PROCESS FOR OBTAINING KANPHEROL FROM AGRO-INDUSTRIAL RESIDUES OF AGAVE SISALANA

Description

/ 18 PROCESS FOR OBTAINING KANPHEROL FROM AGRO-INDUSTRIAL RESIDUES OF Agave sisalana Field of invention

[001] The present invention relates to the technical field of processes for obtaining kaempferol-rich extract from the agro-industrial residue of Agave sisalana. This natural compound is known for its bioactive properties, making it of great interest as an input for the chemical and pharmaceutical industries.

[002] More specifically, this invention relates to a process that uses sequential separation methods to obtain kaempferol-rich extract, such as: fermentation, macroporous resin and liquid-liquid extraction. Fundamentals of the invention

[003] Flavonoids are natural products that are of great importance to the industrial production chain due to their wide range of biological activities, thus enabling their transformation into high value-added commercial products.

[004] The extraction and purification of flavonoids from plant sources faces significant challenges, such as the use of toxic solvents, low yield and high process cost.

[005] Agave sisalana is a natural source for fiber production. In this process, after the leaves are defibered, agro-industrial sisal residue is generated, which corresponds to about 95% of the total weight of the original material. It is estimated that, for the production of sisal fiber, about millions of metric tons of this residue are discarded into the environment annually.

[006] Scientific literature reports the presence of several bioactive compounds in sisal agro-industrial residue, among them kaempferol. This compound, belonging to the flavonoid class, is of great industrial importance due to the difficulty in obtaining it, which is reflected in its high market value. Recognized for its Petition 870250058734, dated 10 / 07 / 2025, page 8 / 42 / 18 proven biological activities, such as antioxidant, immunomodulatory, anthelmintic, antitumor and antimicrobial properties, kaempferol has significant therapeutic potential for the prevention and treatment of various diseases.

[007] Considering that A. sisalana residue is a material produced abundantly and frequently discarded, with low economic value and containing kaempferol, it is necessary to describe a protocol for obtaining kaempferol-rich extract from sisal residue, for its supply as an input in various sectors of industry.

[008] In the case of sisal residue, obtaining the kaempferol-rich extract is hampered by the high production of sugars by the plant matrix. Fermentation emerges as an economically viable and low-environmental-impact technique that can be employed as a strategic solution in this context. This approach is based on the digestion of sugars by microorganisms. This process not only increases the concentration of kaempferol, but also functions as an initial clean-up step, simplifying subsequent purification steps.

[009] The use of macroporous resins as a subsequent step to fermentation increases the efficiency in recovering enriched kaempferol from sisal residue. Through the adsorption process, the resin selectively removes residual sugars and other phytochemical compounds present in the sisal matrix, resulting in an extract enriched in kaempferol.

[0010] Macroporous resins are designed to make the purification process more efficient, combining economic viability with technical robustness. This synthetic polymer adsorbent stands out for its excellent mechanical properties, polarity-based selectivity, well-defined porous structure, and large surface area, characteristics that make it highly effective in the separation and concentration of bioactive compounds.

[0011] Due to the combination of high recovery capacity and low operating cost, macroporous resins are consolidating themselves as the most efficient solution for the large-scale purification of flavonoids. Petition 870250058734, dated 10 / 07 / 2025, page 9 / 42 / 18

[0012] The development of this invention not only enhances low-cost purification techniques, but also encourages the sustainable use of by-products from the Brazilian semi-arid region, highlighting the economic and biotechnological potential of this region for the pharmaceutical industry.

[0013] The scientific literature reports that the fermentation process presents favorable results for obtaining flavonoids, such as: Lin, S., et al. Production of quercetin, kaempferol and their glycosidic derivatives from the aqueous-organic extracted residue of litchi pericarp with Aspergillus awamori. Food Chemistry. 145, 2014; Ajila, CM, et al. Solid-state fermentation of apple pomace using Phanerocheate chrysosporium release and extraction of phenolic antioxidants. Food Chemistry. 126, 1071-1080, 2011; Gulsunoglu, Z., et al. Enhancement of phenolic antioxidants in industrial apple waste by fermentation with Aspergillus spp. Biocatalysis and Agricultural Biotechnology, 25, 101562, 2020. These publications demonstrated a significant increase in the levels of non-glycosylated phenolics (aglycones) from agro-industrial residues, through the process of consuming the sugars linked to the respective phenolics via fermentation.

