Objective: The aim of this study was to obtain kahai protein concentrate from Caryodendron orinocense karst cultivated in the region Amazonia of Ecuador and characterizes its gastric and duodenal hydrolysates using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis method and the reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) method.Methods: Kahai seeds (C. orinocense karst) were utilized to obtain kahai protein concentrate at pH 5.0 using the isoelectric precipitation method and then subject to gastric hydrolysis with pepsin enzyme (2000 U/mg of protein) at pH 1.2, pH 2.0, and pH 3.2 at 37Â°C for 2 h with agitation in simulated gastric fluids and then to duodenal hydrolysis with pancreatin (mix enzymes) at pH 7.0 at 37Â°C for 3 h with agitation in simulated intestinal fluid. Gastric and duodenal hydrolysates from kahai were characterized using the SDS-PAGE electrophoresis method and the RP-UHPLC chromatography method.Results: Proteins obtained from kahai (C. orinocense karst) were hydrolyzed with pepsin, only one protein with molecular weight of 100 kDa presented resistance to hydrolysis with pepsin at all pHs assayed. All proteins from kahai protein concentrate were totally hydrolyzed with pancreatin in in vitro conditions.Conclusion: This study suggests that kahai protein concentrates have a high grade of digestibility in vitro when using the gastroduodenal model of digestion. Kahai protein can be a good source of alternative vegetal proteins to be consumed by animals and humans

