Fermented binding serum

Abstract

This disclosure generally relates to cosmetic compositions, in particular to hair treatment compositions, such as keratin fermentation broth, or fractions or isolates thereof, for use in shampoos and conditioners. The keratin fermentation broth comprises a) keratin and / or hydrolyzed keratin, b) Lactobacillus species, and c) monosaccharides.

Description

[Technical Field]

[0001] Cross-reference of related applications This application is a continuation-in-part application of International Application PCT / US2023 / 029133, filed on 31 July 2023, claiming priority thereto, and also claims priority to U.S. Provisional Application 63 / 498,920, filed on 28 April 2023, each of which is incorporated herein by reference in whole.

[0002] This disclosure generally relates to cosmetic compositions, and more particularly to hair treatment compositions, such as keratin fermentation broth for use in shampoos and conditioners. [Background technology]

[0003] Hair is formed from layers of keratin protein, which is a high molecular weight. Hair damage can occur with age, particularly from environmental factors such as UV light, ozone, and humidity. Damage can also occur from various hair treatments, including physical treatments (e.g., heat from straightening or curling) and chemical treatments (e.g., colorants and hair relaxers).

[0004] Keratin-containing cosmetic formulations, such as shampoos, are thought to strengthen hair by reinforcing and repairing the keratin structure. (See WO1995 / 017157A2, WO2004 / 047774A1, and WO2012 / 025615A2 for examples of keratin-containing cosmetic formulations.) However, formulations containing keratin fermentation broth have not been reported. [Overview of the Initiative]

[0005] This disclosure provides a keratin fermentation broth comprising a) keratin and / or hydrolyzed keratin, b) Lactobacillus species, and c) monosaccharides or fractions or isolates thereof. Furthermore, the disclosure relates to a cosmetic composition comprising keratin fermentation broth.

[0006] The disclosure also relates to a method for treating hair fibers, the method comprising contacting the hair fibers with a hair treatment composition comprising a) keratin and / or hydrolyzed keratin, b) Lactobacillus acidophilus, and c) monosaccharides or fractions or isolates thereof, in a keratin fermentation broth.

[0007] This disclosure further relates to a method for producing keratin fermentation broth, wherein the method is

[0008] a) Combining keratin and / or hydrolyzed keratin, Lactobacillus acidophilus, and monosaccharides in an aqueous fermentation solution,

[0009] b) The method comprising fermenting an aqueous fermentation solution at a temperature of approximately 2 to approximately 53°C and a pH of approximately 4.5 to approximately 6.5 to produce a keratin fermentation broth.

[0010] This disclosure further relates to keratin fermentation products produced by exposing keratin and / or hydrolyzed keratin to the fermentation of monosaccharides by Lactobacillus species.

[0011] This disclosure also relates to products comprising keratin and / or hydrolyzed keratin exposed to monosaccharide fermentation by Lactobacillus species.

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. Methods and materials similar to or equivalent to those described herein may be used in the practice of the present invention, but preferred methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. In case of any conflict, this specification shall prevail, including definitions. In addition, materials, methods, and examples are illustrative and not intended to be limiting.

[0013] Details of one or more embodiments of the present invention are described in the accompanying drawings and the following description. Other characteristics, purposes, and advantages of the present invention will become apparent from the description and drawings, and from the claims. [Brief explanation of the drawing]

[0014] [Figure 1] The average contact angles for the 20% SLES and 0.1% keratin fermentation broth simple solution treatments in Example 5 are shown in the figure. [Figure 2] The remaining time for treatment with 20% SLES and 0.1% keratin fermentation broth simple solution in Example 5 is shown in the figure. [Figure 3] The average fracture levels for treatment with 20% SLES and 0.1% keratin fermentation broth simple solution in Example 6 are illustrated. [Figure 4] Regarding the integrity in Example 7, the average enthalpy of water evaporation for treatment with 20% SLES and 0.1% keratin fermentation broth simple solution is shown in the figure. [Figure 5] The DSC curve showing the enthalpy of keratin denaturation for the 20% SLES (control) treatment in Example 7 is shown. [Figure 6] The DSC curve showing the enthalpy of keratin denaturation for treatment with a 0.1% simple keratin fermentation broth solution in Example 7 is shown. [Figure 7] The gloss values ​​for CTRP, CTRN, and 0.1% keratin fermentation broth simple solution treatment in Example 8 are shown in the figure. [Figure 8A] The fluorescence microscope image of a cross-section of relaxed MB hair immersed for 24 hours in a control (after purification) containing only 0.1% dye, illustrating the natural hair fluorescence in the green channel in Example 9. [Figure 8B] This is a fluorescence microscope image of a cross-section of relaxed MB hair immersed for 24 hours in a control (after purification) containing only 0.1% dye, illustrating the absence of dye in the red channel in Example 9. [Figure 9A]A fluorescence microscope image of a cross-section of relaxed-treated MB hair immersed in 0.1% labeled fermented binding serum for 24 hours, illustrating natural hair fluorescence in the green channel in Example 9. [Figure 9B] A fluorescence microscope image of a cross-section of relaxed-treated MB hair immersed in 0.1% labeled fermented binding serum for 24 hours, in the red channel in Example 9. Visible penetration of the fermented binding serum into the cortex was detected. [Figure 10A] A fluorescence microscope image of a cross-section of relaxed-treated MB hair immersed in 0.1% labeled Keratec™ IFP PE (Croda Inc.) for 24 hours, illustrating natural hair fluorescence in the green channel in Example 9. [Figure 10B] A fluorescence microscope image of a cross-section of relaxed-treated MB hair immersed in 0.1% labeled Keratec™ IFP PE (Croda Inc.) for 24 hours, in the red channel in Example 9. Keratec™ was concentrated in the cuticle and minimal outer cortex penetration was seen in some fibers. [Figure 11] Illustrates the average corrected total fluorescence from penetration of the fluorescently labeled peptide into the hair fiber after 24 hours of treatment in Example 9. Asterisks (*) indicate statistical significance (p < 0.05). [Figure 12] Illustrates the strength of the unfermented hydrolyzed keratin control and the fermented binding serum (n = 18, 6 panelists × 3 evaluations). [Figure 13] Illustrates the preference for the unfermented hydrolyzed keratin control and the fermented binding serum (n = 18, 6 panelists × 3 evaluations). [Figure 14] Illustrates a spider chart representing the general profile and sensory comparison of the unfermented hydrolyzed keratin control and the fermented binding serum. [Figure 15] Illustrates a graph comparison of the concentrations (μg / m3) of each chemical group for the unfermented hydrolyzed keratin control and the fermented binding serum.

Modes for Carrying Out the Invention

[0015] Keratin fermented broth This disclosure describes a keratin fermentation broth comprising a) keratin and / or hydrolyzed keratin, b) Lactobacillus species, and c) monosaccharides or fractions or isolates thereof.

[0016] Specifically, keratin fermentation broth can be referred to by its International Nomenclature for Cosmetic Ingredients (INCI) name Lactobacillus / honey / keratin ferment, mono ID 37496.

[0017] Keratin and / or hydrolyzed keratin can be extracted from wool. Hydrolyzed keratin is more water-soluble than unhydrolyzed keratin. Many methods for hydrolyzing keratin are known, including those disclosed in WO2010 / 114938A1 by KERAPLAST TECHNOLOGIES, LTD, which are incorporated herein by reference.

[0018] In various embodiments, the fermenting organism is Lactobacillus acidophilus.

[0019] The carbohydrate source for fermentation is any monosaccharide suitable for Lactobacillus fermentation, or may include such monosaccharide. In at least one embodiment, the monosaccharide is provided by honey, for example, the carbohydrate source for fermentation is provided by honey.

