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    • 专利摘要:
    This invention describes a process for the extraction of biosurfactants from the solids contained in the corn washing liquors, by extraction with buffered saline solution. Applying this process, a biosurfactant extract is obtained in the form of a white powder with foaming capacity, capable of reducing the surface tension of water by 20 units. Until now, no biosurfactant has been described, extracted from a waste stream of the corn fractionation industry by wet means, by means of aqueous extraction. The biosurfactant extract under study presents differentiating biomarkers from the other biosurfactants studied to date for its application in formulations related to the cosmetic, food, pharmaceutical, agrochemical and environmental industries. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2795574A1
    申请号:ES201900084
    申请日:2019-05-21
    公开日:2020-11-23
    发明作者:Menduina Ana Belén Moldes；Freire José Manuel Cruz；Lopez Lorena Rodriguez；Prieto Alejandro Lopez
    申请人:Universidade de Vigo；
    IPC主号:
    专利说明:

    [0002] Process for the aqueous extraction of biosurfactants from corn washing liquors or "Corn steep liquor"
    [0004] Technical sector
    [0006] The present invention belongs to the sector of obtaining detergents of natural origin, called biosurfactants.
    [0008] The main object of the invention is to obtain a biosurfactant extract obtained from corn washing liquors (CSL), also called "Corn steep liquor" generated during the wet fractionation of corn, using a buffered saline aqueous solution or a buffer solution as the sole extracting agent.
    [0010] Background of the invention
    [0012] Corn washing liquors (CSL) are a residue of the wet corn fractionation industry (Singh and Eckhoff, 1996, Yan et al 2005, Ramírez et al 2008). These liquors are composed of water and solids (Ramírez et al 2008). The concentration of solids in these liquors can reach up to 50% by weight, when they are subjected to a process of water evaporation. In fact, most of the corn washing liquors that are marketed as a low-cost nutritional supplement, in biotechnological processes or for animal feed, contain around 50% solids (see the percentage in solids of the washing liquors of corn). corn sold by Sigma Aldrich, Santa Cruz Biotechnology or FeedStimulants). The solid fraction of these liquors is formed mainly by microbial biomass that dies during the maceration process as well as by substances that have not reached the necessary degree of solubility from corn. It is known that during the maceration process of corn, lactic bacteria grow, among other microorganisms, for its wet fractionation (Yang et al 2005), which is why corn washing liquors contain a high concentration of lactic acid.
    [0014] On the other hand, in recent years the existence of biosurfactants in corn washing liquors (CSL) has been demonstrated in several publications and patents (Vecino et al 2014, Vecino et al 2015, Rodriguez-López et al 2017, Patent WO2014 / 044876 A, Patent ES 2424 399, Patent ES 2 527 366 B1), from the spontaneous fermentation of these liquors by microorganisms that grow during the maceration process of corn. So far, the only process described for obtaining these biosurfactants from CSL consists of a liquid-liquid extraction, mainly using organic solvents such as chloroform or ethyl acetate (Patent WO2014 / 044876). Vecino et al 2015 also collect the use of other solvents such as Isoamyl alcohol; Tributyl Phosphate (TBP); Methyl Butyl Ether (MTBE); Trichlorethylene (TCE); Dichloromethane; Xylene; Hexane Heptane, plus chloroform and ethyl acetate. Figure 1 shows a flow diagram of the process followed so far, to obtain biosurfactants from corn washing liquors.
    [0016] It should be pointed out that the use of organic solvents can be a drawback when applying this biosurfactant extract in certain industrial sectors, mainly in the pharmaceutical sector, so it would be interesting to use alternative methods that allow obtaining biosurfactants from these liquors, using more biocompatible extractants with less environmental impact.