[0014] However, there are no records in the scientific literature on the enrichment of phenolic derivatives, especially kaempferol, in A. sisalana through fermentation. Most research related to fermentation in Agave sp. plants has focused on the traditional production of mezcal and ethanol, especially in Agave tequilana and Agave salmiana (see Santiago-Gómez, et al. Ethanol production from Agave salmiana leaves by semi and simultaneous saccharification and fermentation at high temperature using Kluyveromyces marxianus. Biocatalysis and Agricultural Biotechnology, 50, 102703, 2023).

[0015] Patent documents involving the species A. sisalana and fermentation include the following numbers: BR102023020458-9, BR102018014447-2, BR102018005222-5, BR102017027916-2 and BR102017024444-0.

[0016] Patent application BR102023020458-9 describes an agricultural nematicide obtained from the liquid residue of A. sisalana, produced during the defibration of the leaves. This residue, after pressing and removal of the solid part, Petition 870250058734, dated 10 / 07 / 2025, page 10 / 42 / 18 maintains its nematicidal activity even after natural fermentation. The final product is a wettable powder stable at room temperature, effective against various plant-parasitic nematodes.

[0017] Patent application BR102018014447-2 describes the production of xylitol and arabitol from the lignocellulosic biomass of A. sisalana by means of yeast fermentation. The solutions can be hydrolyzed before fermentation, and after the process, the yeast cells are separated and the pH adjusted. The solution with the sweeteners is recovered and crystallized, resulting in crystalline xylitol and arabitol.

[0018] Patent application BR102018005222-5 describes the production of arabitol from A. sisalana biomass using biotechnological fermentation with yeasts of the genera Candida and Debaryomyces. The solutions can be hydrolyzed before fermentation. After fermentation, the yeast cells are separated, and the arabitol solution is recovered and crystallized.

[0019] Patent application BR102017027916-2 describes the production of ethanol and xylitol from sisal fiber and bagasse (A. sisalana), which are rich in cellulose and hemicellulose. The process includes a chemical pretreatment with 2.5% diluted sulfuric acid at 120°C for 1 hour, followed by fermentation of the resulting liquor using Candida tropicalis yeast.

[0020] Patent application BR102017024444-0 describes obtaining activated carbon from sisal bagasse to minimize environmental impacts and reuse waste. The bagasse is dried, carbonized at 550°C, activated with steam, and treated with distilled water. The resulting activated carbon, with high surface area and porosity, is effective in adsorbing fermentation inhibitors, such as hydroxymethylfurfural and furfural, improving the viability of fermentation processes.

[0021] Patents listed internationally as GB907025A, WO8505126A1, CN113943771A describe methods for obtaining sapogenins from A. sisalana using fermentation processes. However, no results were found in searches for methods of obtaining flavonoids through fermentation using this same matrix. Petition 870250058734, dated 10 / 07 / 2025, page 11 / 42 / 18

[0022] GB907025A describes a process for obtaining hecogenin from the fermented juice of A. sisalana. After fermentation, the sediment is separated from the liquid and dried to a solids content of 90-100%. Then, the sediment is heated to 80-150°C, preferably 110-120°C, for 8 to 15 hours. Finally, the sediment is converted to hecogenin by known methods.

[0023] WO8505126A1 describes the process for producing hecogenin from the anaerobic fermentation of sisal juice. The process maintains a controlled pH in an anaerobic environment at 37°C, with agitation at 50-70 rpm for one week. Sapogenins, such as hecogenin and tigogenin, are purified by crystallization using a solvent of ethyl acetate and cyclohexane (1:1). The process also generates useful secondary compounds such as ethanol, acetic acid, cells, CO2, H2, and methane.

[0024] CN113943771A The invention describes a method for preparing tigogenin through the fermentation of A. sisalana. The process includes inoculating a culture medium with filamentous fungi and, after fermentation, filtering to obtain crude tigogenin hydrolase; mixing the crude hydrolase with sisal residue or juice in a liquid culture medium for fermentation. After fermentation, the liquor is decanted, centrifuged, and the precipitates are collected, dried, and ground to obtain crude tigogenin.

[0025] In contrast, patents listed internationally as CN115404242A, CN116473223A and CN113801084A, among others, utilize fermentation to obtain better yields in the extraction of phenolic compounds from different agro-industrial residues.

[0026] CN115404242A describes an environmentally friendly method for producing flavonoids from agricultural waste, specifically orange peels. The process involves depulping and sterilizing the peel to obtain pulp, followed by aerobic fermentation with a mixture of Aspergillus niger, Corynespora aspera, and Lactobacillus fermentum in equal proportions.

