BR102020005993A2 - AÇAÍ PULP MICROCAPSULAS (EUTERPE OLERACEA MART.), AÇAÍ MICROCAPSULA PROCESSING PROCESS (EUTERPE OLERACEA MART.) AND USES - Google Patents

Abstract

microcapsules from açaí pulp (euterpe oleracea mart.), process of obtaining açaí microcapsules (euterpe oleracea mart.) and uses. Açaí pulp microcapsules, the process for obtaining passion fruit microcapsules and uses are proposed. the production of açaí pulp microcapsules uses standardized açaí pulp, containing active ingredients (anthocyanidins and flavonoids) in açaí microcapsules that are coated with biocompatible and biodegradable encapsulants, followed by drying using a spray-dryer technique, presenting in form of microparticles here also called microcapsules of açaí pulp. Açaí pulp microcapsules have potential application as auxiliary agents for disease prevention and to maintain well-being, in addition to their beneficial health properties. Compositions of açaí pulp microcapsules in solid form have beneficial properties for health, due to their antioxidant activity and in the prevention of pre-diabetes, diabetes, due to their hypoglycemic properties, as well as combating obesity, dyslipidemia and hypertension. Additionally, açaí pulp microcapsules can be used to prepare food products such as ice creams, mousses, cold drinks, cakes, cookies, sports food supplements or even nutraceutical capsules.

Description

AÇAÍ PULP MICROCAPSULAS (EUTERPE OLERACEA MART.), AÇAÍ MICROCAPSULA PROCESSING PROCESS (EUTERPE OLERACEA MART.) AND USES

[001] The invention proposes to develop a composition of AÇAÍ PULP MICROCAPSULAS (Euterpe oleracea Mart) with nutritional, functional and nutraceutical functions, due to the various medicinal properties of açaí, such as antioxidant, anti-inflammatory, hypoglycemic, cardioprotective action, antihypertensive, cancer prevention, among others. However, açaí pulp has some negative aspects, such as easy degradation due to the incidence of light, thermo-degradation, easy fermentation when stored at room temperature and that can hinder its application in the development of compositions, in nutraceutical foods and frozen drinks, ice cream and Mousse. Therefore, its modification is essential, such as its microencapsulation with suitable encapsulating agents, in order to mask and stabilize the phenolic compounds and their lipid constituents. In addition, it is intended to obtain compositions with immediate release in the gastrointestinal tract and physicochemically stable. Açaí pulp microencapsulates use protein food adjuvants, have potential application as auxiliary agents for disease prevention and to maintain well-being, in addition to their beneficial properties for health. Finally, the objective is to develop solid state açaí pulp compositions with beneficial properties for health, with attractive sensory characteristics, with practical applicability in nutraceutical foods, and which can also be added to ice cream, frozen drinks, mousses, in addition to produce pharmaceutical capsules containing the active ingredients of açaí or as food supplements for high-performance athletes due to its antioxidant properties and in the prevention of diabetes and pre-diabetes due to its hypoglycemic properties.

FUNDAMENTALS OF THE INVENTION

[002] The açaizeiro is a typical palm found in the floodplains along the estuary of the Amazon River that presents bunches with globose drupe with a diameter ranging from 1 to 2 cm, with epicopter of a yellowish and brown seed, hard and very thin layer which can be green, known as white açaí, or purple known as dark açaí, depending on the variety, and a mesocarp 1 to 2 mm thick (NERI-NUMA, IA et al. Small Brazilian wild fruits: Nutrients, bioactive compounds , healthpromotion properties and commercial interest. Food research international, v. 103, p. 345-360, 2018).

[003] Although in common terminology it is better known as açaí, other names are frequently used in areas of occurrence in the Brazilian Amazon, but in a more restricted way, with the following names standing out: açaí do Pará, açaí from the lower amazons, açaí of tussock, plant açaí, juçara and tussock juçara. The last two names are widely used in the State of Maranhão. It should also be noted that the other species, of the same generic taxon, occurring in the Amazon, are also called açaí, hence E. oleracea is also called, in some places, true açaí (DE OLIVEIRA, MS; DE CARVALHO, JEU ; DO NASCIMENTO, WMO Açaí (Euterpe oleracea Mart.). Funep, 2000).

[004] According to the Legislation of the Ministry of Agriculture (BRASIL, Ministry of Agriculture. Coordination of Plant Inspection. Decree No. 2,314, of September 4, 1997, art. 87, item II. Technical Regulations for Establishing Identity and Quality Standards for pulp of the following fruits: acerola, cocoa, cupuaçu, soursop, açaí, passion fruit, cashew, mango, guava, cherry, grape, papaya, caja, melon, mangaba, and for juice of the following fruits: passion fruit, cashew, high content cashew açaí fruit is classified according to amount of total solids in: coarse (Type A), which has 14% total solids and very dense appearance; in medium (Type B), which has 11% to 14% total solids and dense appearance; and fine (Type C), which contains 8 to 11% total solids and has a thin appearance.

[005] With the growth of the market, this product started to be consumed also in large Brazilian capitals, reaching even some countries such as Japan, China and the United States (MENEZES, EMS; TORRES, AT; SRUR, AUS Nutritional value of açaí pulp (Euterpe oleracea Mart) freeze-dried. Acta Amaz, v. 38, n. 2, p. 311-316, 2008). All parts of the açaí can be used, both the stone, which corresponds to 85% of the total weight, and the pulp, which represents 15%, and the fruit is not only used in the food and pharmaceutical industries, but also in the cosmetics and automobile industries.

