WO2023096505A1 - Method for obtaining a composition and a colloidal suspension of flavonoids from peels of fruits of the family rutaceae and the genus citrus, and the composition and colloidal suspension thus obtained - Google Patents

Method for obtaining a composition and a colloidal suspension of flavonoids from peels of fruits of the family rutaceae and the genus citrus, and the composition and colloidal suspension thus obtained Download PDF

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WO2023096505A1
WO2023096505A1 PCT/PE2021/050024 PE2021050024W WO2023096505A1 WO 2023096505 A1 WO2023096505 A1 WO 2023096505A1 PE 2021050024 W PE2021050024 W PE 2021050024W WO 2023096505 A1 WO2023096505 A1 WO 2023096505A1
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composition
obtaining
concentration
flavonoids
flavonoid composition
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PCT/PE2021/050024
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Spanish (es)
French (fr)
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Miguel Enrique Jesús MALNATI RAMOS
Moises Alexander ORTEGA NUÑEZ
Michelle Antoinette ARANDA CASAVERDE
Jesaya Alison RIOS MENDOZA
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Bio Natural Solutions S.A.C.
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Priority to PCT/PE2021/050024 priority Critical patent/WO2023096505A1/en
Priority to PE2022000077A priority patent/PE20231028A1/en
Publication of WO2023096505A1 publication Critical patent/WO2023096505A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/75—Rutaceae (Rue family)
    • A61K36/752—Citrus, e.g. lime, orange or lemon

Definitions

  • the present invention includes optimal methods for the extraction of flavonoids, from fruit peels, preferably from fruits of the Rutaceae family and Citrus genus, and their subsequent colloidal suspension, with which it has been reported that the composition of flavonoids that contains quercetin, Nahngenin and catechin, in a synergistic combination, improves the preservation of fresh foods, such as fruits, vegetables, tubers, rhizomes, and roots, by reducing postharvest stress and generating a fungistatic functional barrier against food spoilage. fresh by protecting their freshness and extending their shelf life.
  • This invention falls within the technical field of the food industry, bioindustry, biotechnology, bioengineering and agro-industrial derivatives.
  • the components are inorganic materials that include SiO2, SiO, MgO, CaO, T ⁇ O2, ZnO, MnO that have inorganic substances that are harmful to human health among their composition.
  • the deterioration of the quality of an asparagus occurs when moisture migrates from the surface to the atmosphere and oxygen from the atmosphere into the interior of the plant material, resulting in a dry and wrinkled consistency and an oxidized appearance due to the change in color. color from bright green to brown.
  • polyphenolic compounds such as flavonoids, preferably nahngenin (C27H32O14 8H2O), present in citrus peel, have been granted GRAS status as a flavoring agent in food, have a role as a metabolite, antineoplastic and anti-inflammatory agent.
  • exerting a variety of pharmacological effects such as antioxidant, lipid-lowering, anticancer activity and inhibition of certain cytochrome P450 enzymes, including CYP3A4 and CYP1A2 that can lead to various drug interactions in vitro, belongs to the category of food additive, flavoring, flavor and bitterness enhancing agent.
  • this background is not capable of extracting flavonoids of the quercetin, naringenin and catechin type, in synergistic combination, in a concentration of at least 80%, in addition It lacks purification by ultrafiltration and odor removal by activated carbon, moreover, such background could not be used as a coating agent for food preservation.
  • the extraction time is short, the extraction temperature exceeds 50°C and uses a large amount of hydroalcoholic solvent, in addition it is not capable of extracting flavonoids such as quercetin, naringenin and catechin, in a synergistic combination, in a concentration of at least minus 80%.
  • Fungal infections can cause the following conditions: tinea capitis, tinea corporis, tinea pedis, tinea barbae, tinea cruris, tinea versicolor, onychomycosis, perionychomycosis, pityriasis versicolor, tinea unguium, oral candidiasis, vaginal candidiasis, tract candidiasis respiratory, biliary candidiasis, eosophageal candidiasis, urinary tract candidiasis, systemic candidiasis, mucocutaneous candidiasis, mycetoma, cryptococcosis, American aspergillosis, mucormycosis, blastoccidiomycosis, histoplasmosis, coccidioidomycosis, or sporotrichosis.
  • Another patent document that can be considered as precedent is CN104642533A, published on May 27, 2015; entitled “Fruit and Vegetable Coating Preservative and Method of Preparation Thereof”;
  • the invention refers to a method comprising the following components in percentage by weight: 0.8-1.5% chitosan, 1.5-2% citric acid, 0.2-0.5% sodium alginate, 0.3-0.5% xanthan gum, 1 - 1.5% Tween-60, 2-3% tea polyphenol, and 92-93% pure water and even stirring, however, this background only employs tea polyphenols and not citrus peel flavonoids, so this background is not suitable for use with organic waste, and the extraction of flavonoids of the quercetin, nahngenin and catechin type is not shown.
  • Another article considered as precedent is the one entitled "Improvement of shelf life and sensory quality of pears, used as a specialized edible coating” by Cruz et al. (2015); in which an edible coating functionalized with 0.015% pomegranate polyphenols mixed with 3% candelilla wax, 4% gum arabic, 0.15% jojoba oil is described to improve the quality of useful life of pears.
  • this antecedent uses pomegranate polyphenols, it does not use a primary extraction of it and it could not be used with flavonoids from citrus peel, so its use with organic waste is not suitable, in addition the composition does not include sodium alginate and glycerol.
  • catechin is an antioxidant that comes from plants in which it appears as a secondary metabolite, it helps prevent cell damage, reducing the formation of free radicals in the body, which protects cells and molecules from damage.
  • Patent ES2443547B2 is an antecedent that provides a few-stage process for obtaining polyphenolic extracts, including catechin in a percentage of 1.14 and 1.28 mg/g of dry bagasse from white grape residues.
  • the procedure uses non-polluting materials, allowing the contribution of bioactive polyphenolic extracts that can be used at an industrial level, mainly in the cosmetic, pharmaceutical and / or food sectors, the process begins with a) preparing a mixture that includes a residue of white grape and a dispersant, b) add a solvent, c) elute and d) collect the eluates for their correct use, however it does not offer a combination of flavonoids of the quercetin, nahngenin and catechin type, nor the stages of the present invention.
  • Patent W02010/027572A2 presents a mixture of compounds such as quercetin, vitamin B3 and vitamin C to reduce plasma cholesterol levels in human patients, but it is only limited to this.
  • Patent CN113501892A describes a combined extraction method in citrus peels to obtain different functional components and their application as additive in food, medicine, cosmetics or health products.
  • limonin, pectin and flavonoid substances stand out.
  • US20100159100A1 the innovative method consists in the recovery of hespehdin, narirutin, didymin, flavonoid compounds, from citrus bagasse and peel after juice extraction to incorporate them into an orange juice concentrate.
  • a similar invention is US20100159115A1, which details a process for extracting flavonoids from citrus juice by-product, the aim of which is to decrease the level of limonin and other bitter compounds in the juice and, in turn, use them to create an independent extract.
  • US20040081734A1 refers to a general selective extraction method for obtaining aqueous extracts rich in antioxidants and their balanced composition of simple phenols, polyphenols, bioflavonoids, flavones and flavonones to control product oxidation processes. citrus.
  • the inventions of the state of the art relate extraction processes of bioactive compounds obtained from by-products of fruit processing, preferably bagasse and citrus peel for their application in beverages, cosmetics, medicine and other similar; however, the application of a concentrated compound, rich in flavonoids, in the agricultural sector has not been considered, especially in an invention that incorporates the concentrated composition and a colloidal suspension to achieve postharvest preservation of fruit and vegetable products.
  • This composition of flavonoids contains quercetin, naringenin and catechin which, in synergistic combination, improves the preservation of fresh foods, such as fruits, vegetables, tubers, rhizomes and roots.
  • the present invention proposes a novel process for obtaining a composition and a colloidal suspension of flavonoids from fruit peels of the Rutaceae family and genus Citrus and the composition and colloidal suspension thus obtained, where the flavonoid composition contains quercetin, nahngenin and catechin. , in synergistic combination, in a concentration of at least 80% of the concentrated compound, and this is applied for the preservation of fresh foods.
  • the present invention refers to a compound and/or colloidal suspension with a protective effect for fruit and vegetable products, such as fruits, vegetables, tubers, rhizomes and roots, and their subsequent extension of useful life.
  • This invention contains a composition of flavonoids that have a phenyl-benzo-y-pyrone (or phenyl-Y-chromone) structure, products of the secondary metabolism of the peels of fruits of the Rutaceae family and Citrus genus used as raw material, which They are found in the free state or in the form of heterosides.
  • These heterosides are generally soluble in water, while their genins or aglycones, the unsweetened part of the heteroside, are only slightly so.
  • the flavonoids of this invention are composed of a 3-ring structure with a skeleton of diphenylpropane formula I.
  • This single ring is synthesized by the condensation of 3 moles of malonyl-coenzyme A derived from glucose metabolism and produces a formula II chalcone synthase.
  • flavonoids have a C6-C3-C6 carbon skeleton - formula III, where the C6 components are aromatic rings joined by three carbon atoms that may or may not form a third pyran or pyrone ring (AC rings).
  • the different classes of flavonoids differ in the saturation concentration and in the substituents of the C ring, while individual compounds, within each of these groups, are distinguished by the different substitution of the A and B rings.
  • Quercetin biosynthesis in its initial stage, passed through the phenylpropanoid pathway, where phenylalanine was converted to 4-cumahl-CoA by the catalytic action of phenylalanine ammonia lyase, cinnamate-4-hydroxylase, and 4-cumahl CoA ligase.
  • Chalcone synthase then catalyzed the combination of 4-coumaroyl-CoA with three malonyl-CoA molecules (3), leading to the formation of tetrahydroxy chalcone.
  • Tetrahydroxy chalcone was converted to naringenin using chalcone isomerase and nahngenin was converted to ehodictyol by the action of the enzyme flavanone-3[3-hydroxylase.
  • the ehodictyol formed was converted to dihydroquercetin by the action of flavanone-3[3-hydroxylase, which was finally converted to quercetin, formula IV by flavonol synthase.
  • catechin biosynthesis begins with a 4-hydroxycinnamoyl CoA initiation unit that undergoes chain extension by adding three malonyl-CoAs through a PKSIII pathway.
  • 4-Hydroxycinnamoyl CoA is biosynthesized from L-phenylalanine via the Shikimate pathway.
  • L-phenylalanine is deaminated first by phenylalanine ammonia lyase (PAL) forming cinnamic acid which is then oxidized to 4-hydroxycinnamic acid by cinnamate 4-hydroxylase.
  • PAL phenylalanine ammonia lyase
  • Chalcone synthase then catalyzes the condensation of 4-hydroxycinnamoyl CoA and three malonyl-CoA molecules to form chalcone.
  • the chalcone is then isomehzada nahngenin by chalcone isomerase which is oxidized aeriodictyol by flavonoid 3'-hydroxylase and then oxidized to taxifolin by flavanone 3-hydroxylase.
  • Taxifolin is then reduced by dihydroflavanol 4-reductase and leucoanthocyanidin reductase to produce catechin.
  • nahngenine formula V
  • phenylalanine was converted into 4-cumahl-CoA by the catalytic action of phenylalanine ammonia lyase, cinnamate-4-hydroxylase and 4- cumahl CoA ligase.
  • Chalcone synthase then catalyzed the combination of 4-coumaroyl-CoA with three malonyl-CoA molecules, leading to the formation of tetrahydroxy chalcone.
  • tetrahydroxy chalcone was converted to nahngenin using chalcone isomerase.
  • the object of the invention is the process for obtaining a concentrated flavonoid composition, from fruit peels of the Rutaceae family and Citrus genus, such as fresh fruit peels, which comprises at least 80% of quercetin-type flavonoids. , naringenin and catechin, which group the following stages a) primary extraction and elimination of volatile terpenes b) efficient extraction with polar aqueous solvents, c) ultrafiltration and adsorption with ecological activated carbon, d) concentration and biosynthesis of the flavonoid composition.