[0027] CN116473223A describes a method for increasing the value of Morinda citrifolia fermentation residues by focusing on flavonoid production. The process Petition 870250058734, dated 10 / 07 / 2025, p. 12 / 42 / 18 involves sterilizing fresh fruit, mixing the pulp with fermentation residue, and adding a yeast extract for fermentation.

[0028] CN113801084A describes the extraction of polymethoxylated flavonoids from the bottom sludge of orange vinegar fermentation. The method involves using an organic solvent to extract the sludge, centrifuging, collecting the organic phases, and concentrating under reduced pressure to obtain the flavonoids.

[0029] Therefore, given what has been described above, the objective of this invention is to provide an economically viable process using green chemistry resources to obtain kaempferol from the agro-industrial residue of A. sisalana.

[0030] The present invention addresses the technical problem, unsolved by the prior art, of efficiently extracting kaempferol from the agro-industrial residue of A. sisalana using fermentation followed by macroporous resin and other chromatographic techniques. Unlike existing patents that focus on the extraction of sapogenins, this innovative method aims to overcome the limitations of conventional kaempferol extraction, offering a more efficient and environmentally sustainable process. This provides significant advantages, such as higher yield and reduced environmental impact, enhancing applications in the pharmaceutical, nutraceutical, and cosmetic industries.

[0031] The agro-industrial residue of Agave sisalana represents a source of high commercial value, particularly for sectors such as pharmaceuticals and cosmetics, due to the presence of kaempferol, which exhibits various biological activities. However, the efficient extraction of this compound faces considerable challenges, especially due to the non-extractable form in which this compound is found (glycosylated kaempferol). The conjugation of kaempferol reduces the extraction yield, making the process commercially unviable for large-scale production.

[0032] Attempts to overcome this barrier include fermentation, which helps to release kaempferol from its sugars, followed by the use of macroporous resins to obtain enriched extracts and, finally, the application of conventional and unconventional phytochemical techniques for kaempferol purification. Petition 870250058734, dated 10 / 07 / 2025, page 13 / 42 / 18

[0033] The invention provides a significant increase in yield, as the combination of fermentation with macroporous resins allows the flavonoid to be obtained in free form (aglycone) with a yield far superior to that of conventional methods, which cannot achieve this efficiency.

[0034] Furthermore, the method is highly sustainable, as it adopts the principles of green chemistry, minimizing the use of toxic solvents and promoting the reuse of agro-industrial waste.

[0035] Another advantage is its applicability on an industrial scale: by using a low-cost purification process with macroporous resins, the method achieves commercial viability that meets industrial demands. Demonstrating that the invention has advantages over the prior art. Brief description of the drawings

[0036] Figure 1 presents the results of monitoring representative aliquots from the preliminary fermentation assay at fermentation times (0, 96, 144 and 192 hours), analyzed by thin-layer chromatography (TLC) after extraction with ethyl acetate from the liquid sisal residue, in co-elution with the analytical standard kaempferol.

[0037] Figure 2 shows the chromatogram obtained by High Performance Liquid Chromatography coupled to a Diode Array Detector (HPLC-DAD) of the analyses of the crude extract fermented for extended periods, corresponding to fermentation times of 24, 240, 480 and 720 hours.

[0038] Figure 3 shows the HPLC-DAD chromatogram of the kaempferol-enriched extract, obtained by purification with macroporous resin as described in step “e”, performed at a wavelength of 360 nm, accompanied by the respective absorption spectrum in the ultraviolet region.

[0039] Figure 4 shows the HPLC-DAD chromatogram of the kaempferol analytical standard, with a peak at a retention time of 22.4 minutes, accompanied by its Petition 870250058734, dated 10 / 07 / 2025, page 14 / 42 / 18 respective UV spectrum (absorption maxima at 265 nm and 366 nm) serving as a reference for confirming the identity of the compound in the analyzed samples.

[0040] Figure 5 shows the HPLC-DAD chromatogram of the kaempferol-enriched extract, obtained by purification in liquid-liquid extraction as described in step “e”, performed at a wavelength of 360 nm, accompanied by the respective absorption spectrum in the ultraviolet region. Description of the invention

[0041] In this description, the term “may” is used to indicate the possibility of carrying out a specific treatment or not. Similarly, the terms optionally or optionally indicate that subsequent events or circumstances may or may not occur, including both cases where these events occur and those where they do not.