[006] Açaí has been considered a superfruit due to its nutritional composition, coming to be considered a functional food, rich in proteins, fibers, lipids, vitamin E, and minerals such as magnesium, iron, copper, boron and chromium. amount of carbohydrates such as glucose, fructose and sucrose is relatively low between 2.96% and 3.55% of the total recommendation for the day (MATIAS DOS SANTOS, G. et al. Correlation between antioxidant activity and bioactive compounds from commercial açaí pulps (Euterpe oleácea Mart) Archivos latinoamericanos de nutricion, v. 58, n. 2, p. 187-192, 2008; DE LIMA YAMAGUCHI, KK et al. Amazon acai: Chemistry and biological activities: A review. 179, p. 137-151, 2015).

[007] Dias-Souza et al., 2018 (DIAS-SOUZA, MV et al. Euterpe oleracea pulp extract: Chemical analyses, antibiofilm activity against Staphylococcus aureus, cytotoxicity and interference on the activity of antimicrobial drugs. Microbial pathogenesis, v. 114 , p. 29-35, 2018) in their study found that the carbohydrate and protein content of the samples were within the values established by Brazilian legislation, 0.21 ± 0.13 g/g for carbohydrates and 1.4 ± 0.75 g/g for proteins.

[008] The high lipid content of açaí gives the product a high energy value, representing 70-90% of the total calories found in the juice, represented by oleic acid and palmitic acid. The high content of lipids gives açaí juice an energy value twice as high as those found in milk (50 kcal/100 mL) (MENEZES, EMS; TORRES, AT; SRUR, AUS Nutritional value of açaí pulp (Euterpe oleracea Mart) Acta Amaz, v. 38, no. 2, p. 311-316, 2008; DE SOUZA, MO et al. Diet supplementation with acai (Euterpe oleracea Mart.) pulp improves biomarkers of oxidative stress and the serum lipid profile in rats. Nutrition, v. 26, n. 7, p. 804-810, 2010). Thus, consumption of this fruit provides a good intake of mono- and polyunsaturated foods.

[009] Table 1 shows the proximate composition of the fruit found by several authors.

NEIDA, S.; ELBA, S. Caracterización del acai o manaca (Euterpe oleracea Mart.): un fruto del Amazonas. Archivoslatinoamericanos de nutrición, v. 57, n. 1, p. 94, 2007; MENEZES, E.M.S; TORRES, A.T; SRUR, A.U.S. Valor nutricional da polpa de açaí (Euterpe oleracea Mart) liofilizada. Acta Amaz, v. 38, n. 2, p. 311-316, 2008; FERNANDES, E.T.M. B. et al. Physicochemical composition, color and sensory acceptance of low-fat cupuaçu and açaí nectar: characterization and changes during storage. Food Science and Technology(Campinas), v. 36, n. 3, p. 413-420, 2016).[010] The different values in the published results are probably due to industrial processing, but another reason could be the high genetic variability of the species, the soil in which it was planted, seasonality, harvest time, maturation time, among others.
Table 1 - Proximate composition of açaí (Euterpe oleracea) by different authors in 100g of the fruit.

NEIDA, S.; ELBA, S. Characterization of acai or manaca (Euterpe oleracea Mart.): a fruit from the Amazon. Archivoslatinoamericanos de Nutrición, v. 57, no. 1, p. 94, 2007; MENEZES, EMS; TOWERS, AT; SRUR, AUS Nutritional value of freeze-dried açaí (Euterpe oleracea Mart) pulp. Acta Amaz, v. 38, no. 2, p. 311-316, 2008; FERNANDES, ETMB et al. Physicochemical composition, color and sensory acceptance of low-fat cupuaçu and açaí nectar: characterization and changes during storage. Food Science and Technology(Campinas), v. 36, no. 3, p. 413-420, 2016).

[011] The pulp of the fruit for presenting nutritional and therapeutic functions, has become increasingly object of study, mainly attributed to its antioxidant action, which seems to contribute to the prevention of disease. Among the antioxidants that stand out and are present in the fruit pulp are anthocyanins, flavonoids and phenolic compounds (MATIAS DOS SANTOS, G. et al. Correlation between antioxidant activity and bioactive compounds from commercial açaí pulps (Euterpe oleácea) Archivoslatinoamericanos de nutricion, v. 58, n. 2, p. 187- 192, 2008; VELASQUE, LFL; LOBO, ACM Literature review on the therapeutic effects of açaí and its importance in nutrition. Biohealth, v. 18 , no. 2, p. 97-106, 2017).

[012] E. oleracea is chemically characterized by the presence of bioactive substances. About 90 substances have been described, of which approximately 31% are composed of flavonoids, followed by phenolic compounds (23%), and anthocyanins (9%) (DE LIMA YAMAGUCHI, KK et al. Amazon acai: Chemistry and biological activities: A review. Food chemistry, v. 179, p. 137-151, 2015).

[013] Mulabagual and Calderon (MULABAGAL, V.; CALDERÓN, AI Liquid chromatography/mass spectrometry based fingerprinting analysis and mass profiling of Euterpe oleracea (açaí) dietary supplement raw materials. Foodchemistry, v. 134, n. 2, p. 1156 -1164, 2012), using coupled liquid chromatography technique and mass spectrometry, developed a fingerprinting analysis method, identified several flavonoids, phenolic acids, anthocyanidins and fatty acids, and among the substances identified, flavonoids appear in greater quantity. The main compounds identified were: vanillic acid, protocatecheutic acid, scoparin, chrysoeriol, taxifolin, orientin, isorientin, vitexin, isovitexin, quercetin, quercetin-3 rutinoside, quercetin-3 glucoside, kaempferol, kaempferolraminoside, kaempferoline, kaempferolrtin , quercitrin and isoquercitrin.

[014] Kang et al. (KANG, J. et al. Anti-oxidant capacities of flavonoid compounds isolated from acai pulp (Euterpe oleracea Mart.). FoodChemistry, v. 122, n. 3, p. 610-617,2010) in their study observed the presence of five flavonoids in the fruit of Euterpe oleracea among them are (2S,3S)-dihirokaempferol 3-ObD-glucoside, (2R,3R)-dihydrokaempferol 3-ObD-glucoside, isovitexin, velutin and 5,40-Dihydroxy-7.30 .50 -trimethoxyflavone.

