Contemporary Engineering Sciences, Vol. 11, 2018, no. 80, 3991 - 3998 HIKARI Ltd, www.m-hikari.com https://doi.org/10.12988/ces.2018.85244

Effect of the Antioxidant Activity of the Essential

Oil of mollis G in a Meat Product

Type Sausage

Clemente Granados-Conde1*, Alex Iván Arrieta-Llamas1, Miladys Torrenegra-Alarcón2,4, Glicerio León-Méndez3,4, Nerlis Paola Pájaro-Castro5 and Vanessa Villalba-Vimos3

1 Faculty of Engineering, University of Cartagena. Research Group in Engineering, Innovation, Food Quality and Health-INCAS,

2 Center of Commerce and Services, Regional Bolivar, SENA, Research in Innovation and Biotechnology Group (GIBEI), Colombia

3 Grupo de Investigación CIPTEC, Fundación Universitaria Tecnológico Comfenalco - Cartagena, Cra. 44 #30A, 91, Cartagena Bolívar, Colombia Affiliation ID60110627

4 Doctorate Student in Engineering, University of Cartagena Cartagena, Bolívar 130015, Colombia

5 Pharmaceutical Chemistry, Master of Pharmaceutical Sciences Faculty of Sciences of the Health, University of Sucre Medical and Pharmaceutical Sciences Group, Colombia

Copyright © 2018 Clemente Granados-Conde et al. This article is distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Introduction: Griseb belonging to the genus Minthostachys known commonly as Muña, a native of Colombia, , and . Objective: To evaluate the effect of the antioxidant activity of the essential oil of Minthostachys mollis G in a type sausage meat product. Materials and methods: Determined the composition chemical by chromatography of gases/spectrometer of mass, the activity antioxidant is evaluated by the method DPPH. In addition, we 3992 Clemente Granados-Conde et al.

studied the effects of essential oil on lipid oxidation of a meat product type sausage, analyzing the formation of 2-thiobarbituric acid reactive substances, as indicators of oxidation. Results: Found high levels of oxygenated monoterpenes (Thymol and carvacrol); the antioxidant activity of essential oil Minthostachys mollis (IC50 70,5±0,33 μg/mL). Demonstrating that the AE has the capacity of inhibiting the process oxidative of a matrix food as the sausage. Conclusions: It is established that the AE of M. mollis is a promising natural antioxidant, which can be used in the food industry as a possible substitute for synthetic antioxidants.

Keywords: Muña, antioxidant, Minthostachys

Introduction

Several strategies have been developed for years in order to prevent oxidative deterioration in the different products of meat origin. Most of these new alternatives have focused on limiting the access of oxygen to the components of the meat susceptible to undergo oxidation phenomena such as lipids and proteins. One of these ways to reduce the occurrence of oxidation phenomena in meat products is the use of antioxidants, whose basic function is to prevent or avoid the formation of free radicals in the product [1,2]. However, the current trend of consumers has been skewed by the consumption of natural foods, rich in antioxidants particularly phenolic compounds. Among these foods are some that possess essential oils (OE) with active principles with potential biological and industrial activity, which make our country a very interesting region for the research and development of new products Natural [2-6]. The OE are products obtained from the Vegetal kingdom, in which are concentrated flavors and characteristic aromas. They consist of complex mixtures of hydrocarbons, oxygenated compounds and non-volatile residues. In general, AE can be defined as oily liquids obtained from different parts of such as flowers, buds, seeds, leaves, branches, barks, wood, fruits and roots. [2-11]. Because of the above, it is interesting to study the antioxidant activity of the essential oils (OE) of native plants, to recommend their potential use as natural additives in food, therefore, evaluated the effect of the activity antioxidant of the essential oil of Minthostachys mollis G in a sausage type meat product.

Methodology

The essential oil of Muña was acquired from the laboratory of the research group in engineering, innovation, food quality and health-INCAS- of the University of Cartagena. (Storage conditions for AE were 4 ± 5°C).