  • the primary extraction and elimination of volatile terpenes from the peels of fruits of the Rutaceae family and Citrus genus can comprise the following processes:
  • the extracted terpenes are separated from the process because they present low polar solubility and decrease the purity of the main compounds.
  • the fresh shells with a lower percentage of water are reserved for the following processes.
  • the efficient extraction with polar aqueous solvents is carried out by means of a heat-assisted extraction at 30-50°C, for a ratio of 1-4 hours, using aqueous solutions in a proportion between 10-40% sugarcane ethanol in matter solvent ratio. raw between 1:8 - 1:20, based on the nature and state of maturation of the raw material.
  • the ultrafiltration and adsorption with ecological activated carbon of the extract of fruit residues of the Rutaceae family and Citrus genus comprises the use of an ultrafilter with a particle size of 0.04 - 0.1 um and an activated carbon column to retain typical odors, through which the fluid with the assistance of a tangential pump at a pumping pressure between 50 kPa and 100 kPa.
  • the ecological activated carbon is obtained from the conditioning of the filtered solid residues of the fruits of the Rutaceae family and Citrus genus with a 20-25% phosphoric acid solution carbonized at 426-526 °C for a time of 3- 6 hours.
  • the concentration and biosynthesis of the flavonoid composition is carried out by vacuum distillation in a rotary evaporator under vacuum between 12.7 mmh to 50.8 mmHg at 30-50°C for 40-70 minutes, adding in a first reaction phosphate buffer (50 mM) and sequentially the citrus extract used as processing aid, until reaching a concentration between 10-35%.
  • a first reaction phosphate buffer 50 mM
  • the object of the invention also includes the preparation of a colloidal suspension and/or film with a protective effect for fruit and vegetable products, such as fruits, vegetables, tubers, rhizomes and roots, and its subsequent extension of useful life, thanks to the chelating effect and antioxidant of efficiently extracted and concentrated flavonols, which includes the following postulates:
  • a colloidal material such as sodium alginate, xanthan gum, gum arabic, guar gum, and/or any related gum in a proportion between 2-5%
  • additives that complement its barrier effect such as a food grade plasticizer between 2-5%, waxes of vegetable origin, such as carnauba or candelilla wax between 5-12%.
  • composition of flavonoids concentrated from fruit residues of the Rutaceae family and Citrus genus can be applied directly to fruit and vegetable products, in a concentration by weight between 2-15%, through a solution with purified water in relation, based on the nature of the product, in order to apply a first fungistatic functional barrier.
  • composition of flavonoids concentrated from fruit residues of the Rutaceae family and Citrus genus can be applied as a processing aid with an acidifying function in a matrix if it is applied in a concentration by weight between 1-5%, through a solution with purified water.
  • the flavonoid composition includes flavonoids of the quercetin, nahngenin and catechin type, in synergistic combination, in a concentration of at least 80%.
  • the aqueous colloidal suspension includes a proportion of 1-30% flavonoid composition, 2-5% food grade colloid, 2-5% food grade plasticizer, and 5-12% vegetable wax.
  • the aqueous colloidal suspension includes a proportion of 1-30% flavonoid extract and the remainder contains colloids and excipients for the formation of the suspension itself. Another advantage of the invention is that in its application it manages to maintain the quality of various fresh fruit and vegetable products over time, up to over 200% of its useful life, and its enrichment with active agents from fruit residues of the Rutaceae family and genus Citrus, and its integral use in the production process.
  • Another objective of the invention is to obtain a composition of flavonoids with antioxidant power of high phenolic concentration, from residues of the citrus agroindustry, whose effect between the major compounds quercetin, naringenin and catechin present an unexpected positive synergistic effect on the useful life of perishable fresh products, thanks to its chelating power that reduces postharvest stress of fruits and its fungistatic functional effect not previously reported on the flavonoid composition.
  • the invention represents an improvement in the state of the art, since, from different points of view, the patent is credited with a positive impact on fresh agricultural products, protecting them during their processing, storage, transport and preserving their sensory quality, due to the antioxidant capacity provided by the extracted and concentrated flavonoids mentioned above. Based on an ecological vision, it responds to the need to generate biotechnologies that reduce the amount of non-biodegradable polymehc packaging, which pollutes the environment.
  • FIG. 1 Flowchart of the process of the invention in which its four main stages are detailed.
  • Figure 2 Average percentage of total soluble solids (%TSS) in 'Kent' type mango.
  • Figure 3 Dynamics of soluble solids (°Bhx) in pears of the 'Packham's Triumph' type.
  • Figure 4 Minimum Inhibitory Concentration of the formulated invention.
  • PREFERRED EXAMPLES (OR PREFERRED EMBODIMENT OF THE INVENTION) A. Process for obtaining a composition of flavonoids from fruit peels of the Rutaceae family and Citrus genus for the preservation of fresh foods and the colloidal suspension thus obtained and its application.
  • a preparation of the composition of flavonoids from fruit peels of the Rutaceae family and Citrus genus for the preservation of fresh foods and the colloidal suspension thus obtained, of the invention involves the selection, washing and disinfection of the peels of Citrus reticulata fruit in early season with 100 ppm sodium hypochlorite diluted in filtered water or a related food sanitizer; then the fresh shells go through a primary extraction assisted with microwave waves at a power preferably between 200 to 800 W for 40 minutes, without using any organic solvent. Next, the shells are efficiently extracted with aqueous solvents and heat at 40°C for a ratio of 4 hours, in a proportion of 20% ethanol in a raw material solvent ratio between 1:20.
  • Figure 2 shows that the application of the covering referring to the invention statistically generates less weight loss in cold-stored mangoes.
  • Coverages of different alginate formulations such as the one proposed by Rastegar et al., (2019) report higher weight losses, around 10%, at 12°C storage, as well as no effect on the evolution of the percentage of soluble solids, unlike what was obtained with the coverage referring to the present invention, where the average loss of total soluble solids (% TSS) in mango is between 2 to 3%.
  • the flavonoid-rich composition was evaluated by spectrophotometric analysis in order to quantify the total flavonoid compounds (CFT) and their antioxidant capacity, by the DPPH radical method.
  • CFT total flavonoid compounds
  • CPT total polyphenolic compounds
  • the trials reported values from 190.45 ⁇ 0.26 umol/g to 500.08 ⁇ 0.33 umol/g of sample, in Trolox equivalents for DPPH; a content of total flavonoids that includes ranges of 257.03 ⁇ 0.18 mg/g and 1000.78 ⁇ 0.23 mg/g of sample, in quercetin standard and finally levels of 80.67 ⁇ 5.9 mg / g, in gallic acid standard (GAE) for cpt.
  • the minimums and maximums are based on the extraction parameters used for the recovery of the bioactive compounds, mentioned above.
  • the present invention increases the extraction efficiency of flavonoid compounds present in citrus by-products that are not obtained with those previously exposed by more than 100%.
  • strains of C. gloeosporioides and A. niger were sown in Petri dishes with Sabouraud Agar, and 60 uL of the formulated invention was placed (flavonoid mixture, as Belomil positive control and distilled water, as negative control).
  • the tests are carried out in triplicate at least twice, thus obtaining 3 treatments and 18 plates in total for each of the fungi.
  • the formulation of the invention was submitted to the plate micro dilution test to determine its Minimum Inhibitory Concentration (MIC).
  • Figure 4 shows the results of the Minimum Inhibitory Concentration (MIC) of the invention formulated against the phytopathogens of Aspergillus niger and Colletotrichum gloesporioides. Concentrations of the present invention ranged from 10 mg/ml to 0.02 mg/ml. Finally, the MIC for A. niger was 0.24 mg/ml and 0.12 mg/ml for C. gloesporioides.
  • MIC Minimum Inhibitory Concentration
  • the formulated invention showed effective antifungal activity with a MIC of 0.12 mg/ml for Colletotrichum gloesporioides, a result that presented highly significant advantages over various studies that confronted extracts with the mentioned strain; for example, Paredes et. al (2021) reported a MIC of 0.14mg/ml for the methanolic extract of Samanea saman, while Aguilar et al (2013) reported a MIC of 150mg/L and 300mg/L for ethanolic extracts of rue and lollipop, respectively.

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Abstract

The present invention relates to a method for obtaining a composition of flavonoids from fruit peels, preferably fruit of the family Rutaceae and the genus Citrus, and to the subsequent colloidal suspension thereof. It has been reported that the flavonoid composition contains quercetin, naringenin and catechin, in synergistic combination, and improves the preservation of fresh foods, such as fruit, vegetables, tubers, rhizomes and roots, by reducing post-harvest stress and generating a fungistatic, functional, chelating, antioxidant barrier against the deterioration of the fresh foods, thereby protecting the freshness thereof and extending the shelf life of same.

Description

PROCESO PARA LA OBTENCIÓN DE UNA COMPOSICIÓN Y UNA SUSPENSIÓN COLOIDAL DE FLAVONOIDES DE CASCARAS DE FRUTAS DE LA FAMILIA RUTACEAE Y GÉNERO CITRUS Y LA COMPOSICIÓN Y SUSPENSIÓN COLOIDAL ASÍ OBTENIDA PROCESS FOR OBTAINING A COMPOSITION AND A COLLOIDAL SUSPENSION OF FLAVONOIDS FROM FRUIT PEELS OF THE RUTACEAE FAMILY AND CITRUS GENUS AND THE COMPOSITION AND COLLOIDAL SUSPENSION OBTAINED THUS
CAMPO TÉCNICO TECHNICAL FIELD
La presente invención incluye métodos óptimos de extracción de flavonoides, a partir de cáscaras de frutas, de preferencia de frutas de la familia Rutaceae y género Citrus y su posterior suspensión coloidal, con lo cual se ha reportado que la composición de flavonoides que contiene quercetina, nahngenina y catequina, en combinación sinérgica mejora la conservación de alimentos frescos, tales como frutas, vegetales, hortalizas, tubérculos, rizomas y raíces, a partir de la reducción del estrés postcosecha y la generación de una barrera funcional fungiestática contra el deterioro de los alimentos frescos protegiendo su frescura y extendiendo su vida útil. The present invention includes optimal methods for the extraction of flavonoids, from fruit peels, preferably from fruits of the Rutaceae family and Citrus genus, and their subsequent colloidal suspension, with which it has been reported that the composition of flavonoids that contains quercetin, Nahngenin and catechin, in a synergistic combination, improves the preservation of fresh foods, such as fruits, vegetables, tubers, rhizomes, and roots, by reducing postharvest stress and generating a fungistatic functional barrier against food spoilage. fresh by protecting their freshness and extending their shelf life.
Esta invención está comprendida dentro del campo técnico de la industria alimentaria, bioindustria, biotecnología, bioingeniería y derivados agroindustriales. This invention falls within the technical field of the food industry, bioindustry, biotechnology, bioengineering and agro-industrial derivatives.
ESTADO DE LA TÉCNICA STATE OF THE ART
En la actualidad existen productos comestibles de revestimiento hortofrutícolas que forman una barrera contra la humedad y el oxígeno para dar como resultado una capa comestible y protectora a un alimento, debido a su naturaleza perecedera en el tiempo. Generalmente los componentes son materiales inorgánicos que incluyen SiO2, SiO, MgO, CaO, T¡O2, ZnO, MnO que presentan entre su composición sustancias inorgánicas nocivas para la salud humana. Por ejemplo, el deterioro de la calidad de un espárrago ocurre cuando la humedad migra de la superficie a la atmósfera y el oxígeno de la atmósfera al interior del material vegetal, dando como resultado una consistencia seca y arrugada y un aspecto oxidado por el cambio de color de verde brillante a pardo. Currently, there are edible fruit and vegetable coating products that form a barrier against moisture and oxygen to result in an edible and protective layer for food, due to its perishable nature over time. Generally, the components are inorganic materials that include SiO2, SiO, MgO, CaO, T¡O2, ZnO, MnO that have inorganic substances that are harmful to human health among their composition. For example, the deterioration of the quality of an asparagus occurs when moisture migrates from the surface to the atmosphere and oxygen from the atmosphere into the interior of the plant material, resulting in a dry and wrinkled consistency and an oxidized appearance due to the change in color. color from bright green to brown.