[015] In chromatographic analysis of the açaí pulp identified the presence of a significant amount of 2 anthocyanins, cyanidin-3 glucoside and cyanidin-3 rutinoside considering them the most predominant in the fruit (PACHECO-PALENCIA, LA, DUNCAN, CE, TALCOTT , ST Phytochemical composition and thermal stability of two commercial açai species, Euterpe oleracea and Euterpe precatoria. Food chemistry, v. 115, n. 4, p. 1199-1205, 2009; DE LIMA YAMAGUCHI, KK et al. Amazon acai: Chemistry and biological activities: A review. Food chemistry, v. 179, p. 137-151, 2015; DE MOURA, RS; RESENDE, Â.C. Cardiovascular and metabolic effects of açaí, an Amazon plant. Journal of cardiovascular pharmacology, v. 68, no. 1, p. 19-26, 2016).

[016] In another study, it was possible to identify six anthocyanins in freeze-dried cellulose of the fruit, among which are: cyanidin 3-glucosides; cyanidin 3-rutinoside; cyanidin-3-sambubioside; peonidin-3-rutinoside; pelargonidin-3-glucosides, and delphinidin-3-glucosides (CARDOSO, LM, et al. Chemical composition, characterization of anthocyanins and antioxidant potential of Euterpe edulis fruits: applicability on genetic, dyslipidemia and hepatic steatosis in mice. Hospital nutrition: Official organization de la Sociedad española de nutrición parenteral and enteral, v. 32, n. 2, p. 702-709, 2015).

[017] The anthocyanins present in açaí can offer a new source of these pigments and, thus, a study of ways to obtain it becomes important, aiming at increasing yield, reducing the use of chemical products and greater stability.

[018] The biological properties of açaí are related to anti-proliferative, anti-inflammatory, antioxidant and cardioprotective activity, several studies demonstrate their actions.

[019] Cordeiro et al.(2017), (CORDEIRO, VSC et al. Euterpe oleracea Mart. seed extract protects against renal injury in diabetic and spontaneously hypertensive rats: role of inflammation and oxidative stress. European journal of nutrition, v. 57 , no. 2, p. 817-832, 2017) observed that the results of their study indicated that the açaí pulp extract substantially reduced kidney damage, preventing kidney dysfunction by reducing inflammation, oxidative stress and improving the barrier of renal filtration, providing a nutritional resource for the prevention of diabetic and hypertensive patients related to nephropathy.

[020] A study with rats, related to obesity and hepatic steatosis induced by a high-fat diet with and without supplementation of açaí extract, there was a reduction in lipogenesis, increased cholesterol excretion and improved oxidative stress in the liver, when compared to group that was not supplemented (DE OLIVEIRA, PRB et al. Euterpe oleracea Mart.-derived polyphenols protect mice from diet induced obesity and fatty liver by regulating hepatic lipogenesis and cholesterol excretion. PloSone, v. 10, n. 12, p. e0143721, p. e0143721, p. 2015).

[021] Açaí is one of the foods that has also shown these beneficial effects, in experimental models in the prevention and control of diseases related to metabolic syndrome, in a study with overweight adults, observed that the supplementation of 200g açaí pulp per day , in overweight individuals and after one month of administration of açaí pulp, there was a decrease in the mean levels of fasting glucose, serum insulin, total cholesterol and LDL-cholesterol (low-density lipoprotein) (UDANI, JK et al. Effects of Acai (Euterpe oleracea Mart.) berry preparation on metabolic parameters in a healthy overweight population: a pilot study. Nutrition journal, v. 10, n. 1, p. 45, 2011).

[022] The impact of the properties of açaí was also observed in a study where there was a reduction in total cholesterol, LDL-cholesterol and triglycerides in the blood, but there was no change in HDL (high density lipoprotein) and VLDL (very low lipoprotein) cholesterol density) (YUYAMA, LKO et al. Physicochemical characterization of acai juice of Euterpe precatoria Mart. from different amazonian ecosystems. ActaAmazonica, v. 41, n. 4, p. 545-552, 2011).

[023] Fernando (2013)(FERNANDO, FSL Evaluation of the effect of açaí drinks on the lipid and glycemic profile in wistar rats. São Carlos: Dissertation (Masters), UFSCar, 2013), in a research carried out with rats that ingested the pulp of açaí daily for forty-five days, detected that its daily consumption promotes reductions in serum glucose levels, but there is no significant difference in this reduction, contrasting the study by Udani et al. (2011) (UDANI, JK et al. Effects of Acai (Euterpe oleracea Mart.) berry preparation on metabolic parameters in a healthy overweight population: a pilot study. Nutrition journal, v. 10, n. 1, p. 45, 2011 ).

[024] With the increasing incidence of chronic diseases and their complications it is important to investigate the potential beneficial effect of the acai berry, and metabolic pathways involved. Although there are many reports of beneficial effects of anthocyanins, studies testing physiologically relevant doses of these compounds are still scarce.

[025] In order to protect the oxidation, degradation and drying of solid suspensions, the food industry invested in the technique of spray dryer drying, currently this procedure is applied mainly to bioactive and probiotic compounds (GOUIN, S. Microencapsulation: industrial appraisal of existing Technologies and trends. Trends in food Science & technology, v. 15, n. 7-8, p. 330-347, 2004).

[026] The Spray dryer is an alternative food drying process that has been widely used in the microencapsulation of ingredients susceptible to deterioration by external agents, and consists of the encapsulation of an active agent in a polymeric matrix, to protect it from conditions (TONON, RV; BRABET, C.; HUBINGER, MD Anthocyanin stability and antioxidant activity of spraydried açai (Euterpe oleracea Mart.) juice produced with different carrier agents. Food Research International, v. 43, n. 3, p. 907 -914, 2010).

