in the composition is below limits of detection by method ISO 14362-1 :2017. The term “synthetic leucoindigo salt mixture” refers to a leucoindigo salt mixture, wherein the leucoindigo is obtained from indigo which is produced using commonly known chemical synthesis routes. A differentiation between synthetic indigo or leucoindigo salt and non-synthetic indigo or leucoindigo salt is possible by using carbon isotope-based analytical methods.

[00117] Finally the present invention relates to a textile dyed with the aqueous nonsynthetic leucoindigo salt solution according to the invention, or as produced in steps (A) to (C) of the method according to the invention, or with the leucoindigo salt composition according to the invention.

EXAMPLES

[00118] Four indigo samples from commercially available natural indigo source and two indigo samples from commercially available microorganism indigo source with different purity were used in the following Examples and subjected to hydrogenation, purification, and standardization trials (see Indigo sample overview table 1).

[00119] Indigo purity was determined by applying UV-Vis analysis after a small quantity of sample was dissolved in organic solvent. Synthetic indigo was used as reference sample. In the following, the determination of the indigo purity is disclosed for sample NI-20200801. This procedure was used also for the determination of the purity of all other samples accordingly:

• Synthetic indigo (DENISOL pure 30% from Archroma; used as standard) with high purity and natural indigo sample NI-20200801 were each separately dissolved in an organic solvent e.g. selected from DMSO, DMF, NMP, THF or dichloromethane (0.1 -0.5 mg/100ml)

• UV-VIS analysis at A = 604 nm gave two linear curves with different slopes (Figure 1)

• In the Figure 2 the synthetic indigo was corrected for its purity (98.3 wt% determined by HPLC) • Setting the purity of natural indigo on 56.2 wt% resulted in the same slope as for the synthetic indigo

• This means that based on UV-Vis analysis in the indigo form, the natural indigo sample NI-20200801 contains 56.2 wt% indigo; i.e. the purity is 56.2 wt% based on the total weight of the sample

[00120] Sample overview table 1 :

Example 1

A) Suspension preparation and pre-filtration

[00121] In a 1 I glass reactor equipped with an intensive stirrer and a bottom valve 250 g water, 5 g lignin sulfonate (dispersing agent) and 40 g of a 50% NaOH were charged and homogenized. Under stirring and in portion of 5 g each, 150 g indigo of batch NI20200803 (purity: 65.7 wt%) was added and homogenized. Finally, another 40g of 50% alkali were charged and mixed well. The thus obtained indigo suspension was than prefiltered by circulating it by a pump through a filter with pore sizes of 2 mm first, then 1 mm and finally fine-filtered through a filter with a pore size of 0.5 mm.

B) Hydrogenation in 1 I stainless steel autoclave with anchor stirrer and cross blades

[00122] After addition of 15 g Raney Ni catalyst the suspension was transferred to the 1 I stainless steel autoclave, equipped with an anchor stirrer for hydrogenation. After receiving the indigo suspension, the autoclave was purged and pressurized with inert gas. After releasing the pressure, stirrer was started, indigo suspension in cascade heated to 80-90°C and hydrogen supply started. Hydrogenation is executed under 4 - 6 bar hydrogen pressure and a duration of 5h. The thus obtained leucoindigo salt mixture is transferred to an inert separation funnel and catalyst and insoluble material are separated.

C) Standardization of the non-synthetic leucoindigo salt mixture

[00123] The non-synthetic leucoindigo salt mixture is then under inert gas fine filtrated with filter of different and fine pore size and standardized in inert environment with NaOH and water to 20% liquid strength; i.e. a concentration of 20wt% leucoindigo salt (based on the complete non-synthetic leucoindigo salt mixture). The resulting 20 wt% non-synthetic leucoindigo solution is subjected to test procedures: a) determination of the dyeing strength versus the above defined synthetic standard; b) long-term storage test and c) measurement of the aniline and N-methyl aniline content. In test a) the dyeing strength of the non-synthetic leucoindigo solution is comparable to the dyeing strength of the synthetic standard. In test b) no crystallization was observed after 6 months of storage under inert condition and at room temperature of 23-28°C. In test c) no aniline and N-methylaniline was detected, i.e. the amount of aniline and N-methylaniline was below limit of detection according to ISO 14362-1 :2017. The purity is more than 90 wt%.

Example 2

[00124] Same procedure as in example 1 was conducted, wherein indigo quantity of batch NI20200803 was increased to 228 g and water accordingly reduced to 172 g with target to achieve a 30% concentration. In the end, a 29.8 wt% concentrated non-synthetic leucoindigo salt solution was obtained. A sample thereof was subjected to tests a) to c) described above in Example 1. In test a) the dyeing strength of the non-synthetic leucoindigo solution is comparable to the dyeing strength of the synthetic standard. In test b) no crystallization was observed. In test c) no aniline and N-methylaniline was detected, i.e. the amount of aniline and N-methylaniline was below limit of detection according to ISO 14362-1 :2017. The purity is more than 90 wt%.