Por ello, las películas comestibles en forma de suspensión coloidal amplían las oportunidades de nuevos alimentos y aumentan las expectativas del consumidor de una variedad de alimentos frescos al reducir los conservantes químicos generalmente utilizados en la industria. Asimismo, el uso de tecnologías ecológicas reduce la cantidad de embalaje innecesario para preservar los alimentos en forma de plásticos u otras alternativas no biodegradables. El desarrollo de barreras al vapor de agua y oxigeno se ha centrado en compuestos que incluyen principalmente celulosa, lípidos y proteínas. Las barreras que contienen lípidos, son menos permeables al vapor de agua que las barreras que contienen proteínas. Sin embargo, las barreras que contienen proteínas no son prácticas para algunos productos, como las frutas y/o vegetales frescos, por contener productos no adecuados para la ingesta humana. Therefore, edible films in the form of a colloidal suspension expand the opportunities for novel foods and increase consumer expectations of a variety of fresh foods by reducing the chemical preservatives generally used in the industry. Also, the use of green technologies reduces the amount unnecessary packaging to preserve food in the form of plastics or other non-biodegradable alternatives. The development of barriers to water vapor and oxygen has focused on compounds that mainly include cellulose, lipids and proteins. Lipid-containing barriers are less permeable to water vapor than protein-containing barriers. However, protein-containing barriers are not practical for some products, such as fresh fruits and/or vegetables, as they contain products not suitable for human ingestion.
Por otro lado, el campo de los extractos naturales presenta un abanico infinito de posibilidades para enriquecer las suspensiones coloidales y/o recubrimientos dirigidos a los productos agrícolas frescos. Es así, que compuestos polifenólicos como los flavonoides, de preferencia la nahngenina (C27H32O14 8H2O), presente en la cáscara de cítricos, se le ha otorgado el estatus GRAS como agente aromatizante en los alimentos, tiene un papel como metabolite, agente antineoplásico y antiinflamatorio, ejerciendo una variedad de efectos farmacológicos tales como actividad antioxidante, hipolipemiante, actividad anticancerígena e inhibición de determinadas enzimas del citocromo P450, incluidas CYP3A4 y CYP1A2 que pueden dar lugar a varias interacciones medicamentosas in vitro, pertenece a la categoría de aditivo alimentario, saborizante, agente mejorador del sabor y amargor. Además, se puede utilizar como pigmento natural, como insumo para producir medicamentos de control de enfermedades cardiovasculares, cerebrovascu lares y antiinflamatorias. Tiene mayor actividad biológica como reductor de grasa en la sangre, alivio de la tos y resolución de flemas, regulación de azúcar en sangre y reductor de colesterol; no obstante, no se han reportado investigaciones que asocien al compuesto como agente para mejorar la vida útil de productos agrícolas frescos. On the other hand, the field of natural extracts presents an infinite range of possibilities to enrich colloidal suspensions and/or coatings for fresh agricultural products. Thus, polyphenolic compounds such as flavonoids, preferably nahngenin (C27H32O14 8H2O), present in citrus peel, have been granted GRAS status as a flavoring agent in food, have a role as a metabolite, antineoplastic and anti-inflammatory agent. , exerting a variety of pharmacological effects such as antioxidant, lipid-lowering, anticancer activity and inhibition of certain cytochrome P450 enzymes, including CYP3A4 and CYP1A2 that can lead to various drug interactions in vitro, belongs to the category of food additive, flavoring, flavor and bitterness enhancing agent. In addition, it can be used as a natural pigment, as an input to produce medicines to control cardiovascular, cerebrovascular and anti-inflammatory diseases. It has greater biological activity as a blood fat reducer, cough relief and phlegm resolution, blood sugar regulation and cholesterol reducer; however, no research has been reported that associates the compound as an agent to improve the shelf life of fresh agricultural products.
En el estado de la técnica existe el artículo titulado “Métodos ecológicos para la extracción de flavonoides de material vegetal: impacto de sus condiciones operativas en el rendimiento y las propiedades antioxidantes” de Rodríguez et al. (2020); en el cual se describe una extracción asistida por microondas sin disolvente como pretratamiento entre 100 a 900 W, asimismo, se menciona la extracción mediante métodos convencionales con un mezcla de agua y etanol entre (30-90%), 20-60 °C y 2-72 h según la materia prima que se utilice para extracción de flavonoides. Sin embargo, este antecedente no es capaz de extraer flavonoides del tipo quercetina, naringenina y catequina, en combinación sinérgica, en una concentración de al menos 80%, además carece de una purificación por ultrafiltración y eliminación de olores por carbón activado, además, dicho antecedente no se podría emplear como agente de recubrimiento para la conservación de alimentos. In the state of the art there is an article entitled "Ecological methods for the extraction of flavonoids from plant material: impact of their operating conditions on yield and antioxidant properties" by Rodríguez et al. (2020); in which a microwave-assisted extraction without solvent is described as a pretreatment between 100 to 900 W, likewise, extraction by conventional methods with a mixture of water and ethanol between (30-90%), 20-60 °C and 2-72 h depending on the raw material used for flavonoid extraction. However, this background is not capable of extracting flavonoids of the quercetin, naringenin and catechin type, in synergistic combination, in a concentration of at least 80%, in addition It lacks purification by ultrafiltration and odor removal by activated carbon, moreover, such background could not be used as a coating agent for food preservation.
Otro artículo considerado como antecedente, es el titulado “Extracción de flavanonas de orujos inmaduros de Citrus unshiu: optimización de procesos y evaluación de antioxidantes”; de Kim y Lim (2020). En el cual se describe un método de extracción optimizado para hesperidina y narirutina, donde las condiciones de extracción para los rendimientos más altos de flavonoides basados en una metodología de superficie de respuesta fueron 80.3 ° C, 58.4% (concentración de etanol), 40 mL / g (solvente / alimento) y 30 min, donde los rendimientos de hesperidina y narirutina fueron 66.6% y 82.3%, respectivamente, teniendo en cuenta un pretratamiento que no ha sido mencionado. Si bien el tiempo de extracción es corto la temperatura de extracción excede los 50°C y utiliza una gran cantidad de solvente hidroalcohólico, además no es capaz de extraer flavonoides del tipo quercetina, naringenina y catequina, en combinación sinérgica, en una concentración de al menos 80%. Another article considered as precedent is the one entitled "Extraction of flavanones from immature Citrus unshiu pomace: optimization of processes and evaluation of antioxidants"; from Kim and Lim (2020). In which an optimized extraction method for hesperidin and narirutin is described, where the extraction conditions for the highest yields of flavonoids based on a response surface methodology were 80.3 °C, 58.4% (ethanol concentration), 40 mL / g (solvent / food) and 30 min, where the yields of hesperidin and narirutin were 66.6% and 82.3%, respectively, taking into account a pretreatment that has not been mentioned. Although the extraction time is short, the extraction temperature exceeds 50°C and uses a large amount of hydroalcoholic solvent, in addition it is not capable of extracting flavonoids such as quercetin, naringenin and catechin, in a synergistic combination, in a concentration of at least minus 80%.
Otro antecedente es el documento de patente CN101704867B, titulado “Método de preparación de naringenina o hesperidina”, este antecedente propone un método de extracción eficiente para adoptar un solvente orgánico o una solución acuosa alcalina en condiciones de extracción de reflujo o lixiviado a presión normal ahorrando el consumo del disolvente obteniendo una alta eficiencia de extracción de la naringenina en sinergia con otros flavonoides. En la patente europea EP1845964A1 , la invención sobre una composición que incluye naringenina en cantidades de 0.001 % a 50%, son eficaces para tratar o prevenir una infección por hongos en un sujeto, a quien se le administra una composición en una cantidad eficaz para tratar una infección fúngica. Las infecciones por hongos (micosis) pueden causar las siguientes afecciones: tinea capitis, tinea corporis, tinea pedis, tinea barbae, tinea cruris, tinea versicolor, onicomicosis, perionicomicosis, pitiriasis versicolor, tinea unguium, candidiasis oral, candidosis vaginal, candidiasis del tracto respiratorio, candidosis biliar, candidosis eosofágica, candidosis del tracto urinario, candidosis sistémica, candidosis mucocutánea, micetoma, criptococosis, aspergilosis americana, mucormicomicosis, blastoccidiomicosis, histoplasmosis, coccidioidom ¡cosis o esporotricosis. Otro documento de patente que se puede considerar como antecedente es CN104642533A, publicado el 27 de mayo del 2015; titulado “Conservante de revestimiento de frutas y verduras y método de preparación del mismo”; la invención se refiere a un método comprende los siguientes componentes en porcentaje en peso: 0.8- 1.5% de quitosano, 1.5-2% de ácido cítrico, 0.2-0.5% de alginato de sodio, 0.3-0.5% de goma xantana, 1 -1 .5% de Tween-60 , 2-3% de polifenol de té y 92-93% de agua pura y agitación uniforme, sin embargo, este antecedente solo emplea polifenoles de té y no flavonoides de cáscara de cítricos, por lo que este antecedente no es adecuado para uso con desechos orgánicos, y no se muestra la extracción de flavonoides del tipo quercetina, nahngenina y catequina. Another background is the patent document CN101704867B, entitled "Method for the preparation of naringenin or hesperidin", this background proposes an efficient extraction method to adopt an organic solvent or an alkaline aqueous solution under reflux extraction conditions or normal pressure leaching, saving the consumption of the solvent obtaining a high efficiency of extraction of naringenin in synergy with other flavonoids. In the European patent EP1845964A1, the invention on a composition that includes naringenin in amounts of 0.001% to 50%, are effective to treat or prevent a fungal infection in a subject, to whom a composition is administered in an effective amount to treat a fungal infection. Fungal infections (mycoses) can cause the following conditions: tinea capitis, tinea corporis, tinea pedis, tinea barbae, tinea cruris, tinea versicolor, onychomycosis, perionychomycosis, pityriasis versicolor, tinea unguium, oral candidiasis, vaginal candidiasis, tract candidiasis respiratory, biliary candidiasis, eosophageal candidiasis, urinary tract candidiasis, systemic candidiasis, mucocutaneous candidiasis, mycetoma, cryptococcosis, American aspergillosis, mucormycosis, blastoccidiomycosis, histoplasmosis, coccidioidomycosis, or sporotrichosis. Another patent document that can be considered as precedent is CN104642533A, published on May 27, 2015; entitled "Fruit and Vegetable Coating Preservative and Method of Preparation Thereof"; The invention refers to a method comprising the following components in percentage by weight: 0.8-1.5% chitosan, 1.5-2% citric acid, 0.2-0.5% sodium alginate, 0.3-0.5% xanthan gum, 1 - 1.5% Tween-60, 2-3% tea polyphenol, and 92-93% pure water and even stirring, however, this background only employs tea polyphenols and not citrus peel flavonoids, so this background is not suitable for use with organic waste, and the extraction of flavonoids of the quercetin, nahngenin and catechin type is not shown.
Otro artículo considerado como antecedente, es el titulado “Mejora de la vida útil y la calidad sensorial de las peras, usado como un revestimiento comestible especializado” de Cruz et al. (2015); en el cual se describe un recubrimiento comestible funcionalizado con 0.015% polifenoles de granada en mezcla con 3% cera de candelilla, 4% goma arábiga, 0.15% aceite de jojoba para mejorar la calidad de vida útil de las peras. Sin embargo, este antecedente emplea polifenoles de granada, no emplea una extracción primaria de ello y no podría ser empleado con flavonoides de cáscara de cítricos, por lo que no es adecuado su uso con desechos orgánicos, además la composición no comprende alginato de sodio y glicerol. Another article considered as precedent is the one entitled "Improvement of shelf life and sensory quality of pears, used as a specialized edible coating" by Cruz et al. (2015); in which an edible coating functionalized with 0.015% pomegranate polyphenols mixed with 3% candelilla wax, 4% gum arabic, 0.15% jojoba oil is described to improve the quality of useful life of pears. However, this antecedent uses pomegranate polyphenols, it does not use a primary extraction of it and it could not be used with flavonoids from citrus peel, so its use with organic waste is not suitable, in addition the composition does not include sodium alginate and glycerol.