[027] The powdered fruit juice obtained by this technique can present some difficulties, such as viscosity, hygroscopicity and low solubility. In addition, the adherence of droplets to the walls of the drying chamber decreases the process yield (SANTANA, AA et al. Influence of different combinations of wall materials on the microencapsulation of jussara pulp (Euterpe edulis) by spray drying. Food chemistry, v 212, p. 1-9, 2016).

[028] One way to avoid these problems is to add agents before spray drying, because their high molecular weight increases the product's glass transition temperature (SANTANA, AA et al. Influence of different combinations of wall materials on the microencapsulation of jussara pulp (Euterpe edulis) by spray drying. Food chemistry, v. 212, p. 1-9, 2016).

[029] The most common carrier agents used in this technique of drying fruit pulps are starches and their derivatives, some gums, lipids and proteins, mainly due to their high solubility and low viscosity, which are important conditions for the drying process by spray dryer (QUEK, SY; CHOK, NK & SWEDLUND, P. The physicochemical properties of spray-dried water melon powders. Chemical Engineering and Processing: Process Intensification, v. 46, n. 5, p. 386-392, 2007) .

[030] The use of whey protein is an interesting product for encapsulation because its wall material has good desired emulsifying property, and the largest fraction of these proteins are capable of building water-insoluble thermal gels, and stabilizing anthocyanins in acidic conditions (pH ≥ 1.5) (BETZ, M.; KULOZIK, U. Microencapsulation of bioactive bilberry anthocyanins by means of whey protein gels. Procedia Food Science, v. 1, p. 2047-2056, 2011).

[031] Regarding the açaí powder produced by spray dryer, the high content of lipids in the açaí pulp leads to problems during this process, causing incrustation of the spray nozzle. In a study by Tonon, Brabete Hubinger (2008) (TONON, RV; BRABET, C.; HUBINGER, MD Influence of process conditions on the physicochemical properties of açai (Euterpe oleraceae Mart.) powder by spray drying. , v. 88, n. 3, p. 411-418, 2008), the açaí pulp was previously filtered, in order to reduce the lipid content and obtained satisfactory results for the formation of powder.

[032] Research and development of encapsulation of anthocyanidins, astaxanthin, other beta-carotenoids, extracts enriched with anthocyanidins with other fruit pulps and açaí pulp were obtained using different techniques, including spray drying. Research by SHEN & QUEK (2014) developed commercial product microencapsulates, Bioastina® enriched with 5% astaxanthin, and encapsulating agents sunflower oil, wheyprotein, sodium casein and 70 soluble corn fiber. In this composition, the researchers did not use gelatin or aerosil in the composition (SHEN, Q.; QUEK, SY Microencapsulation of astaxanthin with soft milk protein blend and fiber by spray drying. Journal of Food Engineering, v. 123, p. 165-171, 2014.)

[033] Fermented drink containing Juçara pulp, used as a probiotic containing Lactobacilli reuteri LR92, the influence of different carriers (gum arabic, maltodextrin and gelatin) on the survival of lactobacilli after drying by Spray-dryer was studied (GUERGOLETTO, KB; BUSANELLO, M.; GARCIA, S. Influence of carrier agents on the survival of Lactobacillus reuteri LR92 and the physicochemical properties of fermented juçara pulp produced by spray drying. LWT-Food Science and Technology, v. 80, p. 321-327, 2017.) The spray-dryer powder as a preserving agent for Lactobacilli reuteri LR92 was different from our invention, as whey protein, aerosil and sodium casein were not incorporated. In addition, the beverage included gum arabic and maltodextrin agents not used in the disclosed invention.

[034] Study of stability of anthocyanidins and antioxidant activity of açaí spray-dryer microcapsules using different carrier agents (maltodextrin 10 DE, maltodextrin 20 DE, gum arabic and tapioca starch). Maltodextrin 10 DE was the carrier agent that showed greater protection and greater antioxidant activity for the anthocyanidins of the açaí spray-dryer extract (TONON, RV; BRABET, C.; HUBINGER, MD Anthocyanin stability and antioxidant activity of spray-dried açai (Euterpe oleracea Mart.) juice produced with different carrier agents. Food Research International, v. 43, n. 3, p. 907-914, 2010).

[035] Microencapsulation study of anthocyanidins using whey protein gel and other emusifying agents such as phosphatidyl choline without lecithins and Span 80 showed particle sizes from 20 µm to 70 µm, with a mean size of 50 µm (BETZ, M.; KULOZIK, U) . Microencapsulation of bioactive bilberry anthocyanins by means of whey protein gels. Procedia Food Science, v. 1, p. 2047-2056, 2011).

[036] Study of the influence of the combination of different wall materials (carrier agents) on the microencapsulation of Jussara (açaí) pulp by Spray-dryer used a ternary mixture of gum arabic, modified starch, concentrated whey protein or isolated soy protein. The combination of gum arabic, modified starch and proteins provides better microencapsulation results (SANTANA, AA et al. Influence of different combinations of wall materials on the microencapsulation of jussara pulp (Euterpe edulis) by spray drying. Food chemistry, v. 212, p. 1-9, 2016).

[037] The evaluation of the effect of operating conditions on the yield and nutritional quality of açaí powder combined with maltodextrin (Maltodextrin MOR-REX 1910) obtained by fluidized bed. The increase in temperature decreases the content, humidity, air flow rate, influenced the anthocyanidin degradation rate and optimal operating conditions were established to obtain an energetic nutritional powder, low moisture, excellent fluidity and high anthocyanidin content ( COSTA, RG et al., Effect of operating conditions on the yield and quality of açai (Euterpe oleracea Mart.) powder produced in spouted bed, LWT-Food Science and Technology, v. 64, n. 2, p. 2015).