[0042] The expressions "some embodiments," "other embodiments," "particular / preferred technical solutions," and the like, refer to the fact that a given element (such as a feature, structure, property, and / or function) described in relation to one embodiment is present in at least one of the embodiments described in this document. This element may or may not be present in other embodiments and, moreover, may be appropriately combined with other elements in different embodiments of the invention.

[0043] The present invention describes the process for obtaining a kaempferol-rich extract from the agro-industrial residue of Agave sisalana, characterized by comprising the following steps: a. Collect the residual liquid material resulting from the defibration of Agave sisalana leaves. b. Perform an initial filtration of the liquid residue obtained in step a, and then carry out a fermentation; c. Perform a second filtration of the material obtained in step b; Petition 870250058734, dated 10 / 07 / 2025, page 15 / 42 / 18 d. Centrifuge the material obtained in step c; e. Obtain the kaempferol-rich extract using macropolysaccharides or by liquid-liquid extraction; f. Remove the solvent from the kaempferol-rich extract obtained in step e.

[0044] In one embodiment of the present invention, the liquid residue of A. sisalana is used in the fermentation process. More specifically, step b involves a mesh filtration process, and subsequently the filtered material is subjected to a fermentation process with a controlled temperature between 20°C and 40°C. Fermentation can be promoted by the addition of yeasts, among which Saccharomyces cerevisiae stands out, in concentrations ranging from 1% to 20% in relation to the total volume of the residual material. Alternatively, the process can occur by self-fermentation, without the need for the addition of exogenous yeasts. The fermentation period varies between 36 and 720 hours, and can be carried out with or without agitation, adjusted between 0 and 300 rpm. The process can occur under anaerobic conditions, employing a system that prevents gas exchange with the external environment, or under aerobic conditions, using a closed system that allows gas exchange with the external environment.

[0045] The post-fermentation steps (ced steps) consist of filtration and centrifugation processes. Centrifugation is carried out at a speed of 2000 to 3800 rpm, and the resulting supernatant is then subjected to filtration. In the filtration process, a filter with a porosity between 8 and 45 µm is used. The material resulting from this process is referred to as treated fermentate throughout the description.

[0046] The present invention describes a process for obtaining a kaempferol-enriched extract, in which the fermented material undergoes a specific treatment for the extraction of this compound.

[0047] In one embodiment of the invention, the kaempferol-rich extract is obtained through adsorption and desorption on macroporous resins, as described in step e. Different macroporous resins may be used in this process, varying in polarity (low, medium and high), with the temperature adjusted between 20°C and 40°C. The Petition 870250058734, dated 10 / 07 / 2025, page 16 / 42 / 18 The eluent used for desorption is a mixture of water and ethanol, with the ethanol proportion varying between 10% and 100%. The sample concentration (1 to 8 bed volumes) and the elution flow rate, in both the dynamic or static adsorption process and the dynamic desorption process, vary from 1 to 10 milliliters per minute. In this method, an extract rich in kaempferol is obtained.

[0048] In another embodiment of the invention, the process for obtaining the kaempferol-enriched extract is carried out by liquid-liquid extraction, as described in step e. This method involves separating the components of a liquid mixture by contact with an insoluble solvent, which preferentially dissolves one or more of the components.

[0049] In an illustrative example of this embodiment of the invention, for this liquid-liquid extraction, extraction solvents such as water and / or an organic solvent of medium polarity, more specifically ethyl acetate, may be added to a separatory funnel. In a preferred embodiment, the treated fermented product may be present in a proportion of 10 to 80% relative to the volume of the extraction liquid, preferably between 40 and 50%. This process results in a kaempferol-rich phase (kaempferol-enriched extract) and a kaempferol-poor phase.

[0050] The present invention requires that after step “e”, relating to the separation of kaempferol, the solvents be removed to optimize the purification process, which consists of employing one or more of the techniques described below: lyophilization, rotary evaporation, drying in a fume hood or in an oven. Examples of embodiments of the invention

[0051] The fermentation process promotes changes in the profile of phenolic compounds present in the plant matrix, due to the action of enzymes produced mainly during microbial growth. These enzymes have the ability to degrade the constituents of the plant cell wall, promoting the hydrolysis of ester bonds that keep phenolic compounds anchored to the cell matrix. As a consequence, free phenolic compounds, as well as bound forms, are released more efficiently from the plant matrix. Petition 870250058734, dated 10 / 07 / 2025, page 17 / 42 / 18

[0052] As an example of the present invention, in a preliminary test, the liquid sisal residue (LSR), obtained from the defibration of Agave sisalana leaves, was initially subjected to filtration using a mesh fabric. The resulting liquid filtrate was then subjected to a spontaneous fermentation process, conducted under a controlled temperature of 29 ± 2 °C, for a total period of 240 hours. This preliminary fermentation step aimed to evaluate the increase in the concentration of the flavonoid kaempferol in the LSR.

