"Method for the biosynthesis and extraction of lipophilic compounds in fungal fermentation" DESCRIPTION

The present invention relates to a method for producing an extract obtained by fungal fermentation and the various applications of such an extract .

The economic importance of fungal biotechnology is indisputable .

The natural metabolic capacities of filamentous fungi are highly diverse ; the products obtained today from fungal fermentations range from organic acids , chemicals , drugs such as antibiotics , proteins and enzymes , alternatives to meat , vitamins , polyunsaturated fatty acids and other nutraceuticals , up to composites and vegan leathers .

This great metabolic variety opens the door to several industrial applications of molecules synthesi zed by fermentation .

Generally, to obtain the intracellular lipophilic molecules of interest , the use of cell destruction treatments is necessary, suited for the release of the molecules outside the cell .

The treatment of the cells , aimed at obtaining the molecules of interest , thus inevitably leads to the end of the culture cycle .

Furthermore , given the chemical behavior of lipophilic molecules , the extraction must be performed through the massive use of organic solvents , which are dangerous for the environment and operators . Furthermore , to promote the biosynthesis of the secondary metabolites of interest , new stimulation methods must be developed so as to make the production processes more convenient .

Always to this end, it is also imperative to identi fy new culture techniques adapted to reduce the costs associated with the collection and treatment of fungal biomass , in order to obtain the molecules of interest .

The publication to Lee J J L et al ("Engineering Rhodosporium toruloides with a membrane transporter facilitates production and separation of carotenoids and lipids in a bi-phasic culture , Appl . Microbiology and Biotechnology, vol . 100 , no . 2 , 3 November 2015 , pages 869- 877 ) describes the use of a culture substrate represented by an oil phase , which is hydrolyzed and metaboli zed during the fermentation of yeasts engineered for the production of carotenoids .

The publication to Ningyang L et al ("Production and excretion of astaxanthin by engineered Yarrowia lipolytica using plant oil as both the carbon source and the biocompatible extractant" Appl Microbiology and Biotechnology, vol . 104 , no . 16 , 29 June 2020 , pages 6977- 6989 ) describes the use of a vegetable oil , to the extent of about 20% , as a carbon source in the production of astaxanthin .

The publication to Minglue H et al . ("Releasing intracellular production to prepare whole cell biocatalyst for biosynthesis of Monascus pigments in water-edible oil two-phase system, " Bioprocess and Biosystems Engineering, vol 39 , no . 11 , 28 July 2016 , pages 1785- 1791 ) describes a fermentation process in a system comprising an oil phase represented by olive oil , which is about 20% of the culture medium volume .

Chinese patent application CN 106636249 describes the use of surfactants represented by surfactants of the Tween, Span, PEG, saponins or Triton X- 100 series or by mono- , di- and triglycerides , in a fermentation process in order to increase the production in a yellow pigment .

Summary of the invention

The authors of the present patent application have surprisingly developed a method for fermenting fungi , which solves the above problems .

Furthermore , such a method allows obtaining an extract rich in compounds of interest having several applications .

Obj ect of the invention

In a first obj ect , the present invention describes a method for fermenting fungi and for obtaining an extract represented by a pigmented oil .

The thus-obtained pigmented oil rich in lipophilic compounds is a second obj ect of the present invention .

In accordance with a third obj ect , a method for extracting compounds of interest from the pigmented oil of the present invention is described .

In a fourth obj ect the compounds of interest are described, isolated or in mixture obtained by the puri fication and extraction method of the invention . The applications of the compounds of interest are further obj ects of the present invention .

A particular obj ect of the present invention is a method for dyeing plastics and polymers , as well as natural materials .

Detailed description of the invention

In accordance with a first obj ect , the present invention describes a method for fermenting fungi and for obtaining an extract represented by a pigmented oil .

For the purposes of the present invention, the fermentation step comprises culturing the fungi in suitable bioreactors .

The culture is preferably carried out in liquid; alternatively, it can be carried out in solid as well , for example on suitable substrates containing lipophilic extractants .

As far as the culture medium is concerned, this preferably comprises one or more of : a carbon source , a nitrogen source and micronutrients .

In particular, the carbon source can comprise monosaccharides , disaccharides , oligosaccharides or polysaccharides or mixtures thereof , in a concentration of about 1 - 100 g/ 1 .

Alternatively, other carbonaceous sources can be used such as : glycerol and other polyalcohols such as sorbitol ; other alcohols such as methanol , ethanol ; VOCs (volatile organic compounds ) such as acetic acid, propionic acid and butyric acid, or by-products or scraps of various origin having a high organic carbon content such as molasses , high sugar syrups . In a preferred aspect, glucose or sucrose is used in a concentration of about 20-60 g/1.