En cuanto a otro compuesto flavonoide presente en los residuos cítricos se encuentra la catequina, un antioxidante que procede de las plantas en las cuales aparece como un metabolite secundario, este ayuda a prevenir el daño celular, reduciendo la formación de radicales libres en el cuerpo, lo que protege a las células y las moléculas del daño. La patente ES2443547B2 es un antecedente que brinda un proceso de pocas etapas para la obtención de extractos polifenólicos entre ellos catequina en un porcentaje de 1 ,14 y 1 ,28 mg / g de bagazo seco a partir de residuos de uva blanca. El procedimiento utiliza materiales no contaminantes, permitiendo el aporte de extractos heos en polifenoles bioactivos que pueden ser utilizados a nivel industrial, principalmente en los sectores cosmético, farmacéutico y / o alimentario, el proceso inicia con a) preparar una mezcla que comprende un residuo de uva blanca y un dispersante, b) agregar un solvente, c) eluir y d) recoger los eluidos para su correcto uso, sin embargo no ofrece una combinación de flavonoides del tipo quercetina, nahngenina y catequina, ni las etapas de la presente invención. De la misma manera, un flavonoide como la quercetina cuya actividad antioxidante y antiinflamatoria es reportada ampliamente en el estado del arte del compuesto, con aplicaciones interesantes para combatir células cancerosas, controlar niveles de azúcar en la sangre y hasta para prevenir cardiópatas (Ou, Zheng, Zhao & Lin W, 2020). La patente W02010/027572A2 (Lines, 2010), presenta una mezcla de compuestos como la quercetina, vitamina B3 y vitamina C para reducir los niveles de colesterol plasma en pacientes humanos, pero solo se limita a ello. As for another flavonoid compound present in citrus residues, catechin is an antioxidant that comes from plants in which it appears as a secondary metabolite, it helps prevent cell damage, reducing the formation of free radicals in the body, which protects cells and molecules from damage. Patent ES2443547B2 is an antecedent that provides a few-stage process for obtaining polyphenolic extracts, including catechin in a percentage of 1.14 and 1.28 mg/g of dry bagasse from white grape residues. The procedure uses non-polluting materials, allowing the contribution of bioactive polyphenolic extracts that can be used at an industrial level, mainly in the cosmetic, pharmaceutical and / or food sectors, the process begins with a) preparing a mixture that includes a residue of white grape and a dispersant, b) add a solvent, c) elute and d) collect the eluates for their correct use, however it does not offer a combination of flavonoids of the quercetin, nahngenin and catechin type, nor the stages of the present invention. In the same way, a flavonoid such as quercetin whose antioxidant and anti-inflammatory activity is widely reported in the state of the art of the compound, with interesting applications to fight cancer cells, control blood sugar levels and even to prevent heart disease (Ou, Zheng , Zhao & LinW, 2020). Patent W02010/027572A2 (Lines, 2010), presents a mixture of compounds such as quercetin, vitamin B3 and vitamin C to reduce plasma cholesterol levels in human patients, but it is only limited to this.
Por otro lado, se han reportado procesos de reaprovechamiento de residuos, como en la patente US8481099B2 se describe un proceso para la conversión de cáscaras de cítricos en fibra, jugo, naringenina y aceite, dado que el procesamiento de cítricos genera desechos sustanciales en forma de cáscaras, semillas y pulpa, estos se pueden procesar para obtener aditivos alimentarios o para piensos. Otras formas de procesamiento de los subproductos de los cítricos se han mencionado en la patente estadounidense 4.225.625 donde se explica un método para separar cáscaras de cítricos en componentes separados de albedo y flavedo, los cuales son regiones comprendidas en la cáscara de cítricos, siendo el albedo, una fuente de fibra dietética que incluye azúcar, celulosa y sustancias pécticas; mientras tanto el flavedo, es fuente de vitamina A, carotenos y xantofilas, utilizándose como agente colorante en alimentos, agente enturbiante en bebidas de zumos de fruta y fortificante de alimentos para ganado y humano. En otra de las patentes estadounidenses como en la patente US 7.060.313, tanto la cáscara y pulpa de cítricos sin jugo son procesados para recuperar componentes de alto valor como, melaza, orujo de pectina, D-limoneno y masa de cáscara de calidad alimentaria. Otra conversión de subproductos de cítricos se lee en la patente US7.629.010 que menciona un método para recuperar fibra cítrica de vesículas cítricas utilizando disolventes orgánicos para obtener un aditivo alimentario para bebidas, productos horneados, carne o emulsiones cárnicas, confitería mermeladas y gelatinas. El uso de solventes orgánicos también se enseña en el documento de patente US6.183.806 para la elaboración de extractos de cáscara de cítricos y harina. Otra de las formas de recuperar los compuestos es la obtención de aceite de cítricos como se enseña en la patente estadounidense US6.151.799. On the other hand, waste reuse processes have been reported, such as patent US8481099B2 describing a process for converting citrus peels into fiber, juice, naringenin and oil, since citrus processing generates substantial waste in the form of shells, seeds and pulp, these can be processed to obtain food or feed additives. Other ways of processing citrus by-products have been mentioned in US patent 4,225,625 where a method for separating citrus peels into separate albedo and flavedo components is explained, which are regions comprised in the citrus peel, being albedo, a source of dietary fiber that includes sugar, cellulose, and pectic substances; meanwhile, flavedo is a source of vitamin A, carotenes and xanthophylls, being used as a coloring agent in food, a clouding agent in fruit juice drinks and fortifying food for livestock and humans. In another of the US patents, such as US patent 7,060,313, both the citrus peel and pulp without juice are processed to recover high-value components such as molasses, pectin pomace, D-limonene and food-grade peel mass. . Another conversion of citrus by-products is read in patent US7,629,010 which mentions a method for recovering citrus fiber from citrus vesicles using organic solvents to obtain a food additive for beverages, baked goods, meat or meat emulsions, confectionery, jams and jellies. The use of organic solvents is also taught in patent document US6,183,806 for the preparation of citrus peel and flour extracts. Another way to recover the compounds is to obtain citrus oil as taught in US patent US6,151,799.
La patente CN113501892A describe un método de extracción combinado en cáscaras de cítricos para la obtención de diferentes componentes funcionales y su aplicación como aditivo en alimentos, medicinas, cosméticos o productos sanitarios. Entre los compuestos destacan, limonina, pectina y sustancias flavonoides. Asimismo, en el documento de patente estadounidense US20100159100A1 el método de innovación consiste en la recuperación de hespehdina, narirutin, didymin, compuestos flavonoides, del bagazo y cáscara de cítricos luego de la extracción del jugo para incorporarlos en un concentrado de jugo de naranja. Una invención similar es US20100159115A1 , el cual detalla un proceso de extracción de flavonoides del subproducto de jugo de cítricos, el objetivo es disminuir el nivel de limonina y otros compuestos amargos en el jugo y su vez, utilizarlos para crear un extracto independiente. Semejante al documento de patente estadounidense US9195089A1 , en la que se describe un proceso de extracción de compuestos amargos presentes en los subproductos de proceso de extracción de jugo de cítrico. Bajo esa misma premisa, la patente estadounidense US20040081734A1 , se refiere a un método general de extracción selectiva para la obtención de extractos acuosos ricos en antioxidantes y su composición equilibrada de fenoles simples, polifenoles, bioflavonoides, flavonas y flavononas para controlar procesos de oxidación de productos cítricos. Patent CN113501892A describes a combined extraction method in citrus peels to obtain different functional components and their application as additive in food, medicine, cosmetics or health products. Among the compounds, limonin, pectin and flavonoid substances stand out. Likewise, in the US patent document US20100159100A1, the innovative method consists in the recovery of hespehdin, narirutin, didymin, flavonoid compounds, from citrus bagasse and peel after juice extraction to incorporate them into an orange juice concentrate. A similar invention is US20100159115A1, which details a process for extracting flavonoids from citrus juice by-product, the aim of which is to decrease the level of limonin and other bitter compounds in the juice and, in turn, use them to create an independent extract. Similar to US patent document US9195089A1, which describes an extraction process for bitter compounds present in the by-products of the citrus juice extraction process. Under the same premise, US patent US20040081734A1 refers to a general selective extraction method for obtaining aqueous extracts rich in antioxidants and their balanced composition of simple phenols, polyphenols, bioflavonoids, flavones and flavonones to control product oxidation processes. citrus.
Por lo antes expuesto, las invenciones del estado del arte relacionan procesos de extracción de compuestos bioactivos obtenidos a partir de subproductos del procesamiento de frutas, de preferencia bagazo y cáscara de cítricos para su aplicación en bebidas, cosméticos, medicina y otros semejantes; sin embargo, no se ha considerado la aplicación de un compuesto concentrado, rico en flavonoides, en el sector agrícola, especialmente en una invención que incorpore la composición concentrada y una suspensión coloidal para lograr la conservación poscosecha de productos hortofrutícolas. Esta composición de flavonoides contiene quercetina, naringenina y catequina que, en combinación sinérgica, mejora la conservación de alimentos frescos, tales como frutas, vegetales, hortalizas, tubérculos, rizomas y raíces. Por tanto, es necesario optar por un proceso eficiente de extracción de flavonoides con alto poder antioxidante proveniente de residuos orgánicos, y de fácil proceso sin aditivos químicos artificiales como el hidróxido de amonio o agroquímicos en el producto final, que contribuyan a conservar y/o mejorar las características nutricionales y organolépticas en alimentos frescos, dirigidos a humanos y animales, de grado alimentario. Due to the above, the inventions of the state of the art relate extraction processes of bioactive compounds obtained from by-products of fruit processing, preferably bagasse and citrus peel for their application in beverages, cosmetics, medicine and other similar; however, the application of a concentrated compound, rich in flavonoids, in the agricultural sector has not been considered, especially in an invention that incorporates the concentrated composition and a colloidal suspension to achieve postharvest preservation of fruit and vegetable products. This composition of flavonoids contains quercetin, naringenin and catechin which, in synergistic combination, improves the preservation of fresh foods, such as fruits, vegetables, tubers, rhizomes and roots. Therefore, it is necessary to opt for an efficient process for the extraction of flavonoids with high antioxidant power from organic waste, and an easy process without artificial chemical additives such as ammonium hydroxide or agrochemicals in the final product, which contribute to conserving and/or improve the nutritional and organoleptic characteristics in fresh foods, aimed at humans and animals, food grade.
DESCRIPCIÓN DE LA INVENCIÓN La presente invención propone un novedoso proceso para la obtención de una composición y una suspensión coloidal de flavonoides de cascaras de frutas de la familia rutaceae y género citrus y la composición y suspensión coloidal así obtenida, donde la composición de flavonoides contiene quercetina, nahngenina y catequina, en combinación sinérgica, en una concentración de al menos del 80% del compuesto concentrado, y este es aplicado para la conservación de alimentos frescos. DESCRIPTION OF THE INVENTION The present invention proposes a novel process for obtaining a composition and a colloidal suspension of flavonoids from fruit peels of the Rutaceae family and genus Citrus and the composition and colloidal suspension thus obtained, where the flavonoid composition contains quercetin, nahngenin and catechin. , in synergistic combination, in a concentration of at least 80% of the concentrated compound, and this is applied for the preservation of fresh foods.
Asimismo, la presente invención se refiere un compuesto y/o suspensión coloidal con efecto protector para los productos hortofrutícolas, tales como frutas, hortalizas, tubérculos, rizomas y raíces, y su posterior extensión de vida útil. Likewise, the present invention refers to a compound and/or colloidal suspension with a protective effect for fruit and vegetable products, such as fruits, vegetables, tubers, rhizomes and roots, and their subsequent extension of useful life.
Esta invención contiene una composición de flavonoides que tienen estructura de fenil- benzo-y-pirona (o fenil-Y-cromona), productos del metabolismo secundario de las cáscaras de frutas de la familia Rutaceae y género Citrus utilizados como materia prima, los cuales se encuentran en estado libre o en forma de heterósidos. Estos heterósidos son generalmente solubles en agua, mientras que sus geninas o agliconas, parte no azucarada del heterósido, lo son sólo ligeramente. This invention contains a composition of flavonoids that have a phenyl-benzo-y-pyrone (or phenyl-Y-chromone) structure, products of the secondary metabolism of the peels of fruits of the Rutaceae family and Citrus genus used as raw material, which They are found in the free state or in the form of heterosides. These heterosides are generally soluble in water, while their genins or aglycones, the unsweetened part of the heteroside, are only slightly so.
