Accepted Manuscript

Potential application of epazote (Chenopodium ambrosioides L.) as natural

antioxidant in raw ground pork

Luz H. Villalobos-Delgado, Edith Graciela González-Mondragón, Alma Yadira Salazar

Govea, Juana Ramírez Andrade, J. Tenoch Santiago-Castro

PII: S0023-6438(17)30401-2
DOI: 10.1016/j.lwt.2017.05.076
Reference: YFSTL 6292

To appear in: LWT - Food Science and Technology

Received Date: 23 December 2016

Revised Date: 24 May 2017
Accepted Date: 30 May 2017

Please cite this article as: Villalobos-Delgado, L.H., González-Mondragón, E.G., Salazar Govea,
A.Y., Andrade, Juana.Ramí., Santiago-Castro, J.T., Potential application of epazote (Chenopodium
ambrosioides L.) as natural antioxidant in raw ground pork, LWT - Food Science and Technology (2017),
doi: 10.1016/j.lwt.2017.05.076.

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 ACCEPTED MANUSCRIPT

1 Title

2 Potential application of epazote (Chenopodium ambrosioides L.) as natural

3 antioxidant in raw ground pork

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5 Author names and affiliations

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6 Luz H. Villalobos-Delgado*, Edith Graciela González-Mondragón, Alma Yadira

7 Salazar Govea, Juana Ramírez Andrade, J. Tenoch Santiago-Castro.

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8 Instituto de Agroindustrias, Universidad Tecnológica de la Mixteca, Carretera a

9 Acatlima Km 2.5, 69000, Huajuapan de León, Oaxaca, Mexico

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11 Authors' email addresses:

12 vidluz@mixteco.utm.mx; edith@mixteco.utm.mx; almasalazar@mixteco.utm.mx;

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13 jramirez@mixteco.utm.mx; tenochsc@mixteco.utm.mx.
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14
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15 Corresponding autor. Tel.: +52 953 53 20399 ext 400.

16 E-mail address: vidluz@mixteco.utm.mx (Luz H. Villalobos-Delgado)

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17

18 Abstract
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19 The antioxidant effect of epazote infusion (EI) and ethanolic extract of epazote
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20 (EE) was evaluated in raw ground pork stored at 4 ºC for 9 days. EI and EE were

21 characterised by evaluating total phenolic content (TPC), total flavonoid content

22 (TFC) and antioxidant activity (AA). The phenolic compounds present were

23 identified using ultra-high performance liquid chromatography-quadrupole time-of-

24 flight (UHPLC-qTOF). TBARS were analysed in the raw ground pork with EI (MEI)
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25 and EE (MEE) as well as meat without EI and EE as the control (CTL) and sodium

26 ascorbate (NaASC) as positive control. For instrumental colour, pH, microbiological

27 status and the sensorial analysis, only MEI, MEE and CTL were considered. EI

28 showed higher TPC and TFC while EE showed a higher percentage of some

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29 phenolic compounds, but AA was the same for both samples. TBARS values for

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30 MEI and MEE were lower than the CTL. MEI and MEE had higher values in a* and

31 a*/b*, but the pH and microbial count was lower for MEE. Finally, MEI showed the

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32 highest score for acceptability in colour and appearance, while CTL showed the

33 lowest score in all the attributes evaluated. In conclusion, epazote has potential as

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a natural antioxidant in meat and meat products.
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35

36 Key words:
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37 Epazote, Chenopodium ambrosioides L., natural antioxidant, ground pork, TBARS.

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38
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39 1. Introduction

40 Microbial growth and oxidation of both lipids and oxymyoglobin (OxyMb), are the
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41 main causes of loss of quality in meat and meat products (Min & Ahn, 2005;

42 Morrisey & Kerry, 2004), which is manifested by adverse changes in taste, colour,
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43 texture, nutritional value and the production of toxic compounds (Gray, Gomma, &
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44 Buckley, 1996; Kanner, 1994).