As for the nitrogen source, organic or inorganic nitrogen sources can be used.

The following can be used as organic sources, for example: animal or vegetable protein extracts, protein hydrolysates (e.g., caseins) , serum proteins, urea, amino acids, complex proteincontaining sources such as peptone, yeast or meat extracts (e.g., beef) , corn steep liquor, animal flours; or mixtures thereof.

The following can be used as inorganic sources, for example: nitrate salts, such as calcium or sodium nitrates, or ammonium salts, such as potassium, chloride, sulfate, ammonium phosphate; or mixtures thereof.

Alternatively, mixtures of macro- and meso-elements derived from agricultural fertilizers can be used.

In a preferred aspect, nitrogen can be used in a concentration up to about 50 g/L and preferably about 0.4-30 g/L (concentration referring to the nitrogen source used) or about 0-5000 mg/L N (concentration referring to nitrogen) .

As far as micronutrients are concerned, these are preferably represented by B, Fe, Zn, Cu, Mn, Mo.

In a preferred aspect, such micronutrients are in concentration up to about 1 g/1 (overall) and preferably about 250 mg/ 1.

For the purposes of the present invention, the culture medium is represented by a bi-phase system comprising an oil phase. Said oil phase comprises an oil chosen from natural or synthetic or mineral oils (or fats) , or mixtures thereof.

Natural oils or fats can be chosen from the group comprising vegetable oils such as: olive oil, seed oil, palm oil; or they can be chosen from the group comprising animal fats such as: suet, lard .

Alternatively, other natural products of animal origin can be used, such as: beeswax, poultry fat, tallow, etc.

Alternatively, spent oils, mineral oils, paraffin waxes, petroleum jelly, white oils, waxes and other fractions obtained from refining petroleum can be used.

In a preferred aspect, the oil phase consists of seed oil.

According to the present invention, the oil phase can be between 5-70% ( volume /volume ) and preferably up to 15%

( volume /volume ) and more preferably about 10% (volume /volume) with respect to the culture medium.

The pH of the culture is preferably maintained in a range of about 2-9 and preferably is about 3-8.

During the process of the present invention the culture medium is aerated.

The aeration of the culture medium is maintained with suitable systems so as to maintain an aeration of about 0-8 vvm and preferably about 0-2 vvm (volume of gas blown into the fermenter, on volume of fermentation broth per minute) .

The aeration is carried out with air. A stirring of about 0-800 rpm and preferably about 0-200 rpm (repetitions per minute) is maintained during the fermentation.

The fermentation method is conducted at a temperature of about 18-32°C and preferably about 22-26°C.

Starting from the inoculation of the culture medium by means of a conidial suspension or mycelium portions, the fermentation can be continued for up to 90 days.

For the purposes of the present invention, the fermentation can preferably be continued for 1, 2 or 3 days.

The fermentation method described above can be applied to all fungi .

In a particular aspect, such fungi are chosen from the fungi of the orders: Hypocreales, Eurotiales, Sordaliales, Pleosporales .

In a preferred aspect, the fungi are of the order Hypocreales and Eurotiales .

In a particularly preferred aspect, the present invention describes fermentation according to the above process of fungi selected from the group comprising: Fusarium oxysporum, Fusarium solanl, Fusarium keratoplasticum, Fusarium sp. , Aspergillus floccosus, Aspergillus niger, Aspergillus sp. , Penicillium citrinum, Penicillium sp. , Trichoderma sp., Metarhizium carneum.

From the above-described fermentation step an extract is thus obtained represented by a pigmented oil, which must be separated from the culture medium.

According to an aspect of the invention, the purification step can comprise a sterilization step. In a later step, the culture medium is left to decant and the oil phase is separated therefrom .

Through a centri fugation or filtration or decantation step, the suspended solids are separated and then removed, thus obtaining a pigmented oily extract rich in the compounds of interest .

The thus-obtained pigmented oil rich in pigmented compounds is a second obj ect of the present invention .

In accordance with a third obj ect , a method for extracting compounds of interest from the pigmented oil of the present invention is thus described .

For the purposes of the present invention, the extraction comprises a first step of treating the pigmented oil with a base .

The extraction of the pigment can then be performed on the basi fied pigment obtained by the treatment with said base .