    [0017] Below are some bibliographically proven uses of biosurfactants obtained from corn washing liquors by liquid-liquid extraction with organic solvents: i) at the level of soil and sludge treatment it has been observed that the use of this biosurfactant extract favors the solubilization and subsequent biodegradation of polycyclic aromatic hydrocarbons (Vecino et al 2014, Vecino et al 2015); ii) At the cosmetic level, tests have been carried out with this biosurfactant extract on human hair (Rincón-Fontán et al 2016, Rincón Fontán et al 2017), observing an improvement in the physical characteristics of the hair covering, after treating it with biosurfactant extract obtained from (CSL), compared to the chemical surfactant sodium dodecyl sulfate (SDS); iii) the use of the biosurfactant extract in the formulation of gold and silver nanoparticles has also been evaluated (Gómez-Graña et al 2017), observing that this extract not only has surfactant capacity, replacing the cationic surfactant Cetyltrimethylammonium bromide (CTAB), but it also has a reducing capacity avoiding the use of synthetic reducing agents in the formulation of these nanoemulsions. Gold particles are introduced into many cosmetic formulations in order to promote the nourishment, radiance and vitality of the skin. In addition, Rodríguez-López et al 2019 has also corroborated the improvement of the permeation of active principles through the skin, in the presence of the biosurfactant extract obtained from corn washing liquors, by extraction with organic solvents. On the other hand, López-Prieto et al 2019 have applied this same biosurfactant extract in the formulation of drinkable yogurts in order to improve their rheological properties, the probiotic biomass contained in the yogurt being not affected.
    [0019] As already mentioned above, corn wash liquors have a high solids content, generally 50% w / w. A high percentage of these solids is represented by the microbial biomass that grows during the maceration process of corn (Ramírez et al 2008). Within the microbial flora present in corn washing liquors, there are, among others, lactic acid bacteria. Various studies have shown the presence of biosurfactants adhered to the plasma membrane of this type of bacteria (Velraeds et al 1996, Rodríguez-López et al 2018, Vecino et al 2018), although so far the extraction of biosurfactants from the solid fraction of the corn washing liquors, by solid-liquid extraction, has not been contemplated.
    [0021] On the other hand, it should be noted that there is no evidence to date of obtaining biosurfactants from corn washing liquors using an aqueous saline solution as an extractant.
    [0023] As mentioned before, biosurfactants can be applied in different industrial fields and each biosurfactant extract can be considered unique, because each microorganism produces biosurfactants in a very specific way. Thus, small changes in the composition of the fermentation medium or in the operating variables cause the microorganisms to produce different biosurfactants at the compositional level, varying in many cases the length of the fatty acids or the amino acids that make them up (Mata-Sandoval et al. 2001, Singh et al 2014, Vecino et al 2017). In fact, it has been shown that, even under the same operating conditions, the same microorganism can produce different biosurfactants, depending on the growth phase in which it is found (Velraeds et al 1996, Angelo et al 2003).
    [0025] Currently, regarding the uses of biosurfactants, the industry that can best deal with their inclusion in formulations is the cosmetic industry, given the high added value of its products. This is because the biotechnological production of biosurfactants, through controlled fermentations, requires large investments. Table 1 lists the biosurfactants included so far in the European list of cosmetic Ingredients (Coslng).
    [0026] Table 1. List of inescapable biosurfactants in Coslng and their functions
    [0028] Biosurfactant Microorganism Functions Pseudomonas Emollient Rhamnolipids
    [0029] aeruginosa Emulsifier Soforolipids obtained Candida bombicola Conditioner using as substrate Protective palm hydrolysates Antioxidant Detergent Antoseborrheic Soforolipids Starmerella bombicola Cleaner Emulsifier Surfactant Antimicrobial Soforolipids obtained Cleaner Using as substrate Candida bombicola Deodorant Rapeseed Surfactant Cleaner Subdsorbent Gel Rapeseed Surfactant Cleaner Sodium Cleaner
    [0031] Explanation of the invention
    [0033] A process is proposed to obtain low-cost biosurfactants, from the solid fraction of the corn washing liquors, generated during the wet fractionation of corn, using as the only extracting agent a buffered saline aqueous solution or a solution buffered. After the analysis of the biosurfactant extract, using electrospray-type mass spectrometry, it is observed that the biosurfactant extract obtained by this route presents differentiating biomarkers from the other biosurfactants collected in the bibliography and also presents a different aspect and surfactant capacity to the biosurfactant extract obtained from starting from corn washing liquors by extraction with organic solvents, therefore it can be deduced that corn washing liquors contain different types of biosurfactants, probably produced by different classes of microorganisms.
