Torularhodin extraction method

Abstract

A process for the extraction of Torularhodin from biomass comprising in succession (a) a first extraction phase (F1), in which a dry biomass of lysed cells of microorganisms suitable for the synthesis of Torularhodin is subjected to the extractive action of CO2, in supercritical conditions, as the sole solvent for the extraction of apolar carotenoids; and (b) a second extraction phase (F2), in which the biomass obtained from the first extraction phase is subjected to the extractive action of CO2, in supercritical conditions, together with a polar co-solvent for the extraction of Torularhodin.

Description

[0001] "TORULARHODIN EXTRACTION METHOD"

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This Patent Appl ication claims priority from Italian Patent Application No . 102023000018729 filed on September 12 , 2023 , the entire disclosure of which is incorporated herein by reference .

[0004] FIELD OF THE ART

[0005] The present invention relates to a method of selective extraction of Torularhodin using the supercritical CO2 technique .

[0006] BACKGROUND ART

[0007] Torularhodin is a carotenoid belonging to the class of xanthophylls and is characterised by the presence of a carboxyl group ( COOH) .

[0008] Torularhodin has long been studied for its important biological activities such as anti-inflammatory, anticholesterolaemic, antioxidant , antimicrobial and antitumour .

[0009] Torularhodin is synthesised exclusively by fungi , in particular red yeasts as described in patent applications CN109401988A, JP2018038344A and EP3124597A1 .

[0010] For the production of Torularhodin, red yeasts are generally cultured within bioreactors on synthetic liquid substrates or obtained using secondary raw materials , as reported in patent application EP3124597A1 , in which the growth of an isolate of Sporobolomyces roseus on citrus waste is described .

[0011] Next , the yeast cells are lysed and subj ected to an extraction step . Generally, this extraction step represents the most signi ficant cost of the entire Torularhodin production process .

[0012] The most common extraction techniques for obtaining carotenoids from red yeasts involve the use of organic solvents such as acetone , petroleum ether, hexane , chloroform, ethanol , methanol and / or mixtures thereof . However, the extract obtained is a mixture of carotenoids and other substances and needs a separation step generally using chromatographic techniques . As a person skilled in the art is aware , such techniques can result in the production of potentially harmful and highly flammable waste that is di f ficult to manage .

[0013] In light of the above , it is evident that the prior art of Torularhodin extraction techniques entails clear disadvantages in both economic and environmental terms .

[0014] In addition, a further disadvantage of prior art extraction techniques relates to the fact that residues of the solvents used may remain in the final product , making it unsuitable for particular applications , such as pharmaceuticals . DISCLOSURE OF THE INVENTION

[0015] A need has thus been perceived for a method that capable of extracting Torularhodin without incurring the disadvantages of the aforementioned prior art .

[0016] The inventors of the present invention have devised a method, which by exploiting selective extraction with supercritical CO2 , succeeds in ful filling the above requirement .

[0017] Extraction using supercritical fluids , i . e . fluids that have operating conditions above critical pressure and temperature during extraction, has long been used to extract and isolate molecules from natural matrices .

[0018] Among supercritical fluids , carbon dioxide ( CO2-SFE ) is the most widely used in biotechnological applications .

[0019] It is an obj ect of the present invention to provide a process for the extraction of Torularhodin from biomass ; said method being characterised in that it comprises in succession ( a ) a first extraction phase , in which a dry biomass of lysed cells of microorganisms suitable for the synthesis of Torularhodin is subj ected to the extractive action of CO2 , in supercritical conditions , as the sole solvent for the extraction of apolar carotenoids ; and (b ) a second extraction phase , in which the biomass obtained from the first extraction phase is subj ected to the extractive action of CO2 , in supercritical conditions , together with a polar co-solvent for the extraction of Torularhodin .

[0020] Here and in the following, dry biomass is defined as biomass containing an amount of water less than and equal to 8 % by weight .

[0021] The auxiliary and secondary characteristics are set out in dependent claims 2 - 10 .

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] For a better understanding of the present invention, three embodiments are hereinafter described for illustrative and non-limiting purposes with the aid of the accompanying figures , wherein :

[0024] - Figure 1 illustrates in schematic form an example of an extraction plant of the process according to the present invention;

[0025] -Figure 2 shows a block diagram representing the process according to the present invention;

[0026] - Figure 3 is a chromatogram of the extract obtained from a first extraction phase of a first embodiment reported below;

[0027] - Figure 4 is a chromatogram of the extract obtained from a second extraction phase of the first embodiment reported below;

[0028] - Figure 5 is a chromatogram of the extract obtained from the biomass used in the first embodiment reported below using a comparison extraction process . PREFERRED EMBODIMENT OF THE INVENTION

[0029] In Figure 1 , 1 indicates as a whole an example of a plant for carrying out the process according to the present invention .