[0020] Lactobacillus fermentation has been used for thousands of years to process and preserve food, but it has not yet been applied to animal keratin. Surprisingly, improved conditioning, hair treatment, hair styling, manageability, and other properties have been found to be achieved by fermented wool keratin. Honey contains enzymes (such as diastase, invertase, glucose oxidase, catalase, glucosylceramidase, α-amylase, α-glucosidase, β-glucosidase, and protease) that may have an unknown effect on the keratin structure and / or influence the fermentation process, contributing to the resulting benefits. Furthermore, hydrolyzed keratin fermentation broth has been found to have reduced or improved odor compared to unfermented hydrolyzed keratin.

[0021] In various embodiments, the keratin fermentation broth includes aqueous fermentation broth.

[0022] Keratin fermentation broth is typically acidic. Lactobacillus fermentation generally acts on carbohydrates to produce lactic acid. Therefore, basic amino acids and / or amino alcohols can help adjust the pH to a suitable level (i.e., less acidic).

[0023] In various embodiments, the keratin fermentation broth described herein may have a pH of about 7 or less, for example, about 6.5 or less, about 6 or less, about 5.5 or less, about 5 or less, about 4.5 or less, about 4 or less, or about 3 or less. In some embodiments, the keratin fermentation broth described herein may have a pH of about 3 to about 7, about 3 to about 6, about 3 to about 5, about 3 to about 4, about 4 to about 7, about 4 to about 6, or about 4 to about 5.

[0024] In various embodiments, the keratin and / or hydrolyzed keratin in the keratin fermentation broth have an average molecular weight of about 1 kDa to about 10 kDa, about 3 kDa to about 8 kDa, or about 4 kDa to about 5 kDa. In at least one embodiment, the keratin and / or hydrolyzed keratin have an average molecular weight of less than 10 kDa, for example, about 1 kDa to 10 kDa. In at least one embodiment, the keratin and / or hydrolyzed keratin have an average molecular weight of about 2.5 kDa, about 3 kDa, about 4.5 kDa, or about 5 kDa. In at least one example, the keratin and / or hydrolyzed keratin in the keratin fermentation broth have an average molecular weight of greater than 1 kDa and 20 kDa or less, or greater than 1 kDa and 15 kDa or less.

[0025] Lactobacillus fermentation method This disclosure further relates to a method for producing keratin fermentation broth, wherein the method is

[0026] a) Combining keratin and / or hydrolyzed keratin, Lactobacillus acidophilus, and monosaccharides in an aqueous fermentation solution,

[0027] b) The method comprising fermenting an aqueous fermentation solution at a temperature of approximately 2°C to approximately 53°C and a pH of approximately 4.5 to approximately 6.5 to produce keratin fermentation broth.

[0028] Lactobacillus fermentation does not require any special equipment. It is a conventional process used in various foods (dairy products, vegetable pickles, sausages, and meat fermentation). It is used to ferment materials used in cosmetic compositions, and many methods are publicly known in the art (see, for example, U.S. Patent No. 9295704, U.S. 2021 / 0052486A1, KR101045310B1, KR101452770B1, and KR20000039570A). Fermentation can be carried out in almost any container (container or vessel), on any scale, and more or less under ambient conditions. Lactobacillus fermentation generally involves the action of carbohydrates to produce lactic acid.

[0029] Lactobacillus species, monosaccharides, and keratin and / or hydrolyzed keratin are combined in a container. In some embodiments, the Lactobacillus species is Lactobacillus acidophilus, and the monosaccharides are provided by honey.

[0030] In some embodiments, fermentation is carried out in an aqueous solution. Typically, keratin and / or hydrolyzed keratin are made sufficiently soluble in the aqueous fermentation solution or culture medium, for example, to about 2 to about 15% by weight of keratin and / or hydrolyzed keratin. Hydrolyzed keratin is more soluble in water than keratin, and therefore fermentation at higher concentrations is possible if keratin is hydrolyzed first. Fermentation can be carried out at a temperature of about 2°C to 53°C and a pH of about 4.5 to about 6.5. Lower pH levels may be used. In this fermentation, the temperature and pH conditions suitable for Lactobacillus growth are typically about 30°C to about 40°C and a pH of about 5.5 to about 6.2. The broth can be fermented for up to about 4 days or more, usually about 1 to about 12 hours. The broth can then be pasteurized and packaged. Pasteurization is optional as it kills any remaining live Lactobacillus species in the composition. Pasteurization can occur through a heat shock at at least 70°C for 15 minutes, or through a rapid change in pH.

[0031] This disclosure also relates to isolates or fractions of the keratin fermentation broth obtained. For example, the broth may be subjected to post-fermentation treatment, which includes separating the fermentation biomass (i.e., bacteria of the Lactobacillus species) from the rest of the broth to form a fraction that is essentially free of particulate matter and essentially contains only soluble organic compounds produced by fermentation, and optionally, if there are any residues in the broth, contains soluble residual substrate components such as sugars. Any Lactobacillus species can be removed or separated from the rest of the broth by filtration or another preferred method for producing its fraction (e.g., filtrate). Furthermore, any individual components of the keratin fermentation broth can be isolated to produce their isolates. For example, this may be isolated keratin and / or hydrolyzed keratin after exposure of monosaccharides to fermentation by Lactobacillus species. In these and other embodiments, the aqueous keratin fermentation broth can be post-treated, for example, by spray drying to remove water, to obtain a powdered organic solid (e.g., anhydrous fermentation product or "dried fermentation broth fraction"), which can be rehydrated in an aqueous solution before being added to a cosmetic formulation.

[0032] Keratin fermentation broth may also contain added preservatives. For example, about 2 to about 50% by weight of a biostatic solvent can be added to the fermentation broth, its fraction, or isolate to prevent residual Lactobacillus species or other bacteria from growing in the aqueous composition. Suitable biostatic solvents may be selected from the group consisting of glycerin, propanediol, butylene glycol, 1,2-hexanediol, pentylene glycol, and other water-soluble diols.

[0033] In some cases, keratin fermentation broth contains more than 5% by weight, more than 8% by weight, or more than 10% by weight of keratin and / or hydrolyzed keratin relative to the total weight of the keratin fermentation broth. In some cases, keratin fermentation broth contains more than 5% by weight, more than 8% by weight, or more than 10% by weight of hydrolyzed keratin relative to the total weight of the keratin fermentation broth. Additionally or alternatively, keratin fermentation broth may contain less than 10% by weight of monosaccharide sources, for example, less than 5% by weight, less than 3% by weight, less than 2% by weight, or less than 1% by weight. The remainder of the keratin fermentation broth usually contains water or other suitable solvent. In at least one example, the keratin fermentation broth contains about 5% to about 10% by weight of keratin and / or hydrolyzed keratin, about 0.01% to about 0.1% by weight of Lactobacillus species (e.g., Lactobacillus acidophilus), and about 0.1% to about 3% by weight of monosaccharide sources.

[0034] Cosmetic composition containing keratin fermentation broth This disclosure further relates to cosmetic compositions comprising keratin fermentation broth as described elsewhere in this application.

[0035] The composition can be formulated in a variety of suitable forms, including, for example, low to medium viscosity liquids, lotions, milks, mousses, sprays, gels, creams, shampoos, and conditioners. In various embodiments, the composition described herein is formulated as a hair conditioner or shampoo.

[0036] In various embodiments, the compositions described herein may have a pH of about 7 or less, about 6.5 or less, about 6 or less, about 5.5 or less, about 5 or less, about 4.5 or less, about 4 or less, or about 3 or less. In some embodiments, the compositions described herein may have a pH of about 3 to about 7, about 3 to about 6, about 3 to about 5, about 3 to about 4, about 4 to about 7, about 4 to about 6, or about 4 to about 5.