Analysis of OE by gas chromatography/mass spectrometer (CG/EM) A CG/EM 7890A/5975C Agilent (United States) team was used in an interphase with a HP5973 Network mass-selective detector connected in line with an HP-MS Effect of the antioxidant activity of the essential oil of … 3993

ChemStation system and the NIST-2008 database. The operating conditions were: HP-5MS Capillary column (5% phenyl methyl Silox, 30 m x 250 μm x 0.25 μm), initial temperature 45°C, transfer line temperature 280 °c and Injection Volume 1.0 mL in Split mode (20:1), with injector temperature of 250°C [5, 6, 12,13]. The detection of the compounds was performed by comparison of the mass spectrum, in each retention time, with those reported in the database NIST-2008.

DPPH assay The activity getter of free radicals DPPH•, was determined using the method described by Silva et al. [14] with some modifications 75 µ L of sample were added to 150 µL of a methanolic solution of DPPH• (100 µg/mL) and incubated at room temperature for 30 min, after which it was determined spectrophotometrically the disappearance of the radical DPPH• A 550 nm in Ex Multiskan microplate Reader (Thermoscientific). Ascorbic acid was used (25 µg/mL as positive control for the capture of the DPPH• radicals).

(퐴 −퐴 ) 퐼푛ℎ𝑖푏𝑖푡𝑖표푛 = 0 푓 ∗ 100 (1) 퐴0

Where A0 and Af are the absorbance values of the target (solution of DPPH in alcohol) and the sample (solution of DPPH more antioxidant dissolved in alcohol), respectively.

Meat formulation and of the sausage elaboration A meat formulation was prepared for the preparation of Frankfurt sausages using pork and beef lean meat, pork bacon, ice, salt, nitratal, phosphates, isolated soy protein, Frankfurt sausage condiment and oregano oil as an antioxidant. Natural. It should be noted that the concentrations of the latter in this formulation were varied with values of 0, 50, 100 and 150 µg/mL

Determination of the content of reactive substances with 2-thiobarbituric acid (TBARS) The determination of the content of reactive substances with acid 2-thiobarbituric (TBARS) was carried out by the procedure of Rojano et al [15]. 5 mL of TBA reagent (2-thiobarbituric acid 0.7% in glacial acetic acid at 99.9%) are placed in a test tube, and 0.4 g of specimen and stirred for 10 s and taken to a water bath at 90 °C for 1 hour. The tubes, cooled in water, were centrifuged at 5000 rpm for 20 min after which the supernatant was eliminated. A portion of the sample was taken to a glass cuvette and its absorbance was read at 532 nm. The results were expressed as mg of Malonadialdehído/kg (MDA) sample using a standard curve. The standard curve was constructed by making appropriate dilutions of an aqueous solution of 1, 1, 3, 3 Tetraetoxipropano 1,0x10-3 M. These solutions were treated with TBA reagent (2-thiobarbituric acid 0.02 M in glacial acetic acid at 90%) and absorbance was measured at 532 nm. 3994 Clemente Granados-Conde et al.

Statistical analysis

All trials were conducted in triplicate following the previously established protocols, the results were expressed as the average ± the standard error of the mean (ESM) and were analyzed by Student t test or ANOVA followed by the test of Dunnett or Tukey, or depending on the case. Values of P < 0.05 were considered significant. For the statistical analyses the package GraphPad Prism V 5.00 for Windows.

Results and analysis

The identification of the components, retention times and percentages of EA abundance of M. mollis (Muña) by CG/MS are reported in table 1. The majority compound found is the thymol with a relative abundance percentage of 73.36%.

Table 1. Major components detected in M. mollis OE.