[0030] The plant 1 comprises a container 2 arranged to house compressed CO2 ; a pair of pumps 3 and 4 adapted to compress the CO2 and the co-solvent to operating pressures , respectively; a furnace 5 housing a flow preheater 6 and extraction reactor 7 , loaded with pretreated yeast biomass and glass beads ; and a separator 8 in which the extracts are recovered . The CO2 and co-solvent flows are regulated by means of manual valves V, while pressure and temperature are monitored with the probes PC and TC, respectively .

[0031] As illustrated by the block diagram in Figure 2 , the process according to the present invention comprises a preliminary step known as FP, in which the source cells of Torularhodin are lysed and dried, a first extraction step Fl , from which the apolar carotenoids are extracted without any trace of residual solvents , and a second extraction step F2 , from which Torularhodin with a purity percentage greater than or equal to 95% of the total carotenoids is extracted .

[0032] As will be punctually reported in the following examples , in the first extraction step Fl the lysed cells are subj ected to the extractive action of supercritical CO2 as the sole solvent, at a pressure comprised between 200 and 500 bar and a temperature comprised between 40 and 60 °C; while in the second extraction step F2, the lysed cells from the first extraction step Fl are subjected to the extractive action of supercritical CO2 together with a polar co-solvent, again at a pressure comprised between 200 and 500 bar and a temperature comprised between 40 and 60 °C.

[0033] Example 1

[0034] ELP2022, a red yeast belonging to the Rhodotorula genus, was cultured in stirred liquid culture using a 5-litre fermenter with a working volume of 4 litres. The entire fermentation process was monitored and controlled using a Biostat B setting the pH to 6.8 and the temperature to 26 °C. Stirring was ensured by using Rushton paddles in the range of 120 to 240 rpm. Filtered air (0.22 pm filter) was blown at 0.7 litres per minute from the bottom by means of a sparger. If necessary, pure oxygen was introduced by means of a gas mixer in order to guarantee a value of 40% dissolved oxygen. YPD liquid culture medium consisting of 10 g / L yeast extract, 20 g / L peptone and 20 g / L glucose was used as a substrate .

[0035] After 144 hours of fermentation, the cells were recovered from the culture broth by centrifugation at 7,000 rpm for 10 minutes and then lysed by a combination of mechanical and chemical methods.

[0036] More specifically, the cells were resuspended in 2 litres of sterile sodium bicarbonate ( 0 . 1 M) and trans ferred into a sterile reaction chamber represented by a 10-litre glass bottle filled with 500 grams of 1 -mm diameter glass beads . Subsequently, the flask was placed hori zontally in an orbital shaker at a rotation of 96 rpm for 16 hours at room temperature .

[0037] After pre-treatment , the cell suspension was separated from the glass beads and the biomass was recovered by centri fugation at 9 , 000 rpm for 10 minutes and dried by freeze-drying .

[0038] Starting with the yeast matrix thus obtained, extraction was conducted using the bench-scale supercritical fluid plant schematically depicted in Figure 1 .

[0039] More speci fically, approximately 3 g of biomass was introduced into a 50 mL extraction reactor . In order to prevent agglomeration of the biomass and increase the contact surface area, 60% of the volume of the extraction reactor was filled with glass microspheres ( 3 mm diameter ) . The temperature and process pressure were accurately monitored using WIKA software .

[0040] The extraction was carried out in two stages by setting a CO2 flow rate of 6 litres per minute : the first extraction phase was conducted for 60 minutes , at a pressure of 300 bar and a temperature of 40 the second extraction step was conducted for 30 minutes , at a pressure of 400 bar and a temperature of 60 ° C with the addition of ethanol as co-solvent at a ratio of 3% (w / w) to CO2 .

[0041] In each phase , individual vials immersed in a thermostatic bath maintained at a temperature of 25 ° C were used as separators . The product collected in the individual vial was characterised using an Agilent 1200 series HPLC system in order to determine and quanti fy the percentages of individual carotenoids present .

[0042] Figure 3 and Figure 4 show the chromatograms of the extract obtained from the first and second extraction steps , respectively .