[038] None of the patent documents described discloses açaí pulp microcapsule preparations or microcapsules containing standardized açaí pulp extract or microcapsules loaded with açaí extract containing the ingredients cited in these compositions herein. Açaí pulp microcapsules have as ingredients, whey protein, casein and gelatin and aerosil, as well as açaí pulp microcapsules containing sweet potato and aerosil.

[039] Another innovative feature of these microencapsulates is the increase in thermal stability (thermostability) and increased photostability of bioactive substances present in the açaí pulp. Encapsulating agents protect against the action of heat and light, preventing the easy degradation of substances present in the açaí pulp, especially anthocyanidins. Another innovative feature of these microencapsulates is the use of water-soluble encapsulating agents, facilitating solubilization in gastrointestinal fluids and promoting the easy and quick absorption of bioactive compounds (flavonoids and anthocyanidins). Another innovative feature of these microencapsulates is that protein encapsulants can bring other benefits when combined with bioactive compounds from açaí pulp, such as being used by gym and high performance athletes to prevent diseases, maintain well-being and ensure greater training capacity due to the properties of protein encapsulants associated with açaí pulp antioxidants. In this way, the modalities of the invention described in this document present considerable advantages compared to the state of the art. The encapsulation process did not affect the antioxidant activity of the constituents present in the açaí pulp. The açaí microcapsules demonstrated antioxidant activity using different methods with DPPH and FRAP. The encapsulation process did not harm the biological activity and maintained the ability to reduce glucose levels and cholesterol and triglyceride levels, thus contributing to dyslipidemia processes and diabetes control and hypertension.

[040] The invention proposed in this document seeks to fill a technological gap in terms of the application of açaí pulp as an active element of nutraceutical or pharmaceutical compositions of immediate release to be used as a food supplement, solid food fortification, beverage fortification or in the treatment and prevention of diseases, proposing a form of immediate release of the microencapsulated açaí pulp. Açaí pulp microcapsules can also be used in the composition of functional foods, espresso coffee, traditional coffee, as well as supplements for the composition of green coffee and its derivatives in order to fortify the composition of phenolic compounds and antioxidants in these compositions powders. vitamins and minerals for dairy drinks and juices, farinaceous products (breads, cakes, biscuits, cookies) in powders, sports food supplements (whey-protein, caseinates, albumins) in powders, for frozen dairy food formulas (ice cream, mousses, shakes ). Thus, we propose açaí pulp microcapsules, processes for obtaining açaí pulp microcapsules and uses of these products to prepare medicines with anti-dyslipidemic, hypoglycemic, hypocholesterolemic and hypo-triglycidemic antioxidant action and improve the sensory characteristics of the products based on coffee, chocolate, green coffee, farinaceous products, sports food supplements, iced dairy foods based on açaí pulp.

[041] It should be noted that the use of açaí pulp microcapsules is a determining feature in innovation, in terms of propolis technologies, since anthocyanidins, flavonoids and phenolic compounds have a characteristic composition, which will have a primary role in the activity in prevention and treatment of diseases.

[042] Still, as an essential differential against the state of the art, the açaí pulp microcapsules proposed in the present invention, use compounds in an intermediate matrix of bioactive coating that contain inert, non-toxic, biocompatible, low cost, stable pharmaceutical excipients with rheological characteristics for pharmaceutical solids or nutraceutical solids, immediate release and an external matrix containing usual nutraceutical excipients. Thus, the composition disclosed here can be used to solve semi-industrial and industrial technical problems in the food and pharmaceutical areas, as well as some therapeutic applications.

[043] In addition to the already highlighted objective of proposing an immediate release açaí pulp microcapsule product based on bioactive substances from açaí pulp containing wheyprotein, casein, gelatin, sweet potato starch and aerosil, the present invention proposes a methodology for the obtaining these açaí pulp microcapsules, and the uses for such açaí pulp based products.

SUMMARY OF THE INVENTION

[044] The present invention presents açaí pulp microcapsules a type of immediate release açaí pulp microcapsules, composed of açaí pulp from the bioactive extract, encapsulant and a fluidity promoter. Such dispersant system is composed of an encapsulation system, which, due to its composition, allows the immediate release of the active ingredient. The invention also deals with the process of obtaining açaí pulp microcapsules, using the Spray-Dryer technique. Additionally, the embodiment of the invention is presented in terms of the composition containing the microcapsules in solid formulations, preferably in the form of powders. Finally, uses for modified release of açaí pulp microcapsules and for the compositions in question are presented.

BRIEF DESCRIPTION OF THE FIGURES

[045] The modality of the invention, together with additional advantages thereof can be explained and understood by reference to the attached drawings and the following description:

[046] The attached Figure 1 shows Identification of anthocyanidins by UV-vis method at different concentrations of açaí pulp extract: (A) Pulp extract at different dilutions and different concentrations of HCl. (B) Açaí pulp after a clean-up process by solid phase extraction at different dilutions.

[047] The identification of anthocyanidins by UV-vis spectrophotometry demonstrates that the best wavelength for identification and quantification of anthocyanidins using UV-vis spectrophotometry comprises the range of 510nm and 540nm, since the wavelengths of 220nm and 280nm are wavelengths for quantification of other non-anthocyanin flavonoids.

[048] The attached Figure 2 shows chromatograms (A) Chromatogram of açaí pulp extract using Fluorescence (Ex: 250nm - Em:450nm) detecting the presence of anthocyanidins (1) and flavonoids (2-Chlorogenic acid, 3-Orientin, 4 - Quercitrin, 5-Apigenin); (B) Chromatogram of 30% MEOH SPE extract detecting the presence of cyanidin hydrochloride with retention time of 22.6 minutes and maximum wavelengths (236nm, 279nm and 518nm).

[049] The attached Figure 3 shows a Scanning Electron photomicroscopy of microencapsulated 1 with field division (A) of 50 µm, (B) of 20 µm and (C) of 10 µm. Scanning Electron Photomicroscopy of microencapsulated 2 with field division (D) of 100 µm, (E) of 20 µm and (F) of 10 µm. Scanning Electron Photomicroscopy of microencapsulated 3 with field division (F) of 20 µm (G) of 20 µm and (H) of 5 µm.

