BR102013000308A2 - process of obtaining inulin with polymerization degree> = 20 from stevia rebaudiana roots - Google Patents

Abstract

process of obtaining inulin with polymerization degree> = 20 from stevia rebaudiana roots. The use of stevia rebaudiana roots as a new source for obtaining high polymerization inulin (gp) from the aqueous extraction methodology is very promising, as it has several advantages over the sources currently used commercially. From the aqueous extract of these roots, a yield of 29.31% was obtained, higher than that observed from the main commercially available sources (15-20%). the obtained molecules presented high gp (~ 30) degree, and are rarer inulin, since the main sources of obtaining present low gp (11-15). The freezing / thawing methodology proved to be an efficient technique for the enrichment of inulin molecules, allowing fractions with different solubilities and high gp, supernatant (21-22) and precipitate (35-40). It is noteworthy that due to the inulin obtained having a high natural gp, without the need for enrichment of its chain, thus eliminating any industrial process of enrichment and, consequently, reducing the steps and costs of its production, represents a promising substitute for commercial sources. . From the data obtained it can be stated that s. Rebaudiana is a promising new source for the commercial procurement of high gp inulin in various applications in the food and pharmaceutical industry.

Description

Table 6. 13 C NMR (75.45 MHz) δ (ppm) data on Inulin Standard D20 and Raw Extract, Supernatant, and Precipitate samples. • Statistical Analysis Statistical analysis of the data was performed using a computer program. The comparison between the means was performed by the t-test for independent samples at 5% significance level. • Determination of Polymerization Degree The calculation of the GP of inulin molecules was performed using three different methodologies for validation and greater reliability of results. In the first methodology, the GP was calculated from the spectrophotometric data, using the formula described by Moerman, Van Leeuwen and Delcour (2004) (Annex 5). In the second methodology, the calculation of the GP was determined by the ratio of the signal integration values obtained for the fructose units H-3 and H-4 hydrogens (Figure 8), by the glucose anomeric hydrogen integration value (H- 1 '), and all these values were obtained from the 1H NMR spectra of the samples, as shown in Figure 6 (CÉRANTOLA et al., 2004; YANG; HU; ZHAO, 2011). In the third methodology, the calculation of GP was based on the values obtained from the enzymatic assay of glucose and fructose (MOERMAN; VAN LEEUWEN; DELCOR, 2004) (Annex 6).

Sample GPs from each methodology are described in Table 7.

Table 7. Polymerization Grade values obtained for the different samples from each methodology. From the comparison of the averages obtained for each methodology (spectrophotometric, enzymatic and 1H NMR) using the t-test for independent samples with p = 0.05, no significant difference was observed between the GP values obtained for each methodology.

This result shows consistency and reliability in the GP data obtained for the samples, because regardless of the methodology of choice for the calculation, the values were very similar.

Thus, these data prove the quality (high GP) of inulin obtained from S. Rebaudiana roots.

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Claims (13)