[0037] In various embodiments, the compositions described herein may further contain a solvent. For example, the solvent may include an aqueous solvent (e.g., water). In these and other embodiments, the total amount of keratin fermentation broth in the composition is typically at least about 0.05% by weight and not more than about 20% by weight. In some embodiments, the total amount of keratin fermentation broth may be about 10% by weight or less. For example, the total concentration of keratin fermentation broth may be about 0.05% to about 10% by weight or about 0.1% to about 10% by weight. In some embodiments, the total amount of keratin fermentation broth may be about 0.05% to about 3% by weight or about 0.1% to about 3% by weight. In some specific embodiments, the total amount of keratin fermentation broth may be 0.05% by weight, about 0.1% by weight, about 1% by weight, about 2% by weight, or about 3% by weight.

[0038] The compositions described herein may further contain one or more additives (e.g., cosmetically acceptable ingredients). Examples of cosmetically acceptable ingredients are those listed in the International Cosmetic Ingredient Dictionary and Handbook and the United States Pharmacopeia. Cosmetically acceptable ingredients include, but are not limited to, preservatives, antioxidants, chelating agents, vitamins, dyes, hair colorants, proteins, amino acids, natural extracts such as plant extracts, humectants, fragrances, perfumes, oils, emollients, lubricants, butters, penetrating agents, thickeners, viscosity modifiers, polymers, resins, hair fixatives, film-forming agents, surfactants, cleansing agents, emulsifiers, opacifiers, volatile substances, propellants, liquid vehicles, carriers, salts, pH adjusters, neutralizing agents, buffering agents, hair conditioning agents, antistatic agents, anti-frizzy agents, anti-dandruff agents, absorbents, and combinations thereof.

[0039] For example, surfactants include a variety of anionic, cationic, nonionic, and amphoteric surfactants. Anionic surfactants include, but are not limited to, those containing carboxylate ions, sulfonate ions, and sulfate ions. Examples of anionic surfactants include sodium, potassium, and ammonium long-chain alkyl sulfonates and alkylaryl sulfonates. Cationic surfactants include, but are not limited to, benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyldimethylbenzylammonium chloride, polyoxyethylene, and quaternary ammonium compounds such as coconutamine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbate, polyoxyethylene octylphenyl ether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated beef tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-β-alanine, sodium N-lauryl-β-iminodipropionate, myristoamphoacetate, lauryl betaine, and lauryl sulfobetaine.

[0040] Examples of emollients include silicone compounds, polyols (e.g., propanediol), and triglycerides.

[0041] Examples of emulsifiers include, but are not limited to, copolymers of unsaturated esters and styrene sulfonic acid monomers, cetearyl alcohol, glyceryl esters, polyoxyethylene glycol ether of cetearyl alcohol, stearic acid, polysorbate-20, ceteareth-20, lecithin, glycol stearate, polysorbate-60, polysorbate-80, and combinations thereof.

[0042] Examples of preservatives include, but are not limited to, glycerin-containing compounds, benzyl alcohol, parabens, sodium benzoate, ethylenediaminetetraacetic acid (EDTA), and potassium sorbate. Examples of antioxidants include tocopheryl, BHT, ascorbic acid, Camellia sinensis leaf extract, ascorbyl palmitate, magnesium ascorbyl phosphate, carotenoids, resveratrol, triethyl citrate, arbutin, kojic acid, tetrahexydecyl ascorbate, superoxide dismutase, zinc, sodium disulfite, lycopene, ubiquinone, and combinations thereof.

[0043] Examples of conditioning agents include silicone-based agents, panthenol, hydrolyzed wheat and / or soy protein, amino acids, rice bran wax, meadowfoam seed oil, mango seed oil, grape seed oil, jojoba seed oil, sweet almond oil, hydroxyethyl behenamidopropyl diimonium chloride, aloe leaf extract, aloe vera juice, phytantriol, panthenol, retinyl palmitate, behentrimonium methosulfate, cyclopentasiloxane, quaternium-91, stearamidopropyl dimethylamine, and combinations thereof.

[0044] Examples of viscosity modifiers include viscous liquids such as polyethylene glycol, semi-synthetic polymers, cellulose derivatives, synthetic polymers, naturally occurring polymers, bentonite, colloidal silicon dioxide, and microcrystalline cellulose, as well as salts such as sodium chloride, and combinations thereof.

[0045] Examples of opacifying agents include, but are not limited to, glycol distearate and ethoxylated aliphatic alcohols.

[0046] In some embodiments, the compositions described herein include at least one of the following: viscosity modifiers (e.g., xanthan gum or equivalent), preservatives (e.g., phenoxyethanol), emollients (e.g., propanediol), conditioning agents (e.g., stearamidopropyldimethylamine, behentrimonium methosulfate, and / or sunflower oil), or emulsifiers (e.g., cetearyl alcohol).

[0047] How to use A further aspect of the present invention is a method for treating hair fibers, the method comprising contacting the hair fibers with a hair treatment composition comprising a) keratin and / or hydrolyzed keratin, b) Lactobacillus acidophilus, and c) monosaccharides or fractions or isolates thereof, in a keratin fermentation broth.

[0048] The hair treatment composition may be any keratin fermentation broth or cosmetic composition described herein.

[0049] Hair treatment compositions can be formulated as topical compositions including low-to-medium viscosity liquids, lotions, milks, mousses, sprays, gels, creams, shampoos, conditioners, and the like.

[0050] Hair treatment compositions may be applied directly to the hair or skin, or as part of a kit or series of hair treatments, particularly before, during, or after hair coloring, bleaching, or straightening. Such treatments may be chemical treatments, meaning treatment with oxidizing agents and / or reducing chemicals or enzymes that chemically modify the hair or applied dye. Such hair treatments may be based on applying heat or mechanical force to the hair. The compositions herein may be formulated as leave-in treatments or compositions to be rinsed off after a suitable treatment time.

[0051] Unless otherwise specified, all percentages are based on the total weight of the composition. When multiple compositions are used in a single treatment, the total weight considered is the total weight of all compositions applied to the hair simultaneously (i.e., the weight measured "on the head"), unless otherwise specified. Unless otherwise specified, all ratios are weight ratios.

[0052] As used herein, the terms “hair” and “hair fibers” to be processed may refer to “living” (i.e., on a biological surface) or “non-living” (i.e., wigs, hairpieces, or aggregates of non-living keratin fibers), and in some embodiments, mammalian hair, particularly human hair. However, wool, fur, and other keratin-containing fibers are suitable base materials for the compositions according to the present invention.

[0053] When used in this application, including in the attached claims, the singular forms "a," "an," and "the" are used synonymously with "at least one" and "one or more," including plural references, unless otherwise explicitly indicated by the context. Relative terms such as "about," "substantially," and "approximately" are used to indicate a possible variation of ±10% of the stated number or range.

[0054] The present invention will be further described in the following examples, but these will not limit the scope of the present invention as described in the claims. [Examples]

[0055] The present invention is described in conjunction with its detailed description and accompanying drawings, but the foregoing description is illustrative of the scope of the invention, not limiting it, and it will be understood that the scope of the invention is defined by the scope of the accompanying claims. Other aspects, advantages, and modifications are included in the scope of the following claims.

[0056] Example 1: Fermentation Process To initiate fermentation, 3 kg of hydrolyzed wool keratin (Keraplast, Islington, Christchurch, New Zealand, 10% solution) was added to a glass fermenter and heated to 35°C. Next, 0.4 grams of Lactobacillus acidophilus LA-14 strain (200 billion CFU / g) and 30 grams of edible honey were added. The ingredients were gently mixed and fermented for 3 hours. The mixture was pasteurized and packaged.

[0057] Technicians were provided with 100 ml jars each of Keraplast's CoreTX-pep™—an unfermented hydrolyzed keratin solution and fermented wool keratin—and asked to compare their odors at room temperature. The CoreTX-pep™ product was described as having an animal-like odor. Words such as chicken coop, horse, and cow were used to describe the smell. On the other hand, the fermented wool keratin was described as having a noticeably milder odor, less animal-like, and far more pleasant and tolerable. A dramatic change in odor indicates a substantial chemical change in the material.