[050] The attached Figure 4 shows the DPPH radical scavenging capacity as a function of açaí pulp concentrations and spray-dryer formulations containing açaí pulp.

[051] The attached Figure 5 presents biochemical analysis of Tribolium castaneum larvae in different days of incubation supplemented with microencapsulated formulations of açaí.

[052] The attached Figure 6 presents biochemical analysis of Tribolium castaneum adults on different incubation days supplemented with microencapsulated formulations of açaí.

DETAILED DESCRIPTION OF THE INVENTION

[053] Açaí pulp microencapsulates, also called açaí pulp microcapsules, their processes and uses described in the present invention can be better detailed and understood by referring to the figures in this document and the following description:

Samples

[054] The açaí (Euterpe oleracea) pulps were obtained from an açaí supplier in the municipality of Boca da Mata/AL. After collection, 2 Kg of samples from the same batch were sent to the food analysis laboratory of the Faculty of Pharmacy in a Styrofoam box with ice and placed in a freezer at -20°C until the time of preparation of microencapsulates.

Preparation of açaí extract for chromatographic tests

[055] Initially, 200mL was separated from the fruit pulp, centrifuged in a refrigerated centrifuge Cientec brand CT500E, at a temperature of 1°C at 5000rpm using 15mL falcon tubes, then removed the supernatant that resulted in 80mL of extract. The extract was reduced to a volume of 20 ml using a Fisatom brand rotaevaporator, evaporated under the following conditions, water bath temperature less than 40°C, rotation speed of 100 rpm and pressure 600 mmHg.

[056] The concentrated extract of açaí (20mL) was placed in an amber bottle with a lid under refrigeration at 5°C in a refrigerator until the moment of analysis.

[057] This same procedure was performed with a volume of 1Kg of açaí pulp, and the extract obtained was separated in ampicillin bottles for lyophilization procedure, which was used in the experiment with Tribolium castaneum insects.

Identification of anthocyanidins by UV-vis method

[058] It was performed by the method of Lee et al. (2005) (LEE, J.; DURST, RW; WROLSTAD, RE Determination of total monomeric anthocyanin pigment content of fruit juices, beverages, natural colorants, and wines by the pH differential method: collaborative study. Journal of AOAC international, v. 88, n. 5, p. 1269-1278, 2005) with modifications, in which a volume of 100µL or 200 µL of the concentrated açaí extract was transferred to a 10mL volumetric flask. Then, the flask was diluted to 10mL with 0.01% or 0.037% HCl and submitted to UV-vis spectrophotometry analysis in the range of 200nm to 750nm to obtain the UV-vis spectrum.

Solid phase extraction chromatography for identification of flavonoids and anthocyanidins

[059] The concentrated açaí extract that was stored in the refrigerator was weighed 130 mg and transferred to a solid phase extraction cartridge (EFS) C18 for clean-up extraction test.

[060] The EFS cartridge was packed with 10mL methanol then packed 10mL with 0.010% hydrochloric acid (HCl). After the conditioning step, 130mg of the concentrated açaí extract was applied to the C18 cartridge and then eluted with 20mL of different proportions of the 0.01% HCl:methanol mobile phase system; starting with 90:10 (0.01% HCl: methanol) followed by the proportions 80:20, 70:30, 60:40, 50:50, 40:60, and 100% methanol, the latter was used for cleaning the cartridge, following the methodology of Cheynier et al. (1994) with modifications.

[061] The EFS extracts obtained in different in different proportions were concentrated in a rotaevaporator, under the same conditions described above, to obtain a final volume of 1mL and then stored in the refrigerator until the moment of analysis.

Identification of flavonoids and anthocyanidins using high performance liquid chromatography (HPLC)

[062] Performed by high performance liquid chromatography (HPLC) according to the method of Ieri et al. (2011), the mobile phase used was: solvent A (0.1% formic acid) and solvent B (acetylnitrile). The chromatographic column was maintained at a temperature of 33°C C18 column, brand Phenomenex, model Kinetex (100 x 4.6 mm: 5 µm) for analysis. Two UV-vis detectors were used at wavelengths of 275nm, 290nm, 320nm, 354nm and 515 nm, and a fluorescence detector in three excitation and emission lengths (250nm:450nm; 290nm:450nm; 320nm:450). The mobile phase was pumped at a flow of 0.4mL in a gradient system starting at 3%.

[063] The flavonoid standards used were vitexin, chlorogenic acid, orientin, quercetin, apigenin, luteolin, myricetrin, rutin, quercitrin, cacetin, chrysin, hisperidin, for anthocyanins were cyanidin hydrochloride purchased from extrasynthese® (Lyon Nord, France).

Obtaining açaí spray-dryer microencapsulated

[064] Initially, all components of the formulations were weighed according to Table 1.
Table 1. Composition of açaí spray-dryer microenpsulates

[065] The açaí pulp, after the filtration process in a stainless steel sieve, was subjected to solid mass determination, using an infrared scale for moisture determination at a temperature of 120°C for a period of 20 minutes or until weight was obtained constant. The determination of soluble solid mass was expressed in terms of percentages and determined in triplicate.

[066] In formulation 1, initially the pulp was filtered through a stainless sieve to remove excess gum, reserving the filtered pulp in a 200mL beaker. In another beaker, the sweet potato powder was sieved in a 300µm particle size sieve, and then the quantity was weighed according to table 1. The sieved sweet potato powder was solubilized with distilled water under manual stirring and temperature heating at 45°C for 15 minutes. It was left to rest for 15 minutes where there was precipitation of a part of the sweet potato powder. This suspension was filtered through a stainless steel sieve to obtain a clear solution, to which the filtered açaí pulp, the alcohol, and finally the aerosil were added.