1. PROCESS OF OBTAINING POLYMERIZED INULINS> 20 From Stevia rebaudiana roots, comprising a set of techniques for aqueous extraction, freezing and thawing of Stevia rebaudiana roots and showing a high degree of polymerization when compared. other known sources of this polysaccharide. 2. PROCESS OF OBTAINING POLYMERIZATION INULINS> 20 From the roots of Stevia rebaudiana according to claim 1, characterized by the fact that for the crude extract the degree of polymerization observed was 30, a value naturally obtained without any process. of enrichment. 3. PROCESS OF OBTAINING POLYMERIZATION INULINS> 20 From Stevia rebaudiana's roots, according to claim 1, characterized in that the physical freezing and thawing technique used for the separation of inulin molecules is based on the fact that Since inulin is a product with variable solubility at different temperatures, it allows to separate the lower mass (low GP) inulin from the higher mass (high GP) with cooling. 4. PROCESS OF OBTAINING POLYMERIZATION> 20 FROM STevia rebaudiana roots according to claim 1, characterized by obtaining in the supernatant fraction more soluble inulin with high solubility in GP 21-22 and in the precipitated fraction with high solubility. GP 35-40. 5 PROCESS OF OBTAINING POLYMERIZATION INULINS> 20 FROM STevia rebaudiana roots, characterized by the fact that the main technical characteristics to be protected are the roots of Stevia rebaudiana as an innovative source for obtaining inulin. PROCESS FOR OBTAINING POLYMERIZATION> 20 FROM STevia rebaudiana ROOTS 1. GOAL. APPLICATION AND FIELD OF USE This invention relates to obtaining the inulin fructooligosaccharide from the aqueous extract of Stevia rebaudiana roots. Fructooligosaccharides (FOS) are the most abundant reserve polysaccharides found naturally in a wide variety of plants and bacteria. Inulin is the basic representative of this category and can be found in many vegetables, fruits and cereals, such as onions, garlic, bananas, chicory roots, artichokes, dahlias, dandelion, wheat and barley (MEYER et al. !., 2011; EVAGELIOU et al., 2010), but its obtaining from the roots of S. rebaudiana was reported only by Oliveira et al. (2011). Inulin occurs naturally as a homologous series of oligo and polysaccharides of different chain lengths. These chains are formed by fructose units linked together by 3-2.1 bonding and by a terminal glucose unit via cr-1.2 bonding, which can be represented by the general form GFn and Fm, where F represents fructose units and G glucose units, n refers to the number of terminal glucose-linked fructose and the number of fructose linked together to form the carbohydrate chain (MOERMAN; VAN LEEUWEN; DELCORUR, 2004; GLIBOWSKI, 2010 ). There are numerous applications of these polysaccharides, which can be used as a source of dietary fibers, prebiotics, in technological applications in the food industry, such as fat and sugar substitute, thickener, gelling agent, humectant (RINALDONI; CAMPDERRÓS; PADILHA, 2011), in the pharmaceutical industry (FARES; SALEM; KHANFAR, 2011), and recently finds application in biofuel production (ZHANG et al., 2010; WANG etal., 2011). In the food industries, inulin is used to formulate innovative and healthy foods. Dietary fibers, due to their low calorie value, have found increasing use in improving the nutritional value of cereal products, being used as a substitute for fat and sucrose, thus allowing an improvement in taste and texture (ARAVIND et al. , 2011; EVAGELIOU et al., 2010). Some benefits of soluble fiber such as inulin include the prevention of diseases such as gastrointestinal and respiratory infections, reduced colorectal cancer, obesity, cardiovascular disease, and type II diabetes (POINOT et al., 2010; SILVA et al., 2011). Associated with the fermentation of fibers in the gut is also its prebiotic activity, which is the ability to selectively stimulate desirable colonic bacteria, instigating the growth and / or activity of these bacteria (bifidobacteria and lactobacilli) that benefit the host as well as the colon. inhibit the growth of pathogenic and harmful microorganisms (TÁRGA; ROCAFULL; COSTELL, 2009; GOMES; PENNA, 2010). For these reasons these soluble fibers are currently widely used in the human diet (DE VUYST; LEROY, 2011). Inulin also has the ability to increase intestinal absorption of calcium, magnesium and iron, among other minerals, positively affecting their bioavailability (LOBO et al., 2009; SAAD, 2006). Increased calcium absorption has been used to prevent osteoporosis in the elderly (RASCHKA; DANIEL, 2005; JUDPRASONG, 2011). Inulin can also be used for the development of an iron-carrying system, which is based on its great biocompatibility and biodegradability in the intestinal tract, as well as ensuring an increase in iron absorption, reducing the side effects of oral iron administration. unchelated (PITARRESI et al., 2008). Due to the characteristic of soluble fiber inulin having neutral flavor and minimal influence on the organoleptic characteristics of a product (ARAVIND et al., 2011), it has been used in a growing number of applications in the food and animal feed market. for example in dairy products, bakery products, beverages, cereals, sour cream, confectionery products, and meat products such as sausage and bologna (MEYERetal., 2011; MORRIS; MORRIS, 2012). In addition to these food applications, inulin