45 Ground meat is more susceptible to oxidation because the grinding process alters

46 the integrity of the muscular membranes and exposes the membrane lipids. This

47 facilitates the interaction of pro-oxidant molecules with unsaturated fatty acids,

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48 encouraging the generation of free radicals and the propagation of oxidative

49 reactions (Devatkal & Naveena, 2010).

50 Lipid oxidation in meat products can be minimized or inhibited through the use of

51 synthetic antioxidants (Decker, 1998). However, their use is limited to certain

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52 amounts, and they have been identified as toxicological agents and carcinogens

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53 (Kumar, Yadav, Ahmad, & Narsaiah, 2015; Lorenzo, Sineiro, Amado, & Franco,

54 2014). Therefore, there have been efforts to identify new antioxidants from natural

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55 sources used to preserve and improve the quality of meat and meat products

56 (Hygreeva, Pandey, & Radhakrishna, 2014; Karre, Lopez, & Getty, 2013; Shah,

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Don Bosco, & Mir, 2014). Natural antioxidants have been obtained from fruits,
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58 vegetables, spices and herbs, which contain antioxidant-like biocompounds, for

59 example phenolic diterpenes, flavonoids, tannins and phenolic acids (Falowo,

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60 Fayemi & Muchenje, 2014; Jiang & Xiong, 2016; Kumar et al., 2015; Zhang, Xiao,
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61 Samaraweera, Lee & Ahn, 2010).

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62 Chenopodium ambrosioides L. (Chenopodiaceae; syn: Dysphania ambrosioides

63 (L.) Mosyakin & Clements; Teloxys ambrosioides (L.) W. Weber) is a plant known
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64 in Mexico as 'epazote' (Castillo-Juárez et al., 2009; Kliks, 1985), and its infusions

65 and decoctions of leaves, roots and inflorescences have been used as condiments
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66 and in traditional medicine. This species is rich in flavonoids (Barros et al., 2013;
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67 Kokanova-Nedialkova, Nedialkov, & Nikolov, 2009; Vysochina, 2010), suggesting

68 that the epazote plant can be used as a source of natural antioxidants in the food

69 industry. Therefore, the objective of this research was to evaluate for the first time

70 the effect of the incorporation of C. ambrosioides L. (infusion and ethanolic extract

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71 of epazote) on lipid oxidation, instrumental colour, pH, microbiological status and

72 sensorial acceptance in raw ground pork stored under refrigeration.

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74 2. Material and methods

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75 2.1. Chemicals

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76 Solvent and water used for UHPLC-qTOF were OptimaTM LC/MS grade, Fisher

77 Chemical and HPLC grade obtained from Merck (Darmstadt, Germany). Radical

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78 DDPH• (1,1-Diphenyl-2-picrylhydrazyl), Folin-Ciocalteu reagent, trichloroacetic acid

79 (TCA), 1,1,3,3-tetraethoxypropane (TEP), butylated hidroxytoluene (BHT),

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hydroxide ammonium (NH4OH) and p-coumaric, ferulic, gallic, caffeic acids and
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81 kaempferol reference standards were purchased from Sigma Aldrich (St. Louis,

82 MO, USA). 4,6-Dihydroxy-2-mercatopyrimidine (2-Thiobarbituric Acid thiobarbituric)

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83 (TBA) was acquired from Alfa Aesar (Ward Hill, MA, USA). Sodium hydroxide
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84 (NaOH), aluminium chloride (AlCl3), sodium nitrite (NaNO2), ethyl acetate,

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85 methanol anhydrous, ethanol, ethyl ether and acetic acid were purchased from J.T.

86 Baker (Xalostoc, Mexico State, Mexico). Sodium carbonate (Na2CO3) and sodium
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87 ascorbate (C6H7NaO6) were purchased from Meyer (Mexico City, Mexico).

88 Petroleum ether was purchased from Golden Bell (Mexico City, Mexico).
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89 Acetonitrile LiChrosolv was acquired from Merck (Darmstadt, Germany). Peptone

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90 water and plate count agar were purchased from Difco™ (Sparks, MD, USA).