[0058] Soluble wool proteins and peptides are highly advantageous for hair treatment, but all processed proteins can produce unpleasant odors from amines and other nitrogen-containing compounds. Amine odors occur when amino acids in proteins are cleaved and decarboxylated, leaving behind various types of volatile organic amines. Unpleasant amine odors are present in hydrolyzed wool proteins and peptides. It is important to note that even trace amounts of amine compounds can cause discomfort in cosmetic formulations. All humans have olfactory receptors for trace amounts of amines, which helps us avoid undesirable sources of volatile amines (e.g., rotten meat, decomposed food, feces). Therefore, producing soluble wool peptides with reduced amine odor is highly advantageous, and it was a surprising result that the materials of the present invention significantly improved or reduced odor, with a distinctly different odor profile and reduced amine odor compared to unfermented wool.

[0059] Example 2: Shampoo formulation Table 1 shows the formulation of the shampoo containing the keratin fermentation broth prepared in Example 1. [Table 1]

[0060] This composition is prepared by slurring guar gum in glycerin and adding it with stirring to the aqueous phase consisting of the remaining components of phase A. This composition is hydrated for 10-15 minutes. Then phase B is added and stirred under low shear until combined. Next, this is heated to 75°C. Next, the combined components of phase C are added to phase A / B at 75°C and stirred thoroughly under low shear until melted and homogenized. After stopping the heating, phase D is added at less than 40°C and the product is mixed until homogenized. Next, phase E is added and stirred until homogenized. The composition is kept below 30°C and the pH is adjusted using phase F.

[0061] Example 3: Conditioner formulation Table 2 shows the formulation of the conditioner containing the keratin fermentation broth prepared in Example 1. [Table 2]

[0062] First, combine the components of Phase 1 and disperse the guar hydroxypropyltrimonium chloride until no lumps remain. Then, heat this to 80°C. Next, add the combined components of Phase B to Phase A and stir under high shear until Phase B melts. Continue stirring while cooling, and at 40°C, add the components of Phase C and stir until homogenized. Next, add Phase D and stir until homogenized. Keep the composition below 30°C and adjust the pH using Phase E.

[0063] Example 4: Formulation of leave-in conditioner Table 3 shows the formulation of the leave-in conditioner containing the keratin fermentation broth prepared in Example 1. [Table 3]

[0064] This composition is prepared by slurring guar gum in propanediol and adding it with stirring to the aqueous phase consisting of the remaining components of phase A. This composition is hydrated for 10-15 minutes. Then phase B is added and stirred under low shear until mixed. Next, phase C is added and stirred until homogeneous. Then phase D is added and stirred until homogeneous, and the pH is adjusted using phase E.

[0065] Example 5: Evaluation of the hydrophobicity of hair Hair hydrophobicity is an important property of hair that protects it from excessive moisture or humidity. A reduction in hair hydrophobicity due to chemical, mechanical, or environmental damage can contribute to frizz, tangles, and / or breakage in humid conditions. Therefore, damaged hair needs to be restored to a more hydrophobic state. The following examples demonstrate that hair treated with a 0.1% keratin fermentation broth simple solution has higher hydrophobicity compared to a control.

[0066] One type II bleached hair swatch was prepared for each treatment as described below.

[0067] 1. Control group (CTR): Non-conditioning shampoo 20% SLES (sodium laureth sulfate),

[0068] 2. Treatment group (TRT): 0.1% keratin fermentation broth simple solution. The components of the 0.1% keratin fermentation broth simple solution are shown in Table 4 below.

[0069] [Table 4]

[0070] Before applying the treatment, hair swatches were washed with a non-conditioning shampoo to remove any residue they may have. 20% SLES was applied at a rate of 0.4 mg per gram of hair swatch. The hair was massaged six times from root to tip, and then thoroughly rinsed with lukewarm water for 60 seconds.

[0071] 0.1% keratin fermentation broth simple solution: Apply 0.2g of cream per gram of hair and spread it over wet hair. Massage a hair swatch from root to tip 6 times.

[0072] After applying the test product to each hair swatch, they were dried for 24 hours in a temperature and humidity controlled chamber (22±2℃, 50±5%RH).

[0073] Dry hair swatches were placed horizontally, with the fibers aligned, and water droplets were placed on different areas of the hair swatches. The test was photographed for all treatments. The droplet angle (immediately after the droplet adheres to the hair surface) and the remaining time (until the droplet is absorbed) were calculated to confirm the formation of a protective film on the hair.

[0074] Three water droplets were placed on a strand of hair, and the contact angle was measured three times. The mean values ​​of the evaluation parameters were compared using a Student's t-test (treated vs. controlled). The confidence level considered was 95% (α=0.05).

[0075] Table 5 shows the statistical results observed in the comparison between treatment with "0.1% keratin fermentation broth simple solution" and the control (CTR).

[0076] [Table 5]

[0077] According to the data shown in Table 5 and the statistical comparisons performed, it was observed that the control group showed no difference in contact angle compared to the treatment with a 0.1% keratin fermentation broth simple solution (Figure 1). However, after treatment with a 0.1% keratin fermentation broth simple solution, the hair took longer to absorb water droplets compared to the control group (Figure 2).

[0078] Therefore, after treatment with a 0.1% simple keratin fermentation broth solution, the droplets remained on the hair longer and were therefore more hydrophobic compared to the control.

[0079] Example 6: Evaluation of fracture resistance Hair damaged by chemical, mechanical, and environmental causes can become more prone to breakage, thus requiring hair treatments to help resist hair breakage. The following examples show that treatment with a 0.1% simple keratin fermentation broth solution exhibits 59% higher breakage resistance, twice that of the control.

[0080] Four natural hair swatches of type V were prepared for each treatment as described below.

[0081] 1. Control group (CTR): Unconditioning shampoo 20% SLES

[0082] 2. Treatment group (TRT): 0.1% keratin fermentation broth simple solution. The components of the 0.1% keratin fermentation broth simple solution are shown in Table 4.

[0083] Before applying the treatment agent, all hair swatches were washed with a non-conditioning shampoo to remove any residue. 0.4 ml / g (hair) was applied to the hair swatches and spread over wet hair. The hair swatches were massaged six times from root to tip and rinsed with running water (5±1 L / min, 35±2℃) for 30 seconds.

[0084] A control group swatch was inserted into an automatic brushing machine BLPA101 and tested: 1000 brushing cycles were performed for 8 times, with the fibers counted at the end of each cycle.

[0085] The remaining four hair swatches were processed as follows:

[0086] 0.1% keratin fermentation broth simple solution: Then, 0.2g of cream per gram of hair was applied and spread over wet hair. A hair swatch was massaged 6 times from root to tip.

[0087] After applying the treatment agent, hair swatches were inserted into an automatic brushing machine (BLPA101) and tested.

[0088] The fewer the number of broken fibers, the higher the break resistance provided by the treatment.

[0089] The average number of broken hairs was compared using a Student's t-test with a 95% confidence interval.

[0090] Equation 1 calculates the percentage of fracture resistance compared to the control. Equation 2 calculates the resistance of hair after treatment application compared to the control. These values ​​are shown in the following sections along with the observed statistical results.

[0091] %=(1-TRT / CTR)×100 Formula 1

[0092] Multiple = (TRT / CTR) -1 formula 2

[0093] Table 6 shows the number of broken fibers and statistical results for each cycle of treatment.

[0094] [Table 6]

[0095] Based on the data shown in Table 6 and the statistical comparisons performed, it was observed that treatment with a 0.1% simple keratin fermentation broth solution resulted in a reduction in hair breakage compared to the control (Figure 3).

[0096] Using equations 1 and 2, the following could be observed:

[0097] 1) After applying a 0.1% simple keratin fermentation broth treatment agent, the hair showed 59% higher resistance to breakage.

[0098] 2) Treatment with a 0.1% simple solution of keratin fermentation broth showed approximately twice the tolerance of the control.