also finds use in pharmaceutical applications, such as bulking agent and / or tablet binder, as controlled release coatings of drugs with low availability in vivo, because inulin is a biodegradable polysaccharide and biocompatible in the human body (FARES; SALEM; KHANFAR, 2011), as well as chemical derivatives of inulin are used in industrial applications, such as carboxymethyl inulin is used as an agent in wastewater treatment (MEYER et al., 2011). . Another important application of inulin is its use as a substrate for biofuel production, especially bioethanol (WANG et al., 2011; ZHANG et al., 2010). Given the wide field of application of these molecules, this study reveals the discovery of a new and promising source for commercially obtaining this FOS. Thus, the present invention relates to a process of obtaining polymerization inulin> 20 from S. rebaudiana roots. This is a very promising source when compared to commercially available sources for obtaining inulin, as it has better yield and is obtained with great purity and quality. Priority searches carried out revealed the existence of similar patents, however, there are divergences in some points. In PI 0301192-5, entitled Process for obtaining inulin and its by-products from tubers, the sources of obtaining are different, and do not mention polymerization grade or other product and process specifications, as well as PI 9902934. -0, entitled Process for the manufacture of chicory inulin, hydrolysates and inulin derivatives and improved chicory inulin products, hydrolysates and derivatives, and method of using chicory roots as a source material therein processes. PI 0710867-2 Long chain inulin, process for obtaining same, foodstuff, dietary supplement, cosmetic preparation, use of inulin, aqueous inulin paste and use thereof, refers to a long chain inulin and It is obtained from artichoke roots, and also protects its use in foodstuffs and cosmetic preparations. However, the source is artichokes, which naturally do not have a high degree of polymerization, and not S. rebaudiana. In the patents entitled Processes for Producing Inulin (US 7507558) and Long Chain Inulin (PI 0608781-7), the source and process of obtaining are different. Inulin products with improved nutritional properties (US 7812004) is related to the inulin molecule and not to the source or process of obtaining it. Thus, the present invention - PROCESS OF OBTAINING POLYMERIZATION> 20 FROM S. REBAUDIANA ROOTS - differs from the existing ones and has several advantages related to the efficiency of aqueous extraction of the roots and the degree of polymerization of the inulin. . Regarding the yield of aqueous extraction of S. rebaudiana roots, the observed yield was 29.3%, higher than that obtained in the two species currently used commercially by the industry to produce inulin, the Jerusalem artichoke (Helianthus tuberosus) and the chicory (Cichorium intybus) (CHI et al., 2011), which show inulin extraction yields of 16-20% and 15-20%, respectively (GALANTE, 2008; PASEEPHOL, 2008; MEYER, et al. 2011). Table 1 shows that the yield (inulin content) of S. rebaudiana roots is also higher when compared to other sources of this polysaccharide. Table Λ. Inulin and FOS content in plants used for human nutrition (% fresh weight). ND - Data not available (PASEEPHOL, 2008). These data demonstrate that the potential for application of S. reabaudiana roots as a new source of inulin production is promising, since it initially has a higher yield than most plants currently known for obtaining this FOS, showing high productivity. The methodology used for extraction is a simple and low cost technique, which increases the industrial interest due to the low price and high productivity. Regarding the advantages regarding the Degree of Polymerization (GP). this refers to the average length of the inulin chain. The technological properties, as well as the functional and prebiotic properties of interest of these molecules, are related to their average GP-chain length of the molecule (TÁRGA; ROCAFULL; COSTELL, 2009; BAYARRI; CHULIÁ; COSTELL, 2010; MORRIS; MORRIS, 2012 ). According to GP, inulin has different properties such as digestibility, prebiotic activity and health promotion, caloric value, sweetening power and water binding capacity, thickener, gelling and fat substitution (MOERMAN; VAN LEEUWEN; DELCOR, 2004; RINALDONI; CAMPDERRÓS; PADILHA, 2011; MORRIS; MORRIS, 2012). GP depends on many factors, such as source plant, climate and growing conditions, harvest maturity and storage time after harvest. Inulin molecules with different GPs can be found, ranging from 2-60 (VILLEGAS; COSTELL, 2007; GLIBOWSKI, 2010) to 11-65 (MORRIS; MORRIS, 2012) fructose units, with the average GP of native inulin. ranges from 10-15 (VILLEGAS; COSTELL, 2007; GLIBOWSKI, 2010; DE VUYST; LEROY, 2011; MORRIS; MORRIS, 2012). For example, chicory and yacon potato inulin have a low average polymerization grade (12) (VILHENA; CAMERA; KAKIHARA, 2000; CHI et al., 2011; MOSCATTO; PRUDENCIO-FERREIRA; HAULY, 2004), already inulin. slightly larger GPs (22-25) are found in artichoke (Cynara scolymus), globe thistle (Echinops ritró) and broom (Viguiera discolor) (CHI et al., 2011). High GP inulin can be industrially produced from native inulin by applying specific enrichment / purification techniques such as precipitation, ultrafiltration, crystallization, which