A, Barrionuevo

E, Vilcacundo

J, Greffa

W, Carrillo

English

Innovare Academic Sciences: E-Journals

Vol 11, Issue 6, 2018Online - 2455-3891 Print - 0974-2441GASTROINTESTINAL DIGESTION OF KAHAI PROTEIN CONCENTRATE (CARYODENDRON ORINOCENSE KARST)GREFFA J2, BARRIONUEVO A1, VILCACUNDO E1, CARRILLO W1* 1Research Department, Bolívar State University, Campus Laguacoto, Guaranda, Ecuador. 2Department of Research, Escuela Superior del Chimborazo, Panamericana Sur km 1 1/2, Riobamba, Ecuador. Email: wcarrillo@ueb.edu.ec/wi.carrillo@uta.edu.ec Received: 31 May 2017, Revised and Accepted: 26 March 2018ABSTRACTObjective: The aim of this study was to obtain kahai protein concentrate from Caryodendron orinocense karst cultivated in the region Amazonia of Ecuador and characterizes its gastric and duodenal hydrolysates using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis method and the reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) method.Methods: Kahai seeds (C. orinocense karst) were utilized to obtain kahai protein concentrate at pH 5.0 using the isoelectric precipitation method and then subject to gastric hydrolysis with pepsin enzyme (2000 U/mg of protein) at pH 1.2, pH 2.0, and pH 3.2 at 37°C for 2 h with agitation in simulated gastric fluids and then to duodenal hydrolysis with pancreatin (mix enzymes) at pH 7.0 at 37°C for 3 h with agitation in simulated intestinal fluid. Gastric and duodenal hydrolysates from kahai were characterized using the SDS-PAGE electrophoresis method and the RP-UHPLC chromatography method.Results: Proteins obtained from kahai (C. orinocense karst) were hydrolyzed with pepsin, only one protein with molecular weight of 100 kDa presented resistance to hydrolysis with pepsin at all pHs assayed. All proteins from kahai protein concentrate were totally hydrolyzed with pancreatin in in vitro conditions.Conclusion: This study suggests that kahai protein concentrates have a high grade of digestibility in vitro when using the gastroduodenal model of digestion. Kahai protein can be a good source of alternative vegetal proteins to be consumed by animals and humans.Keywords: Kahai, Proteins, Hydrolysis, Kahai protein concentrate, Pepsin, Pancreatin.INTRODUCTIONCaryodendron orinocense Karst is a native fruit of the Amazonic region belonging to the family Euphorbiaceae, this plant has about 60 genus and 529 species [1], namely, kahai, inchi, and nuez de barinas. This fruit has a high content of proteins (17.78%, lipids (28.29%), and carbohydrates (34.60%) [2,3]. Lipid from kahai seeds are used to elaborate different products pharmaceutics and cosmetics, but its proteins are not used. The indigenous of Amazonia of Ecuador and Colombia have used kahai oil in the preparation of ointments and different foods. Different Amazonic fruits have a high content of lipids such as Sacha inchi (Plukenetia volubilis) and Ungurahui (Oenocarpus bataua). After the extraction of oil, a byproduct named (tort or flour) is obtained, this byproduct is rich in protein [4-6]. This byproduct can be used to feeding and animal or human nutrition for their qualities. In the food industry, different hydrolysates are used for their functional properties such as hydrolysate of soybean, hydrolysate of whey protein, and hydrolysate of caseins [7,8].  Hydrolysate of soybean is used in the infant formula milks for their high digestibility and good composition of amino acids [8,9].  In the literature, different hydrolysates obtained from vegetable proteins have been described. Among them are quinoa, amaranth, sesamo, macadamia, sacha inchi, chia and tocte [9-18]. The aim of this study was to obtain kahai protein concentrate from C. orinocense karst cultivated in the region Amazonia of Ecuador and characterizes its gastric and duodenal hydrolysates using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis method and the reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) method.METHODSProtein concentrate obtained from kahai flourKahai protein concentrate was prepared according to Martínez and Añón [19] with modifications. The defatted flour was suspended in water in a 1:10 w/v, and the suspension was adjusted to pH 8.0 by adding 2M NaOH. The suspension was stirred during 1 h and then centrifuged at 4500 g for 30 min at 25°C. The supernatant was adjusted to pH 5.0 with 2 N HCl and centrifuged for 20 min at 4500 g. The pellet was suspended in a small volume of water, neutralized with 0.1 M NaOH, and lyophilized, and then frozen at −20°C. The content of protein in kahai protein concentrate was determined using the bicinchoninic acid (BCA) and Dumas methods with values of 50.65% and 48.32%, respectively [20,21].SDSPAGE analysisSDS-PAGE of kahai protein concentrate was analyzed according to the method proposed by Laemmli [22]. The SDS-PAGE system uses 12% polyacrylamide gel/100 mL of resolving gel; 4 g acrylamide/100 mL