[0043] As can be seen from the chromatograms in Figures 3 and 4 , a mixture of apolar carotenoids such as Torulene , y- Carotene and p-Carotene can be extracted from the first extraction step, while Torularhodin in virtually pure form can be extracted from the second extraction step . In this way, there is no need for any separation step using, for example , chromatographic techniques , with the obvious productivity and environmental advantages that this entails .

[0044] The method of the present invention envisages that the first extraction step ends once the extraction of apolar carotenoids such as Torulene , Y_Carotene and p-Carotene has been completed . This is established by veri fying the gradual decrease and then absence of the aforementioned apolar carotenoids in the extract .

[0045] COMPARISON EXAMPLE 1

[0046] For comparison, a part of the same biomass used in Example 1 was extracted according to the prior art with a mixture of methanol and acetone . Figure 5 shows the chromatogram obtained . As can be seen from the chromatogram in Figure 5 , with the method of the prior art , Torularhodin is extracted in a mixture with other carotenoids and, thus , there is a need for a separation step with the obvious disadvantages in terms of productivity and disposal of organic solvents as mentioned above .

[0047] For the sake of suf ficiency of description, the HPLC method used for analysing the extracts is briefly described below .

[0048] Aliquots of 20 pL of the extracts resuspended in ethanol and filtered were inj ected through an automatic sampler into the HPLC system . The carotenoids in the extracts were separated using a C- 18 reverse-phase analytical column ( Zorbax RX-C18 4 . 6 x 250 mm, 5 pm) and analysed using a diode array detector ( DAD) . The chromatographic run was conducted in gradient using acetone and water as mobile phase at a flow rate of 1 mL per minute and a temperature of 25 ° C .

[0049] Direct absorbance detection was performed at the characteristic wavelength of p-carotene ( 453 nm) and spectra of the corresponding signals were acquired between 350 and 650 nm. The main peaks were identified by comparing the spectra and relative wavelengths for maximum absorbance (X max) with literature data. In order to quantify the concentration, in terms of p-carotene equivalents (pg / mL) , of the main peaks, a calibration curve was determined using synthetic p-carotene as a standard. All data were collected and analysed using OpenLAB CDS Chemstation Edition Rev. C.01.10 (201) software.

[0050] Example 2

[0051] DSM444 of Rhodotorula glutinis, purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) , was cultured in agitated liquid culture using a 5-litre fermenter with a working volume of 4 litres. The entire fermentation process was monitored and controlled using a Biostat B setting the pH to 7.1 and the temperature to 28 °C. Stirring was ensured by using Rushton paddles in the range of 120 to 240 rpm. Filtered air was blown at 0.7 litres per minute from the bottom by means of a sparger. If necessary, pure oxygen was introduced by means of a gas mixer in order to guarantee a value of 40% dissolved oxygen. A liquid mineral culture substrate consisting of 5 g / L ammonium nitrate, 2 g / L yeast extract, 3 g / L basic sodium phosphate, 1.5 g / L monobasic potassium phosphate, 0.5 g / L magnesium sulphate and 20 g / L glucose was used. After 168 hours of fermentation, the cells were recovered from the culture broth and subsequently lysed and dried as shown in Example 1 .

[0052] At this point , extraction was conducted using the benchscale supercritical fluid plant described in Example 1 .

[0053] More speci fically, 3 g of biomass was introduced into a 50 mL extraction reactor with 60% of the volume filled with glass microspheres ( 3 mm diameter ) .

[0054] The extractions were carried out in two phases by setting a CO2 flow of 6 litres per minute :

[0055] • the first phase was conducted for 60 minutes , at a pressure of 300 bar and a temperature of 40 °C ;

[0056] • the second phase was conducted for 30 minutes at a pressure of 300 bar and a temperature of 40 ° C with the addition of ethanol as co-solvent in a ratio of 3% (w / w) to CO2 .

[0057] In each phase , individual vials immersed in a thermostatic bath maintained at a temperature of 25 ° C were used as separators . The product collected in the individual vials was analysed using the method described in Example 1 .

[0058] Example 3

[0059] DSM111740 of Sporidi obol omyces roseus, purchased from DSMZ , was cultured in 1 L Erlenmeyer flasks containing 250 ml of YPD consisting of 10 g / L yeast extract , 20 g / L peptone and 20 g / L glucose . Erlenmeyer flasks inoculated with a starter culture of the yeast isolate were incubated in an orbital shaker at 28 °C and 115 rpm. 120 hours after incubation, the cells were separated from the culture liquid by centrifugation at 9000 rpm for 10 minutes. Next, for cell lysis, the biomass recovered from 250 mL of culture was resuspended in 16 mL of 0.1 M NaHCOs inside 50 mL Falcon tubes previously filled with 8 g of 1 mm diameter glass beads. The suspension was stirred at 130 rpm at 40 °C for 16 hours. In order to avoid bacterial contamination, the materials used for pre-treatment were previously sterilised.

