WO2020186326A1 - Cosmetic formulations formed by a nutritive mixture from a fermentative process - Google Patents

Abstract

The present invention describes a cosmetic formulation containing a nutrient-rich medium obtained by means of a fermentative process using genetically engineered bacteria. The microorganism engineered for the process was Corynebacterium glutamicum as it is of industrial interest since it produces high yields and is easy to work with. The nutritive medium produced is rich in various biomolecules, such as vitamins, organic acids and amino acids, including aminolevulinic acid. The compounds found in the nutritive mixture are not harmful to human health. Said medium is incorporated in cosmetic skin formulations capable of moisturizing and delaying aging in skin, preventing wrinkles and blemishes. One of the fastest growing cosmetic industry sectors encompasses the use of fermented substrates in product compositions. As they are biosynthesized, said compounds are considered to be more natural and are being increasingly accepted by the market.

Description

COSMETIC FORMULATIONS CONTAINED BY A NUTRITIONAL MIXTURE FROM A FERMENTATIVE PROCESS

FIELD OF THE INVENTION

[001] The present invention relates to the production of a nutrient broth by means of fermentative processes using a genetically modified bacterial strain. With the application of genetic engineering techniques, a strain of Corynebacterium glutamicum was developed that promotes the production of a nutrient medium containing organic acids, sugars and amino acids, such as aminolevulinic acid. This nutritive medium is incorporated into the composition of cosmetics to promote healthier skin, preventing wrinkles and blemishes. The area of cosmetics containing fermented extracts has been growing in recent years, and these compounds are an alternative to more natural ingredients in cosmetics.

HISTORY OF THE INVENTION

[002] The fermentative processes use microorganisms for the production of several biomolecules that are of commercial interest. The microorganisms metabolize the substrate present in the reaction, generating primary metabolites, such as amino acids, and secondary ones, such as antibiotics. These processes are currently being widely used in the food, pharmaceutical and cosmetic industries. This technology being applied in the production, on an industrial scale, of amino acids, vitamins, organic acids, among other compounds.

[003] Greater knowledge of microorganisms was only possible with the development of new techniques for the analysis of the genome. These techniques made it possible to apply genetic and metabolic engineering to the carrying out specific mutations. Through the use of these techniques, it is possible to guarantee a better production of biomolecules of commercial interest.

[004] The possibility of mutations in specific genes using genetic engineering tools prevents unwanted changes in the microbial genome. Therefore, with the genomic information of the microorganisms and the techniques of genetic engineering, it is possible to carry out modifications that aim to increase or decrease the expression of proteins or even the insertion and expression of genes from different organisms.

[005] The use of microorganisms capable of carrying out fermentations has been growing in the cosmetics industry since 1960. There are already products being commercialized that have in their composition ingredients generated by fermentations of bacteria and yeasts. Cosmetics containing fermented extracts have grown a lot, becoming one of the largest cosmetic groups (KR101320045).

[006] One of the most well-known products is SK-II, widely marketed in Asia. This product features a fermented broth obtained by yeast as an active ingredient (KR101320045 and WO20171 13263). Another company of Korean origin also presents products with fermentation technology (KR101320045). The main microorganisms used in these fermentations are lactobacilli or yeasts, as they are already well known, do not cause harmful effects to humans and because they are more effective in fermentations. There are still limitations in this area and more research is being done on the use of active compounds from fermentation in cosmetics, since there is a market demand for increasingly natural compounds (KR101320045 and WO20171 13263).

[007] 5-Aminolevulinic acid (5-ALA) is an amino acid that can be found in animals and plants. It is also possible to identify this amino acid in some foods, such as tea and red wine (KR 101 180266). 5- ALA is generally biosynthesized from succinyl-CoA and glycine by of the enzyme 5-aminolevulinic synthase (US 9333156). This molecule is an important intermediary in cell biosynthesis, also acting on energy metabolism, providing hydration when applied to the skin, in addition to delaying skin aging, avoiding blemishes and wrinkles. The amino acid incorporated in cosmetics also has a pharmacological action, being effective to treat acne, dermatitis, used in the treatment of skin cancer and photodynamic therapy, one of the applications being the treatment of alopecia (KR 101 180266, KR 20080090741 and US 5520905).