[0053] During the RLS fermentation process, several physicochemical parameters were monitored in order to track and optimize the fermentation process, the main purpose of which is to generate kaempferol enrichment in the extract. These fermentation parameters included: °Brix, pH, extract weight, total sugar and reducing sugar content, and total phenolic and flavonoid content. Optical density was measured to provide a detailed analysis of changes in the properties of the fermented residue and to evaluate kaempferol enrichment in the fermented liquid residue.

[0054] The results of the physicochemical parameters evaluated during spontaneous fermentation in RLS are shown in Table 01. This preliminary assay resulted in a significant reduction in sugar content and a significant increase in the levels of phenolic compounds and total flavonoids. These results, observed throughout the 240 hours of the process, demonstrate the effectiveness of fermentation as a strategic step for enriching flavonoids in the Agave sisalana residue matrix. Petition 870250058734, dated 10 / 07 / 2025, page 18 / 42 12 / 18 Table 1. Influence of Spontaneous Fermentation on the Physicochemical Parameters of Agave sisalana Liquid Residue. Soluble solids content (°Brix), Hydrogen ion potential (pH), Dry Extract Weight (PE), Total Sugars Content (AT), Total Phenolic Content (FEN), Total Flavonoid Content as a function of Fermentation Time and in the anaerobic fermentation system. TIME (DAYS) VAR 0 1 2 3 4 5 6 7 8 9 10 °BRIX 8.03 Aa 7.30 Ab 6.53 Ad 6.67 Acd 6.77 Ad 6.50 Acde 6.30 Ae 6.33 Ae 6.37 Ace 6.33 Ae 6.37 Ae 6.38 Ae 4.31 Ph Ab 3.71 Acdef 3.64 Ag 3.65 Bfg 3.63 Bg 3.67 Befg 3.68 Bdefg 3.74 Ac 3.73 Bed 3.71 Bcde PE (mg) 14.2 Af 23.37 Adf 7.13 Af 31.53 Acdf 5.63 Abe 58.67 Abe 69.63 Aae 74.80 Aae 81.13 Aa 33.93 Acd 48.60 Abc AT (mg / mL GLI) 0.27 Aa 0.12 Ab 0.05 Ab 0.06 Ab 0.07 Ab 0.07 Ab 0.06 Ab 0.07 Ab 0.05 Ab 0.07 Ab FEN (mg / mL EAG) 21.70 Ab 90.87 Ab 102.62 Ab 321.08 Ab 321.49 Ab 629.03 Ab 604.41 Aa 602.1 Aa 671.33 Aa 673 Aa 602.1 AA 671.33 Ab (mg / mL 172.96 Aabc 187.78 Aab 125.56 Aa 176.67 Aabc 61.11 Ad 85.55 Abcd 61.11 Bb 71.48 Acd 96.30 Abcd 99.26 Bbcd 102.2 EQd Aabc) Means followed by the same letters in the rows are not significantly different from each other by Tukey's test (p < 0.05).

[0055] Soluble solids content (°Brix) and pH, as a function of time, showed typical values ​​for this matrix, ranging around 8 for °Brix and 4 for pH. From the second day onwards, a decreasing trend was observed in these variables.

[0056] The total phenolic content in RLS at the beginning of the process (fermentation time zero) was 21.70 mg / mL Gallic Acid Equivalents (GAE). During the fermentation process, a notable enrichment of approximately 1600% was observed in 96 hours (321.40 mg / mL GAE) and more than 3000% in 192 hours (629 mg / mL GAE), with levels maintained at high levels until the end of fermentation. In the case of total flavonoids, Tukey's test (p < 0.05) confirmed a significant increase in the averages in the last 48 hours of monitoring.