[0099] Therefore, it can be concluded that treatment with a 0.1% simple keratin fermentation broth solution shows 59% higher fracture resistance, twice that of the control.

[0100] Example 7: Differential Scanning Calorimetry Hair integrity, a characteristic related to hair health, can be measured by differential scanning calorimetry (DSC). The following examples demonstrate that treatment with a 0.1% simple solution of keratin fermentation broth increases hair integrity compared to a control.

[0101] One type II bleached hair swatch was selected for each treatment as described below.

[0102] 1. Control group (CTR): Unconditioning shampoo 20% SLES

[0103] 2. Group TRT1: 0.1% keratin fermentation broth simple solution. The components of the 0.1% keratin fermentation broth simple solution are shown in Table 4.

[0104] Before applying the treatment agent, hair swatches were washed with a non-conditioning shampoo to remove any residue they may have. 20% SLES was applied at a rate of 0.4 mg per gram of hair swatch, and the hair was massaged six times from root to tip, followed by thorough rinsing with lukewarm water for 60 seconds.

[0105] Subsequently, the treatment agent was applied as follows.

[0106] Control: 0.4 mg of 20% SLES per gram of hair was applied, the hair was massaged 6 times from root to tip, and then thoroughly rinsed with lukewarm water for 60 seconds.

[0107] 0.1% keratin fermentation broth simple solution: Apply 0.2g of cream per gram of hair and spread it over wet hair. Massage a hair swatch from root to tip 6 times.

[0108] Hair integrity analysis was performed using DSC (Differential Scanning Calorimetry), and the enthalpy of keratin denaturation was obtained and compared between treatments.

[0109] DSC curves for keratin denaturation heat flow and enthalpy were obtained from treated swatches. Three measurements were performed for each treatment, and a summary of the results can be found in the following section. The mean values ​​of keratin denaturation enthalpy were compared using Student's t-test (treatment vs. control). Higher enthalpy indicates higher hair fiber integrity. The confidence level considered was 95% (α=0.05).

[0110] Equation 3 calculates the percentage compared to the control. Equation 4 calculates the multiplier that hair exhibits after treatment application compared to the control. When the treatment differs from the control group, these values ​​are shown along with the observed statistical results in the following sections.

[0111] %=(1-TRT / CTR)×100 Formula 3

[0112] Multiple = (TRT / CTR) -1 formula 4

[0113] Table 7 shows the statistical results observed in the comparison between treatment with a 0.1% simple keratin fermentation broth solution and the control (CTR). [Table 7]

[0114] According to the data shown in Table 7 and the statistical comparisons performed, treatment with a 0.1% simple keratin fermentation broth solution showed an enthalpy of keratin denaturation approximately 42% higher than that of the control (Figures 4-6).

[0115] Therefore, treatment with a 0.1% simple solution of keratin fermentation broth showed higher integrity compared to the control.

[0116] Example 8: Evaluation of gloss Shiny hair is a highly desirable quality among consumers and generally indicates good hair condition. The following examples demonstrate that treatment with a 0.1% keratin fermentation broth simple solution restores shine to hair compared to a negative control, even when both hair samples are contaminated with artificial sebum.

[0117] Six natural type I swatches were isolated for each treatment, as described below.

[0118] 1. Positive control group (CTRP): Unconditioning shampoo (20% SLES).

[0119] 2. Treatment group (TRT): 0.1% keratin fermentation broth simple solution. The components of the 0.1% keratin fermentation broth simple solution are shown in Table 4.

[0120] Since the swatches were first coated with artificial sebum (negative control group - CTRN), they all showed the same level of soiling.

[0121] After the staining process, the swatches underwent their respective coatings as described below.

[0122] CTRP: 4 ml / g (hair) was applied to a swatch and spread over wet hair. Each hair swatch was massaged 6 times from root to tip, and then rinsed with running water (5 ± 1 L / min, 35 ± 2°C) for 30 seconds.

[0123] 0.1% keratin fermentation broth simple solution: Then, 0.2g of cream per gram of hair was applied and spread over wet hair. Each hair swatch was massaged 6 times from root to tip.

[0124] After applying the treatment agent, swatches were photographed in a raster chamber. The images were analyzed using ImageJ®, and the light intensity of each image was quantified.

[0125] Specular and diffuse reflectance were calculated from the quantification of the light intensity profile, and gloss was calculated using these values ​​according to Equation 5.

[0126] B ALL =(D Stamm / S Gaussian )×(I MAX / w 1 / 2 ) Equation 5

[0127] In the formula, B ALL is the gloss data (internally developed) developed internally to correlate with the sensory perception of gloss from the image obtained within the raster chamber, and D Stamm is the diffuse reflection proposed by Stamm and Fochs from the light intensity profile, and S Gaussian is the first specular reflection, and I MAX is the maximum intensity of the profile, and w 1 / 2 is the full width at half maximum of the intensity profile (or the emission bandwidth of the image). For each switch, the gloss was calculated and the average value was compared between the non-conditioning shampoo and the treatment with the intensive shampoo and conditioner.

[0128] Equation 6 calculates the percentage compared to the non-conditioning shampoo. Equation 7 calculates the multiple shown by the hair after application of the treatment agent compared to the non-conditioning shampoo. If the treatment is different from the control group, these numerical values are shown together with the statistical results observed in the following section.

[0129] %=(1-TRT / CTR)×100 Equation 6

[0130] Multiple=(TRT / CTR) -1 Equation 7

[0131] The following section shows the observation results.

[0132] Table 8 shows the statistical results observed in the comparison between the treatment group and the control group.

Table 8

Table 9

[0133] According to the data shown in Table 8 and the statistical comparison shown in Table 9, the following was observed.

[0134] 1) Treatment with a 0.1% simple solution of keratin fermentation broth showed approximately 69% higher gloss than the negative control (CTRN).

[0135] 2) Treatment with a 0.1% simple keratin fermentation broth solution showed no significant difference compared to non-conditioning shampoo (CTRP).

[0136] Compared to the negative control group, shine was significantly increased, and there was no difference between the 0.1% keratin fermentation broth simple solution treatment and the positive control group. Therefore, it can be concluded that the 0.1% keratin fermentation broth simple solution product restores shine to hair (Figure 7).

[0137] Example 9: Visualization of the penetration of specific peptides into hair fibers using a fluorescence microscope. Hair is formed from layers of polymerized keratin protein, and keratin-containing cosmetic formulations are thought to strengthen hair by reinforcing and repairing the keratin structure. However, for these formulations to be successful, keratin that can penetrate the hair fibers is necessary.

[0138] Key points We successfully labeled hydrolyzed keratin peptides in a fermentation-binding serum with a fluorescent probe and visualized their penetration into relaxed hair fibers.

[0139] The fermented serum peptides penetrated the entire cortex of the relaxed-treated hair fibers.

[0140] The penetration of fermentation-bound serum peptides into the cortex of relaxed hair fibers was significantly higher compared to the penetration of high molecular weight peptides (Keratec®, Croda Inc.).

[0141] Keratec® peptide (high MW, Croda Inc.) accumulates only in the cuticle and, rarely, may reach the outermost part of the cortex in certain areas of relaxed-treated hair fibers where the cuticle is most likely to be damaged.

[0142] Materials and methods Base material: 10 mg of relaxed-treated hair fibers (approximately 30 hair fibers).

[0143] product:

[0144] Fermentation-bound serum (Actera Ingredients, Lot No. 0462), approximately 4,500 Da MW). This material was prepared according to the process described in Example 1.

[0145] Keratec® IFP PE (Croda Inc., batch number 22A26B105), approximately 50,000 Da MW.

[0146] sample:

[0147] Control relaxed-treated hair fibers - treated with a control solution containing only purified pigments.

[0148] Relaxed hair fibers treated with labeled fermentation binding serum

[0149] Relaxed treated hair fibers treated with the Keratec® trademark.