[008] 5-ALA is mainly prepared by chemical synthesis, however this type of synthesis has disadvantages, such as high cost, low conversion and environmental pollution. An alternative to this process is the genetic modification of microorganisms, ensuring that they start to produce, through fermentation, the amino acid in a more efficient and viable way (CN 104450812).

[009] There are microorganisms very well known and of great commercial interest, as they are easier to work with, safer, have less contamination and reach higher productions on a large scale. The idea is to introduce new genes in these microorganisms so that they generate several metabolites of interest that are more difficult to be produced on a large scale by their original microorganisms.

[010] Corynebacterium glutamicum is one of those microorganisms of industrial interest. This non-pathogenic bacterium is already used in industry for the large-scale production of L-glutamate, among other amino acids, with the potential for the production of organic acids and biofuels. C. glutamicum has a great role in the biotechnology industry and has been used for the production of 5-ALA, since L-glutamate is a precursor to ALA and this microorganism is considered safe, presenting no risks to humans (YU et al, 2015). [01 1] An advantage of producing active ingredients for cosmetics by fermentation is obtaining a fermented broth that contains not only 5- ALA, but also other amino acids, vitamins, organic acids, sugars, among other compounds. The incorporation of fermented in cosmetics guarantees a greater result, being cheaper, and the active ingredient becomes the broth rich in nutrients. In these fermentates there are already natural hydration factors that are responsible for promoting skin metabolism, preventing wrinkles and blemishes, and have antioxidant action (WO 20171 13263 and US 9333156).

STATE OF ART

[012] Different patents report the use of fermented broth or a substrate fermented by microorganisms, in the composition of cosmetics for topical use, as in publication WO No. 20171 13263. This publication mentions the use of acetic bacteria, Gluconacetobacter xylinus, and a substrate which features wood, glucose and yeast extract. The culture is carried out in two stages, each with a different temperature. After fermentation, a purification step is required to remove the cellulose membrane formed in the medium. Activated charcoal can be used in this step to obtain the fermented broth that will be incorporated into the cosmetic with a concentration of around 5% (based on weight).

[013] KR patent No. 101320045 mentions the use of a bacterial lysate in the formulation of a cosmetic for the skin. The active compound in this lysate is bacterioclorophyll obtained from the lysis of the bacterium Rhodobacter sphaeroides. After lysis of the cells, the solution obtained is centrifuged to remove the present biomass.

[014] The present invention differs from the two cases cited in that the nutrient medium to be incorporated as an active ingredient in cosmetics is different, since the microorganism used and the substrate to be fermented are not the same, generating a different final product. Having this nutrient medium in its composition: sugars, like glucose; organic acids, such as citric acid; vitamins; amino acids, such as aminolevulinic acid. Promoting healthier skin, moisturizes and prevents aging, in addition to having pharmacological actions, it can be used to treat acne, dermatitis and alopecia, with photodynamic therapy.

[015] US patent No. 9333156 cites a product that contains 5-ALA as an active ingredient for moisturizing the skin. KR patent No. 101325553 reports the possibility that 5-ALA, used as an active compound in the reported formulation, originates from a fermentation process, but does not mention what the substrate, the microorganism or the purification process would be like. The invention described here differs from the documents already published in that it presents a different cosmetic formulation, where the active compound is not only 5-ALA, but a nutrient medium coming from the fermentation of C. glutamicum, genetically modified, containing sugars, vitamins, minerals and different amino acids.

[016] KR patent No. 101814888 mentions the production of 5-ALA by means of a genetically modified strain of C. glutamicum, but does not cover an application or formulation of any cosmetic. The present invention has as a difference from the cited patents the use of the genetically modified bacterium C. glutamicum for the production of a fermented medium rich in nutrients for the purpose of formulating a cosmetic for use on the skin promoting a healthier and more hydrated skin, delaying the aging, avoiding spots and wrinkles. In addition to the cosmetic can be used for its pharmacological action being effective to treat acne, dermatitis and alopecia.