Therefore , according to a first aspect , the extraction method of the invention according to Method A comprises the steps of :

1A) adding a base to the pigmented oil ,

2A) stirring until color change ,

3A) separating the oil phase from the basi fied pigment with possible reuse of the oil ,

4A) washing the basi fied pigment with a suitable washing solvent to remove any residual oil ,

5A) suspending the basi fied pigment in an appropriate suspending solvent ,

6A) adj usting the pH to the desired pH value . For the purposes of the present invention, sodium or potassium hydroxide, sodium or potassium carbonate, sodium or potassium bicarbonate, sodium acetate are used in step 1A) .

In a preferred aspect, a base preferably represented by sodium hydroxide is added.

Preferably, an amount of 20 pl of 4 M NaOH is added.

For the purposes of the present invention, in step 3A) a suitable separation technique of the basified pigment is used.

For this purpose, centrifugation or filtration can for example be performed.

In an aspect of the invention it is possible to perform a centrifugation at 2000xg for a time of about 5 minutes.

For the purposes of the present invention, in step 4A) the washing solvent is preferably represented by acetone.

For the purposes of the present invention, in step 5A) is the suspension solvent used is represented by hot water or by a mixture of water and a suitable surfactant.

A suitable surfactant is selected from the group comprising: sodium dodecyl sulfate (SDS) or sodium lauryl ether sulfate (SLES) , or analogous natural or synthetic surfactants; or mixtures thereof .

Preferably, the surfactant can be used in a concentration of about 0.1-5 g/1. Preferably, SDS is used in a concentration of about 1 g/1.

Alternatively, the pigment is extracted from the solution obtained after having basified the pigmented oil. In accordance with such an alternative, the extraction method of the invention according to Method B comprises the steps of:

IB) adding a base to the pigmented oil,

2B) stirring until color change,

3B) adding a suitable solvent,

4B) liquid-liquid extraction,

5B) removing the oil phase and possible reuse,

6B) collecting the solvent containing the pigment,

7B) adjusting the pH to the desired pH value.

For the purposes of the present invention, sodium or potassium hydroxide, sodium or potassium carbonate, sodium or potassium bicarbonate, sodium acetate are used in step IB) .

In a preferred aspect, a base preferably represented by sodium hydroxide is added.

Preferably, an amount of 20 pl of 4 M NaOH is added.

For the purposes of the present invention, in step 3B) the solvent is selected from water, methanol, ethanol or another solvent immiscible with the oil phase.

For the purposes of the present invention, step 4B) can for example be carried out by centrifugation, mixing or decantation.

In a preferred aspect of the invention, in step 4B) a centrifugation is carried out at 2000xg for a time of about 5 minutes. In a fourth object the compounds of interest are described, isolated or in mixture obtained by the purification and extraction method of the invention. The applications of the compounds of interest are further objects of the present invention.

In particular, such applications are intended for the pigmented oil obtained from the fermentation step, as well as for the product obtained from the purification and extraction step, represented for example by the aqueous solution comprising the surfactant, both as such and as individual pigmented compounds or mixtures thereof.

In particular, such a pigmented oil, such a solution, such individual pigmented compounds or mixtures thereof, can be used in a method for dyeing and/or staining polymer materials, plastics, resins and natural materials.

More in general, such polymer materials, plastics and resins, comprise :

- phenolic resins (e.g., bakelite) ,

- amide resins (e.g., melamine resins) ,

- epoxy resins (e.g., based on bisphenol A and epichlorohydrin) ,

- polyurethane resins,

- unsaturated polyester resins,

- silicone resins,

- alkyd resins,

- acrylic resins (such as acrylic polymers, polyacrylonitrile) , polycarbonates (polycarbonate) , - polyester resins (polyethylene terephthalate, polylactic acid, polyhydroxyalkanoates, polybutylene terephthalate) ,

- vinyl (polyvinyl fluoride, polyvinylchloride) ,

- polyolefins (polyethylene, polypropylene) ,

- polyamides (for example: PA6, PA66, PA11, PA12, PA46) .

Natural materials are represented by: wood, leather and hides, paper and cardboard, rubber.

Therefore, in an aspect of the invention, the use of the pigmented oil, the solution, the individual pigmented compounds, or mixtures thereof, in the field of paints and impregnating agents is described.

According to a particularly preferred aspect of the present invention, the application of the pigmented oil, of the solution, of the individual pigmented compounds, or mixtures thereof, obtained from the above extraction and purification in the textile industry is described.

In fact, such lipophilic pigments are used for staining and/or dyeing natural and synthetic fibers.

Among natural fibers, vegetable fibers can be dyed, such as cotton, linen, hemp, jute, bamboo, while among animal fibers, silk and wool can be dyed.

Among synthetic fibers, polyamides (such as nylon) and polyester can be dyed.

For the purposes of the present invention, dyeing the fibers with the pigments of the invention can be carried out by immersion in a suitable bath. Such a bath can be both aqueous and oily.