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92 2.2. Description of sample

93 The raw ground pork was obtained from a mixture of two different fresh meat cuts

94 (lean pork shoulder and pork leg) purchased from a local market in Huajuapan de
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95 León, Oaxaca, Mexico, at 24 h post mortem. No details were available on the

96 animal breeds. The raw ground pork was immediately transported to the laboratory

97 in an ice-filled cooler and then it was kept at 4 °C until used. The epazote plant

98 (Chenopodium ambrosioides L.) used in this study was obtained from a local

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99 market in the same locality, collected from crops in the village of Santa Cruz

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100 Tacache de Mina, Oaxaca, Mexico, during the flowering stage.

101

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102 2.3. Identification of the epazote plant

103 Complete epazote plants (stem, leaves and flowers), were deposited in the

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National Herbarium of Mexico (MEXU), Department of Botany, Institute of Biology,
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105 National Autonomous University of Mexico.

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107 2.4. Preparation of the epazote plant

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108 After washing with tap water, leaves, flowers and part of the stem were disinfected
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109 by immersion with 0.35% colloidal silver in gelatin (Microdyn™, Mexico) diluted

110 with potable water (1:300, v/v). Excess water was drained and drying was
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111 performed for 8 days at room temperature (25 ± 2 ºC) until 14% humidity was

112 reached. The epazote was then ground (Moulinex, Colombia), and passed through
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113 a 40-mesh sieve (Montinox, Mexico), obtaining a particle size of 420 µm. This
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114 sample was stored in airtight plastic bottles at 4 ± 1 °C until use.

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116 2.4.1. Preparation of the infusion and ethanolic extract of epazote

117 The epazote infusion (EI) was prepared according to Karabacak and Bozkurt

118 (2008), with some modifications. The epazote powder was mixed with potable
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119 water (1:20, w/v) and heated at 40 °C with constant stirring for 1 h. The mixture

120 was filtered with Whatman No. 41 filter paper, the filtrate was collected and stored

121 in freezing at -20 °C, with no exposure to light until use. The ethanolic extract of

122 epazote (EE) was obtained according to Biswas, Chatli, and Sahoo (2012), with

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123 some modifications. The powder was macerated with food grade ethanol (1:20,

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124 w/v) for 10 min at room temperature (25 ± 2 °C) and was shaken at 2,000 rpm for

125 10 min and then centrifuged at 10,000 rpm for 10 min at 25 °C. The supernatant

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126 was recovered by filtration using Whatman No. 41 filter paper and collected in an

127 amber glass bottle with an airtight seal. A second extraction was carried out on the

128
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solid residue following the same methodology. Both extracts were mixed and
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129 stored at -20 °C until use. EI and EE were prepared 24 h prior to incorporation into

130 the raw ground pork.

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131
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132 2.5. Preparation of the raw meat samples

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133 Raw ground pork was prepared with epazote infusion (MEI) and ethanolic extract

134 of epazote (MEE), using as control (CTL) meat without EI or EE and positive
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135 control ground pork with a solution of 0.01 g/mL of the antioxidant sodium

136 ascorbate (NaASC). EI, EE and NaASC solutions were incorporated at a rate of 50
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137 mL/kg of ground pork meat by manually mixing thoroughly until the solutions were
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138 homogeneously dispersed throughout the ground pork meat (5 min aprox.). Then,

139 0.25 kg of each treatment were stored in retail mode for 9 days: polystyrene trays

140 covered with a PVC plastic film at 4 ± 1 °C and exposure to fluorescent light. Three

141 replicates of the four treatments were carried out. The TBARS content,

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142 instrumental colour, pH, microbial count and sensorial analysis were evaluated on

143 days 0, 3, 6 and 9 in triplicate.