[0150] Sample preparation:

[0151] 1. Both products were dialyzed against phosphate buffer (PBS) for at least 16 hours using a dialysis cassette and a floating device. The molecular weight cutoff (MWCO) of the cassette was 2,000 Da. This step ensures the removal of amine-containing substances that may interfere with fluorescent labeling.

[0152] a. Inject 10 mL of the peptide solution into a pre-moistened cassette (Slide-A-Lyzer, Thermo Scientific) according to the manufacturer's protocol.

[0153] b. The dialysis cassette was immersed in 2 liters of fresh PBS at room temperature for 2 hours using a floating device, while gently agitating the buffer.

[0154] c. Replace the buffer with 2 L of fresh PBS and dialyze for another 2 hours at room temperature.

[0155] d. Replace the buffer solution with fresh PBS, place in the refrigerator, and dialyze at 4°C for 16 hours.

[0156] 2. The protein concentration of the dialysis peptide solution is evaluated using a BCA assay (ThermoFisher Scientific).

[0157] a. Determine the protein concentration in the dialysis peptide solution relative to a standard curve of known protein concentrations.

[0158] 3. A peptide solution equivalent to approximately 10 mg of protein was used to perform the fluorescent labeling reaction.

[0159] 4. Optimal fluorescent labeling requires a weakly basic pH solution. Therefore, the pH of the solution was adjusted using 1 M sodium bicarbonate buffer.

[0160] 5. For the fluorescent labeling of the peptides, 5(6)-carboxytetramethylrhodamine N-succinimidyl ester (Sigma Aldrich) was used. This amine-reactive rhodamine dye has an approximate excitation / emission maximum at about 546 / 579 nm, which helps to avoid autofluorescence of hair fibers that can be observed at 350–450 nm.

[0161] 6. A working solution of the dye was prepared in DMSO at a concentration of 10 mg / mL.

[0162] A volume corresponding to 7.1 mg of the dye was slowly added to the dialysis peptide solution while continuously stirring. A control containing only the dye was prepared in the same manner by adding 1 mg of the dye to sodium bicarbonate buffer (without the peptide).

[0163] 8. The conjugation reaction was carried out at room temperature with continuous shaking for 1 hour.

[0164] 9. The labeled peptide solutions were further purified using a centrifuge at 1,000 MWCO for the fermentation-bound serum peptide solution and 10,000 MWCO for the Keratec® peptide solution. This step removes unbound dyes present in the solution and concentrates the labeled peptides.

[0165] a. 5 mL of the labeled peptide solution was loaded into each MWCO centrifugation device (Microsep® Advance Centrifugal Devices, Fisher Scientific, equipped with an Omega membrane).

[0166] b. The centrifuge device was centrifuged at 6,500 × g, 24°C, and for 20 minutes. This step was repeated 15 times, using 1 × PBS for washing after each rotation cycle. After 15 cycles, the flow-through was visibly clear.

[0167] c. The purified labeled peptide solution was resuspended in 10 mL of fresh 1×PBS and used for immersion treatment and infiltration evaluation.

[0168] 10. Approximately 30 relaxed MB hair fibers were bundled together and immersed in 1.5 mL of labeled peptide solution (0.1% labeled peptide solution).

[0169] 11. The hair samples were incubated at room temperature for 24 hours with gentle shaking.

[0170] 12. After 24 hours, the hair strands were removed from the solution, rinsed with deionized water for 20 seconds, and dried with a hairdryer set to a cold setting for 10 seconds.

[0171] 13. Hair fibers were prepared for freeze-sectioning, and 10 μm cross-sections were collected for imaging using a light microscope and a fluorescence microscope.

[0172] Microscopic view of treated hair fibers: fluorescently labeled peptides Bright-field microscopy and fluorescence imaging were collected using an Eclipse TE2000-U microscope (Nikon Instruments Inc.) equipped with a Kiralux compact scientific camera (ThorLabs Inc.), a Prior Lumen 200 Fluorescence Illumination System, and a Nikon DS-Qi2 monochrome camera. Cross-sections (10 μm) of 15–20 hairs were analyzed at 20x and 100x magnification. Fluorescence images were acquired using NIS Elements software with a red channel (CY3 filter, excitation: 560 / 40 nm, emission: 630 / 75 nm) at 20x and 100x magnification, with exposure times of 10 ms and 5 ms for the red channel (CY3) and 50 ms for the green channel, respectively. Spontaneous fluorescence of hair fibers was recorded in the green channel. Labeled peptides, on the other hand, fluoresced in the red channel.

[0173] In addition to fluorescence microscopy, semi-quantitative total fluorescence analysis was performed. This data is reported in bar graph form.

[0174] result After dialysis, the peptide concentration relative to a standard curve of known concentrations was determined using a BCA protein quantification assay. This step was performed before fluorescent labeling to calculate the volume required for 10 mg of "protein" for properly controlled labeling.

[0175] Control results

[0176] Representative images of each sample and treatment are provided in this report, and the images were collected using the same parameters for all groups. In this experiment, a control containing only the dye was purified in parallel with the labeled peptide solution, and its permeability was evaluated. As expected, no fluorescence was detected in the red channel of the control containing only the dye after centrifugal purification using a centrifugal column device (Figures 8A-8B). This result confirms the effectiveness of the washing protocol for removing free / unbound dye from the sample solution.

[0177] Results of fermentation-binding serum penetration

[0178] After confirming that no free pigment remained in the solution according to the washing protocol, fluorescence in the red channel could be attributed solely to the labeled peptide, providing a direct evaluation of peptide penetration into the hair fibers. The fermentation-bound serum peptide penetrated throughout the cortex of the relaxed-treated hair fibers. Strong and uniform fluorescence intensity was observed across cross-sections of all hair evaluated in this project, and the peptide could be identified throughout the interior of the cortex (Figures 9A-9B).

[0179] Keratec (trademark) penetration results

[0180] Unlike fermentation-bound serums, Keratec® concentrates only in the cuticle of hair fibers, with minimal penetration into the outermost region of the cortex observed in one or two cross-sections (Figures 10A-10B). The manufacturer specified that Keratec® contains hydrolyzed keratin peptides in the molecular weight range of 40,000–60,000 daltons. However, due to the manufacturing process, Croda Inc. warns that peptides in the molecular weight range of 3,000–4,000 daltons are present. The centrifugation device selected to purify labeled Keratec® had a molecular weight cutoff of 10,000 Da to mitigate this potential discrepancy. Labeled peptides above 10,000 Da MW will concentrate, while all lower ones should be washed away.

[0181] The substrate used was relaxed-treated hair. This means that the cuticle can be damaged by chemical treatment. A possible explanation for the outer cortical penetration region of this high-MW peptide is that the cuticle is sufficiently damaged in those regions, allowing some penetration of Keratec®.

[0182] Total fluorescence analysis as a semi-quantitative evaluation of peptide penetration into hair fibers. Figure 11 shows a semi-quantitative analysis of the total fluorescence intensity of hair cross-sections after 24 hours of treatment with labeled peptides. Values ​​were corrected for the image background. Results are reported as mean ± standard deviation. The control refers to hair that was purified and then immersed in a solution of only the pigment for 24 hours. The corrected total fluorescence values ​​corresponding to the penetration of the fermentation-bound serum were significantly higher than those of Keratec®, further supporting the increased penetration of the fermentation-bound serum peptide into the hair cortex observed after 24 hours of treatment. [Table 10]

[0183] Conclusion: We successfully labeled hydrolyzed keratin peptides in a fermentation-binding serum with a fluorescent probe and visualized their penetration into relaxed hair fibers.

[0184] The fermented serum peptides penetrated the entire cortex of the relaxed-treated hair fibers.

[0185] The penetration of fermentation-bound serum peptides into the cortex of relaxed hair fibers was significantly higher compared to the penetration of high molecular weight peptides (Keratec®, Croda Inc.).