DEFINITIONS [017] The terms "increased expression" or "gene overexpression" are used throughout the text and have similar meanings.

[018] The term "microorganism" described in this patent preferably refers to, but is not limited to, bacteria of the species Corynebacterium glutamicum.

[019] The term "genetic modification" refers to any type of change in the genome of the target organism, or insertion of DNA in the form of plasmids or analogues to them. The inserted DNA can consist of genes from the same or other organisms, constituting a genetically modified organism.

[020] The term “gene” refers to any deoxynucleotide sequence that codes for a particular protein.

[021] The genes described here can be expressed preferentially from chromosomal DNA, with the possibility of being expressed also by plasmid DNA. The genes described here can have their activity modified as long as it accumulates mutations that code for the enzyme with the desired optimization.

[022] The coding sequences of the described template genes can be obtained by PCR using high-fidelity enzymes with a low rate of incorporation of mutations, or by chemical synthesis performed by companies specialized in the service.

[023] The term “glucose” refers to the carbon source used, varying only the concentration of C ₆ H ₁₂ O ₆ in its composition.

[024] The term “xylose” refers to the carbon source used, varying only the concentration of C ₅ H ₁₀ O ₅ in its composition.

[025] The term “arabinose” refers to the carbon source used, varying only the concentration of C ₅ H ₁₀ O ₅ in its composition.

[026] The term "glycerol" refers to the carbon source used, varying only the concentration of C ₃ H ₈ O ₃ in its composition. [027] The term "glycine" refers to the amino acid of the chemical formula C2H5N02 and CAS number 50-40-6 specifically for the L isomer.

[028] The term “serine” refers to the amino acid of the chemical formula C3H7N03 and CAS number 56-45-1 specifically for the L isomer.

[029] The terms "5-amino levulinic acid", "5-aminolevulinate" or "5-ALA" refer to the same molecule, characterized by being a non-protein amino acid of the chemical formula C5H9N03 and CAS number 160-60-5 specifically for the L isomer.

[030] The deoxynucleotide sequence identified in the genome of strain C. glutamicum 13032 as NCgl2476 is defined by the sucD gene, among deoxynucleotides 2,724,476 to 2,725,360, according to the reference genome (Ikeda, 2003).

[031] The sequence of deoxynucleotides identified in the genome of strain C. glutamicum 13032 as NCgl2477 is defined by the sucC gene, among deoxynucleotides 2,725,382 to 2,726,578, according to the reference genome (Ikeda, 2003).

[032] The sequence of deoxynucleotides identified in the genome of strain C. glutamicum 13032 as NCgl2810 is defined by the Idh gene, among deoxynucleotides 3.1 12.447 to 3.1 13.391, according to the reference genome (Ikeda, 2003).

[033] The deoxynucleotide sequence identified in the genome of strain C. glutamicum 13032 as NCgl2657 is defined by the pta gene, among deoxynucleotides 2,936,506 to 2,937,495, according to the reference genome (Ikeda, 2003).

[034] The deoxynucleotide sequence identified in the genome of strain C. glutamicum 13032 as NCgl2656 is defined by the ackA gene, among deoxynucleotides 2,935,313 to 2,936,506, according to the reference genome (Ikeda, 2003). [035] The sequence of deoxynucleotides identified in the genome of strain C. glutamicum 13032 as NCgl0954 is defined by the glyA gene, among the deoxynucleotides 1,050,624 to 1,051,928, according to the reference genome (Ikeda, 2003).

[036] The sequence of deoxynucleotides identified in the genome of strain R. capsulatus SB1003 as RCAP_rcc01447 is defined by the hemA gene, among deoxynucleotides 1,563,630 to 1,564,859, according to the reference genome (Strnad et al., 2010 ).

[037] The serA gene is defined as the deoxynucleotide sequence identified in the genome of strain C. glutamicum 13032 as NCgl1235, among deoxynucleotides 1,350,855 to 1,352,447, according to the reference genome (Ikeda, 2003).