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145 2.6. Characterisation of the infusion and ethanolic extract of epazote

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146 2.6.1 Sample preparation

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147 Chlorophyll was removed from the two samples by liquid-liquid extraction using

148 petroleum ether and ethyl ether (1:1, v/v). A second liquid-liquid extraction using

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149 ethyl acetate (1:1, v/v) was carried out for the infusion. The dissolvent was

150 removed from both extracts using a rotary evaporator at 45 ºC, and redissolved in

151
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methanol. It was placed into a Supelclean LC18 column, previously equilibrated
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152 with 100% methanol, and eluted with 100% acetonitrile, completely dried in a rotary

153 evaporator at 45 °C and re-suspended in methanol for total phenolic content (TPC)
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154 and antioxidant activity (AA), and ethanol for total flavonoid content (TFC). For the
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155 identification of the phenolic compounds by UHPLC-qTOF, the extracts were re-
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156 dissolved in methanol:NH4OH (99.99:0.1, v/v), filtered using a 0.2 µm nylon

157 membrane, and placed in a 2 mL glass vial for analysis.

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158

159 2.6.2. Total phenolic content (TPC)

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160 The method according to Shahwar and Raza (2012) was used with some
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161 modifications. A 50 µL aliquot of the extract were added to 3 mL of distilled water

162 and 250 µL of 1N Folin-Ciocalteu reagent. After 5 min, 750 µL of 20% Na2CO3 and

163 950 µL of distilled water were added. The reaction mixture was homogenized and

164 allowed to stand for 40 min. The absorbance was measured at 765 nm and the

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165 concentration was determined using a calibration curve with gallic acid as a

166 reference standard, dissolved in methanol (0 to 1000 µg/mL).

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168 2.6.3. Total flavonoid content (TFC)

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169 TFC was determined using a method described by Ferreira et al. (2012) with some

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170 modifications. One millilitre of the extract was added to 4 mL of distilled water, 300

171 µL of NaNO2 at 5% and 300 µL of AlCl3 at 5%. After 1 min, 2 mL of NaOH 1 M and

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172 2.4 mL of distilled water were added. The reaction mixture was homogenized and

173 the absorbance was measured at 330 nm. The concentration was determined

174
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using a calibration curve with quercetin as a reference standard dissolved in
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175 ethanol (0 to 1000 µg/mL).
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176

177 2.6.4. Phenolic compounds identification

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178 Experiments were carried out using Waters® Acquity UPLC® Class I equipment
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179 connected to a Xevo® G2 XS Qtof equipped with an electrospray ionization source

180 (ESI) (Waters Corp., Milford, MA, USA). MassLynx 4.1 software was used for data
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181 acquisition and processing. Acquity UPLC HSS T3 C18 Column of 2.1x100 mm,

182 1.8 µm was used and maintained at 40°C. The mobile phase A was water:acetic
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183 acid (99.9:0.1, v/v), and the mobile phase B was acetonitrile (100%) with a linear
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184 gradient elution: 97% A (0 min), 60% A (15 min), 3% A (16 min), 97% A (16.5 min),

185 flow rate of 0.2 mL/min, and injection volume of extract or infusion was 5 µL at 7

186 °C. The washing solvent and purge was water:acetonitrile (1:1, v/v), and the seals

187 wash with water:acetonitrile (90:10, v/v). The mass spectrometry (MS) system was

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188 operated in a negative ionization (ESI) and sensitivity mode. Capillary voltage was

189 2.5 kV, the source temperature was maintained at 120 °C, the solvation

190 temperature at 400 °C, and the cone voltage was set at 25 V. Nitrogen gas was

191 used in the cone and desolvation gas flow was the 1000 L/h. MS data were

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192 collected in full scan mode (m/z 100-1200) in continuous format 0.5 s, using

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193 leucine/enkephalin ([M-H]- = 554.2615) as a reference standard. Also, commercial

194 standards for p-coumaric, ferulic, gallic, caffeic acids and kaempferol were used at

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195 0.1 g/mL in methanol:NH4OH (99.99:0.01, v/v). The identification of compounds

196 with no reference standards was obtained from the high-resolution mass spectrum,

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considering that the time-of-flight allows very accurate mass detection. Firstly, from
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198 the molecular formula reported for the compounds present in plants of genus

199 Chenopodium, the monoisotopic molecular mass was calculated in negative mode,
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200 which was used for analysis of elemental composition. The molecular masses
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201 suggested for each compound were accepted when the mass error was up to 3
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202 mDa.