% Relative Abundance, (tR, Min) Compound M. mollis α-pineno 0,05 (5,24)

β-mirceno 0,17 (6,27) α -terpineno 0,17 (6,48) p-cimeno 7,28 (7,05) 4-careno 0,53 (41) β -felandreno 0,17 (6,48) Carvacrol 10,94 (7,76) Timol 73,36 (13,17) Pulegona 7,00 (13,55)

Cano et al. [16], they found as a major component of the essential oil from Muña to Pulegone (36.68%). Highlight that this compound is present in the Chromatographic profile shown in table 1. However, a small variation is visible with respect to the minority components found by Cano et al. [16], caused by the effect of different environmental factors on the content of compounds in medicinal plants. The intensity of light and photoperiod, which vary from region to region; Harvest time and age of plants affect essential oil composition. Effect of the antioxidant activity of the essential oil of … 3995

The antioxidant activity of Muña OE, was evaluated by the method of DPPH• obtaining a value of IC50 70,5 ± 0,33 μg/mL. Antioxidants can act by multiple mechanisms, depending on the reaction system or the source Radicalaria or oxidant used [17.18], the results are expressed as antiradical activity or IC50, which is defined as the concentration of Antioxidant that decreases the absorption of the radical to 50% of the initial amount [6, 17,18]. Free radicals have a significant effect on the oxidation of unsaturated lipids; The DPPH radical was used as a stable free radical to determine antioxidant activity of natural compounds. The method is based on the reduction of the alcoholic solution of DPPH in the presence of an antioxidant to donate hydrogen due to the formation of the non-radical DPPH – H. Figure 1 It shows the effect of the different concentrations of essential oil, ascorbic acid and white on the formation of reactive substances to thiobarbituric acid (TBARS) in the samples of Frankfurt sausages over time. A continuous increase in TBARS was observed in all samples during storage time. White exhibited the highest values of TBARS at all stages of analysis during the storage.

Figure 1. Changes in TBARS in Frankfurt sausages at different concentrations of essential oil of M. mollis, ascorbic acid 0.02 and without any additive.

Treatments with the essential oil of M. Mollis reduced the oxidation of Frankfurt sausage during incubation at 30 °c, demonstrating an inhibitory effect on the formation of TBARS and indicating a potential protection of oxidation for sausage. The increase in TBARS concentration was slow and similar for all samples until Day 3, from which the formation in the target and the concentrations of 100 and 250 μg/mL were accelerated until the last day. The increase in the content of TBARS was more intense and linear behavior for white. Samples of 750 μg/mL and ascorbic acid showed no significant differences in their behaviour. According to Villanueva et al. [19] ascorbic acid yields a hydrogen, producing a double-link electron transfer to the oxygen that suffered the loss of an electron, repeating the same action in the next oxygen atom, which suffered the loss of the hydrogen atom, until Establish the balance of energy. According to this reaction, 3996 Clemente Granados-Conde et al.

ascorbic acid yields two hydrogens, thus explaining its efficiency as an excellent antioxidant. Other effects that act on the mechanisms of inhibiting lipid peroxidation of ascorbic acid on hydroperoxides in the meat product, is the reduction of hydroperoxides to more stable hydroxyl compounds, as they do not allow them to continue Chain reactions that are characteristic of the mechanism of the free radicals [19], another alternative is that the ascorbic acid can act like a chelating agent of metals, or like a system remover of the oxygen singuete which also contributes to the peroxidation [6].

Conclusions

The essential oil of Minthostachys mollis has a high content of oxygenated monoterpenes being the thymol which is found with a higher percentage of relative abundance, which are expressed through its high antioxidant capacity. Therefore, it is established that the AE of Muña is a promising natural antioxidant, which can be used in the food industry as possible substitutes for synthetic antioxidants.

Acknowledgments. Authors thanks to Fundacion Universitaria Tecnologico Comfenalco, Universidad de Cartagena and SENA – Centro de Comercio y Servicios for all Support work.

Conflict of interest: Authors declare no conflict of interest for this work.

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Received: June 3, 2018; Published: September 18, 2018