197D

[038] The serA gene is defined as the variant of the serA gene in which the deoxynucleotide sequence consists of only 1005 nucleotides, so that after gene expression, a truncated protein with 197 amino acids excluded from the carboxy-terminal region is generated.

[039] The xylA gene is defined as the deoxynucleotide sequence identified in the genome of E. coli K12 MG1655 as b3565, among deoxynucleotides 3,729,433 to 3,730,765, according to the reference genome (Blattner et al., 1997).

[040] The deoxynucleotide sequence identified in the E. coli K12 MG1655 strain genome as b3564 is defined by the xylB gene, among deoxynucleotides 3,727,917 to 3,729,371, according to the reference genome (Blattner et al., 1997).

[041] The deoxynucleotide sequence identified in the genome of E. coli K12 MG1655 strain as b0062 is defined by the araA gene, among deoxynucleotides 66,835 to 68,337, according to the reference genome (Blattner et al., 1997). [042] The deoxynucleotide sequence identified in the E. coli K12 MG 1655 strain genome as Ò0064 is defined by the araB gene, among deoxynucleotides 68,348 to 70,048, according to the reference genome (Blattner et al., 1997).

[043] The deoxynucleotide sequence identified in the E. coli K12 MG1655 strain genome as b0061 is defined by the araD gene, among deoxynucleotides 65,855 to 66,550, according to the reference genome (Blattner et al., 1997).

[044] The deoxynucleotide sequence identified in the genome of the E. coli K12 MG1655 strain as b2841 is defined by the araE gene, among deoxynucleotides 2,980,674 to 2,982,182, according to the reference genome (Blattner et al., 1997).

[045] The glox gene is defined as the deoxynucleotide sequence identified in the E. coli K12 MG1655 genome as b3926 and located between nucleotides 4.1 15.714 to 4.1 17.222 in the reference genome (Blattner et al., 1997).

[046] The depynucleotide sequence identified in the genome of E. coli K12 MG1655 strain as b3927 and located between nucleotides 4.1 17.245 to 4.1 18.090 in the reference genome is defined as the glpF gene (Blattner et al., 1997).

[047] The depynucleotide sequence identified in the genome of strain E. coli K12 MG 1655 as b3426 and located between nucleotides 3,562,013 to 3,563,518 in the reference genome is defined as the glpD gene (Blattner et al., 1997) .

DESCRIPTION OF THE INVENTION

[048] The present invention aims to describe in detail the process of genetic modification for the production of strains of bacteria from species C. glutamicum capable of producing 5-aminolevulinic acid. In addition, the present invention brings as an innovation the use of the fermented broth of any of the said strains of C. glutamicum as a mixture rich in nutrients, thus being the main component of different cosmetic formulations. These formulations have the ability to hydrate, nourish, protect and repair skin cells.

[049] In order to make the present invention viable, the C. glutamicum strain ATCC 13032 was used as a chassis strain for the genetic modifications described here, because it is a wild strain and its genome is completely sequenced and annotated in databases (Ikeda, 2003).

[050] Also, any strain of bacteria of the genus Corynebacterium, as long as they are not pathogenic, can be used as chassis strains for the development of organisms capable of producing molecules of commercial interest, such as 5-aminolevulinic acid, and be used in processes fermentative in order to generate a fermented broth to be used as a mixture rich in nutrients for the formulation of cosmetics.

[051] Originally strains of C. glutamicum are excellent platforms for the natural production of amino acids, such as glutamic acid, for example. In addition, there are commercially genetically modified strains for the production of amino acids such as L-lysine and L-threonine on a large scale. The same is true for the production of aminolevulinic acid, which currently has its production on an industrial scale from chemical or chemo-enzymatic synthesis. For C. glutamicum to be able to produce the aforementioned amino acid, it is necessary that the chassis strain be genetically modified by altering it, in order to alter the metabolic flow of the microorganism in order to produce this amino acid.