of stacking gel was used in a Mini-Protean Tetra Cell electrophoresis system (Bio-Rad, Hercules, CA, USA) with the run of 220 V constant during 30 min. Polypeptide bands were stained in Coomassie Brilliant Blue G-250 for 12 h. Relative molecular masses of proteins were determined by comparison to molecular weight markers 6.5–200 kDa kaleidoscope prestained standards (Bio-Rad) containing myosin (198 kDa), β-galactosidase (125 kDa), BSA (88 kDa), carbonic anhydrase (37 kDa), STI (31 kDa), LYS (17 kDa), and aprotinin (6.5 kDa). Molecular weight calibration kit for SDS electrophoresis (GE Healthcare, Uppsala, Sweden) containing phosphorylase B (97 kDa), BSA (66 kDa), OVA (45 kDa), carbonic anhydrase (37 kDa), trypsin inhibitor (20.1 kDa), and LA (14.4 kDa) was used. Polypeptide bands were stained in Coomassie Brilliant Blue G-250 for 12 h with constant agitation. Then, the gels were washed out with a solution of methanol with acetic acid.Gastric and duodenal digestion of kahai protein concentrateKahai Protein Concentrate obtained at pH 5.0 (10 mg/ml) was subjected at simulation gastrointestinal digestion. The environment of the stomach and duodenum was reproduced as close as possible. © 2018 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2018.v11i6.20374Research Article398Asian J Pharm Clin Res, Vol 11, Issue 6, 2018, 397-400 Greffa et al. This study uses a model which emulates the environment of human gastrointestinal tract with its pH, the presence of enzymes, bile salts, temperature, and movement.Gastric digestionWe used pepsin in a concentration of 2.000 U/ml of sample of protein at different pHs: pH 1.2, pH 2.0, and pH 3.2 in (simulated gastric fluid [SGF] 0.35 M NaCl), for 2 h at 37°C with agitation. The pepsin reaction was stopped with heat at 90°C for 10 min. After the hydrolysates were lyophilized and stored at −20°C.Duodenal digestionWe used pancreatin in a concentration of 100 U/ml of sample protein in simulated intestinal fluid (SIF). The composition is a monobasic potassium phosphate solution of pancreatin with bile salt 10 mm and CaCl2 1.5 M. 1 mL of gastric digestion at pH 1.2, pH 2.0, and pH 3.2 was mixed with 1 mL of solution of pancreatin in medium SIF for 3 h at 37°C with agitation. The reaction was stopped with heat at 90°C for 10 min. After the hydrolysates were lyophilized and stored at −20°C.Analysis of hydrolysates from kahai protein concentrate using RP-UHPLCGastric and duodenal hydrolysates of kahai lyophilized were analyzed using RP-UHPLC on Agilent 1200 infinity series UHPLC system (Agilent Technologies, Waldbronn, Germany). The variable wavelength detector was 214 nm. The column used was EC C18 (Agilent Poroshell 120, 4.6 × 50 mm × 2.7 µm of particle size). Samples were eluted at 1.0 mL/min with a linear gradient from 0% to 70% of solvent B (acetonitrile and trifluoroacetic acid [TFA], 1000: 0.270 v/v) in solvent A (water and TFA, 1000: 0.370 v/v) during 10 min. The injection volume was 100 µL for each duplicated sample.RESULTSSDS-PAGE electrophoresis analysis of gastric digestionThe gastric hydrolysates of kahai protein concentrate at pH 1.2; pH 2.0, and pH 3.2 hydrolyzed with pepsin were analyzed using SDS-PAGE electrophoresis in reducing conditions (2-mercaptoethanol). Proteins of kahai were hydrolyzed with pepsin at pH 1.2, pH 2.0, and pH 3.2. Only one protein presented resistance at the hydrolysis with pepsin at all pHs assayed. This protein presented high molecular weight with approximate 100 kDa (Fig. 1).SDS-PAGE electrophoresis analysis of duodenal digestionThe gastric hydrolysates of kahai protein at pH 1.2, pH 2.0, and pH 3.2 hydrolyzed with pepsin were subject to duodenal hydrolysis with pancreatin at pH 7.0. After hydrolysis, the digestion of kahai was analyzed using SDS-PAGE electrophoresis in reducing conditions (2-mercaptoethanol). All proteins of kahai protein concentrate were hydrolyzed with pancreatine (duodenal digestion). Proteins from kahai have a high digestibility in in vitro conditions (Fig. 2).RP-UHPLC chromatography analysis of gastro-duodenal hydrolysis of kahaiGastric and duodenal hydrolysates of kahai were analyzed with the RP-UHPLC method. Kahai protein concentrate obtained at pH 5.0 isoelectric precipitation without hydrolysis was used as comparative control (Fig. 3e). Kahai protein concentrate presents four majoritarian fractions. Kahai gastric hydrolysate at pH 1.2, pH 2.0, and pH 3.2 presents similar profile of fractions (peptides) (Fig. 3a-c). Only kahai gastric hydrolysate at pH 3.2 presented less intensity in the chromatogram. The first and the second fractions (F1 and F2) presented resistance to hydrolysis with pepsin. This result is in accordance with the SDS-PAGE electrophoresis analysis as in the gel we observe one band of protein without hydrolysis. The third and the fourth fractions were totally hydrolyzed with the pepsin enzyme in in