In an aspect of the present invention, such a dye bath does not comprise mordant agents or, alternatively, prior treatment with mordant agents of the fibers to be dyed is not included.

Mordant agents which can be used for the purposes of the present invention comprise: rock alum or other aluminum salts, cream of tarter (KC4H5O6 or potassium bitartrate) , tannins, copper salts, iron salts, soda, sodium chloride.

According to an alternative aspect of the present invention, once the pigmented solution is obtained, possibly free of surfactant, this is brought to a desired pH (between 1 and 12) inside which the material to be dyed is immersed.

The bath is then heated to about 90°C for a time of about 30 minutes-5 hours or at a temperature of about 120°C for a time of 20 minutes.

After cooling, the material is extracted.

The process of the present invention allows staining with a very wide range of shades of brown, red, pink, orange, yellow, ochre and purple.

Other applications for the pigmented oil, solution or compounds of the present invention can be in one or more of the following industries: pharmaceutical, agrochemical (as a pesticide, insecticide, herbicide or the like) , of biosensors (due to the sensitivity of the compound to pH variation) , food, e.g., as a food and beverage colorant, cosmetics. In this regard, the pigmented oil , solution, individual pigmented compounds , or mixtures thereof , are described for use against lignin pathogens , contributing to the protection and preservation of wood .

The invention will be better described below by means of some explanatory and non-limiting examples .

EXAMPLE 1

Fungal fermentation in oil phase

A fermentation is initiated by inoculating Fusari um oxysporum in a liquid culture containing 15% (v/v) seed oil .

The culture medium further comprises : glucose at a concentration of 20 g/L .

The culture is maintained under the following conditions :

- pH 3 aeration 2 vvm stirring 0 rpm temperature of 25 ° C for a period of 3 days .

At the end of the fermentation period, the oil phase is separated by decantation .

The solids suspended in the oil are removed by centri fugation, centri fuging at 2000xg .

Finally, a pigmented oily extract without residues is obtained .

EXAMPLE 2

Pigment extraction

Method. A 20 pl of 4 M NaOH are added per ml of pigmented oily extract obtained from Fusarium oxysporum .

It is subjected to Vortex for 20 seconds and a color change of the oil is noted, from deep red to dark purple.

Upon complete color change it is centrifuged at 10,000xg for 5 min .

The supernatant (oil) is removed, which can be reused in further fermentation .

The pellet is suspended in 1 ml extraction solvent.

It is subjected to Vortex for a few seconds

Centrifuge 10,000xg for 5 min.

The supernatant is removed.

The pellet is suspended in the desired volume of an aqueous solution containing a suitable surfactant in a concentration of 1 g/1.

Method A can be carried out on a pigmented oily extract obtained from Fusarium solani:

3 ml of pigmented oil are added with a drop of 4 M NaOH. The mixture is stirred by phase inversion (until color change, from red to purple) and centrifuged for 5 min at least at 2000xg. Remove the supernatant (oil) and rinse the pellet, with acetone for example, centrifuge and resuspend the pellet in hot water. The pH is corrected around pH=7 and the solution turns red/pink.

Method. B

20 pl of 4 M NaOH are added per ml of pigmented oily extract obtained from Aspergillus floccosus . It is subjected to Vortex for 20 seconds and a color change of the oil is noted, from yellow to ochre.

0.5 ml of water per ml of pigmented oily extract is added after complete color change.

Centrifuge at 2,000xg for 5 min.

The supernatant (oil) is removed, which can be reused in further fermentation .

The aqueous phase containing the pigment is recovered.

The pH is changed to the desired values.

Method B can be carried out on a pigmented oily extract obtained from Metarhizium carneum:

3 ml of pigmented oil are added with a drop of 4 M NaOH. The mixture is stirred by phase inversion. Then 3 ml of 96% ethanol is added and it is centrifuged at least at 2250xg for 5 minutes. Finally, the yellow pigmented upper alcohol phase is collected. Method B can be carried out on a pigmented oily extract obtained from Fusarium keratoplasticum:

3 ml of pigmented oil are added with a drop of 4 M NaOH. The mixture is stirred by phase inversion (until color change, from red to purple) then distilled water, for example 3 ml, is added and it is centrifuged at least at 2250xg for 5 minutes. Finally, the underlying aqueous phase pigmented purple is collected. The pH is corrected to the desired levels (around pH=7 the solution turns red) .

