Protocol for obtaining a biosurfactant extract from corn steep liquor by means of liquid-solid extraction, using polymer solid matrices

Abstract

The present invention discloses the protocol to follow to obtain a biosurfactant extract from corn steep liquor by means of separating it using different polymer solid matrices (liquid-solid extraction) and subsequently desorbing the biosurfactant extract contained in the polymer matrix by means of solid-liquid extraction. The solvent used to regenerate the polymer and elute the biosurfactant extract is separated by means of distillation or evaporation, producing a solvent-free biosurfactant extract that is capable of reducing the surface tension of water by more than 15 units and having a critical micelle concentration (CMC) of less than 200 mg / L under more favourable conditions.

Description

[0001]

[0002] PROTOCOL FOR OBTAINING A BIOSURFACTANT EXTRACT FROM CORN WASHING LIQUORS, BY LIQUID-SOLID EXTRACTION, USING SOLID POLYMERIC MATRICES

[0003] Separation of biosurfactants from corn wash liquors using solid polymer matrices involving liquid-solid extraction processes and subsequent elution of the biosurfactant extract with different eluents.

[0004] TECHNICAL SECTOR

[0005] The present invention falls within the scope of obtaining surfactants of natural origin from fermented liquid streams.

[0006] The main objective of this invention is to obtain a biosurfactant extract from corn washing liquors by liquid-solid extraction using different polymeric matrices and subsequent desorption of the biosurfactant extract using different eluents.

[0007] BACKGROUND OF THE INVENTION

[0008] Corn wash liquors, produced as a by-product or secondary raw material during wet corn processing, constitute an interesting source of bioactive compounds, including compounds with surfactant capacity which, when produced from a spontaneously fermented natural stream, are connotated as biosurfactants. Biosurfactant compounds, unlike chemical surfactants, are made up of natural compounds produced by microorganisms or extracted from plant cells. In recent years, several methods for the extraction of these biosurfactants have been collected, obtaining different extracts depending on the proposed separation process. The extraction processes are collected in several patents (ES-2424399_B2, ES-2435324_B2, EP 28262624_B1-

[0009] WO2014044876_A1 , ES-2527366_B1 , ES-2739050_B2, ES-2795574_A1). The first publications related to the surfactant activity of corn wash liquors were included in a patent (ES-2424399_B2) filed in 2012 and subsequently published in 2014 in the Journal Agricultural and Food Chemistry (Vecino et al. 2014). These publications do not refer to or propose extraction methods for the biosurfactant extracts contained in corn wash liquors. They simply provide data that demonstrate the surface-active capacity of corn wash liquors, claiming, in patent ES-2424399_B2, their direct use as a surface-active agent, detergent or surfactant.

[0010] Subsequently, in national patent EP-28262624_B1 and European patent WO2014044876_A1, the production of biosurfactant extracts from corn wash liquors is proposed, using different extracting agents including the following solvents: isoamyl alcohol, chloroform, 1,2-dichloroethane, ethyl acetate, tetrahydrofuran, tributyl phosphate, methyl butyl ether, diethyl ether, trichloroethene, hexane, heptane, xylene, dichloromethane. The process involves producing biosurfactant extracts by liquid-liquid extraction and subsequent separation of the biosurfactant extract and recovery of the solvent by distillation. The uses of these biosurfactant extracts are included in several publications with potential application in the cosmetics industry, the pharmaceutical industry and the industry related to floor cleaning.The biosurfactant extracts obtained through this process are composed of lipopeptides, phospholipids, and other biocompounds, including antioxidants. The characterization of these extracts was published in 2020 in the journal Separation and Purification Technology (Rodríguez-López et al. 2020); additionally, information related to the composition of these extracts is included in patent ES-2527366_B1 . The extract obtained, as reported in the literature, has an oily, orange-brown appearance (Knoth et al. 2019, International Journal of Pharmaceutics).

[0011] On the other hand, patent ES-2795574_A1 describes the extraction of biosurfactants from corn wash liquors using different buffered aqueous solutions, avoiding the use of organic solvents. In this patent, the extraction of the biosurfactant extract is carried out from the solid fraction contained in the corn wash liquors, which includes microbial biomass, which contains bioactive compounds adhered to its plasma membrane and are extracted with the applied buffered solutions. After the extraction of the biosurfactant compounds, the extract separated from the biomass is subjected to a dialysis process to eliminate salts and other low molecular weight compounds. After dialysis, the aqueous extract containing the biosurfactant compounds can be lyophilized for better conservation and handling.The extract obtained, once lyophilized, is a white-yellow powder with a different composition, as stated in said patent, than that obtained following liquid-liquid extraction with organic solvents.

[0012] Furthermore, the presence of phospholipids and lecithin, natural compounds with surfactant capacity, has been detected in corn wash liquors. These compounds are the extraction process described in patent ES-2739050_B2. This patent claims the use of corn wash liquors as a source of lecithin and phospholipids for use in the cosmetic and food sectors. The process applied in this invention to obtain a biosurfactant extract based on lecithin and phospholipids consists of extraction with organic solvents (chloroform, ethyl acetate, etc.), followed by distillation or evaporation of the solvent and subsequent precipitation of the biosurfactant extract with acetone. Furthermore, and subsequent to the date of application for this patent, a paper has been published in the journal Separation and Purification Technology (Rodríguez-López et al.2020) on the extraction and characterization of this phospholipid and lecithin-based biosurfactant extract extracted from corn washing liquors.