vitro conditions. Kahai duodenal digestion was analyzed, and we can observe that the four fractions were hydrolyzed with pancreatin because the profile is different. All peaks presented different retention times when compared to retention times of fractions of gastric digestion (Fig. 3d).DISCUSSIONVegetal proteins are being the object of many studies to evaluate their use such as source of protein isolate and protein concentrate. Soy protein isolate is the product more used in the food industry for different purposes such as infant formula, nutraceutics products, functional foods, milks, and beverages [23-25]. There is extensive research looking for new sources of vegetal proteins (amaranto, quinoa, pea, and lupinus). Digestibility of animal and plant proteins is very important for their use in the food industry, pharmaceutical, and medical purposes. Plant proteins present high digestibility in in vitro and in vivo conditions. For this reason, plant proteins are more consumed and recommended for children and adults [26]. Furthermore, it is important to know their content of possible allergens. The allergens present a high resistance to hydrolysis in vitro with pepsin. For this reason, the FAO and WHO recommend assays in vitro of the digestibility with pepsin to predict the presence of possible allergens in the foods. Hen proteins have been evaluated as allergens with assays of digestion in vitro with pepsin using SFG, this method being recommended by American pharmacopeia. Fig. 1: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of gastric hydrolysates of kahai. Line 1: Standard molecular weight, Line 2: Kahai gastric digestion at pH 1.2, Line 3: Kahai gastric digestion at pH 2.0, Line 4: Kahai gastric digestion at pH 3.2, and Line 5: Kahai protein concentrate obtained at pH 5.0 of precipitation without hydrolysisFig. 2: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of duodenal digestion of kahai. Line 1: Standard molecular weight, Line 2: Kahai duodenal digestion of gastric digestion of pH 1.2, Line 3: Kahai duodenal digestion of gastric digestion of pH 2.0, Line 4: Kahai duodenal digestion of gastric digestion of pH 3.2, and Line 5: Kahai protein concentrate obtained at pH 5.0 of precipitation without hydrolysis399Asian J Pharm Clin Res, Vol 11, Issue 6, 2018, 397-400 Greffa et al. Astwood et al. [27] reported a relation between the stability of a protein in SGF and its status as a food allergen. SGF is defined as reagents held under specific conditions (0.32% pepsin, pH 1.2, 37°C) and was developed to represent human gastric conditions in the stomach of a healthy adult. Lysozyme and ovoalbumine from hen chicken were evaluated with digestion in vitro with pepsin, presenting resistance to hydrolysis with pepsin. One characteristic of food allergens is the resistance to gastrointestinal hydrolysis [28-30].Kahai protein concentrate presents a high grade of digestibility with pepsin and pancreatin when used in in vitro conditions. Only one protein presents resistance to hydrolysis with pepsin but was hydrolyzed with pancreatin. It is probable that kahai proteins have no allergens. These kahai hydrolysates can have bioactive peptides. Future research could evaluate different biological activities and possibly identify the sequence of bioactive peptides from kahai seeds. In the developed countries, the consume of vegetal proteins is a good alternative of good proteins, vegetal protein is cheaper, and their quality is excellent. Some vegetal proteins have a good amino acids composition such as amaranth and quinoa. New sources of vegetal proteins are necessary to evaluate their digestibility to find out their possible allergenicity.CONCLUSIONSKahai protein concentrate presents high in vitro digestibility with pepsin and pancreatin enzymes using the SGF and SIF methods of hydrolysis, these methods are recommended to assay the in vitro digestibility. It is possible that kahai seeds have no allergens proteins. Gastric and duodenal hydrolysates from kahai can have bioactive peptides that can be used to elaborate functional foods and nutraceuticals products. Kahai seeds can be a good source of proteins to countries in development such as Ecuador and a good crop to indigenous for their high contents of lipids and proteins.REFERENCES1. Alfaro Mde J, Alvarez I, El Khor S, de Padilla FC. Functional properties of a protein product from Caryodendron orinocense (Barinas nut). ALAN 2004;54:223-8.2. Alfaro JM. Physico-chemical characteristics of the Barinas nut (Caryodendron orinocense Karst. 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GASTROINTESTINAL DIGESTION OF KAHAI PROTEIN CONCENTRATE (CARYODENDRON ORINOCENSE KARST)

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GASTROINTESTINAL DIGESTION OF KAHAI PROTEIN CONCENTRATE (CARYODENDRON ORINOCENSE KARST)

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