[0186] Keratec® peptide (high MW, Croda Inc.) accumulates only in the cuticle and, rarely, may reach the outermost part of the cortex in certain areas of relaxed-treated hair fibers where the cuticle is most likely to be damaged.

[0187] Example 10: Odor analysis using VDI3882 test methodology

[0188] Sample preparation for sensory analysis (VDI3882 and characterization)

[0189] Half a milliliter (0.5 ml) of each sample (CoreTX-Pep® - unfermented hydrolyzed keratin control, or fermented binding serum - fermented hydrolyzed keratin prepared according to the process described in Example 1) was placed in a 250 ml vial and heated at 35°C for 24 hours to promote the release of odor substances. The samples were then presented to panelists for sensory evaluation.

[0190] In the evaluation tests for odor intensity, palatability, and properties, panelists directly evaluated the samples at room temperature. Samples were presented to individuals in a blinded manner in sets of three, and each panelist evaluated three different parts of the same sample.

[0191] Analysis of odor intensity, palatability, and characteristics.

[0192] The intensity, palatability (characteristics related to the pleasantness or unpleasantness of the odor), and properties of the samples were analyzed. A panel of six members was formed. The panelists were calibrated according to the UNE-EN 13725:2004 “Air Quality - Determination of odor concentration by dynamic olfactometry” standard and trained based on the German standard VDI3882.

[0193] To determine the potential for unpleasantness of a particular odor, both intensity and preference parameters were evaluated independently. These parameters were assessed based on a predefined scale, as established in the VDI3882 criteria. For the intensity parameter, the evaluation scale ranged from a value of 0 (imperceptible) to a value of 6 (very strong), with an intermediate value of 3 representing a distinguishable intensity (Table 11). [Table 11]

[0194] Regarding palatability, the scale ranges from a negative value of -4 (extremely unpleasant) to a positive value of +4 (extremely pleasant), with a value of 0 representing a situation where the odor is neither pleasant nor unpleasant (Table 12). This classification allows for direct comparison of samples during testing by directly inhaling the odor emitted by each sample. [Table 12]

[0195] These tests determined whether the odors emitted by various samples produced the perception of odors that panel members considered relevant / low intensity and / or (unpleasant) pleasant. Furthermore, these tests enabled the definition of sample-specific properties and the identification of similarities or differences that may be related to VOCs as determined by GC-sniffing MS.

[0196] result

[0197] Sensory evaluation of odor intensity and palatability

[0198] A group of six calibrated and trained panelists evaluated the odor intensity and pleasantness / unpleasantness level of each sample by direct sniffing. This assay was performed according to the German standard VDI3882. In addition to the measurements mentioned, the same panelists also described the odor characteristics produced by each sample during the assay. The panelists were provided with a guide of odor descriptors for each sample related to the sample's characteristics. The evaluation was performed in triplicate. A summary of these results is shown in Table 13 and in Figures 12 and 13. [Table 13]

[0199] After heating at 35°C, the odor intensity of both samples was similar, with a score of nearly 3.5, rated as "distinct to strong." Regarding palatability, both samples had a value close to -1.5 (standard deviation less than 1), indicating a significant difference in odor attributes, rated as "somewhat unpleasant to unpleasant." The main odor characteristics of the unfermented hydrolyzed keratin control sample were described as fermented / rotten, pungent / sour, and toasty / cereal, while the main odor characteristics of the fermented bound serum were described as animalistic, phenolic, and hay / dried herb.

[0200] Student's t-test was used to assess the statistical significance of paired comparisons between samples in relation to scores provided by the sensory panel. The p-values ​​were determined and used for hypothesis testing (n<20). The samples were considered significantly different with α=0.05 (two-sided). Differences in intensity and palatability between samples when heated to 35°C were not statistically significant (p-value 0.16 for intensity, p-value 0.18 for palatability).

[0201] Student's t-test revealed no significant differences between samples in terms of both intensity and palatability (p>0.05).

[0202] General perception of smell

[0203] To better represent the information, individual odors perceived during the analysis were grouped according to a more general description, thus obtaining six new categories. The general intensity values ​​were obtained by summing the intensity values ​​of all odors belonging to the new categories. A summary of this information is shown in Table 14. Table 14 represents an attempt to simplify the sensory information obtained for each sample in order to provide an evaluation of the general sensory profile. Note the differences between samples and the sensory characteristics where the differences are more pronounced. Figure 14 shows the information shown in Table 14 in a graph (spider chart). [Table 14]

[0204] The most prominent odor categories differed significantly between the two samples: the unfermented hydrolyzed keratin control was reported as fermented / rotten, pungent / sour, and toasty / cereal, while the fermented bound serum was reported as animalistic, phenolic, and hay / dried herb.

[0205] Example 11: GC-ToF-MS test methodology

[0206] Sample preparation for GC-ToF-MS analysis

[0207] Sample preparation for high-resolution GC-ToF-MS analysis involved adding 2 ml of sample material (CoreTX-pep(trademark) - unfermented hydrolyzed keratin control, or fermented bound serum - fermented hydrolyzed keratin) and introducing it into separate 114 ml microchambers. After closing, the microchamber conditions were adjusted to 35°C to maintain each sample at this temperature to promote the release of volatile substances. This simulated the application conditions of the sample in the final product. Next, two adsorption tubes (Tenax / carbograph) were attached to the top lids of each microchamber, and the volatile substances released by each sample were collected in the gas within the tubes, with a volume of 1000 ml per sample. The tubes were then sealed with caps.

[0208] High resolution GC-ToF-MS analysis

[0209] After collecting VOCs in adsorption tubes, the tubes were inserted into a thermal desorption unit connected to a GC-(TOF)MS. The instrumentation consisted of a gas chromatograph (Agilent 7890, USA), a mass spectrometer with a time-of-flight analyzer (BenchTOF-dx, Almsco, Germany), and a thermal desorption unit (Unity2, Markes, UK). The amount of desorption from the tubes was optimized according to a specific temperature program to ensure good analytical resolution.

[0210] After removal from the tube by thermal desorption, the volatile compounds were trapped in a cold trap at low temperatures (-20°C to 10°C) by thermoelectric cooling. Subsequently, the cold trap was heated to 300°C to 350°C according to a programmed temperature profile to release all volatile substances to the GC for subsequent chromatographic separation. At the end of the GC column, once separated, the compounds reached the mass detector with different retention times, where they were ionized. A time-of-flight (TOF) selector allowed for high-precision determination of the ion mass, enabling compound identification.

[0211] Next, the data provided by each sample was analyzed. This analysis was partially automated using advanced software and compound libraries. System calibration was performed in-house using a wide range of compounds. The analysis in this test was performed semi-quantitatively using toluene-d8 as a reference solution to prioritize full-scan quantification of all VOCs present in the sample.

[0212] result

[0213] High resolution GC-ToF-MS analysis

[0214] The unfermented hydrolyzed keratin control showed nearly twice the number of VOCs as the fermented conjugated serum. A total of 67 VOCs were identified in the tested samples: unfermented hydrolyzed keratin control (CoreTX-Pep®) (55), fermented hydrolyzed keratin (fermented conjugated serum) (28).

[0215] Comparative VOC composition by chemical group

[0216] Table 15 summarizes the VOC concentrations measured in the samples based on the total concentration for each chemical group of compounds. The information provided in Table 15 is graphically represented in Figure 15. Total VOC concentration in unfermented hydrolyzed keratin control (4378.6 μg / m³) 3The ) level was higher than in the fermentation-bound serum sample (824.4 μg / m3). The main family of alcohols in the samples was alcohol, accounting for 77% in the unfermented hydrolyzed keratin control and 64% in the fermentation-bound serum. Some of the main alcohols included ethanol, 2-phenoxy-(control), isopropyl alcohol, and linalool (fermentation-bound serum).