[0057] In order to carry out a preliminary assessment of the enrichment of the flavonoid kaempferol from RLS during the fermentation process, a liquid-liquid partition extraction was carried out using ethyl acetate as solvent, in samples collected at 0, 96, 144 and 192 hours of fermentation. Petition 870250058734, dated 10 / 07 / 2025, page 19 / 42 / 18

[0058] The main objective of this step was to facilitate the detection of kaempferol using thin-layer chromatography (TLC), since the complexity of the plant matrix could mask the presence of the compound. Ethyl acetate was chosen as a solvent due to its selectivity for compounds of medium polarity, such as flavonoids, which favors sample fractionation and allows for a clearer visualization of kaempferol release and concentration throughout the fermentation process.

[0059] Additionally, we sought to observe the possible correlation between the increase in kaempferol content and the reduction in sugar content over the fermentation time.

[0060] For each liquid-liquid partition extraction, 30 mL of the RLS sample (at times 0, 96, 144, and 192) were added to 20 mL of water and 10 mL of ethyl acetate in a separatory funnel. This procedure was repeated three times for each sample, resulting in separation into two phases: an organic phase, containing ethyl acetate, and an aqueous phase.

[0061] The organic phase, rich in phenolic compounds, was then subjected to an ultrasonic treatment for 5 minutes to remove any residual emulsion and then concentrated in a rotary evaporator. This procedure resulted in extracts enriched in kaempferol, at times of 0, 96, 144 and 192 hours, suitable for further analysis and applications.

[0062] Analysis of the partitioned extracts at a concentration of 10 mg / mL by TLC, after development with a 5% methanolic aluminum chloride solution and visualization under ultraviolet light at 366 nm, shown in Figure 1, revealed fluorescence and a blue-green coloration, indicating the presence of compounds from the flavonol class. The identity of the compound was confirmed by comparing the chromatographic profile of the samples with the kaempferol standard, by means of co-elution.

[0063] The observation of a blue fluorescent band with Rf 0.33, whose intensity progressively increased in the fermented samples—all prepared at the same concentration—suggests an enrichment of the flavonoid kaempferol. This increase in intensity over the fermentation time, in the samples extracted with ethyl acetate, comparatively demonstrates the increase in kaempferol concentration over time. Petition 870250058734, dated 10 / 07 / 2025, page 20 / 42 / 18 of the fermentative time of RLS, reinforcing the positive impact of spontaneous fermentation of RLS as an important step for the enrichment of kaempferol in the matrix.

[0064] Considering the strategic role of fermentation in the enrichment of kaempferol in liquid residues of Agave sisalana (RLS), the material was subjected to a prolonged fermentation process, with a total duration of 720 hours, aiming to evaluate the continuity or possible exhaustion of the release and concentration of this flavonoid over time. This new assay was conducted under the same experimental conditions as the fermentation process applied in the preliminary study, ensuring the comparability of the results.

[0065] To this end, aliquots were collected at 24, 240, 480, and 720 hours and analyzed by HPLC-DAD under the same chromatographic conditions. Figure 2 shows the chromatogram corresponding to the crude extract, without partitioning, obtained after 720 hours of fermentation, illustrating the kaempferol enrichment profile at this advanced stage of the process. As a reference, the kaempferol analytical standard was also subjected to analysis under the same conditions, through co-elution, allowing confirmation of the compound's identity based on retention time and absorption spectrum in the UV region, with absorption maxima (λmax) at 265 and 370 nm.

[0066] The results of the fermentation process analyses show a progressive increase in peak area with a retention time of 23.7 ± 0.01 minutes, as highlighted in Figure 2. This increase over the different collection times indicates a continuous enrichment of kaempferol in the extract, with the peak being practically non-existent or of low intensity in the initial phases and significantly more pronounced after 720 hours of fermentation.

[0067] In another example of this invention, the kaempferol-enriched extract was obtained according to the procedures described in step e, using the purification of the fermented RLS by macroporous resin.

[0068] In this example, 70 g of a non-ionic, macroporous polymethacrylic adsorbent resin was used, with a particle size between 350 and 1200 µm and a diameter of Petition 870250058734, dated 10 / 07 / 2025, page 21 / 42 / 18 pore of 120 Å, which occupied a bed volume of 115 mL. For the adsorption step, 300 mL of the fermented RLS for 30 days were subjected to static agitation in a shaker for 6 hours, at a rotation of 25 rpm.