[0060] After pre-treatment, the cell suspension was separated from the glass beads and the cells were then recovered from the suspension by centrifugation. Subsequently, the cell biomass thus recovered was dehydrated by freeze-drying. At this point, extraction was conducted using the bench-scale supercritical fluid plant described in Example 1.

[0061] More specifically, 0.3 g of biomass was introduced into a 50 mL extraction reactor filled with glass microspheres (3 mm diameter) occupying 60% of the volume.

[0062] The extractions were carried out in two phases by setting a CO2 flow of 6 litres per minute:

[0063] • the first was conducted for 60 minutes, at a pressure of 500 bar and a temperature of 40 °C;

[0064] • the second was conducted for 30 minutes, at a pressure of 400 bar and a temperature of 40 °C with the addition of ethanol as co-solvent in a ratio of 6 % (w / w) to C02.

[0065] In each phase , individual vials immersed in a thermostatic bath maintained at a temperature of 25 ° C were used as separators . The product collected in the individual vials was analysed using the method described in Example 1 .

[0066] From the above , it is evident that the process according to the present invention has the following advantages over processes of the prior art : ( i ) no use of harmful organic solvents ; ( ii ) no residual solvents in the extracts ; ( iii ) minimal waste production; ( iv) no need for chromatographic separation techniques .

[0067] These advantages necessarily lead to higher productivity and greater safety both for the environment and for the workers involved in the process . These advantages are even more important when considering the industrialscale application of the process .

[0068] In this respect , it should be noted that in a circular economy perspective , red yeasts can be cultured on substrates obtained from agro-industrial waste , without , therefore , being tied to the seasonality of the plants in which the carotenoids are present .

[0069] Furthermore , it should be noted that the CO2 recovered downstream of the process can be reused again, with the obvious advantages in terms of savings and sustainability . Finally, a further advantage of the present invention lies in the fact that the carotenoids extracted from the first extraction step may have commercial value in themselves , thus conferring further productivity advantages to the process of the present invention .

Claims

CLAIMS1. Torularhodin extraction process from biomass; said method being characterized by the fact that it comprises in succession (a) a first extraction phase (Fl) , in which a dry biomass of lysed cells of microorganisms suitable for the synthesis of Torularhodin is subjected to the extractive action of CO2, in supercritical conditions, as the sole solvent for the extraction of apolar carotenoids; and (b) a second extraction phase (F2) , in which the biomass obtained from the first extraction phase is subjected to the extractive action of CO2, in supercritical conditions, together with a polar co-solvent for the extraction of Torularhodin .

2. Process according to claim 1, characterized in that said polar co-solvent is an absolute alcohol or aqueous mixtures thereof.

3. Process according to claim 2, characterized in that said alcohol is absolute ethyl alcohol or in 96% v / v aqueous solution .

4. Process according to one of the previous claims, characterized by the fact that in said second extraction phase (F2) the polar co-solvent is present in a % w / w of between 2 and 8 compared to CO2 in supercritical conditions.

5. Process according to one of the previous claims, characterized by the fact that in said second extractionphase (F2) the polar co-solvent is present in a % w / w of between 3 and 6 compared to CO2 in supercritical conditions.

6. Process according to one of the previous claims, characterized by the fact that said first extraction phase (Fl) and said second extraction phase (F2) take place at a pressure between 200 and 500 bar.

7. Process according to one of the previous claims, characterized by the fact that said first extraction phase (Fl) and said second extraction phase (F2) take place at a pressure between 350 and 450 bar.

8. Process according to one of the previous claims, characterized in that said first extraction phase (Fl) and said second extraction phase (F2) take place at a temperature between 40 and 60 °C.

9. Process according to one of the previous claims, characterized in that said biomass includes yeasts of the Rhodotorula (Rhodosporidium) and Sporobolomyces (Sporidiobolus) genera.

10. Process according to one of the previous claims, characterized by the fact that in said first extraction phase (Fl) and in said second extraction phase (F2) the dry biomass is housed inside an extraction reactor together with spheres of glass with a diameter between 1 and 3 mm; said spheres occupying from 60 to 70% of the volume of the extraction reactor .

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