    [0035] Detailed explanation of the invention
    [0037] Corn washing liquors were taken, coming from the wet corn fractionation process, and subjected to a centrifugation process, in order to obtain the solid fraction contained in these liquors, which, once the supernatant was removed, It was washed several times with deionized water at room temperature, repeating the centrifugation process between each washing cycle.
    [0039] Subsequently, the solids, coming from the corn washing liquors, once washed and centrifuged, were subjected to an extraction process with a buffered saline aqueous solution (PBS) that may have the following salts: sodium chloride, sodium phosphate, chloride of potassium and potassium phosphate. The extraction process was carried out for at least 1 hr, at room temperature. After extraction, the saline solution was centrifuged and the solid fraction of the corn wash liquors, obtaining a biosurfactant extract and a solid precipitate, which was discarded.
    [0041] Furthermore, extractions were carried out with the same extracting agent but eliminating the NaCl (aqueous buffer solution). In this case, to obtain comparable yields, it was necessary to work at temperatures above 50 ° C, following the same washing and centrifuging protocol as described above.
    [0043] Figure 2 shows, by way of example, the scheme of the new process proposed for obtaining biosurfactants from corn washing liquors, by extraction with PBS.
    [0045] Once the biosurfactant extract was obtained, it was dialyzed and lyophilized, to be subsequently subjected to several analyzes, including elemental analysis, surfactant capacity (based on measurement of surface tension), calculation of the critical micellar concentration, evaluation of the foaming capacity and mass analysis by electrospray mass spectrometry.
    [0047] Brief description of the drawings
    [0049] To complement the description made and in order to help a better understanding of the characteristics of the invention, a set of drawings is attached as an integral part of said description, in which, with an illustrative and non-limiting nature, the following has been represented:
    [0051] Figure 1. Scheme of the process proposed so far to obtain biosurfactants from corn washing liquors. In this scheme, the corn washing liquors, which can contain a percentage of solids between 18 and 50%, are subjected to a liquid-liquid extraction process with organic solvents such as ethyl acetate or chloroform, for 1-2 h, depending on the selected solvent. During this process, the biosurfactants contained in the corn washing liquors pass to the organic phase, which is subsequently separated from the aqueous phase of the liquors and is subjected to a vacuum distillation process, obtaining a biosurfactant extract. The biosurfactant extract obtained has an oily appearance and has no foaming capacity.
    [0053] Figure 2. Scheme proposed in this invention for obtaining biosurfactants from corn washing liquors. In this scheme, we start from the corn washing liquors with 50% solids, which are centrifuged, obtaining a precipitate that is washed several times with water, discarding the supernatant. Subsequently, the precipitate is subjected to an extraction process with PBS (phosphate buffer saline) or simply with phosphate buffer, for at least 2 h between 20 ° C and 70 ° C. After the extraction process, an aqueous phase with biosurfactants is obtained, which is recovered by centrifugation and subjected to a dialysis process. In this way the salts present are eliminated. Finally, the resulting solution is lyophilized, obtaining a white powder with biosurfactant capacity, capable of forming foam in aqueous solution.
    [0055] Figure 3. Mass spectrum of the biosurfactant extract under study, in which a series of biomarkers between 905, 1034 and 1141 m / z are observed that correspond to the mass of the biosurfactant object of this invention and which falls within the range of masses of biosurfactants of a lipopeptide nature.