[052] Aiming to increase the metabolic flow for the production of a certain molecule, several strategies involving metabolic engineering of microorganisms can be used. For example, it is possible to modulate the expression of a given gene by: increasing the number of copies of the gene of interest on the chromosome of the desired strain; use of a regulated or constitutive promoter in order to induce, increase or decrease the expression of the desired gene; alteration of the initiation codon; or use of the gene in a plasmid that has stability when inserted into the strain of interest. For the expert in the art, there are countless other ways to carry out the modulation of gene expression in microorganisms such as C. glutamicum.

[053] As mentioned earlier, the expression of a given gene can occur from chromosomal DNA or from a plasmid. In the case of insertion, deletion or modification of a DNA fragment in the chromosome, the homologous recombination technique based on viral recombinases is commonly used. Associated with this, one can apply the technique of gene modification using the CRISPR system through the use of enzymes such as Cas9, Cpf1, or similar.

[054] In the case of the present invention, the homologous recombination technique associated with the Cpf1 enzyme is used for the insertion of genes in the chromosome of the chassis strain, in a manner adapted from the system employed by Jiang et al., 2017.

[055] The nucleotide sequences corresponding to the promoter regions, genes and terminator regions, as well as other elements, such as restriction enzyme sites, coding sequences for the guide RNA, among others, were obtained from the nucleotide synthesis performed by companies specialized in art.

[056] The assembly of the gene cassettes described in the present invention was carried out by means of a cloning system using restriction enzymes. In this case, the cassettes were assembled using the Xbal and BamHI enzymes, and the cassettes were inserted into the chromosome integration vector using the Apal and Hindlll enzymes. [057] The integration of the gene cassettes into the chromosome of the chassis strain is performed after electroporation of the cells with the plasmid pNBCg_Cpf1 (and its variants) containing the appropriate guide RNA for the target of interest, as well as the gene that codes for the Cpf1 nuclease of Francisella tularensis novicida.

[058] Confirmation of the integration of the gene cassette into the chromosome of the chassis strain is verified by PCR using primers specific to the regions of interest. In this way, positive clones are selected, subcultured and prepared for new stages of gene editing. The genetic modifications performed are described below.

[059] The chassis strain or any other strain of the genus Corynebacterium is not capable of producing 5-aminolevulinic acid naturally. Thus, it is necessary to express the hemA gene that codes for the enzyme 5-aminolevulinate synthase (ALAS), which produces 5-aminolevulinic acid from L-glycine and succinyl-CoA. In the case of the present patent, the nucleotide sequence used comes from the bacteria of the species Rhodobacter capsulatus, optimized for expression in C. glutamicum.

[060] In addition, it is necessary to overexpress the glyA gene of the chassis strain itself, since this gene codes for the enzyme serine hydroxymethyltransferase, which is responsible for producing glycine from the amino acid L-serine and tetrahydrofolate. In this way, the hemA-glyA expression cassette, which has the expression controlled by the constitutive synthetic promoter pTac, generates the strain NB_CgALA01. It is important to note that the insertion of this cassette is performed using the sucD gene as a target, so that its expression is suppressed in order to redirect the metabolic flow for the production of 5-ALA.

[061] In order to generate a strain with increased metabolic flow for the production of the amino acid L-serine, which is a precursor to L-glycine, a gene cassette composed of the serA, serB and serC genes is inserted, which code for the phosphoglycerate synthase, phosphoserine aminotransferase and phosphoserine phosphatase enzymes, respectively. It is important to note that the serA gene contains the deleted carboxy-terminal region, so that the last 591 nucleotides of the sequence are eliminated, in order to generate a variant of the phosphoglycerate synthase protein that does not undergo self-regulation by L-serine. The gene cassette is controlled by the constitutive promoter pTac, and incorporated into the strain NB_CgALA01, thus generating the strain NB_CgALA02. It is important to note that the insertion of this cassette is performed using the sucC gene as a target, so that its expression is suppressed in order to redirect the metabolic flow for the production of 5-ALA.