can separate GP inulin less than 10 from GP inulin greater than 22-25 ( MOERMAN; VAN LEEUWEN; DELCOR, 2004; VILLEGAS; COSTELL, 2007; GLIBOWSKI, 2010). There are few companies that specialize in obtaining and trading inulin that offer them with different characteristics and GP from industrial processing. Some of the commercially available inulin are described in Table 2. Table 2. Types of commercially available inulin and their main characteristics. In the crude extract obtained from aqueous extraction of S. rebaudiana roots, an average GP of 30 was observed. This sample was obtained from the extraction without any processing to enrich the molecules. This data is very significant since the GP of native inulin obtained from different plant sources is approximately 10-15, including the source commercially used for obtaining inulin, which are chicory roots (VILLEGAS; COSTELL, 2007; GLIBOWSKI, 2010; DE VUYST; LEROY, 2011; MORRIS; MORRIS, 2012). A high initial GP of inulin molecules is very important given that these inulin have higher commercial value due to their rarer obtainment and their wide application potential (TÁRGA; ROCAFULL; COSTELL, 2009; ARAVIND et al., 2011 ; RINALDONI; CAMPDERRÓS; PADILHA, 2011; LOBO etal., 2009, MEYERetal., 2011). It is noteworthy that due to the inulin obtained from the roots of S. rebaudiana having high natural GP, without the need for enrichment of its chain, thus eliminating any industrial process of enrichment and, consequently, reducing the stages and costs of its production, represents a promising substitute for existing commercial sources of inulin. The mean GP values obtained for the supernatant (20) and precipitated (36-40) fractions were also very expressive, since even in the most soluble fraction (the supernatant) obtained from the Freeze / Thaw technique, the GP It is very close to that considered high for vegetable inulin, which is 22-25 (TÁRREGA; ROCAFULL; COSTELL, 2009, MORRIS; MORRIS, 2012). The precipitate has the highest GP obtained in this study, demonstrating the advantages that inulin obtained from S. rebaudiana roots represent for the market. This value is considered a high GP and is close to the GPs obtained from synthetic reactions and sophisticated physical methodologies of inulin chain enrichment, where the molecules reach higher polymerization degrees (TÁRGA; ROCAFULL; COSTELL, 2009; MOERMAN; VAN LEEUWEN DELCOR, 2004). The methodology used to obtain the different fractions (supernatant and precipitate) is simple, efficient and inexpensive, demonstrating numerous advantages over the other existing techniques (ultrafiltration, crystallization, precipitation), which are more complex methodologies and require more expensive materials to use. execution (MOERMAN; VAN LEEUWEN; DELCOR, 2004; VILLEGAS; COSTELL, 2007). Thus, the process employed in this work is viable and applicable for obtaining inulin with GP> 20 from S. Rebaudiana roots, according to the need of GP for each specific application (TÁRGA; ROCAFULL; COSTELL, 2009; BAYARRI; CHULIÁ; COSTELL, 2010; MORRIS; MORRIS, 2012). 2. DESCRIPTION OF THE INVENTION-RELATED FIGURES Figure 1 represents a part of Stevia rebaudiana roots after collection and washing. Figure 2 represents the oven dried roots circulating at 40 ° C for 5 days. Figure 3 shows the crushed roots to be routed to the extraction process. Figure 4 illustrates the supernatant and precipitate samples after the Freeze / Thaw technique. Figure 5 illustrates the appearance of fractions after centrifugation separation (A) supernatant and (B) precipitate, both obtained after separation and freeze drying. Figure 6 shows 1H NMR spectra (300.06 MHz, 02O). 1 H NMR spectra were used for sample identification compared to the inulin standard spectrum, and a second complementary data obtained from the spectra is the GP of the samples. Inulin Standard (A), Crude Extract (B), Supernatant (C) and Precipitate (D). Figure 7 shows 13 C NMR spectra (75.45 MHz, D 20). 13 C NMR spectra were also used for sample identification as compared to the Inulin Standard spectrum. Inulin Standard (A), Crude Extract (B), Supernatant (C) and Precipitate (D). Figure 8 shows the general formula of inulin, in which the H-3 and H-4 Hydrogens of Fructose units are highlighted. 3. DETAILED DESCRIPTION OF THE INVENTION • Plant Material The roots of S. rebaudiana Bertoni were collected in November 2011 at the Didactic Garden of Medicinal Plants Professor Irenice Silva of the State University of Maringá (HDPM-UEM) (Figure 1). After collection, the roots were washed with running water to remove dirt and then separated from the shoot. The drying process was performed for five days in a circulating air oven at 40 ° C (Figure 2). After drying, they were reduced in size manually (Figure 3) with the help of scissors, weighed and sent for extraction. • Extraction The aqueous extraction process is the methodology most used by several authors to obtain polysaccharides, based mainly on the solubility of these compounds in water at high temperatures. The methodology used was an adaptation from other authors (LEITE et al., 2004; MIAO et al., 2011; GRZYBOWSKI, 2008). The aqueous extraction was carried out under reflux with the aid of a heating blanket and a condenser. 