    [0056] Preferred embodiment of the invention
    [0058] Extractions were made to corn wash liquors supplied by FeedStimulants (Utrecht, The Netherlands).
    [0060] The extract object of this invention was obtained from corn washing liquors, after centrifuging them and after subjecting the precipitate, once separated from the supernatant, to an aqueous extraction with PBS. Alternatively, phosphate buffered water can also be used at temperatures around 50 ° C-65 ° C. Table 2 shows some of the conditions used during the extraction process by way of example.
    [0062] Table 2. Conditions for obtaining the extract from corn washing liquors.
    [0064] Solvent Extraction Agent Time (h) Temperature (° C)
    [0066] PBS 2 Ambient water
    [0067] PBS water free from NaCl 1.5 65 ° C
    [0069] After extraction, the analysis of the biosurfactant extract (dialyzed and lyophilized) revealed that it is capable of reducing the surface tension of the medium by 16-20 units and that it presents a critical micellar concentration (CMC) of around 420 mg / L, although CMC may vary depending on the degree of purity achieved during the extraction process. Analysis by mass spectrometry (Figure 3) reveals that the biosurfactant produced is a lipopeptide.
    [0070] After characterization, it is observed that the biosurfactant extract obtained has a different appearance and composition to the biosurfactant extracted from the corn washing liquors by extraction with organic solvents. Thus, the biosurfactant extract obtained by extraction with organic solvents has a yellow color, is viscous, has an oily appearance and does not have the ability to form foam. However, the biosurfactant extract, object of this invention, has the appearance of a white powder and has the ability to form foam. Regarding its elemental composition, the extract object of this invention is composed of 12.5% of N, 42.5% of C, 6.2% of H and less than 0.3% of sulfur.
    [0072] Both the composition, the appearance and the physicochemical properties of the biosurfactant extract object of this invention are different from the biosurfactant extract extracted from the previously used corn washing liquors (Patent WO2014 / 044876 A, Patent ES 2424399, Patent ES 2527 366 B1) . Probably the difference in the appearance and composition of this new biosurfactant extract is due to the fact that in the previous inventions the biosurfactant extract is obtained from the liquid fraction, where the biosurfactants present in it are produced extracellularly, by microorganisms. that grow spontaneously there, while the biosurfactant under study is probably attached to the plasma membrane of the microorganisms present in the corn washing liquors, changing its nature. There are even strains of microorganisms that produce extracellular biosurfactants when they are in a vegetative state and non-extracellular biosurfactants when they grow in a sporulated way, and their characteristics and properties may vary (Angelo et al 2003). On the other hand, the biosurfactant extract obtained by extraction with organic solvents from corn washing liquors, has other substances in its composition (such as free fatty acids, or antioxidants) that give the whole extract different characteristics (Rodriguez-López et al 2016), to the biosurfactant extract under study.
    [0074] In the analysis of the biosurfactant extract, object of this invention, by mass spectrophotometry, by electrospray, several biomarkers are observed at 905, 1034 and 1141 m / z (Figure 3) that can correspond to biosurfactant masses of a lipopeptide nature. It should be noted that microorganisms in general produce very specific biosurfactants depending on the fermentation medium where they grow, being very difficult to find two identical biosurfactants, obtained from different media. Even during the same fermentation process, microorganisms can produce different types of biosurfactants depending on the growth phase in which they are found (Velraeds et al 1996, Angelo et al 2003). In fact, Figure 3 shows several signals above 900 m / z that are probably due to this fact.
    [0076] It should be noted that the biosurfactant object of this invention has biomarkers and masses different from those collected in the literature and that therefore have not yet been applied in any industrial field, including cosmetic, pharmaceutical, environmental, agrochemical or food.