[062] In order to obtain a strain capable of metabolizing glucose and xylose as carbon sources, the genes xylA and xylB of Escherichia coli, as described by Kawaguchi et al., AEM 72: 3418-3428 (2006), and in a way similar to that performed in the patent US939984E2, they are overexpressed in the form of an operon in strain NB_CgALA02. The xylA and xylB genes code for the enzymes xylose isomerase and xylulokinase, respectively. The expression of this gene cassette is performed by the constitutive promoter pTac, generating the strain NB_CgALA03. It is important to note that the insertion of this cassette is performed using the Idh gene as a target.

[063] In order to obtain a strain capable of metabolizing glucose, xylose and arabinose as carbon sources, the E. coli araA, araB, araD and araE genes, in a similar way to that carried out in EP2513302A1, are overexpressed in the form of operon in strain NB_CgALA03. The araA, araB, araD and araE genes code for the enzymes arabinose isomerase, ribulokinase, ribulose-5-phosphate-4-epimerase, and for the arabinose transporter, respectively. The expression of this gene cassette is performed by the constitutive promoter pTac, generating the strain NB_CgALA04. It is important to note that the insertion of this cassette is performed using the pta gene as a target. [064] In order to obtain a strain capable of metabolizing glucose, xylose, arabinose and also glycerol as carbon sources, the genes glpF, glpK and glpD of Corynebacterium diphteriae, in a similar way to that carried out in the patent US8426165B2, are overexpressed in the form of operon in strain NB_CgALA04. The glpF, glpK and glpD genes code for the glycerol carrier protein and for the enzymes glycerol kinase and glycerol-3-phosphodihydrogenase, respectively. The expression of this gene cassette is performed by the constitutive promoter pTac, generating the strain NB_CgALA05. It is important to note that the insertion of this cassette is performed using the ackA gene as a target.

[065] Table 1 represents the strains constructed with their respective modifications.

Table 1 - Genetically modified strains for the production of 5-ALA.

[066] Fermentative processes using different types of microorganisms have a variety of industrial applications. In general, the growth of a genetically modified strain, or not, has the ability to generate a nutritive mixture that can be made up of biomass and fermented juice together or separately, provided that, in the case of biomass, it is prepared in a way that does not contain any trace of the live microorganism. [067] A process for generating such a product is described in patent number US5840358A, which refers to the fermentation process using microorganisms such as E. coli or C. glutamicum genetically modified for the production of L-lysine, so that the final product consists of nutrient-rich biomass and fermented broth, in addition to the L-lysine produced by the microorganisms in conjunction with the biomass itself.

[068] In the case of the present invention, the use of a genetically modified microorganism of the species C. glutamicum capable of producing 5-aminolevulinic acid is described, where, when submitted to a fermentative process, the final product, which may be composed of biomass plus fermented broth, or each individual component, is used as a mixture rich in nutrients, used as the main composition for the formulation of cosmetics.

[069] The fermentation process for the production of the nutrient-rich mixture containing different compounds, such as saccharides, nucleic acids, lipids and amino acids, including 5-aminolevulinic acid, must contain:

[070] a) at least one carbon source, such as glucose, xylose, arabinose, among others, provided that the microorganism is able to metabolize the carbon source in question;

[071] b) at least one source of nitrogen, such as yeast extract, peptone or inorganic salts that are used as a source of nitrogen by said microorganism;

[072] c) at least one source of phosphorus, such as monobasic sodium phosphate, dibasic sodium phosphate, monobasic potassium phosphate, dibasic potassium phosphate, or other inorganic salt composed of phosphorus that is assimilated by said microorganism;

[073] d) inorganic salts derived from minerals or vitamins, such as sodium chloride, calcium carbonate, ammonium sulfate, among others, as long as they are assimilated by the said organism. [074] e) at least one organic acid such as citric acid, succinic acid, acetic acid, among others.

[075] The fermentation process should preferably be carried out in bioreactors with controlled temperature, pH, feeding, agitation and aeration conditions. However, the fermentation process is not limited to the conditions described above, but can be carried out in flasks such as conical flasks, for example, provided the conditions for biomass growth are provided.