[0013] Regarding the use of liquid-solid extraction processes to obtain biosurfactants from corn wash liquors, a paper was published in 2021 in the journal Water (Martí nez-Arcos et al. 2021) that reports the use of calcium alginate polymers and calcium alginate polymers combined with biologically oxidized grape bagasse for the extraction of biosurfactants from corn wash liquors, with extraction percentages of 55%.

[0014] Figure 1 shows, in the form of a flow diagram, the processes according to patents EP-28262624_B1-WO2014044876A1, ES-2795574_A1 for obtaining biosurfactants from corn wash liquors. This diagram shows two types of biosurfactant extracts. A first biosurfactant extract produced extracellularly and extracted from the liquid fraction of the corn wash liquors by liquid-liquid extraction and a second biosurfactant extract obtained from the solid fraction of the corn wash liquors by means of buffered aqueous solutions.

[0015] EXPLANATION OF THE INVENTION

[0016] Different solid matrices from the commercial company Phenomenex are evaluated for the extraction of biosurfactants from corn wash liquors, selecting a weak anion exchange polymer that couples a hydrophobic skeleton with a di-amino ligand containing primary and secondary amines (Strata-X-AW - Phenomenex), as the best option since it allows the complete recovery of biosurfactants present in corn wash liquors.

[0017] DETAILED EXPLANATION OF THE INVENTION

[0018] Firstly, a pre-selection of the solid polymeric matrices from the commercial house Phenomenex (Strata-XL, Strata-XL-C, Strata-XL-CW, Strata-XA, Strata-X-AW, Strata C18-E, Strata Phenyl, Strata NH2) was carried out, whose characteristics are listed in Table 1.

[0019] To do this, the different solid polymeric matrices were placed in contact at room temperature for 5 minutes with the solid-free corn wash liquors, using different solid matrix concentrations between 4 and 20 g / L.

[0020] Any solid-liquid separation process known in the art, such as centrifugation or filtration, may be used to separate the solid phase from the corn wash liquors.

[0021] Table 1. Different solid polymeric matrices used with their characteristics.

[0022] After contact of the corn wash liquors with the different solid polymeric matrices, the surface tension of the liquors was measured to verify the presence or absence of biosurfactants.

[0023] Those matrices capable of extracting biosurfactants from corn wash liquors induced an increase in the surface tension of the corn wash liquors after contact with them. The surface tension of solid-free corn wash liquors is around 50 mN / m, after contact with solid polymeric matrices, this surface tension increased up to 70-72 mN / m. Among the solid polymeric matrices evaluated, those that provided the best results were the Strata-X-AW solid matrices, followed by Strata-XA and Strata XL-C. Table 2 shows the percentages of biosurfactant extraction detected in the corn wash liquors after contact with the different solid polymeric matrices evaluated.

[0024] Table 2. Percentage of biosurfactant trapped by solid polymeric matrices from corn wash liquors.

[0025] As can be seen, the biosurfactant extract can be trapped by both anion and cation exchange matrices, which is in line with its amphoteric nature demonstrated in a previous work (Rodríguez-López et al. 2016) published in the Journal of Surfactants and Detergents. In this work, biosurfactant extracts were extracted from corn wash liquors with chloroform, and after distillation of the organic phase, the biosurfactant extract was diluted in water and placed in contact with Amberlite IRA 400 and Amberlite IR 120 resins, of anionic and cationic nature, respectively. After the liquid-solid extraction process, it was verified that both resins were capable of extracting the biosurfactant compounds present in the aqueous solution, reaching surface tension values ​​of 72 mN / m.It should be noted that in this work, the resins were not applied to the corn wash liquors but to the biosurfactant extract subjected to a chloroform extraction process in order to study its ionic character.

[0026] With the solid matrices that gave the best results, more extensive studies were carried out to optimize the operating conditions, testing different temperatures including room temperature in the range 18-27 °C, as well as 40 °C. Tests were also carried out with different solid-liquid ratios until the solid-liquid ratio was reduced to 0.01 g / mL (1:100 g / mL) and applying different extraction times (5 min, 30 min, 1 h, 4 h, 24 h).

[0027] Once the biosurfactants were trapped in the solid matrix, different eluents were tested in order to recover the biosurfactant extract.

[0028] The eluting agents tested were Milli-Q water, ethyl acetate, chloroform, ethanol, acetone, methanol, methanol + 5% formic acid, methanol + 5% ammonia, which were kept in contact with the solid matrix loaded with biosurfactants for at least 5 minutes at temperatures between 18 and 40 °C, observing that an eluent:solid matrix ratio of 100:1 (mL / g) is sufficient to extract approximately 100% of the biosurfactant extract when the eluent is methanol in combination with ammonia or formic acid. In addition, reuse tests of the solid matrix were carried out after 10 extraction and regeneration cycles, with the losses in efficiency of said solid matrix with respect to the liquid-solid extraction of biosurfactants being less than 25%.