Example 3

[00125] Same procedure as in example 1 was conducted, with indigo quantity of batch NI20200802 and indigo amount increased due to indigo purity of 51.4 wt% and water quantity was accordingly reduced with target to achieve a 20% concentration. A stable 19.8 wt% non-synthetic leucoindigo salt solution was obtained. A sample thereof was subjected to tests a) to c) described above in Example 1. No crystallization tendency was observed during the long-term storage test over 6 months, at room temperature and inert condition. Dyeing strength is comparable to the dyeing strength of the synthetic standard. No aniline and N-methylaniline was detected; i.e. the amount of aniline and N-methylaniline was below limit of detection according to ISO 14362-1 :2017. The purity is more than 90 wt%.

Example 4

[00126] Same procedure as in example 1 was conducted, with indigo quantity of batch NI20200801 and indigo amount increased according to indigo purity of 56.2% and water quantity was accordingly reduced with target to achieve a 20% concentration. A stable 20.2 % non-synthetic leucoindigo salt solution was obtained. A sample thereof was subjected to tests a) to c) described above in Example 1. Dyeing strength is comparable to the dyeing strength of the synthetic standard. No crystallization tendency was observed during the long-term storage test over 6 months, at room temperature and inert condition. No aniline and N-methylaniline was detected; i.e. the amount of aniline and N-methylaniline was below limit of detection according to ISO 14362-1 :2017. The purity is more than 90 wt%.

Example 5

[00127] Same procedure as in example 1 was conducted, with an indigo amount adjusted to 140 g of batch NI20200804 and during 3h hydrogenated at 90-95°C at 5 - 7 bar pressure, reactor was depressurized, and another 141 g indigo were added, and the batch was hydrogenated for another 5 h at 90-95°C. A 20.3 wt% concentrated non-synthetic leucoindigo salt solution was obtained. A sample thereof was subjected to tests a) to c) described above in Example 1. Dyeing strength is comparable to the dyeing strength of the synthetic standard. No crystallization tendency was observed during the long-term storage test over 6 months, at room temperature and inert condition. No aniline and N- methylaniline was detected; i.e. the amount of aniline and N-methylaniline was below limit of detection according to ISO 14362-1 :2017. The purity is more than 90 wt%.

Example 6

[00128] Same procedure as in example 5 was conducted, with an indigo quantity of batch NI20200804 and indigo amount reduced to 210 g indigo. Water quantity was accordingly adjusted to 190 g. Indigo suspension was hydrogenated at 90-95°C, with 5 - 7 bar hydrogen pressure and for 8 hours. A 14.9 wt% concentrated non-synthetic leucoindigo salt solution was obtained. A sample thereof was subjected to tests a) to c) described above in Example 1. Dyeing strength is comparable to the dyeing strength of the synthetic standard. No crystallization tendency was observed during the long-term storage test over 6 months, at room temperature and inert condition. No aniline and N-methylaniline was detected i.e. the amount of aniline and N-methylaniline was below limit of detection according to ISO 14362-1 :2017. The purity is more than 90 wt%.

Example 7

[00129] Same procedure as in example 1 was conducted, using a 0.5 I stainless steel autoclave instead of a 1 I autoclave, with an indigo quantity of batch BI20200704 and indigo amount of 79 g indigo. Water quantity was accordingly adjusted to 121 g. All other ingredients scaled down to 0.5 I volume (1 :2). The thus obtained indigo suspension was hydrogenated at 80-90°C, with 4 - 6 bar hydrogen pressure and for 5 hours. A 30.1 wt% concentrated non-synthetic leucoindigo salt solution was obtained. A sample thereof was subjected to tests a) to c) described above in Example 1. Dyeing strength is comparable to the dyeing strength of the synthetic standard. No crystallization tendency was observed during the long-term storage test over 6 months, at room temperature and inert condition No aniline and N-methylaniline was detected i.e. the amount of aniline and N-methylaniline was below limit of detection according to ISO 14362-1 :2017. The purity is more than 96 wt%.

Example 8

[00130] 150.0 g of the indigo solution obtained in example 7 were added to 0.5 I stainless steel autoclave, were purged with N2 gas, then pressure was adjusted to 350-400 mbar and heated up to distill off 37.5 g water. A 40.5 wt% concentrated non-synthetic leucoindigo salt solution was obtained. A sample thereof was subjected to tests a) to c) described above in Example 1. Dyeing strength is comparable to the dyeing strength of the synthetic standard. No crystallization tendency was observed during the long-term storage test over 6 months, at room temperature and inert condition. No aniline and N- methylaniline was detected i.e. the amount of aniline and N-methylaniline was below limit of detection according to ISO 14362-1 :2017. The purity is more than 90 wt%.