Method B can be carried out on a pigmented oily extract obtained from Aspergillus floccosus: 3 ml of pigmented oil are added with a drop of 4 M NaOH. The mixture is stirred by phase inversion (until color change, from light yellow to ochre) then distilled water, for example 3 ml, is added and it is centrifuged at least at 2250xg for 5 minutes. Finally, the underlying aqueous phase pigmented yellow is collected. Once the pigment is in water, the pH of the solution can be changed (a color change is observed) .

EXAMPLE 3 Cotton dyeing in oil bath

A fragment of white cotton fabric was immersed in the pigmented oily extract obtained after fungal fermentation, for about 16 hours at room temperature, buffering the excess oil.

The fabric was not treated with a mordant.

Three different washing protocols were applied for three different chromatic results:

Protocol A:

The fabric is washed for 10 minutes in cold water, then with a neutral soap solution 1 g/L for 10 min at 95°C. After washing at 95°C for 10 minutes, it was washed with cold water for another 10 minutes. The fabric is air-dried.

A blue-colored fabric is obtained with this protocol.

It was noted that by adding hydrochloric acid, the fabric changes colors towards purple.

Protocol B:

The fabric impregnated with the pigmented oily extract is immersed in a 5% acetone solution for 2 minutes at the temperature 95°C. The solution containing the fabric is subjected to centrifugation at 2000xg for 5 minutes to separate the oil from the fabric. The fabric is dried. A purple fabric is obtained with this protocol. Protocol C:

The fabric impregnated with the pigmented oily extract is immersed in a 5% acetic acid solution for 2 minutes at the temperature 95°C. The solution containing the fabric is subjected to centrifugation at 2000xg for 5 minutes to separate the oil from the fabric. The fabric is dried.

A red-orange fabric is obtained with this protocol.

Protocol D - dyeing cotton fabric in autoclave in oil bath.

Protocol DI :

The cotton fabric is immersed in the pigmented oily extract using 1 ml of oil/cm ² of fabric. Add 20 pl of hydrochloric acid and heat at 120°C for 20 min. Wash in a solution containing 1 g/L SDS at 90°C for 15 minutes and then rinse in water.

A blue-violet fabric is obtained with this protocol.

Protocol D2 :

The cotton fabric is immersed in the pigmented oily extract using 1 ml of oil /cm ² of fabric. Heat up to 50°C for 60 minutes. Wash in a 1 g/L SDS solution at 90°C for 15 minutes and then rinse in water .

A pink fabric is obtained with this protocol.

EXAMPLE 3

Cotton dyeing in pigmented aqueous solution The pH of the pigmented aqueous solution containing the surfactant is brought between 1 and 5 and the fabric (not treated with a mordant) is added.

Heat at 70°C for 1 hour or 120°C for 20 min. Allow to cool and extract the dyed fabric. Rinse with water.

Varying the pH of the pigmented solution and the heating temperature, fabrics of different color among different shades of red and pink were obtained.

The fabrics were washed with neutral soaps at 40°C for 15 minutes without an appreciable loss of color.

EXAMPLE 4

Polyamide dyeing

70 mg of polyamide 66 (PA66) (not treated with a mordant) were immersed in 3 ml of a pigmented aqueous solution containing the surfactant at pH=2 by addition of HC1 6 M.

The dye bath was heated to the temperature of about 80°C for 30 minutes .

The extracted dye PA66 was washed with hot water and neutral soap.

According to this protocol, the PA66 is stained red.

EXAMPLE 5

Wood dyeing

An untreated and unpainted section of a board of fir wood was immersed in a dye bath consisting of the pigmented aqueous solution containing the surfactant. The dye bath containing the wood sample was heated at 120 ° C for 20 min . At the end of the heat treatment the pigmented wood was washed with water and left to air dry until complete drying . Following this process , a red wood was obtained .

From the above description the advantages of fered by the present invention will be clear .

Firstly, the method described utili zes well-established technological structures and does not require reagents or expensive equipment .

By virtue of the possibility of recovering and reusing the oil phase in further fermentation steps , the operating costs can then be reduced .

The presence of the oil phase in fermentation avoids the use of organic solvents which are dangerous to the environment and operators .

By virtue of the use of an oil phase in fermentation, the separation between the lipophilic compounds of interest and the biomass is obtained; thereby, the downstream puri fication steps , which are particularly onerous , can be avoided, and for separating the biomass from the culture broth and the lipophilic compounds of interest .

In addition, the method of the present invention is easily and quickly implementable on an industrial scale , through expedients which can be implemented by those skilled in the art . Furthermore , the method described is quickly adaptable and integrable in already known processes , whereby the industrial transposition can be rapid .

With respect to analogous processes already known in the art , the process of the present invention has very high extraction yields , by virtue of the locali zation of the pigment of the oil phase .