Los flavonoides de esta invención están compuestos por una estructura de 3 anillos con un esqueleto de difenil propano fórmula I.
Figure imgf000008_0001
The flavonoids of this invention are composed of a 3-ring structure with a skeleton of diphenylpropane formula I.
Figure imgf000008_0001
Este único anillo se sintetiza por la condensación de 3 moles de malonil-coenzima A derivada del metabolismo de la glucosa y produce una chalcona sintasa formula II.
Figure imgf000008_0002
This single ring is synthesized by the condensation of 3 moles of malonyl-coenzyme A derived from glucose metabolism and produces a formula II chalcone synthase.
Figure imgf000008_0002
Como estructura básica, los flavonoides presentan un esqueleto carbonado C6-C3-C6 - fórmula III, donde los componentes C6 son anillos aromáticos unidos por tres átomos de carbono que pueden formar o no un tercer anillo pirano o pirona (anillos A-C). Las distintas clases de flavonoides se diferencian en la concentración de saturación y en los sustituyeles del anillo C, mientras que los compuestos individuales, dentro de cada uno de estos grupos, se distinguen por la diferente sustitución de los anillos A y B. De esta forma, hemos identificado 2 flavonoles y un flavonoide en la invención.
Figure imgf000009_0001
As a basic structure, flavonoids have a C6-C3-C6 carbon skeleton - formula III, where the C6 components are aromatic rings joined by three carbon atoms that may or may not form a third pyran or pyrone ring (AC rings). The different classes of flavonoids differ in the saturation concentration and in the substituents of the C ring, while individual compounds, within each of these groups, are distinguished by the different substitution of the A and B rings. Thus, we have identified 2 flavonols and one flavonoid in the invention.
Figure imgf000009_0001
A partir de esta distribución, nuestra invención aisló quercetina presente como (2- (3,4- dihidroxifenil) -3,5,7 trihidroxi-4 H -cromen-4-ona) fórmula IV. From this distribution, our invention isolated quercetin present as (2-(3,4-dihydroxyphenyl)-3,5,7 trihydroxy-4 H -chromen-4-one) formula IV.
La biosíntesis de la quercetina, en su etapa inicial pasó a través de la ruta fenilpropanoide, donde la fenilalanina se convirtió en 4-cumahl-CoA por la acción catalítica de la fenilalanina amoniaco liasa, cinamato-4-hidroxilasa y 4-cumahl CoA ligasa. Luego, la chalcona sintasa catalizó la combinación de 4-cumaroil-CoA con tres moléculas de malonil-CoA (3), lo que condujo a la formación de tetrahidroxi chalcona. La tetrahidroxi chalcona se convirtió en naringenina usando la chalcona isomerasa y la nahngenina se convirtió en ehodictiol por la acción de la enzima flavanona-3[3- hidroxilasa. El ehodictiol formado se convirtió en dihidroquercetina por la acción de la flavanona-3[3-h¡drox¡lasa, que finalmente se convirtió en quercetina, fórmula IV por el flavonol sintasa.
Figure imgf000009_0002
Quercetin biosynthesis, in its initial stage, passed through the phenylpropanoid pathway, where phenylalanine was converted to 4-cumahl-CoA by the catalytic action of phenylalanine ammonia lyase, cinnamate-4-hydroxylase, and 4-cumahl CoA ligase. . Chalcone synthase then catalyzed the combination of 4-coumaroyl-CoA with three malonyl-CoA molecules (3), leading to the formation of tetrahydroxy chalcone. Tetrahydroxy chalcone was converted to naringenin using chalcone isomerase and nahngenin was converted to ehodictyol by the action of the enzyme flavanone-3[3-hydroxylase. The ehodictyol formed was converted to dihydroquercetin by the action of flavanone-3[3-hydroxylase, which was finally converted to quercetin, formula IV by flavonol synthase.
Figure imgf000009_0002
Por su parte, la biosíntesis de catequina comienza con una unidad de iniciación 4- hidroxicinamoil CoA que experimenta una extensión de cadena mediante la adición de tres malonil-CoAs a través de una ruta PKSIII. La 4-hidroxicinamoil CoA se biosintetiza a partir de L-fenilalanina a través de la vía Shikimate. La L-fenilalanina se desamina primero por la fenilalanina amoniaco liasa (PAL) formando ácido cinámico que luego se oxida a ácido 4-hidroxicinámico por la cinamato 4-hidroxilasa. La calcona sintasa luego cataliza la condensación de 4-hidroxicinamoil CoA y tres moléculas de malonil-CoA para formar chalcona. La chalcona luego se isomehza a nahngenina por la chalcona isomerasa que se oxida aeriodictyol por flavonoide 3'-hidroxilasa y luego oxidado a taxifolina por flavanona 3-hidroxilasa. Luego, la taxifolina se reduce mediante dihidroflavanol 4-reductasa y leucoantocianidina reductasa para producir catequina.
Figure imgf000010_0001
For its part, catechin biosynthesis begins with a 4-hydroxycinnamoyl CoA initiation unit that undergoes chain extension by adding three malonyl-CoAs through a PKSIII pathway. 4-Hydroxycinnamoyl CoA is biosynthesized from L-phenylalanine via the Shikimate pathway. L-phenylalanine is deaminated first by phenylalanine ammonia lyase (PAL) forming cinnamic acid which is then oxidized to 4-hydroxycinnamic acid by cinnamate 4-hydroxylase. Chalcone synthase then catalyzes the condensation of 4-hydroxycinnamoyl CoA and three malonyl-CoA molecules to form chalcone. The chalcone is then isomehzada nahngenin by chalcone isomerase which is oxidized aeriodictyol by flavonoid 3'-hydroxylase and then oxidized to taxifolin by flavanone 3-hydroxylase. Taxifolin is then reduced by dihydroflavanol 4-reductase and leucoanthocyanidin reductase to produce catechin.
Figure imgf000010_0001
Mientras que, la nahngenina, fórmula V, en su etapa inicial pasó a través de la ruta fenilpropanoide, donde la fenilalanina se convirtió en 4-cumahl-CoA por la acción catalítica de la fenilalanina amoniaco liasa, cinamato-4-hidroxilasa y 4-cumahl CoA ligasa. Luego, la chalcona sintasa catalizó la combinación de 4-cumaroil-CoA con tres moléculas de malonil-CoA, lo que condujo a la formación de tetrahidroxi chalcona. Finalmente, la tetrahidroxi chalcona se convirtió en nahngenina usando la chalcona isomerasa.
Figure imgf000010_0002
While, nahngenine, formula V, in its initial stage passed through the phenylpropanoid pathway, where phenylalanine was converted into 4-cumahl-CoA by the catalytic action of phenylalanine ammonia lyase, cinnamate-4-hydroxylase and 4- cumahl CoA ligase. Chalcone synthase then catalyzed the combination of 4-coumaroyl-CoA with three malonyl-CoA molecules, leading to the formation of tetrahydroxy chalcone. Finally, tetrahydroxy chalcone was converted to nahngenin using chalcone isomerase.
Figure imgf000010_0002
Finalmente, la presente invención a partir de las cáscaras de frutas de la familia Rutaceae y género Citrus, se extrajo una serie de moléculas generando una composición de flavonoides, fórmula (VI), cuyos compuestos mayoritarios son la quercetina, catequina y nahngenina.
Figure imgf000011_0001
Finally, the present invention from the peels of fruits of the Rutaceae family and Citrus genus, a series of molecules was extracted generating a composition of flavonoids, formula (VI), whose main compounds are quercetin, catechin and nahngenin.
Figure imgf000011_0001
El objeto de la invención es el proceso para la obtención de una composición de flavonoides concentrada, a partir de cáscaras de frutas de la familia Rutaceae y género Citrus como son las cáscaras frescas del fruto, que comprende al menos 80% de flavonoides del tipo quercetina, naringenina y catequina, que agrupan las siguientes etapas a) extracción primaria y eliminación de terpenos volátiles b) extracción eficiente con solventes acuosos polares, c) ultrafiltración y adsorción con carbón activado ecológico, d) concentración y biosíntesis de la composición de flavonoides. The object of the invention is the process for obtaining a concentrated flavonoid composition, from fruit peels of the Rutaceae family and Citrus genus, such as fresh fruit peels, which comprises at least 80% of quercetin-type flavonoids. , naringenin and catechin, which group the following stages a) primary extraction and elimination of volatile terpenes b) efficient extraction with polar aqueous solvents, c) ultrafiltration and adsorption with ecological activated carbon, d) concentration and biosynthesis of the flavonoid composition.
La extracción primaria y eliminación de terpenos volátiles de las cáscaras de frutas de la familia Rutaceae y género Citrus, puede comprender los siguientes procesos: The primary extraction and elimination of volatile terpenes from the peels of fruits of the Rutaceae family and Citrus genus, can comprise the following processes:
- Selección, lavado y desinfección de las cáscaras de frutas seleccionadas. - Selection, washing and disinfection of the peels of selected fruits.
- Extracción primaria, de las cáscaras frescas, asistidas con ondas microondas a una potencia entre 200 - 800 W por 10- 50 minutos, sin usar ningún solvente orgánico, o usando la técnica de arrastre de vapor por 40 - 70 minutos. - Primary extraction, from the fresh shells, assisted with microwave waves at a power between 200 - 800 W for 10 - 50 minutes, without using any organic solvent, or using the steam stripping technique for 40 - 70 minutes.
- Los terpenos extraídos son separados del proceso por presentar baja solubilidad polar y disminuir la pureza de los compuestos principales. Las cáscaras frescas con un menor porcentaje de agua son reservadas para los siguientes procesos. La extracción eficiente con solventes acuosos polares se realiza mediante una extracción asistida con calor a 30-50°C, por un ratio de 1 -4 horas, usando soluciones acuosas en proporción entre 10-40% etanol de caña de azúcar en relación solvente materia prima entre 1 :8 - 1 :20, en base a la naturaleza y estado de maduración de la materia prima. - The extracted terpenes are separated from the process because they present low polar solubility and decrease the purity of the main compounds. The fresh shells with a lower percentage of water are reserved for the following processes. The efficient extraction with polar aqueous solvents is carried out by means of a heat-assisted extraction at 30-50°C, for a ratio of 1-4 hours, using aqueous solutions in a proportion between 10-40% sugarcane ethanol in matter solvent ratio. raw between 1:8 - 1:20, based on the nature and state of maturation of the raw material.
La ultrafiltración y adsorción con carbón activado ecológico del extracto de residuos de frutas de la familia Rutaceae y género Citrus comprende el uso de un ultrafiltrador con tamaño de partícula 0.04 - 0.1 um y una columna de carbón activado para retener olores típicos, por donde pasa el fluido con asistencia de una bomba en forma tangencial a una presión de bombeo entre 50 kPa a 100 kPa. Previamente, el carbón activado ecológico se obtiene a partir del acondicionamiento de los residuos sólidos filtrados de las frutas de la familia Rutaceae y género Citrus con una solución de ácido fosfórico 20- 25% carbonizado a 426-526 °C por un tiempo de 3-6 horas. The ultrafiltration and adsorption with ecological activated carbon of the extract of fruit residues of the Rutaceae family and Citrus genus comprises the use of an ultrafilter with a particle size of 0.04 - 0.1 um and an activated carbon column to retain typical odors, through which the fluid with the assistance of a tangential pump at a pumping pressure between 50 kPa and 100 kPa. Previously, the ecological activated carbon is obtained from the conditioning of the filtered solid residues of the fruits of the Rutaceae family and Citrus genus with a 20-25% phosphoric acid solution carbonized at 426-526 °C for a time of 3- 6 hours.