[0069] The subsequent step consisted of dynamic elution in a glass column packed with the resin previously adsorbed in the previous step. The column bed volume was maintained at 115 mL (1 BV). Initially, washing was performed with 300 mL of water under agitation, followed by conditioning the bed with 10% ethanol. Elution was performed by increasing polarity gradient, using sequentially: 1.5 BV of 30% ethanol (180 mL), 1.5 BV of 50% ethanol (180 mL), 2 BV of 70% ethanol (240 mL), 2 BV of 80% ethanol (240 mL), and 2 BV of 100% ethanol (240 mL). The system was operated at a constant flow rate of 3 BV / h, which corresponds to approximately 5.5 mL / min.

[0070] As a result of the described process, 20 mg of lyophilized extract enriched in kaempferol were obtained. The purity of this extract is shown in Figure 3, which presents the chromatogram of the HPLC-DAD analysis, performed at 360 nm, of the material purified by macroporous resin. The UV absorption spectrum of the peak with a retention time of 22.8 minutes confirms the identity of the compound as kaempferol, showing characteristic absorption bands with maxima at 265 nm and 366 nm, compatible with the analytical standard shown in Figure 4.

[0071] This chromatographic profile, when compared to the chromatogram obtained after 720 hours of fermentation (Figure 2), demonstrates the effectiveness of the purification technique in obtaining an extract rich in kaempferol. This material was subsequently subjected to quantification of the kaempferol content in a solution containing 1 mg / mL of the rich extract obtained in this experiment.

[0072] In another example of this invention, the kaempferol-enriched extract was obtained according to the procedures described in step e, using the purification of the fermented RLS by liquid-liquid extraction.

[0073] In this example, a 300 mL aliquot of the fermented RLS for 30 days was subjected to liquid-liquid extraction with 300 mL of ethyl acetate, repeated three times. The organic phase was separated from the aqueous phase using a separatory funnel. Petition 870250058734, dated 10 / 07 / 2025, page 22 / 42 / 18 Next, the organic phase was concentrated by rotary evaporation, resulting in 2.28 g of ethyl acetate extract. The purity of this extract obtained by the liquid-liquid partition technique is shown in Figure 5. From this material, a sample containing 10 mg / mL of ethyl acetate extract was prepared, which was used in the HPLC-DAD quantification step.

[0074] In order to facilitate yield calculations, a 300 mL aliquot of the fermented RLS after 720 hours was subjected to lyophilization, resulting in a final mass of 9.38 g. From this material, a sample containing 70 mg of the lyophilized fermented RLS was prepared and used in the HPLC-DAD quantification step. In addition to the fermented material, the enriched extracts obtained in “step e” — by purification with macroporous resin and by liquid-liquid partitioning — were also subjected to HPLC-DAD quantification.

[0075] Table 2 presents the analytical parameters for the quantification of kaempferol by HPLC-DAD in different samples of liquid residue from Agave sisalana. The data for the calibration line equation, coefficient of determination (R2), limit of quantification (LOQ), limit of detection (LOD), and the kaempferol content quantified in each sample are shown: fermented liquid residue (720 h), extract enriched by purification with macroporous resin, and extract enriched by liquid-liquid partitioning. The concentrations of the analyzed solutions are indicated in parentheses. Table 2: Analytical parameters for the quantification of kaempferol by HPLC-DAD in different samples of liquid residue from Agave sisalana (RLS). Equation of the line R2 LQ (pg) LD (pg) Kaempferol quantification (pg) Fermented RLS (720 h) - Spontaneous fermentation (70 mg / mL) y = 126.4x + 6.7573 0.9994 0.0498 0.0164 3.180 Enriched extract y = 101.11x - 14.523 - step e - purification by macroporous resin (1 mg / ml) 0.9993 0.0104 0.0034 13.337 Petition 870250058734, dated 10 / 07 / 2025, page 23 / 42 / 18 Enriched extract - step e - purification by liquid-liquid extraction (10mg / ml) y = 126.4x + 6.7573 0.9975 0.0068 0.0022 5,930

[0076] Quantification of kaempferol in the different samples analyzed by HPLC-DAD revealed significant variations in kaempferol levels, reflecting the efficiency of the strategies applied for its enrichment.

[0077] In the RLS fermented for 720 hours, analyzed at a concentration of 70 mg / mL, a content of 3,180 pg was quantified, which corresponds to a relative concentration of approximately 0.0045% in the fermented material. This result represents the endpoint of the prolonged fermentation process, where the gradual enrichment of kaempferol was observed over hours from the plant matrix under spontaneous fermentation.