[067] When solubilized, it was subjected to Spray-dryer processing, in which a coating was performed with the equipment's ambiance, so there is no loss of the sample.

[068] In formulations 2 and 3 the water was heated to a temperature of 35°C to solubilize the proteins (wheyprotein, gelatin and/or casein). The solubilization took place under agitation in an IKA equipment at a speed of 1800rpm, for a period of 10 to 15 minutes. The pulp was filtered through a stainless steel sieve to eliminate excess gums and then added to the soluble protein solution. The filtered açaí pulp was added little by little, keeping the system under constant agitation to avoid precipitation. Then, in another beaker, the sweet potato was solubilized in 50 mL of water under manual stirring for 15 minutes. This system was left at rest and the content of adjuvant 1 was added to the solution of soluble proteins containing açaí pulp and, at the end, the aerosil was added, always keeping it under constant agitation.

[069] When solubilized, it was subjected to Spray-dryer processing, with the same conditions mentioned above.

Scanning Electron Microscopy (SEM)

[070] The morphological analysis of the microenpsulates and the pulp extract was carried out with the aid of a scanning electron microscope of TESCAN VEGA 3, with a working distance of 15.97mm, the samples were fixed on a metallic support with the aid of a tape double-sided carbon and covered with a thin layer of gold. A current of 45 mA was applied for 200 seconds and a voltage acceleration equal to 20 kV, in the Chemistry laboratory of the Federal Institute of Alagoas (IFAL).

Thermal analysis: Thermogravimetry (TG)

[071] Thermal analyzes of the excipients used in the formulations (açaí pulp, sweet potato powder and protein mixture) and of the three formulations obtained by Spray-dryer for comparison purposes were carried out in the Chemistry laboratory of the Federal Institute of Alagoas (IFAL).

[072] The thermal degradation of the samples was carried out in a TGA-50 Shimadzu, with an atmosphere of nitrogen and oxygen at 50 mL/min-1, with a heating rate of 10°C min-1 in the temperature range of 26- 900°C. The mass of the analyzed samples was 5.0 ±10%mg.

Determination of antioxidant activity by DPPH

[073] The antioxidant activity was determined using DPPH solubilized in an alcoholic medium (absolute ethanol) according to Sales (2012). 4 mL of DPPH reagent were weighed and solubilized in a volumetric flask with a capacity of 100 mL, and the solution obtained was stored under refrigeration in an amber flask.

[074] In evaluating the antioxidant activity it was necessary to prepare a stock solution of each of the formulations. Then 130 µg of açaí pulp extract, 260 µg of each powder of the formulation and 300 µL of pulp with açaí acidified with 0.037% HCl were weighed, solubilized in a volumetric flask with a capacity of 10 ml, using 2 ml of distilled water as solvent and alcohol until completing the flask, centrifuged and following with the removal of aliquots of 750 µL, 500 µL, 250 µL, 100 µL, 50 µL, 25 µL and 10 µL, which were transferred to a 5 mL volumetric flask, the analyzes were made in triplicate.

[075] 2 ml of DPPH reagent were added, incubating for 30 minutes protected from light. After the reaction time, the volume of the flask was completed with absolute ethanol, then reading using UV spectroscopy at a wavelength of 518 nm. The data were treated and the antioxidant activity was expressed according to the equation: %AA = 100 –{[(Abstract – Abswhite) X 100] / Abscontrol}.

Determination of antioxidant activity using FRAP

[076] The reduction capacity was determined using the FRAP assay according to the method described by Benzie and Strain (1996). Different concentrations of açaí extract and formulations (10, 25 and 50 µg) were used for the test. The FRAP reagent was prepared on the day of the test, mixing 25 ml of acetate buffer (0.3M, pH 3.6), 2.5 ml of TPTZ solution and 2.5 ml of ferric chloride in aqueous solution.

[077] It was used 90µL of different concentrations of samples, which were added in a 5mL volumetric flask containing 270mL of distilled water. Then, 2.7mL of frap solution was added and incubated for 30 minutes at 37°C in a water bath. Finally, reading using UV spectroscopy with a wavelength of 595 nm. The absorbance obtained was calculated using the calibration curve and expressed in µM of ferrous sulfate/g of açaí. The blank for the samples was FRAP. After exactly 10 min the absorbance (620 nm) was read using an HP Spectrophotometer 8452a.

Biochemical analysis of açaí microencapsulates in Tribolium casteneum insect model

[078] Two experiments were carried out, one with Tribolium castaneume larvae and the other with adult Tribolium castaneum insects.
Preparation of the experiment with Tribolium castaneum larvae
Tribolium castaneum larvae were obtained from a colony of the Laboratory of Biochemistry and Molecular Biology of Insects at the Federal University of Alagoas; larvae were kept at 28 °C with a relative humidity between 70 and 80%, fed ad libitum. Four groups of 30 larvae, three days after hatching, were submitted to the diet in petri dishes with a mixture of 300 µg of açaí extract and of each microencapsulated with 300 µg wheat flour, adapted from Sarro et al. (2005) (SARRO, FB et al. Feeding substrates to black coconut bunch weevil, Homalinotuscoriaceus (Gyllenhal)(Coleoptera: Curculionidae), rearing in laboratory. Neotropical Entomology, v. 34, n. 3, p. 451-457, 2005 ) and another group of 30 larvae was submitted to a diet composed only of wheat flour, in equivalent amounts, and all groups were carried out in triplicate, kept at 28 °C with a relative humidity between 70 and 80%. Biochemical analyzes of larvae were followed for 4, 5, 8 days.