La concentración y biosíntesis de la composición de flavonoides se realiza mediante una destilación al vacío en un evaporador giratorio sometido a vacío entre 12,7 mmh a 50, 8 mmHg a 30-50°C por 40 - 70 minutos, añadiendo en una primera reacción buffer fosfato (50 mM) y de forma secuencial el extracto cítrico usado como coadyuvante tecnológico, hasta lograr una concentración entre 10-35%. The concentration and biosynthesis of the flavonoid composition is carried out by vacuum distillation in a rotary evaporator under vacuum between 12.7 mmh to 50.8 mmHg at 30-50°C for 40-70 minutes, adding in a first reaction phosphate buffer (50 mM) and sequentially the citrus extract used as processing aid, until reaching a concentration between 10-35%.
El objeto de la invención también comprende la preparación de una suspensión coloidal y/o película con efecto protector para los productos hortofrutícolas, tales como frutas, hortalizas, tubérculos, rizomas y raíces, y su posterior extensión de vida útil, gracias al efecto quelante y antioxidante de los flavonoles eficientemente extraídos y concentrados, que comprende los siguientes postulados: The object of the invention also includes the preparation of a colloidal suspension and/or film with a protective effect for fruit and vegetable products, such as fruits, vegetables, tubers, rhizomes and roots, and its subsequent extension of useful life, thanks to the chelating effect and antioxidant of efficiently extracted and concentrated flavonols, which includes the following postulates:
- realizar una extracción primaria de las cáscaras de frutas asistida, y eliminar los terpenos volátiles de las cáscaras de frutas, las cáscaras frescas con un menor porcentaje de agua son reservadas para los siguientes procesos; - carry out a primary extraction of the assisted fruit peels, and eliminate the volatile terpenes from the fruit peels, the fresh peels with a lower percentage of water are reserved for the following processes;
- realizar una extracción eficiente con solventes acuosos polares del producto de la etapa anterior con calor entre 30-50°C; - carry out an efficient extraction with polar aqueous solvents of the product of the previous stage with heat between 30-50°C;
- realizar una ultrafiltración y adsorción con carbón activado ecológico del producto de la etapa anterior; - carry out an ultrafiltration and adsorption with ecological activated carbon of the product of the previous stage;
- realizar una concentración y biosíntesis de la composición de flavonoides del producto de la etapa anterior; - Suspender y/o disolver entre 6-30% del extracto líquido concentrado en un material coloidal como el alginato de sodio, goma xanthan, goma arábiga, goma guar, y/o cualquier goma afín en una proporción entre 2- 5%, junto con aditivos que complementan su efecto de barrera como un plastificante de grado alimentario entre 2-5%, ceras de origen vegetal, como la cera de carnauba o candelilla entre 5-12%. - carry out a concentration and biosynthesis of the flavonoid composition of the product of the previous stage; - Suspend and/or dissolve between 6-30% of the concentrated liquid extract in a colloidal material such as sodium alginate, xanthan gum, gum arabic, guar gum, and/or any related gum in a proportion between 2-5%, together with additives that complement its barrier effect such as a food grade plasticizer between 2-5%, waxes of vegetable origin, such as carnauba or candelilla wax between 5-12%.
- Formación de microemulsiones de la suspensión anterior, a partir de altas revoluciones y esfuerzos de cizalla por encima de 10 000 RPM para una eficiente aplicación sobre la superficie de las frutas. - Formation of microemulsions from the previous suspension, from high revolutions and shear stresses above 10,000 RPM for an efficient application on the surface of the fruits.
- Deshidratación y/o pulverización de la suspensión coloidal anterior para su manipulación en forma sólida. - Dehydration and/or pulverization of the previous colloidal suspension for its handling in solid form.
La composición de flavonoides concentrada de residuos frutas de la familia Rutaceae y género Citrus puede aplicarse directamente sobre productos hortofrutícolas, en una concentración en peso entre 2-15%, a través de una solución con agua purificada en relación, en base a la naturaleza del producto, con el fin de un aplicar una primera barrera funcional fungiestática. The composition of flavonoids concentrated from fruit residues of the Rutaceae family and Citrus genus can be applied directly to fruit and vegetable products, in a concentration by weight between 2-15%, through a solution with purified water in relation, based on the nature of the product, in order to apply a first fungistatic functional barrier.
La composición de flavonoides concentrada de residuos frutas de la familia Rutaceae y género Citrus puede aplicarse como coadyuvante tecnológico con función acidificante en una matriz si se aplica en una concentración en peso entre 1 -5%, a través de una solución con agua purificada. The composition of flavonoids concentrated from fruit residues of the Rutaceae family and Citrus genus can be applied as a processing aid with an acidifying function in a matrix if it is applied in a concentration by weight between 1-5%, through a solution with purified water.
Una ventaja de la presente invención es que la composición de flavonoides (concentrada^ incluye flavonoides del tipo quercetina, nahngenina y catequina, en combinación sinérgica, en una concentración de al menos 80%. An advantage of the present invention is that the flavonoid composition (concentrated) includes flavonoids of the quercetin, nahngenin and catechin type, in synergistic combination, in a concentration of at least 80%.
En una forma de realización, la suspensión coloidal acuosa incluye una proporción de 1 - 30% de la composición de flavonoides, 2 - 5% de un coloide grado alimentario, 2 - 5% plastificante grado alimentario y 5 - 12% de cera vegetal. En otra forma de realización, la suspensión coloidal acuosa incluye una proporción de 1 - 30% de extracto de flavonoides y el resto contiene coloides y excipientes para la formación de la suspensión propiamente dicha. Otra ventaja de la invención es que en su aplicación logra mantener la calidad de productos hortofrutícolas frescos diversos en el tiempo, hasta por encima del 200% de su vida útil, y su enriquecimiento con agentes activos de los residuos de frutas de la familia Rutaceae y género Citrus, y su uso integral en el proceso productivo. In one embodiment, the aqueous colloidal suspension includes a proportion of 1-30% flavonoid composition, 2-5% food grade colloid, 2-5% food grade plasticizer, and 5-12% vegetable wax. In another embodiment, the aqueous colloidal suspension includes a proportion of 1-30% flavonoid extract and the remainder contains colloids and excipients for the formation of the suspension itself. Another advantage of the invention is that in its application it manages to maintain the quality of various fresh fruit and vegetable products over time, up to over 200% of its useful life, and its enrichment with active agents from fruit residues of the Rutaceae family and genus Citrus, and its integral use in the production process.
Otro objetivo de la invención es obtener una composición de flavonoides con poder antioxidante de alta concentración fenólica, a partir de residuos de la agroindustria citrícola, cuyo efecto entre los compuestos mayohtarios quercetina, naringenina y catequina presenten un efecto inesperado sinérgico positivo sobre la vida útil de los productos frescos perecibles, gracias a su poder quelante que reduce el estrés postcosecha de las frutas y su efecto funcional fungiestático no reportado previamente en la composición flavonoide. Another objective of the invention is to obtain a composition of flavonoids with antioxidant power of high phenolic concentration, from residues of the citrus agroindustry, whose effect between the major compounds quercetin, naringenin and catechin present an unexpected positive synergistic effect on the useful life of perishable fresh products, thanks to its chelating power that reduces postharvest stress of fruits and its fungistatic functional effect not previously reported on the flavonoid composition.
La invención supone una mejora en el estado de la técnica, puesto que, desde diferentes puntos de vista, se le atribuye a la patente un impacto positivo en los productos agrícolas frescos, protegiéndolos durante su procesamiento, almacenamiento, transporte y conservando su calidad sensorial, debido a la capacidad antioxidante brindado por los flavonoides extraídos y concentrados mencionados anteriormente. A partir de una visión ecológica, responde a la necesidad de generar biotecnologías que disminuyan la cantidad de empaques poliméhcos no biodegradables, que son contaminantes para el medio ambiente. The invention represents an improvement in the state of the art, since, from different points of view, the patent is credited with a positive impact on fresh agricultural products, protecting them during their processing, storage, transport and preserving their sensory quality, due to the antioxidant capacity provided by the extracted and concentrated flavonoids mentioned above. Based on an ecological vision, it responds to the need to generate biotechnologies that reduce the amount of non-biodegradable polymehc packaging, which pollutes the environment.
BREVE DESCRIPCIÓN DE LAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
Figura 1 : Diagrama de flujo del proceso de la invención en el que se detallan sus cuatro etapas principales. Figure 1: Flowchart of the process of the invention in which its four main stages are detailed.
Figura 2: Porcentaje promedio de sólidos solubles totales (%SST) en mango del tipo ‘Kent’. Figure 2: Average percentage of total soluble solids (%TSS) in 'Kent' type mango.
Figura 3: Dinámica de los sólidos solubles (°Bhx) en peras del tipo ‘Packham’s Triumph’. Figure 3: Dynamics of soluble solids (°Bhx) in pears of the 'Packham's Triumph' type.
Figura 4: Concentración Mínima Inhibitoria de la invención formulada. Figure 4: Minimum Inhibitory Concentration of the formulated invention.
EJEMPLOS PREFERIDOS (O REALIZACIÓN PREFERENTE DE LA INVENCIÓN) A. Proceso para la obtención de una composición de flavonoides de cáscaras de frutas de la familia Rutaceae y género Citrus para la conservación de alimentos frescos y la suspensión coloidal así obtenida y su aplicación. PREFERRED EXAMPLES (OR PREFERRED EMBODIMENT OF THE INVENTION) A. Process for obtaining a composition of flavonoids from fruit peels of the Rutaceae family and Citrus genus for the preservation of fresh foods and the colloidal suspension thus obtained and its application.
Una preparación de la composición de flavonoides de cáscaras de frutas de la familia Rutaceae y género Citrus para la conservación de alimentos frescos y la suspensión coloidal así obtenida, de la invención, a manera de ejemplo, supone la selección, lavado y desinfección de las cáscaras del fruto Citrus reticulata en temporada temprana con 100 ppm de hipoclohto de sodio diluido en agua filtrada o un desinfectante para alimentos afín; luego las cáscaras frescas pasan por una extracción primaria asistida con ondas microondas a una potencia preferiblemente entre 200 a 800 W por 40 minutos, sin usar ningún solvente orgánico. En seguida, las cáscaras se extraen de manera eficiente con solventes acuosos y calor a 40°C por un ratio de 4 horas, en proporción de 20% etanol en relación solvente materia prima entre 1 :20. Una vez culminado se purifica con asistencia de un proceso de ultrafiltración que comprende el uso de un filtrador con tamaño de partícula 0.1 um, por donde pasa el fluido con asistencia de una bomba en forma tangencial a una presión de bombeo a 1 bar, y una columna de carbón activado ecológica elaborada a partir de los sólidos filtrados anteriores carbonizados y acondicionados con 30% de ácido fosfórico, que le confiere una adsorción de los olores y un proceso circular del uso integral de los residuos. La solución se concentra con el método de destilación al vacío en un evaporador sometido a una presión entre 12,7 - 50, 8 mmHg a 50°C por 40 minutos, añadiendo tampón fosfato salino (50 mM) en hasta un pH<3 y lograr una concentración de al menos 10%. A preparation of the composition of flavonoids from fruit peels of the Rutaceae family and Citrus genus for the preservation of fresh foods and the colloidal suspension thus obtained, of the invention, by way of example, involves the selection, washing and disinfection of the peels of Citrus reticulata fruit in early season with 100 ppm sodium hypochlorite diluted in filtered water or a related food sanitizer; then the fresh shells go through a primary extraction assisted with microwave waves at a power preferably between 200 to 800 W for 40 minutes, without using any organic solvent. Next, the shells are efficiently extracted with aqueous solvents and heat at 40°C for a ratio of 4 hours, in a proportion of 20% ethanol in a raw material solvent ratio between 1:20. Once completed, it is purified with the assistance of an ultrafiltration process that includes the use of a filter with a particle size of 0.1 um, through which the fluid passes with the assistance of a tangential pump at a pumping pressure of 1 bar, and a ecological activated carbon column made from the previously filtered solids carbonized and conditioned with 30% phosphoric acid, which gives it an adsorption of odors and a circular process of the integral use of waste. The solution is concentrated with the vacuum distillation method in an evaporator subjected to a pressure between 12.7 - 50.8 mmHg at 50°C for 40 minutes, adding saline phosphate buffer (50 mM) until pH<3 and achieve a concentration of at least 10%.