[0217] As shown in Example 1 above, even trace amounts of amine compounds can cause discomfort in cosmetic formulations. No nitrogen-containing compounds were detected in the fermented binding serum, while 32.0 μg / m² was detected in the unfermented hydrolyzed keratin control. 3 The fact that it was detected was a surprising result. [Table 15]

[0218] Related odor substances identified by GC-ToF-MS

[0219] To understand the "weight" of odor perception, odor activity (OAV) is commonly used. This is calculated by dividing the abundance (concentration) of the odor molecule by its odor threshold (OTV). The odor threshold is understood as the minimum concentration of a particular odorous compound that can be detected by the human sense of smell. In reality, this value is variable because human olfactory sensitivity varies greatly. This uncertainty is addressed by making the OTV traceable against an agreed-upon reference stimulus, as outlined in the European standard for odor measurement EN13725:2003.

[0220] The compounds with the highest OAV values ​​in the unfermented hydrolyzed keratin control were phenol, 2-methoxy- and butanal, 3-methyl-, and their OAV values ​​were considered distinct. These compounds are typically understood to be associated with odor characteristics such as aldehyde, oily, nutty, phenolic, and medicinal. In contrast, the compounds most associated with the fermented conjugated serum sample were linalool and butanal, 3-methyl-, and their OAV values ​​were considered distinct. These compounds are typically understood to be associated with odor characteristics such as aldehyde, oily, bergamot, and floral (Table 16). [Table 16-1] [Table 16-2]

[0221] In Table 16, the concentrations in bold exceed the odor threshold (OTV). The concentrations in italics are 0.01 μg / m³. 3 The values ​​did not exceed the specified range. Values ​​marked with * (1-15) indicate a very weak, almost imperceptible odor. Values ​​marked with ** (15-500) indicate a clear, easily recognizable, but not strong odor. Values ​​between 500 and 5000 indicate a moderate to strong odor. Values ​​between 5000 and 50,000 indicate a very strong odor. Values ​​above 50,000 indicate an extremely strong odor. The concentration of acetaldehyde could not be accurately measured.

Claims

1. It is keratin fermented broth, a) Keratin and / or hydrolyzed keratin, b) Lactobacillus species, and c) monosaccharide; The keratin fermentation broth comprising the fraction or isolate thereof.

2. The keratin fermentation broth according to claim 1, wherein the monosaccharide is provided by honey, and optionally the keratin fermentation broth contains less than 5% by weight or less than 3% by weight of the monosaccharide.

3. The keratin fermentation broth according to claim 1 or 2, wherein the keratin and / or hydrolyzed keratin is extracted from wool, and the keratin fermentation broth contains more than about 5% by weight, more than about 8% by weight, or more than about 10% by weight of keratin and / or hydrolyzed keratin.

4. The keratin fermentation broth according to any one of claims 1 to 3, wherein the Lactobacillus species is Lactobacillus acidophilus.

5. Keratin fermentation broth according to any one of claims 1 to 4, wherein the pH is approximately 7 or less, approximately 6.5 or less, approximately 6 or less, approximately 5.5 or less, approximately 5 or less, approximately 4.5 or less, approximately 4 or less, or approximately 3 or less.

6. Keratin fermentation broth according to any one of claims 1 to 4, wherein the pH is approximately 3 to approximately 7, approximately 3 to approximately 6, approximately 3 to approximately 5, approximately 3 to approximately 4, approximately 4 to approximately 7, approximately 4 to approximately 6, or approximately 4 to approximately 5.

7. The keratin fermentation broth according to any one of claims 1 to 6, wherein the keratin fermentation broth comprises aqueous fermentation broth.

8. The keratin fermentation broth according to any one of claims 1 to 6, wherein the keratin fermentation broth includes dried fermentation broth.

9. The keratin fermentation broth according to any one of claims 1 to 8, wherein the keratin and / or hydrolyzed keratin has an average molecular weight of less than 10 kDa, for example, about 1 kDa to 10 kDa.

10. The keratin fermentation broth according to any one of claims 1 to 9, wherein the keratin and / or hydrolyzed keratin has an average molecular weight of about 2.5 kDa or about 4.5 kDa.

11. A cosmetic composition comprising keratin fermentation broth according to any one of claims 1 to 10, or a fraction or isolate thereof.

12. The cosmetic composition according to claim 11, wherein the composition is formulated as a lotion, milk, mousse, spray, gel, cream, shampoo, or conditioner.

13. The cosmetic composition according to claim 11 or 12, wherein the concentration of the keratin fermentation broth is about 0.05% by weight to about 20% by weight.

14. The cosmetic composition according to claim 11 or 12, wherein the concentration of the keratin fermentation broth is about 10% by weight or less.

15. The cosmetic composition according to claim 11 or 12, wherein the concentration of the keratin fermentation broth is about 0.05% by weight to about 10% by weight.

16. The cosmetic composition according to claim 11 or 12, wherein the concentration of the keratin fermentation broth is about 0.1% by weight to about 10% by weight.

17. The cosmetic composition according to claim 11 or 12, wherein the concentration of the keratin fermentation broth is about 0.05% by weight to about 3% by weight.

18. The cosmetic composition according to claim 11 or 12, wherein the concentration of the keratin fermentation broth is about 0.1% by weight to about 3% by weight.

19. The cosmetic composition according to claim 11 or 12, wherein the concentration of the keratin fermentation broth is about 0.05% by weight, 0.1% by weight, about 1% by weight, about 2% by weight, or about 3% by weight.

20. A cosmetic composition according to any one of claims 11 to 19, wherein the pH is approximately 7 or less, approximately 6.5 or less, approximately 6 or less, approximately 5.5 or less, approximately 5 or less, approximately 4.5 or less, approximately 4 or less, or approximately 3 or less.

21. A cosmetic composition according to any one of claims 11 to 20, wherein the pH is approximately 3 to approximately 7, approximately 3 to approximately 6, approximately 3 to approximately 5, approximately 3 to approximately 4, approximately 4 to approximately 7, approximately 4 to approximately 6, or approximately 4 to approximately 5.

22. The cosmetic composition according to any one of claims 11 to 21, wherein the composition further comprises at least one additive selected from the group consisting of surfactants, vitamins, natural extracts, preservatives, chelating agents, fragrances (perfume), preservatives, antioxidants, proteins, amino acids, humectants, fragrances (fragrance), emollients, penetrating agents, thickeners, viscosity modifiers, hair fixing agents, film-forming agents, emulsifiers, opacifiers, propellants, liquid vehicles, carriers, salts, pH adjusters, neutralizing agents, buffering agents, hair conditioning agents, antistatic agents, anti-friction agents, anti-dandruff agents, and combinations thereof.

23. A method for treating hair fibers, wherein the method comprises contacting the hair fibers with a cosmetic composition according to any one of claims 11 to 22.

24. The method according to claim 23, wherein the cosmetic composition is a lotion, milk, mousse, spray, gel, cream, shampoo, or conditioner, and optionally the composition is a leave-in hair treatment composition.

25. A method for producing keratin fermentation broth, wherein the method is a) Combining keratin and / or hydrolyzed keratin, Lactobacillus acidophilus, and monosaccharides in an aqueous fermentation solution, b) The method comprising fermenting the aqueous fermentation solution at a temperature of about 2°C to about 53°C and a pH of about 4.5 to about 6.5 to produce the keratin fermentation broth.

26. The method according to claim 25, wherein the aqueous fermentation solution is fermented for about 1 to about 12 hours.

27. The method according to claim 25 or 26, wherein step b) is carried out at a temperature of approximately 30°C to approximately 40°C.

28. The method according to any one of claims 25 to 27, wherein step b) is carried out at a pH of approximately 5.5 to approximately 6.

2.

29. Keratin fermentation products produced by exposing keratin and / or hydrolyzed keratin to the fermentation of monosaccharides by the Lactobacillus species.

30. A product comprising keratin and / or hydrolyzed keratin exposed to monosaccharide fermentation by the Lactobacillus species.

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