Example 9

[00131] Same procedure as in example 8 was repeated, only 50.0 ml water was distilled and a 45.7 wt% concentrated non-synthetic leucoindigo salt solution was obtained. A sample thereof was subjected to tests a) to c) described above in Example 1 . Dyeing strength is comparable to the dyeing strength of the synthetic standard. No crystallization tendency was observed during the long-term storage test over 4 months, at room temperature and inert condition. No aniline and N-methylaniline was detected i.e. the amount of aniline and N-methylaniline was below limit of detection according to ISO 14362- 1 :2017. The purity is more than 90 wt%.

ExamplelO

[00132] Same suspension preparation as in example 7 was conducted, with an indigo quantity of batch BI20200708 and indigo amount of 99.2 g indigo. Water quantity was accordingly adjusted to 100.8 g. The thus obtained indigo suspension was hydrogenated at 80-90°C, with 4 - 6 bar hydrogen pressure and for 5 hours. A 30.5 wt% concentrated nonsynthetic leucoindigo salt solution was obtained. A sample thereof was subjected to tests a) to c) described above in Example 1. Dyeing strength is comparable to the dyeing strength of the synthetic standard. No crystallization tendency was observed during the long-term storage test over 6 months, at room temperature and inert condition. No aniline and N-methylaniline was detected i.e. the amount of aniline and N-methylaniline was below limit of detection according to ISO 14362-1 :2017. The purity is more than 90 wt%.

Example'll

[00133] Same suspension preparation as in example 1 was conducted, with an indigo quantity of batch BI20200708 and indigo amount of 99.2 g indigo. Water quantity was accordingly adjusted to 100.8 g. The thus obtained indigo suspension was hydrogenated at 90-95 °C, with 3 - 5 bar hydrogen pressure and for 5 hours. A stable 30.1 wt% nonsynthetic leucoindigo salt solution was obtained. A sample thereof was subjected to tests a) to c) described above in Example 1. Dyeing strength is comparable to the dyeing strength of the synthetic standard. No crystallization tendency was observed during the long-term storage test over 6 months, at room temperature and inert condition. No aniline and N-methylaniline was detected i.e. the amount of aniline and N-methylaniline was below limit of detection according to ISO 14362-1 :2017. The purity is more than 90 wt%. Examples 12a to 12e

[00134] Examples 12a to 12e demonstrate that non-synthetic indigo obtained from the non-synthetic, standardized leucoindigo salt mixture subjected to the method according to invention (i.e. an indigo from microorganism sources were subjected to the method as described in Example 1 ; the then obtained non-synthetic, standardized leucoindigo salt mixture was then oxidized to obtain the indigo as used/analyzed in Examples 12d and 12e) has a significantly higher purity than commonly available indigo. This has the effect, that less amounts of such non-synthetic indigo (respectively non-synthetic leucoindigo salt) in the dyeing bath is needed in order to obtain comparable dyeing results if synthetic indigo

(respectively synthetic leucoindigo salts) is used in a dyeing bath. Thus, less by-products and salts in the downstream wastewater product are present, making the dyeing a more economically and environmentally friendly process.

[00135] The following indigos were examined using the method as described in the introductory part of the Example section. The curves of the UV/Vis Spectrophotometric method are depicted in Figures 5a to 5e:

* In this case of Examples 12a to 12e, the “standard” of Example 12a was set to 100.00% purity. Thus, a value above 100.00% means that the purity is higher, whereas a value below 100.00% means that the purity is lower with reference to the standard as set in Example 12a. Thus, the purity reported with regard to Examples 12b to 12e is a “reference” purity with regard to Example 12a.

Application Example 1 - Dyeing strength

[00136] The non-synthetic leucoindigo salt solution as obtained from the procedure of Example 1 comprising 20 % by weight of leucoindigo from NI20200803 was used in comparison with a synthetic leucoindigo solution (Denisol PURE 30% standard from Archroma). Both solutions were diluted to 6 % by weight and applied in dyeing processes known in the art. Afterwards, the dyeing strength of both dyes was assessed and found to be identical (strength comparison 100.3%). Further, it was found that the non-synthetic leucoindigo solutions provide shadings on the preferred greenish side and possess color properties comparable to the synthetic leucoindigo solutions. The textile materials obtained for comparison are depicted in Figure 3.

Application Example 2 - Washing fastness

[00137] The textile samples dyed with the 6% by weight leucoindigo solutions (non- synthetic and synthetic sources) of application example 1 were used in a washing fastness test according to AATCC 61/2A. The test results may be found in Figure 4 comparing the textile samples before and after the washing fastness test. The samples dyed with the non- synthetic leucoindigo according to the invention shows similar washing fastness compared to the synthetic leucoindigo solutions. [00138] The Application examples demonstrate that the obtained non-synthetic leucoindigo salt solutions from natural sources as provided by the present invention possess comparable properties in application as synthetic leucoindigo solutions already available in the market.