En un proceso aparte, se suspende 10% de la composición de flavonoides en una solución de alginato de sodio al 6% plastificante USP (3%), ácido graso saturado (5%), y agua destilada con movimiento constante a 500 RPM durante 1 hora y se homogeniza a 15,000 RPM formando una microemulsión que forma parte del ejemplo preferente que se aplica sobre productos agrícolas postcosecha frescos, a partir del método de aspersión aplicando entre 1 -1.3 litros de la dispersión por tonelada de producto agrícola, se presentan los resultados a continuación: In a separate process, 10% of the flavonoid composition is suspended in a solution of 6% sodium alginate, USP plasticizer (3%), saturated fatty acid (5%), and distilled water with constant motion at 500 RPM for 1 hour and homogenizes at 15,000 RPM forming a microemulsion that is part of the preferred example that is applied on fresh post-harvest agricultural products, from the spray method applying between 1 -1.3 liters of the dispersion per ton of agricultural product, the results are presented next:
Evaluación de la cobertura referente a la invención en la vida poscosecha de productos hortícolas Se realizaron dos experimentos para evaluar el efecto de la invención propuesta en la vida poscosecha de mangos (Mangifera indica var. ‘Kent’) y peras (Pyrus communis var. ‘Packham's Triumph’) en comparación a un control negativo sin cobertura (Tabla 2). El tratamiento de cobertura concerniente a la invención fue aplicado mediante aspersión sobre la superficie de las frutas y dejada a secar a temperatura ambiente. Posteriormente, se almacenó las unidades experimentales en condiciones específicas para simular condiciones de exportación (Tabla 1 ). Evaluación de resultados Refrigeración Evaluation of the coverage regarding the invention in the postharvest life of horticultural products Two experiments were carried out to evaluate the effect of the proposed invention on the postharvest life of mangoes (Mangifera indica var. 'Kent') and pears (Pyrus communis var. 'Packham's Triumph') compared to a negative control without cover (Table 2 ). The covering treatment according to the invention was applied by spraying on the surface of the fruits and left to dry at room temperature. Subsequently, the experimental units were stored under specific conditions to simulate export conditions (Table 1). Evaluation of results Refrigeration
Tabla 1. Condiciones de almacenamiento
Figure imgf000016_0001
Tabla 2. Resumen de resultados
Figure imgf000017_0001
Table 1. Storage conditions
Figure imgf000016_0001
Table 2. Summary of results.
Figure imgf000017_0001
En la figura 2 se puede observar que, la aplicación de la cobertura referente a la invención genera, estadísticamente, una menor pérdida de peso en mangos almacenados en frío. Coberturas de diferentes formulaciones de alginato como la propuesta por Rastegar et al., (2019) reportan mayores pérdidas de peso, alrededor del 10%, a 12°C de almacenamiento, así como ausencia de efecto en la evolución del porcentaje de sólidos solubles, a diferencia de lo obtenido con la cobertura referente a la presente invención, donde la pérdida promedio de sólidos solubles totales (%SST) en mango está entre 2 a 3%. Figure 2 shows that the application of the covering referring to the invention statistically generates less weight loss in cold-stored mangoes. Coverages of different alginate formulations such as the one proposed by Rastegar et al., (2019) report higher weight losses, around 10%, at 12°C storage, as well as no effect on the evolution of the percentage of soluble solids, unlike what was obtained with the coverage referring to the present invention, where the average loss of total soluble solids (% TSS) in mango is between 2 to 3%.
En la figura 3 se puede observar que la aplicación de la cobertura referente a la presente invención genera, estadísticamente, una menor pérdida de peso en peras almacenados en frío, reduciendo en 1 °Bhx dicha pérdida medida a los 20 días después de la aplicación. Con respecto al antecedente Moraes et al., (2012), a diferencia de lo reportado por estos autores del antecedente, la cobertura de la presente invención genera diferencias significativas en la desaceleración de los sólidos solubles totales en comparación con el control. In figure 3 it can be seen that the application of the coverage referring to the present invention generates, statistically, a lower weight loss in pears stored in cold storage, reducing said loss by 1 °Bhx measured 20 days after the application. Regarding the antecedent Moraes et al., (2012), unlike what was reported by these authors of the antecedent, the coverage of the present invention generates significant differences in the deceleration of total soluble solids compared to the control.
Poder antioxidante de la Invención Antioxidant Power of the Invention
La composición rica en flavonoides se evaluó mediante análisis espectrofotométhcos con la finalidad de cuantificar los compuestos flavonoides totales (CFT) y su capacidad antioxidante, por el método de radical DPPH. Para un estudio más detallado, se realizó también la cuantificación de compuestos poli fenólicos totales (CPT). Los ensayos reportaron, valores desde 190.45±0.26 umol/g hasta 500.08±0.33 umol/g de muestra, en equivalentes de Trolox para DPPH; un contenido de flavonoides totales que comprende rangos de 257.03±0.18 mg/g y 1000.78±0.23 mg/g de muestra, en estándar de quercetina y finalmente niveles de 80.67 ± 5,9 mg /g, en estándar de ácido gálico (GAE) para CPT. Los mínimos y máximos están en función de los parámetros de extracción utilizados para la recuperación de los compuestos bioactivos, mencionados anteriormente. The flavonoid-rich composition was evaluated by spectrophotometric analysis in order to quantify the total flavonoid compounds (CFT) and their antioxidant capacity, by the DPPH radical method. For a more detailed study, the quantification of total polyphenolic compounds (CPT) was also performed. The trials reported values from 190.45±0.26 umol/g to 500.08±0.33 umol/g of sample, in Trolox equivalents for DPPH; a content of total flavonoids that includes ranges of 257.03 ± 0.18 mg/g and 1000.78 ± 0.23 mg/g of sample, in quercetin standard and finally levels of 80.67 ± 5.9 mg / g, in gallic acid standard (GAE) for cpt. The minimums and maximums are based on the extraction parameters used for the recovery of the bioactive compounds, mentioned above.
Autores como Sogi etal., (2013), detallan 188 umol eq.Trolox/g (DPPH), 20.32mg GAE/g (CPT) y no reporta CFT. Diversas investigaciones que utilizan tecnologías tradicionales han sido recopiladas en el ensayo de Rafiq et al., (2018), de los métodos se obtuvo 0.58 mg/g CPT en limón (cv. Meyer), 1.19 mg/g CPT en Limón (cv. Yenben), 1.211 mg/g CPT en mandarinas (cv. Ellendale), 0.736 mg/g CPT en naranjas (cv. Navel) 2.649-6.923 mg/g CPT en mandarinas. Ensayos en naranja Mauritian expresan resultados en estándar de quercetina generalmente superiores a 2 mg/g de muestra. Authors such as Sogi et al., (2013), detail 188 umol eq.Trolox/g (DPPH), 20.32mg GAE/g (CPT) and do not report CFT. Various investigations that use traditional technologies have been compiled in the trial by Rafiq et al., (2018), from the methods 0.58 mg/g CPT in lemon (cv. Meyer), 1.19 mg/g CPT in Lemon (cv. Yenben), 1.211 mg/g CPT in mandarins (cv. Ellendale), 0.736 mg/g CPT in oranges (cv. Navel) 2.649-6.923 mg/g CPT in mandarins. Assays in Mauritian orange express results in quercetin standard generally higher than 2 mg/g of sample.
La presente invención aumenta en más de 100% la eficiencia de extracción de los compuestos flavonoides presentes en los subproductos de cítricos que no se obtienen con los expuestos previamente. The present invention increases the extraction efficiency of flavonoid compounds present in citrus by-products that are not obtained with those previously exposed by more than 100%.
Poder fungistático fungistatic power
Para evaluar el poder fungistático de la presente invención se realizaron pruebas in vitro en hongos fitopatógeno de Colletotrichum gloeosporioides y Aspergillus niger. In order to evaluate the fungistatic power of the present invention, in vitro tests were carried out on phytopathogenic fungi of Colletotrichum gloeosporioides and Aspergillus niger.
Tabla 3. Condiciones para la evaluación del poder fungistático
Figure imgf000018_0001
Pruebas in vitro Table 3. Conditions for the evaluation of the fungistatic power.
Figure imgf000018_0001
in vitro tests
En placas Petri con Agar Sabouraud se sembraron las cepas de C. gloeosporioides y A. niger, se procedió a colocar 60 uL de la invención formulada (mezcla de flavonoides, como control positivo Belomil y agua destilada, como control negativo). The strains of C. gloeosporioides and A. niger were sown in Petri dishes with Sabouraud Agar, and 60 uL of the formulated invention was placed (flavonoid mixture, as Belomil positive control and distilled water, as negative control).
Los ensayos se realizan por triplicado al menos dos veces, obteniéndose de esta manera 3 tratamientos y 18 placas en total para cada uno de los hongos. The tests are carried out in triplicate at least twice, thus obtaining 3 treatments and 18 plates in total for each of the fungi.
Finalmente, se incubaron las placas a 25°C por siete días, tomando como resultado positivo la aparición de un halo de inhibición alrededor de los pocilios. Se observaron las zonas claras de inhibición del crecimiento (halos) y se midieron los diámetros en mm con el calibre Vernier, se registraron y se consideraron como un indicativo de actividad antifúngica. Finally, the plates were incubated at 25°C for seven days, taking as a positive result the appearance of an inhibition halo around the wells. The clear zones of growth inhibition (halos) were observed and the diameters were measured in mm with the Vernier caliper, recorded and considered as an indication of antifungal activity.
Los resultados fueron reportados en las tablas 4 y 5.
Figure imgf000019_0001
The results were reported in tables 4 and 5.
Figure imgf000019_0001
Tabla 4. Análisis estadístico de la media de los halos de inhibición de A. niger. Table 4. Statistical analysis of the mean of the inhibition halos of A. niger.
Inv. Invención de la formulación; C+. Belomil; C-. agua; N. número de repeticiones; DS. Desviación estándar. Inv. Invention of the formulation; C+. Belomil; C-. water; N. number of repetitions; DS. Standard deviation.
*Tukey; letras diferentes en las columnas representan diferencias significativas en un 5% de error.
Figure imgf000019_0002
Tabla 5. Análisis estadístico de la media de los halos de inhibición de C. gloesporioides. Inv. Invención de la formulación; C+. Belomil; C-. agua; N. número de repeticiones; DS. Desviación estándar. *Tukey; different letters in the columns represent significant differences within 5% error.
Figure imgf000019_0002
Table 5. Statistical analysis of the mean of the inhibition halos of C. gloesporioides. Inv. Invention of the formulation; C+. Belomil; C-. water; N. number of repetitions; DS. Standard deviation.
*Tukey; letras diferentes en las columnas representan diferencias significativas en un 5% de error. *Tukey; different letters in the columns represent significant differences within 5% error.
Con estos resultados, la formulación de la invención fue sometida a la prueba de micro dilución en placa para determinar su Concentración Mínima Inhibitoria (CMI). With these results, the formulation of the invention was submitted to the plate micro dilution test to determine its Minimum Inhibitory Concentration (MIC).
La Figura 4 muestra los resultados de la Concentración Mínima Inhibitoria (CMI) de la invención formulada frente a los fitopatógenos de Aspergillus niger y Colletotrichum gloesporioides. Las concentraciones de la presente invención vaharon entre 10 mg/ml y 0.02 mg/ml. Finalmente, la CMI para A. niger fue de 0.24 mg/ml y 0.12 mg/ml para C. gloesporioides. Figure 4 shows the results of the Minimum Inhibitory Concentration (MIC) of the invention formulated against the phytopathogens of Aspergillus niger and Colletotrichum gloesporioides. Concentrations of the present invention ranged from 10 mg/ml to 0.02 mg/ml. Finally, the MIC for A. niger was 0.24 mg/ml and 0.12 mg/ml for C. gloesporioides.