[0029] On the other hand, different contact times between the eluent and the solid polymeric matrix loaded with biosurfactants were tested between 5 and 60 min, observing that 5 min is sufficient time to extract most of the biosurfactants trapped by the solid polymeric matrix.

[0030] The obtained biosurfactant extract presents biomarkers with higher molecular weights than the biomarkers found in the biosurfactant extract protected under patents ES-2435324_B2, EP-28262624B1- WO2014044876A1, ES-2527366_B1, ES-2739050_B2, ES-2795574_A1, giving rise to an extract that is more selective in terms of components.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] To complement the description made and in order to help better understand the characteristics of the invention, a set of drawings is attached as an integral part of said description, in which the following has been represented for illustrative and non-limiting purposes: Figure 1. Diagram of the biosurfactant separation processes applied to corn wash liquors, comprising a liquid-liquid extraction process with organic solvents or a solid-liquid extraction process with buffered aqueous solutions, applied in the latter case to the solid fraction of the corn wash liquors. After the liquid-liquid extraction process, a distillation process necessary to recover the solvent and obtain the biosurfactant extract is included in this diagram.In relation to the solid-liquid extraction process applied to the solid fraction of corn washing liquors, a dialysis process is included that aims to eliminate salts present in the extracting agent and a lyophilization process to obtain the biosurfactant extract in powder form.

[0033] Figure 2. Diagram of the proposed novel extraction process for the separation of biosurfactants from the liquid fraction of corn wash liquors using the Strata-AW solid matrix, composed of a hydrophobic polymer bonded to a di-amino ligand, which has been taken as an example. This diagram starts with corn wash liquors which, after centrifugation or filtration, are subjected to a liquid-solid extraction process with different polymeric matrices that retain the biosurfactant substances contained in the corn wash liquors. Subsequently, the polymeric matrices loaded with the surfactant substances are subjected to a solid-liquid extraction process using a solvent solution based, for example, on methanol and NH3.After the recovery of the biosurfactant substances, the solvent is subjected to a distillation or evaporation process in order to recover the solvent and separate it from the biosurfactant extract object of the invention.

[0034] PREFERRED EMBODIMENT OF THE INVENTION

[0035] Example 1. Obtaining the biosurfactant extract

[0036] Liquid-solid extractions were performed on corn wash liquors supplied by FeedStimulants (Zoetermeer, The Netherlands) from the wet corn fractionation process. The corn wash liquors were then centrifuged or filtered to obtain a solid-free liquid fraction. This fraction was then contacted with various solid polymer matrices, including the weak anionic polymer Strata-X-AW from Phenomenex, for at least 5 minutes at room temperature.

[0037] Figure 2 shows, as an example, the schematic of the new process proposed for obtaining biosurfactants from corn wash liquors using a Strata-AW solid polymer matrix. This schematic includes the elution step of the biosurfactant extract with a solution of methanol and NH3, as well as the recovery of the solvent by evaporation or distillation.

Claims

CLAIMS 1. A process for extracting biosurfactants from corn wash liquors, characterized in that it comprises the steps of separating the liquid fraction of the corn wash liquors by means of separation processes with solid polymeric matrices formed by a hydrophobic main chain and a di-amino ligand containing primary and secondary amines.

2. A process for the extraction of biosurfactants from corn wash liquors characterized in that it comprises the steps of separating the liquid fraction of the corn wash liquors by means of separation processes with solid matrices formed by strong cation exchange matrices, which combine a hydrophobic backbone with a sulfonic acid functional group.

3. A process for extracting biosurfactants from corn wash liquors, characterized in that it comprises the steps of separating the liquid fraction of the corn wash liquors by means of separation processes with solid polymeric matrices formed by a strong anion exchange polymeric matrix, which couples a hydrophobic backbone with a dimethylbutylated quaternary amine functional group.

4. The process for extracting biosurfactants from corn wash liquors according to claim 1, 2 or 3, further comprising elution of the biosurfactants using the following solvents: ethyl acetate, chloroform, ethanol, acetone, methanol, formic acid, ammonia, in combination or separately.

5. The process for extracting biosurfactants from corn wash liquors according to claim 4, further comprising separating and recovering the solvent by distillation or evaporation.

6. Use or application of biosurfactants obtained from the liquid fraction of corn washing liquors, after subjecting them to the separation processes, according to claim 5 as a surfactant and / or emulsifier in the food, cosmetic, pharmaceutical, agrochemical or environmental industry.

7. Use or application of biosurfactants obtained from the liquid fraction of corn washing liquors, after subjecting them to the separation processes, according to claim 5 as a surfactant and / or emulsifier agent in surface cleaning.

8. Use or application of biosurfactants obtained from the liquid fraction of corn washing liquors, after subjecting them to the separation processes, according to claim 5 to improve the permeability of those formulas, which contain active ingredients, through cell membranes, intended for cosmetic, pharmaceutical and agrochemical uses.

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