[0078] In contrast, the enriched extracts obtained by the different strategies of step “e” showed significantly higher kaempferol contents than the RLS fermented for 720h.

[0079] The extract purified by macroporous resin showed 13.337 pg of kaempferol in a solution corresponding to 1 mg of rich extract analyzed, which is equivalent to 1.337% kaempferol. This result demonstrates the efficiency of this technique in obtaining a kaempferol-rich extract from fermented RLS.

[0080] The extract obtained by liquid-liquid partitioning with ethyl acetate showed a content of 5.93 pg of kaempferol in a solution corresponding to 10 mg of the analyzed extract, corresponding to 0.0593% kaempferol in the analyzed extract.

[0081] The reliability of the quantifications was supported by the validation of the chromatographic method, with calibration curves generated from the triplicate analysis of seven kaempferol standard solutions, in different concentration ranges. The equations obtained showed excellent linearity, with correlation coefficients (R2) of 0.9994 for the fermented RLS, 0.9993 for the macroporous resin extract, and 0.9975 for the liquid-liquid partition extract. Petition 870250058734, dated 10 / 07 / 2025, page 24 / 42 / 18

[0082] Furthermore, the limits of detection (LOD) and quantification (LOQ) were adequate for the concentration ranges studied, and the precision of the method was confirmed by relative standard deviations of less than 1% in the repetitions of the standards.

[0083] The results obtained demonstrate that the combination of the fermentation process with the purification techniques applied in the step enables an efficient enrichment of kaempferol from the liquid residue of Agave sisalana, highlighting the biotechnological and industrial potential of the process. Both purification strategies employed resulted in a significant increase in kaempferol concentration compared to the fermented crude extract (0.0045%). Purification with macroporous resin showed the highest absolute yield, reaching a kaempferol concentration of 1.33% in the enriched extract, while liquid-liquid partitioning also proved effective, obtaining 0.0593% kaempferol, although with a lower relative kaempferol concentration. Petition 870250058734, dated 10 / 07 / 2025, page 25 / 42

Claims

1 / 2 CLAIMS 1. Process for obtaining kaempferol from the agro-industrial residue of Agave sisalana, characterized by comprising the following steps: a. Collect the liquid residue from the defibration of Agave sisalana leaves. b. Perform a first filtration of the liquid residue obtained in “step a” and then carry out a fermentation; c. Perform a second filtration of the material obtained in step b; d. Centrifuge the material obtained in step c; e. Obtain the kaempferol-rich extract using macropolysaccharides or by liquid-liquid extraction; f. Remove the solvent from the kaempferol-rich extract obtained in step e.

2. Process according to claim 1, characterized in that, prior to step b, the liquid residue of Agave sisalana is subjected to a first filtration using a mesh sieve, with the aim of separating the liquid fraction from the solid fraction; and in step b, the fermentation occurs under controlled temperature between 20 °C and 40 °C, under anaerobic or aerobic conditions, for a period of 36 to 720 hours, with agitation varying between 0 and 300 rpm.

3. Process according to claim 2, characterized in that the fermentation is carried out through auto-fermentation or with the addition of yeasts (0.10%), such as Saccharomyces cerevisiae. Petition 870250058734, dated 10 / 07 / 2025, page 26 / 42 2 / 2 4. Process according to claim 1, characterized in that in step “c” the filtration occurs in a filter with a pore size of 8 to 45 gm.

5. Process according to claim 1, characterized in that in step “d” the centrifugation occurs at 1000-3800 rpm.

6. Process according to claim 1, characterized in that in step “e” the process of obtaining the kaempferol-rich extract occurs through adsorption and desorption on a macroporous resin.

7. Process according to claim 6, characterized in that step “e” occurs in macroporous resins (low, medium and high polarity), temperature (20 to 40°C), pH (pH 5 to 9), desorption eluent (water and ethanol ratio of 30% to 90% ethanol), sample concentration (2 to 8 bed volumes) and flow rate (1 to 5 mL / min).

8. Process according to claim 1, characterized in that in step “e” the process of obtaining the kaempferol-rich extract occurs by liquid-liquid extraction with the organic solvents ethyl acetate, chloroform and / or dichloromethane in a proportion of 10 to 80% relative to the volume of the extracting liquid.

9. Process according to claim 1, characterized in that the solvent removal step can occur through lyophilization, rotary evaporation, drying in a fume hood or in an oven. Petition 870250058734, dated 10 / 07 / 2025, page 27 / 42

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