Por lo tanto, la invención formulada mostró efectiva actividad antifúngica con una CMI de 0.12 mg/ml para Colletotrichum gloesporioides, resultado que presentó ventajas altamente significativas frente a diversos estudios que enfrentaron extractos a la cepa mencionada; por ejemplo, Paredes et. al (2021 ) reportaron una CMI de 0.14mg/ml para el extracto metanólico de Samanea saman, mientras que Aguilar et al (2013) reportaron un CMI de 150mg/L y 300mg/L para extractos etanólicos de ruda y pirúl respectivamente. En cuanto a Aspergillus niger, en el presente estudio se reportó una CMI de 0.24mg/ml resultado que difiere ventajosamente con diversos estudios, como el de Díaz et al (2019) quienes reportaron una CMI de 3.12 mg/ml para el extracto crudo de ajo. Therefore, the formulated invention showed effective antifungal activity with a MIC of 0.12 mg/ml for Colletotrichum gloesporioides, a result that presented highly significant advantages over various studies that confronted extracts with the mentioned strain; for example, Paredes et. al (2021) reported a MIC of 0.14mg/ml for the methanolic extract of Samanea saman, while Aguilar et al (2013) reported a MIC of 150mg/L and 300mg/L for ethanolic extracts of rue and lollipop, respectively. Regarding Aspergillus niger, in the present study a MIC of 0.24mg/ml was reported, a result that differs advantageously with various studies, such as that of Díaz et al (2019) who reported a MIC of 3.12 mg/ml for the crude extract of garlic.

Claims

REIVINDICACIONES Un proceso para la obtención de una composición de flavonoides de cascaras de frutas de la familia Rutaceae y género Citrus para la conservación de alimentos frescos, caracterizado porque comprende las siguientes etapas: CLAIMS A process for obtaining a composition of flavonoids from fruit peels of the Rutaceae family and Citrus genus for the preservation of fresh food, characterized in that it comprises the following stages:
- realizar una extracción primaria de las cáscaras de frutas asistida, y eliminar los terpenos volátiles de las cáscaras de frutas, las cáscaras frescas con un menor porcentaje de agua son reservadas para los siguientes procesos;- carry out a primary extraction of the assisted fruit peels, and eliminate the volatile terpenes from the fruit peels, the fresh peels with a lower percentage of water are reserved for the following processes;
- realizar una extracción eficiente con solventes acuosos polares del producto de la etapa anterior con calor entre 30-50°C; - carry out an efficient extraction with polar aqueous solvents of the product of the previous stage with heat between 30-50°C;
- realizar una ultrafiltración y adsorción con carbón activado ecológico del producto de la etapa anterior; - carry out an ultrafiltration and adsorption with ecological activated carbon of the product of the previous stage;
- realizar una concentración y biosíntesis de la composición de flavonoides del producto de la etapa anterior. El proceso para la obtención de la composición de flavonoides, según la reivindicación 1 , caracterizado porque previo a la etapa de la extracción primaria de las cáscaras de frutas se realiza una selección, lavado y desinfección de las cáscaras de frutas seleccionadas. El proceso para la obtención de la composición de flavonoides, según la reivindicación 1 , caracterizado porque la etapa de la extracción primaria es asistida con ondas microondas a una potencia entre 200 - 800 w por 10-50 minutos, sin usar ningún solvente orgánico, o usando la técnica de arrastre de vapor por 40 - 70 minutos. El proceso para la obtención de la composición de flavonoides, según la reivindicación 1 , caracterizado porque la etapa de realizar la extracción eficiente con solventes acuosos polares comprende una extracción asistida con calor entre 30-50°C por un ratio de 1 -4 horas, usando soluciones acuosas en proporción en peso entre 10-40% etanol en relación solvente materia prima entre 1 :8 - 1 :20, en base a la naturaleza y estado de maduración de la materia prima. El proceso para la obtención de la composición de flavonoides, según la reivindicación 1 , caracterizado porque la etapa de realizar la ultrafiltración y adsorción con carbón activado ecológico comprende el uso de un filtrador con tamaño de partícula 0.04 - 0.1 um y una columna de carbón activado para retener olores típicos, por donde pasa el fluido con asistencia de una bomba en forma tangencial a una presión de bombeo entre 50 kPa a 100 kPa. - carry out a concentration and biosynthesis of the flavonoid composition of the product of the previous stage. The process for obtaining the flavonoid composition, according to claim 1, characterized in that prior to the stage of primary extraction of the fruit peels, a selection, washing and disinfection of the selected fruit peels is carried out. The process for obtaining the flavonoid composition, according to claim 1, characterized in that the primary extraction stage is assisted with microwave waves at a power between 200 - 800 w for 10-50 minutes, without using any organic solvent, or using the steam stripping technique for 40 - 70 minutes. The process for obtaining the flavonoid composition, according to claim 1, characterized in that the stage of carrying out the efficient extraction with polar aqueous solvents comprises a heat-assisted extraction between 30-50°C for a ratio of 1-4 hours, using aqueous solutions in proportion by weight between 10-40% ethanol in raw material solvent ratio between 1:8 - 1:20, based on the nature and state of maturation of the raw material. The process for obtaining the flavonoid composition, according to claim 1, characterized in that the step of performing ultrafiltration and Ecological activated carbon adsorption comprises the use of a filter with a particle size of 0.04 - 0.1 um and an activated carbon column to retain typical odors, through which the fluid passes with the assistance of a tangential pump at a pumping pressure between 50 kPa to 100kPa.
6. El proceso para la obtención de la composición de flavonoides, según la reivindicación 1 , caracterizado porque el carbón activado ecológico se obtiene a partir del acondicionamiento de los residuos sólidos filtrados de las cáscaras con una solución de ácido fosfórico entre 20-25% carbonizado a 426-526 °C por un tiempo de 3-6 horas. 6. The process for obtaining the flavonoid composition, according to claim 1, characterized in that the ecological activated carbon is obtained from the conditioning of the solid waste filtered from the shells with a phosphoric acid solution between 20-25% carbonized at 426-526 °C for a period of 3-6 hours.
7. El proceso para la obtención de la composición de flavonoides, según la reivindicación 1 , caracterizado porque la extracción primaria se realiza sin deshidratar el residuo fresco ni la adición de solventes. 7. The process for obtaining the flavonoid composition, according to claim 1, characterized in that the primary extraction is carried out without dehydrating the fresh residue or adding solvents.
8. El proceso para la obtención de la composición de flavonoides, según la reivindicación 1 , caracterizado porque la concentración y biosíntesis de la composición de flavonoides comprende una destilación al vacío en un evaporador giratorio sometido a vacío entre 12,7 - 50, 8 mmHg a 30-50°C por 40 - 70 minutos, añadiendo en una primera reacción tampón fosfato salino y de forma secuencial un extracto cítrico usado como coadyuvante tecnológico, hasta lograr una concentración en peso entre 10-35%. 8. The process for obtaining the flavonoid composition, according to claim 1, characterized in that the concentration and biosynthesis of the flavonoid composition comprises vacuum distillation in a rotary evaporator under vacuum between 12.7 - 50.8 mmHg at 30-50°C for 40 - 70 minutes, adding saline phosphate buffer in a first reaction and sequentially adding a citric extract used as processing aid, until reaching a concentration by weight between 10-35%.
9. El proceso para la obtención de una composición de flavonoides según la reivindicación 1 , caracterizado porque no presenta solventes químicos artificiales como hidróxido de amonio, ni agroquímicos en su composición, y la composición de flavonoides es de grado alimentario previniente de residuos frutas de la familia Rutaceae y género Citrus. 9. The process for obtaining a flavonoid composition according to claim 1, characterized in that it does not have artificial chemical solvents such as ammonium hydroxide, or agrochemicals in its composition, and the flavonoid composition is food grade, preventing fruit residues from the family Rutaceae and genus Citrus.
10. Una composición de flavonoides obtenida de acuerdo con el proceso de la reivindicación 1 , caracterizada porque la composición incluye flavonoides del tipo quercetina, nahngenina y catequina en una concentración de al menos 80%. 10. A flavonoid composition obtained according to the process of claim 1, characterized in that the composition includes flavonoids of the quercetin, nahngenin and catechin type in a concentration of at least 80%.
11. La composición de flavonoides de acuerdo con la reivindicación 10, caracterizada porque la composición está en una concentración en peso entre 1- 15%, en una solución con agua purificada. 11. The flavonoid composition according to claim 10, characterized in that the composition is in a concentration by weight between 1-15%, in a solution with purified water.
12. La composición de flavonoides de acuerdo con la reivindicación 11 , caracterizada porque la composición está en una concentración en peso entre 1- 5%, en una solución con agua purificada adecuada para aplicarse como coadyuvante tecnológico con función acidificante en una suspensión coloidal. 12. The flavonoid composition according to claim 11, characterized in that the composition is in a concentration by weight between 1-5%, in a solution with purified water suitable for application as a processing aid with an acidifying function in a colloidal suspension.
13. Un proceso para la obtención de una suspensión coloidal de flavonoides de cáscaras de frutas de la familia Rutaceae y género Citrus para la conservación de alimentos frescos, caracterizado porque incluye: 13. A process for obtaining a colloidal suspension of flavonoids from fruit peels of the Rutaceae family and Citrus genus for the preservation of fresh food, characterized in that it includes:
- realizar una extracción primaria de las cáscaras de frutas asistida, y eliminar los terpenos volátiles de las cáscaras de frutas, las cáscaras frescas con un menor porcentaje de agua son reservadas para los siguientes procesos;- carry out a primary extraction of the assisted fruit peels, and eliminate the volatile terpenes from the fruit peels, the fresh peels with a lower percentage of water are reserved for the following processes;
- realizar una extracción eficiente con solventes acuosos polares del producto de la etapa anterior con calor entre 30-50°C; - carry out an efficient extraction with polar aqueous solvents of the product of the previous stage with heat between 30-50°C;
- realizar una ultrafiltración y adsorción con carbón activado ecológico del producto de la etapa anterior; - carry out an ultrafiltration and adsorption with ecological activated carbon of the product of the previous stage;
- realizar una concentración y biosíntesis de la composición de flavonoides del producto de la etapa anterior; - carry out a concentration and biosynthesis of the flavonoid composition of the product of the previous stage;
- suspender entre 6 - 30% en peso del extracto líquido concentrado en un material coloidal como alginato de sodio, goma xanthan, goma arábiga, goma guar, y/o cualquier goma afín en una proporción entre 2 - 5% en peso, junto con aditivos que complementan su efecto de barrera como plastificantes grado alimentario en concentración entre 2-5% en peso, ceras de origen vegetal en concentración entre 5-12% en peso; - suspend between 6 - 30% by weight of the concentrated liquid extract in a colloidal material such as sodium alginate, xanthan gum, gum arabic, guar gum, and/or any related gum in a proportion between 2 - 5% by weight, together with additives that complement its barrier effect such as food grade plasticizers in a concentration between 2-5% by weight, waxes of vegetable origin in a concentration between 5-12% by weight;
- formar microemulsiones de la suspensión anterior, a partir de altas revoluciones y esfuerzos de cizalla por encima de 10 000 RPM para una eficiente aplicación sobre la superficie de las frutas. - Form microemulsions of the previous suspension, from high revolutions and shear stresses above 10,000 RPM for an efficient application on the surface of the fruits.
14. El proceso para la obtención de la suspensión coloidal, según la reivindicación 13 caracterizado porque luego de la etapa de formar microemulsiones en la suspensión, se deshidrata y/o pulveriza con criodesecación o atomización. Una suspensión coloidal de flavonoides obtenida de acuerdo con el proceso de la reivindicación 13, caracterizada porque la suspensión coloidal está compuesta por una proporción en peso de 1 - 30% de composición de flavonoides, 2 - 5% de un coloide grado alimentario, 2 - 5% plastificante grado alimentario y 5 - 12% de cera vegetal. 14. The process for obtaining the colloidal suspension, according to claim 13, characterized in that after the stage of forming microemulsions in the suspension, it is dehydrated and/or sprayed with freeze-drying or spray drying. A colloidal suspension of flavonoids obtained according to the process of claim 13, characterized in that the colloidal suspension is composed of a proportion by weight of 1 - 30% flavonoid composition, 2 - 5% of a food grade colloid, 2 - 5% food grade plasticizer and 5 - 12% vegetable wax.
PCT/PE2021/050024 2021-11-23 2021-11-23 Method for obtaining a composition and a colloidal suspension of flavonoids from peels of fruits of the family rutaceae and the genus citrus, and the composition and colloidal suspension thus obtained WO2023096505A1 (en)

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