Claims

1. Method for the preparation of a standardized non-synthetic leucoindigo salt mixture comprising no aniline or N-methylaniline according to ISO 14362-1 :2017, wherein the method comprises steps (A) to (C):

2. Method according to claim 1 , wherein the natural indigo source used in step (A) is obtained from plants capable of producing indigo or indigo precursors selected from the group comprising at least Indigofera tinctoria, /satis tinctoria, Amorpha fruticose, polygonum tinctorium, Persicaria tinctoria, Succisa pratensis, Baptisia australis, Wrightia tinctoria, Marsdenia tinctoria or new plants based on a further development of one of above or cross of 2 or more.

3. Method according to claim 1 or 2, wherein the microorganism indigo source used in step (A) is obtained from microorganisms capable of producing indigo or indigo precursors selected from the group consisting of bacteria, yeast or fungi, preferably chosen from the group consisting of Streptomyces, Escherichia, Bacillus, Pseudomonas, Saccharomyces, Aspergillus.

4. Method according to at least one of the claims 1 to 3, wherein the concentration of the at least one natural indigo source, the at least one microorganism indigo source, or mixtures thereof used in step (A) is at least 5% to at most 60% by weight based on the total weight of the mixture. Method according to at least one of the claims 1 to 4, wherein in step (A) and/or (C) an alkali metal source is used, wherein said alkali metal source is selected from the group consisting of lithium hydroxide, sodium hydroxide, potassium hydroxide or mixtures thereof. Method according to at least one of the claims 1 to 5, wherein the dispersing agent is selected from the group consisting of lignin sulfonate, turkey red oil, alkyl acryl sulphonates, alkyl sulphonates, soap powder, or mixtures thereof. Method according to at least one of the claims 1 to 6, wherein after step (A) further steps (I) and/or (II) are conducted:

(I) pre-filtering the mixture obtained in step (A); and/or

(II) fine-filtering the mixture obtained in step (A) or step (I). Method according to at least one of the claims 1 to 7, wherein the temperature in step (B) is at least 20 °C and at most 140 °C, and/or wherein the pressure in step (B) is at least 1 bar and at most 50 bar. Method according to at least one of the claims 1 to 8, wherein the catalyst in step (B) is selected from the group consisting of Raney-Nickel, Palladium on carbon (Pd/C), tetrakis(triphenylphosphine) palladium(O), or a mixture thereof. Method according to at least one of the claims 1 to 9, wherein after step (B) further steps (III) and/or (IV) and/or (V) are conducted:

(III) separating the catalyst used in step (B) from the obtained non-synthetic leucoindigo salt mixture; and/or

(IV) fine-filtering the mixture obtained from step (B) and/or step (III) to remove remaining contaminants; and/or (V) analyzing the obtained mixture from step (B) and/or step (III) and/or step (IV). Method according to at least one of the claims 1 to 10, wherein the standardizing in step (C) is done by at least one of the following procedures: dilution with water, dilution with an alkali metal source, distillation of water. Aqueous non-synthetic leucoindigo salt mixture obtained by the method according to at least one of the claims 1 to 11 , wherein no aniline or N-methylaniline according to ISO 14362-1 :2017 is contained. Aqueous non-synthetic leucoindigo salt mixture according to claim 12, wherein the concentration of the non-synthetic leucoindigo salt contained in the obtained mixture is adjusted to at least 5 % by weight, and at most 60 % by weight, based on the total weight of the non-synthetic leucoindigo salt mixture. Aqueous leucoindigo salt composition comprising or consisting of the aqueous non- synthetic leucoindigo salt mixture as claimed in claims 12 or 13 and aqueous synthetic leucoindigo salt mixture, wherein no aniline or N-methylaniline according to ISO 14362- 1 :2017 is contained. Method of making indigo, comprising step (E):

(E) oxidizing the aqueous leucoindigo salt composition as claimed in claim 14, or the aqueous non-synthetic leucoindigo salt mixture as claimed in at least one of the claims 12 or 13, or as produced according to the method as claimed in at least one of the claims 1 to 11. Method of claim 15, comprising step (D) prior to step (E):

(D) treating a textile with the leucoindigo salt composition as claimed in claim 14, or the non-synthetic leucoindigo salt mixture as defined in at least one of the claims 12 or 13, or as produced according to the method as claimed in at least one of the claims 1 to 11. Textile dyed with the leucoindigo salt composition as claimed in claim 14, or the non-synthetic leucoindigo salt mixture as defined in at least one of the claims 12 or

13, or as produced according to the method as claimed in at least one of the claims 1 to 11 .