7.27g of dried roots were used in the process and the amount of extracting liquid (deionized water) was standardized at 300mL in each extraction. In all, three extractions were performed with a reflux time of four hours for the same material. After each extraction, the material was hot filtered, the solids (roots) returned to the flask and again extracted by reflux, and at the end of the three extractions, the aqueous extract was concentrated by rotary evaporator. Then, in the extraction and concentration process, the concentrated extract was precipitated with ethanol (PA) to obtain crude polysaccharides. The amount of ethanol was standardized to three times the initial volume of crude extract. After precipitation by the addition of ethanol, the extract was kept for 24 hours at 4 ° C in a refrigerator. The suspension was then filtered through a Büchner funnel with the aid of a vacuum pump. The filtrate was discarded and the precipitate was resuspended in the smallest volume of water required for solubilization of the sample, and then frozen in a freezer. subsequently lyophilized to obtain the dry crude extract, and after weighing it was possible to determine its yield (29.3%) (Annex 1). • Enrichment of high GP inulin chains The enrichment methodology was carried out with the objective of separating inulin according to their average chain size, which is related to the GP of the molecule. The technique used was freezing / thawing, which is a simple, low cost, easy to perform method that guarantees good separation (GORIN; BARON, 1988). The fractions submitted to this process were solubilized in deionized water, frozen for 24 hours and then thawed at room temperature. The cold water-insoluble precipitate and the supernatant were separated by centrifugation (9,500rpm / 20min at 5 ° C) (Annex 2). This process was repeated four times until the observation that from the aqueous supernatant no more precipitate formed after freezing and thawing, and from the aqueous precipitate, after attempting solubilization in water, a clear supernatant was obtained. At the end of the process all fractions obtained from the supernatants and precipitated after each centrifugation were pooled, concentrated and lyophilized (Figures 4 and 5). • Extraction Yield Calculations Gross extraction yield calculations were performed based on the initial dry mass of S. reabaudiana roots and the dry mass of the crude extract after freeze drying. The yield of FOS (inulin) was calculated based on the initial dry mass of S. reabaudiana roots and on the values of total sugar assay for the crude extract (Annex 3). For this determination, from 7.27g of dried roots submitted to the extraction process, 2.65g of dry extract was obtained. Considering that 80.37% of this extract is composed of sugars, it can be determined that the gross yield of extraction from S. rebaudiana roots was 36.45%, and when considering only the yield based on the amount of inulin. present was 29.31%. • Fraction yield calculations after the Freeze / Thaw methodology The calculation of the yield to obtain the supernatant and precipitate fractions was performed based on the initial dry mass of the crude extract (2.65g) used for the technique, followed by the obtained values. in dry mass after lyophilization of the supernatant (1.26g) and precipitate (0.70g) (Annex 4). • Chemical Characterization of Samples Chemical assays were performed by methodologies based on colorimetric reactions and spectrophotometer reading. The following chemical assays were performed: total sugar analysis by the Phenol-Sulfuric methodology (DUBOIS et al., 1956), fructose by the Resorcinol methodology (ROE; EPSTEIN; GOLDSTEIN, 1948) and reducing sugar by the p acid hydrazide methodology. benzoic hydroxy (LEVER, 1972). In addition to these methodologies, glucose and fructose were selectively determined by methodologies that use enzymatic reactions (LEPORE et al., 2004; PEREIRA et al., 2008; MOERMAN; VAN LEEUWEN; DELCORUR, 2004). modified Lowry method (HARTEER, 1972). Initially, after extraction and drying of the crude extract and obtaining the other fractions, spectrophotometric assays were performed for a preliminary characterization of the extract and the obtained fractions. This methodology was initially applied in order to perform a qualitative evaluation of the extract to verify the characteristic components of the polysaccharide obtained and, later, through the calibration curves of each methodology, a quantitative analysis of the same components was performed and the results obtained are described. Table 3. Table 3. Assay results by spectrophotometric methodologies. Enzyme analyzes were performed for specific glucose and fructose assays using commercial enzyme kits for spectrophotometric determination of glucose and fructose. The assay results are described in Table 4. Table 4. Enzyme assay results for fructose and glucose. • 1H and 13C Nuclear Magnetic Resonance Analyzes 1H and 13C Nuclear Magnetic Resonance spectroscopy were performed on a Nuclear Magnetic Resonance spectrometer, operating at 300.05 MHz for the 1H core and 75.45 MHz for the core. of 13C. The samples were dissolved in deuterated water (20mg in 0.7ml_ D20). The chemical displacements (δ) obtained from the Hydrogen 1 (Figure 6) and C13 Carbon (Figure 7) spectra were expressed in ppm, whose values are described in Tables 5 and 6. Table 5. δ data (ppm) 1H NMR (300.05 MHz) at D20, Inulin Standard and Raw Extract, Supernatant and Precipitate samples.