    [0078] Thus, Chen et al (2017) have produced a lipopeptide using Bacillus licheniformis with biomarkers at 994, 1008, 1022 and 1036 m / z, while Bechet et al. (2012) also produced a lipopeptide with surfactant capacity using Bacillus thuringiensis, in which biomarkers were identified at 879, 893, 893, and 907 m / z. On the other hand, Madonna et al (2003) produced several homologues of the biosurfactant produced by Bacillus subtilis with biomarkers at 892, 906 and 920 m / z. Furthermore, Li et al (2016) found that Bacillus pseudomycoides produces a lipopeptide with surfactant capacity with a main signal at 933 m / z, and other lower signals at 1533, 1231 and 1024 m / z, which correspond to different biosurfactants produced by the same strain at different stages of growth. Angelo et al (2003) observed that Bacillus globigii produces different bisourfactants depending on whether it is in a vegetative or a sporulated state.
    [0080] It should be noted that the biosurfactants described above are lipopeptides that present biomarkers close to those of the biosurfactant object of this invention, although none entirely coincides with the biosurfactant extracted from the corn washing liquors by means of PBS extraction and object of study. It should also be noted that these biosurfactants were produced in controlled fermentations with fermentation media other than corn washing liquors, unlike the biosurfactant object of this invention. Furthermore, the biosurfactants mentioned in these works have been extracted from the fermentation medium, using methods other than the extraction with aqueous solution proposed in this invention.
    权利要求:
    Claims (5)
    [1]
    1. The procedure for the extraction of biosurfactants from corn washing liquors using an aqueous solution based on phosphate salts and / or sodium salts as extracting agent.
    [2]
    2. The use or application of biosurfactants obtained from corn washing liquors, after subjecting them to an aqueous extraction, according to claim 1, as surfactant and / or emulsifying agent in the food, cosmetic, pharmaceutical industry, agrochemical or environmental.
    [3]
    3. The use or application of biosurfactants obtained from corn washing liquors after subjecting them to an aqueous extraction, according to claim 1 as a surfactant, emulsifier and / or stabilizer of active principles in aqueous solutions and / or oily used for cosmetic, pharmaceutical and agrochemical uses.
    [4]
    4. The use or application of biosurfactants obtained from corn washing liquors after subjecting them to an aqueous extraction, according to claim 1 for cleaning surfaces.
    [5]
    5. The use or application of biosurfactants obtained from corn washing liquors after subjecting them to an aqueous extraction, according to claim 1 to improve the permeability of those formulas, which contain active principles, through the membranes cell phones, intended for cosmetic, pharmaceutical and agrochemical uses.
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    同族专利:
    公开号 | 公开日
    ES2795574B2|2021-06-03|
    CN113874090A|2021-12-31|
    WO2020234501A1|2020-11-26|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    EP2862624A1|2012-06-18|2015-04-22|Universidad de Vigo|Method for separating the surfactants present in the washing liquors of corn, and uses|
    ES2424399B2|2012-03-27|2014-01-20|Universidade De Vigo|Application of corn washing liquors as a surfactant|
    ES2527366B1|2013-07-22|2015-11-02|Universidade De Vigo|Detergent composition comprising a biosurfactant of plant origin|
    法律状态:
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    优先权:
    申请号 | 申请日 | 专利标题
    ES201900084A|ES2795574B2|2019-05-21|2019-05-21|Process for the aqueous extraction of biosurfactants from corn washing liquors or "corn steep liquor"|ES201900084A| ES2795574B2|2019-05-21|2019-05-21|Process for the aqueous extraction of biosurfactants from corn washing liquors or "corn steep liquor"|
    CN202080035458.4A| CN113874090A|2019-05-21|2020-05-19|Method for aqueous extraction of biosurfactants from corn steep liquors|
    PCT/ES2020/070321| WO2020234501A1|2019-05-21|2020-05-19|Process for the aqueous extraction of biosurfactants from corn steep liquor|
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