Preparation of the experiment with Tribolium castaneum adult insects

[079] Tribolium castaneum larvae were obtained from a colony of the Laboratory of Biochemistry and Molecular Biology of Insects at the Federal University of Alagoas; larvae were kept at 28 °C with a relative humidity between 70 and 80%, fed ad libitum. Three groups of 30 larvae, 12 days after hatching, were submitted to a diet with a mixture of 300 µg of each microencapsulated with 300 µg of wheat flour in petri dishes, and another group of 30 larvae was submitted to a diet composed only of wheat flour, in equivalent amounts, and all groups performed in triplicate, kept at 28 °C with a relative humidity between 70 and 80%. Biochemical analyzes of adults were followed for 30, 45 and 60 days.

Biochemical analysis of Tribolium castaneum insects

[080] To assess the levels of glucose, triglycerides, total cholesterol and HDL cholesterol of Tribolium casteneum insects. Larvae and adults were collected and added to saline solution containing phenylthiourea (3-13 mg mL-1) (Sigma Aldrich), macerated in porcelain grade, placed in eppendorf, centrifuged (5 min - 13,000 g) and the supernatant used . The concentration of biochemical levels were determined using the commercial kit, according to the manufacturer's instructions (Bioclin® Colorimetric Enzyme Kit) for each analysis.

[081] All data are expressed as mean ± mean standard error. The comparison between groups and different times of analysis was performed by analysis of variance (two-way ANOVA) using the Bonferroni post tests, with a significance level of 0.05 and 0.001, in which the formulations 1, 2 and 3 were compared in relation to the control group. Unpaired Student t-analysis with a significance level <0.05 two-tailed was used to compare the control group in relation to each formulation for each specific time of glucose, total cholesterol, triglycerides and HDL cholesterol analyses.

Examples

[082] Tables 2 and 3 demonstrate antioxidant activity of açaí pulp and its açaí pulp microcapsules.

[083] Table 2. Determination of IC50 and other statistical parameters from antioxidant activity data using DPPH.

[084] Table 3. Antioxidant activity by the FRAP method of açaí pulp extract and formulations obtained by spraydryer in mmol ferrous sulfate/g açaí pulp.

[085] Table 4 shows the mass loss of açaí pulp, coating materials and formulations by TG.

Claims (11)

Açaí pulp microcapsules (Euterpe oleraceae Mart.) characterized by comprising active principles derived from açaí pulp; Açaí pulp microcapsules (Euterpe oleraceae Mart.), according to claim 1, characterized by the fact that it comprises:

• a) Standardized extract of açaí pulp (Euterpe oleraceae Mart.) from açaí pulp in a proportion between 1 and 90%, preferably between 1 and 80%;
• b) encapsulant in a proportion between 20 and 70%;
• c) and fluidity promoter in a proportion between 0.01 and 15%;

Açaí pulp microcapsules (Euterpe oleraceae Mart.), according to claim 2, characterized in that it comprises a dispersing system composed of a system composed of a) protein encapsulant based on whey protein, gelatin and casein and fluidity promoter based on colloidal silicon dioxide , or; sweet potato starch based encapsulant and colloidal silicon dioxide based flow promoter; Açaí pulp microcapsules (Euterpe oleraceae Mart.), according to claims 1 to 3, characterized in that it is obtained by spray-dryer and has a particle size between 1 and 40 µm and more preferably between 2 and 30 µm; Açaí pulp microcapsules (Euterpe oleraceae Mart.), according to claims 1 to 4, characterized by presenting bioactive substances anthocyanidins and flavonoids identified by HPLC-DAD-UV; Açaí pulp microcapsules (Euterpe oleraceae Mart.), according to claims 1 to 5, characterized by containing, among its components, compounds derived from standardized açaí pulp, preferably standardized extract of açaí pulp, for use with hypoglycemic, anticholesterolemic, in inflammatory processes related to diabetes, hypertension and dyslipidemia, and also with antioxidant properties; Process for obtaining açaí pulp microcapsules (Euterpe oleraceae Mart.), characterized by comprising the following steps:

• i) Obtaining fresh or commercially frozen açaí pulp, preferably frozen;
• ii) Preparation of standardized açaí pulp extract using standardized sieve to control açaí pulp particle size, obtained in i);
• iii) Preparation of the emulsification system using encapsulant and fluidity promoter containing standardized extract of açaí pulp obtained in ii) and distilled water using controlled temperature between 32 and 37°C, preferably 35°C;
• iv) Redispersion of the emulsification system containing standardized extract of açaí pulp in a H2O:Ethanol solvent system, in proportion (1:1, V:V), and prepared in iii);
• v) Drying of the emulsification system obtained in iv) and formation of açaí pulp microcapsules by spray-dryer;
• vi) Characterization of açaí pulp microcapsules (Euterpe oleraceae Mart.) using techniques such as: particle size, TG/DTG, FTIR, SEM, HPLC-DAD-UV, total phenols, total flavonoids, antioxidant activity and biological assays with Tribolium castaneum and monitored by biochemical assays;

Process for obtaining açaí pulp microcapsules (Euterpe oleraceae Mart.), according to claim 6, characterized in that, in step vi), the integrity of the biactive compounds of the standardized extract of açaí pulp is proven using an antioxidant activity test and biological assays with Tribolium castaneum and monitored by biochemical assays; Process for obtaining açaí pulp microcapsules (Euterpe oleraceae Mart.), according to claim 6, characterized in that, in step vi), the hypoglycemic and hypocholesterolemic activity is proven using biological assays with Tribolium castaneum and monitored by biochemical assays; Use of açaí pulp microcapsules (Euterpe oleraceae Mart.), according to claims 1 to 5, characterized by being alone or as an adjuvant, to combat diabetes, obesity, dyslipidemia, hypertension, in inflammatory processes due to dyslipidemia; Use of açaí pulp microcapsules (Euterpe oleraceae Mart.), according to claims 1 to 6, characterized by being incorporated into an active ingredient or adjuvant, for sports food supplement formulas (whey protein, caseinates, albumins), and for dairy food formulas ice creams (ice cream, mousses, shakes) containing active ingredient of açaí pulp microcapsules.

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