[076] Regarding temperature, the fermentation process can be carried out in ambient temperature ranges at 37 ° C, preferably from 25 ° C to 34 ° C, more preferably from 28 ° C to 32 ° C.

[077] Regarding pH, the fermentation process can be carried out in a pH range of 5.0 to 7.5, preferably from 5.5 to 7.0, more preferably from 6.0 to 6.5.

[078] Regarding food, the fermentation process can be started with concentrations of carbon source varying from 10g / L to 60g / L, preferably from 20g / L to 50g / L, more preferably from 30g / L to 40g / L . Furthermore, feeding can be carried out constantly or in pulses, as long as the concentration of the carbon source is maintained at a threshold of 2g / L to 8g / L, preferably from 4g / L to 6g / L, more preferably 5g / L.

[079] Regarding agitation and aeration, the fermentation process can be carried out with an agitation range that varies from 150 to 250 rpm and aeration between 1 to 3 vvm, preferably agitation from 180 to 230 rpm and aeration between 1, 5 to 2 vvm. Eventually, when in cases of high cell density, agitation can be increased to a range of 300 to 600 rpm and aeration from 2 to 2.5 vvm.

[080] In relation to biomass, the fermentation process can be started with an inoculum where the cell concentration corresponds to D.0 ₆ oo in the range of 1 to 10, preferably 5 to 15, more preferably 10 to 20. [081] After the fermentation process, the resulting biomass and the fermented broth, whether together or separately, can be subjected to a complete or partial drying process, vacuum or not, at temperatures of 40 to 80 ° C, preferably 50 to 70 ° C, more preferably 55 to 65 ° C. Also, if the fermented juice is used separately from the biomass, it can be subjected to filtration or ultrafiltration before the drying process.

[082] The final product, consisting of a mixture rich in nutrients, composed of biomass and fermented broth, either together or separately, after the drying process, can be used in the powder stage or be reconstituted through the addition of water, aiming at the formulation of cosmetics.

[083] The final product can also be mixed with other additive molecules that act as an active ingredient. These molecules can be other saccharides, amino acids or peptides, nucleic acids and other organic acids, lipids and enzymes or growth factors, and these molecules may already be present in the fermented broth described here and added in greater quantities, or simply be other molecules of origin other than the fermentative process, being added to the final product, forming a cosmetic composition with other properties in addition to those conferred by the fermented broth containing 5-aminolevulinic acid.

[084] Also, the final product, in order to compose a cosmetic product that is used in ointments, creams, lotions, makeup, or other cosmetic applications, may consist of elements commonly accepted in the formulation of cosmetics, such as emulsifiers, emollients, solvents, excipients, antioxidants, among others.

[085] The example described in the present invention deals only with a representation of the fermentative process and subsequent formulation of a cosmetic cream in order to illustrate one of the various applications of the nutritional mixture, so as not to limit the possible applications of the product described here to just this case.

[086] Example 1:

[087] Preparation of a nutritive mixture by fermentation process using a strain of C. glutamicum producing 5-aminolevulinic acid for cosmetic formulations

[088] First, from a colony isolated from the strain NB_CgALA05, a pre-inoculum is performed in 10mL of medium containing 10g / L of tryptone, 5g / L of yeast extract, 10g / L of sodium chloride. This is incubated at 30 ° C for 16 hours with 200 rpm shaking. Then, the content of the pre-inoculum is diluted in 400mL of the same previous medium, and incubated under the same conditions, also for 16 hours, thus constituting an inoculum. Finally, the content of the inoculum, with a DOeoo of approximately 5.0, is diluted in 1 L of the medium for the 5L bioreactor fermentation process. The culture medium for the fermentation process is initially composed of 40g / L of glucose, 10g / L of tryptone, 5g / L of yeast extract, 10g / L of sodium chloride and 10g / L of L-glycine.

[089] During the process, glucose is added in order to maintain a minimum of 3g / L and a maximum of 30g / L of glucose available in the medium. The fermentation pH is maintained at around 6.5 by the addition of sodium hydroxide when necessary. Fermentation takes place for 48 hours at 30 ° C with 300rpm agitation and constant addition of 2 v.v.m. of oxygen.

[090] At the end of the fermentation process, the biomass and the fermented broth are subjected to drying in a rotary evaporator under 60 ° C until the solution dries completely. [091] An example of cosmetic preparation, but not limited to this, can be performed using the following composition: 77.5% deionized water, 0.1% EDTA, 1% propylene glycol, 0.3% methylparaben , 2% ceto-stearyl alcohol, 0.10% cyclomethicone DC 245, 0.5% silicone 9040, 0.5% hyaluronic acid, 13% glycerin and 5% of the nutrient mixture from the previous fermentation.

[092] All components of the mixture, with the exception of cetostearyl alcohol and silicone 9040, are mixed at 45 ° C until they form a homogeneous solution. The cetostearyl alcohol is heated to 80 ° C and then added to the previous mixture. The mixture is homogenized for about 10 minutes. Then, the silicone 9040 is acidified and the mixture is homogenized again for 10 minutes. After stabilizing the complete mixture and adjusting the pH to 6.5, the final product is formed.

REFERENCES

- YU, X. et al. Engineering Corynebacterium glutamicum to produce 5-aminolevulinic acid from glucose. Microbial Cell Factories, 14: 183 (2015).

- KAWAGUCHI, H. et al., Engineering of a xylose metabolic pathway in Corynebacterium glutamicum. Applied and Environmental Microbiology, 72: 5 (2006).

Referenced patents: CN 104450812; KR 101 180266; KR 101320045; KR 101325553; KR 101814888; KR 20080090741; US 5,520,905; US 9333156; WO 20171 13263; US939984E2; EP2513302A1; US8426165B2; US5840358A.

Claims

1. COSMETIC FORMULATIONS CONTAINED BY A NUTRITIONAL MIXTURE FROM A FERMENTATIVE PROCESS, characterized by a fermentative process that generates a fermented substrate containing a rich mixture of nutrients, such as amino acids, including 5-aminolevulinic acid, carbohydrates, nucleic acids, organic acids , vitamins, peptides, lipids and minerals, initially present in the culture medium and / or from fermentation for the formulation of a compound that has any cosmetic application. 2. COSMETIC FORMULATIONS CONSTITUTED BY A NUTRITIONAL MIXTURE FROM A FERMENTATIVE PROCESS, according to claim 1, characterized by a fermentative process carried out with any genetically modified Corynebacterium glutamicum strain capable of producing 5-aminolevulinic acid. 3. COSMETIC FORMULATIONS CONTAINED BY A NUTRITIVE MIXTURE FROM A FERMENTATIVE PROCESS, according to claims 1 and 2, characterized by using microorganisms such as Escherichia coli and / or Saccharomyces cerevisiae genetically modified for the production of 5-aminolevulinic acid. 4. COSMETIC FORMULATIONS CONTAINED BY A NUTRITIONAL MIXTURE FROM A FERMENTATIVE PROCESS, according to claims 1 to 3, characterized by a fermented medium rich in nutrients containing or not the inactive biomass as part of the final product. 5. COSMETIC FORMULATIONS CONTAINED BY A NUTRITIONAL MIXTURE FROM A FERMENTATIVE PROCESS, according to claims 1 to 4, characterized by a cosmetic formulation containing from 0.05 to 20% of the mixture rich in nutrients obtained from the fermentation used for the treatment of acne, hydration and skin nutrition, inhibiting the progression of the appearance of wrinkles and blemishes. 6. COSMETIC FORMULATIONS CONTAINED BY A NUTRITIVE MIXTURE FROM A FERMENTATIVE PROCESS, according to claims 1 to 5, characterized by a cosmetic formulation containing 0.05 to 20% of the nutrient-rich mixture obtained from the fermentation used with the purpose of constituting a pharmaceutical product used in procedures such as photodynamic therapy, treatment of dermatitis and treatment of alopecia.