203
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204 2.6.5. Antioxidant activity

205 The antioxidant activity was evaluated using the method of Pyo, Lee, and Lee
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206 (2005) modified. Fifty microlitres of extract or infusion were added to 2 mL of 0.1
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207 mM DPPH• solution in methanol. The solution was homogenized and the

208 absorbance at 517 nm was measured after 0 (Ai) and 30 min (Af). The results are

209 reported as the percentage of inhibition of the antioxidant activity.

( − )
% ℎ = ∗ 100

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210

211 2.7. Analysis of the raw ground pork

212 2.7.1. Proximal analysis

213 Proximal analysis was determined on each treatment and only on day 0, following

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214 standard AOAC (1997) methods: moisture (950.46); ash (920.153); crude protein

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215 (928.08); crude fat (991.36).

216

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217 2.7.2. Chemical and instrumental analysis

218 The pH of the meat samples was measured with a digital pH-meter (Oakton 510,

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USA). An amount of 10 g of sample was homogenized with 100 mL of distilled
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220 water using a conventional blender. The resulting suspension was filtered using

221 cotton gauze to remove connective tissue.

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222 Thiobarbituric acid reactive substances (TBARS) concentrations were analysed

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223 using the method described by Nam & Ahn (2003) with some modifications. Briefly,
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224 2 g of meat sample were homogenized in 20 mL of distilled water with an

225 ultraturrax T18 (IKA, Germany) at 13600 rpm for 1 min. Then, 1 mL of meat
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226 homogenate was transferred into a screw cap tube, and 30 µL of butylated

227 hydroxytoluene (7.2%, in ethanol) and 2 mL of thiobarbituric acid:trichloroacetic

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228 acid solution [20 mM TBA and 15% (w/v) TCA] were added. The mixture was
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229 vortexed and then incubated in a water bath at 80 °C for 20 min. After cooling for

230 10 min in cold water, the sample was centrifuged at 2500 rpm for 10 min at 4 °C.

231 The absorbance was measured at 531 nm. TBARS values were calculated from a

232 standard curve of 1,1,3,3-tetraetoxypropane (TEP) and expressed as mg

233 malondialdehyde/kg meat. The colour was evaluated using a UltraScan Vis 1139
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234 colorimeter (HunterLab, USA). Results were expressed as lightness (L*), redness

235 (a*) to yellowness (b*), ratio (a*/b*), chroma (C*) [(a*2 + b*2)1/2] and reflectance

236 ratio at 630 nm and 580 nm (R630/R580) (AMSA, 2012).

237

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238 2.7.3. Microbiological analysis

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239 Total plate count was determined by using the pour plate method as described in

240 Mexican Legislation (Secretaría de Salud, 1994). The total bacterial count (TBC)

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241 was determined on plate count agar (Difco, USA) at 35 °C for 48 h. Microbial

242 colonies were counted and expressed as log10 Colony Forming Units/g meat

243 (CFU/g).
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244

245 2.7.4. Sensory analysis

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246 A hedonic test (Anzaldúa-Morales, 1994) was performed on raw samples in order
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247 to evaluate consumer acceptability of the experimental treatments. Meat samples

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248 were evaluated by 33 non-trained panellists (aged between 20 and 50 years),

249 recruited from staff and students at the Institute of Agroindustry at the
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250 Technological University of the Mixteca (UTM for its Spanish initials; Huajuapan de

251 León, Oaxaca, Mexico), who were selected based on experience in the regular
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252 consumption of raw ground pork. Three sensory evaluation sessions were carried
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253 out for each storage period (0, 3, 6 and 9 days) in the UTM´s testing room, in

254 individual partitioned-off booths. Three different samples, one from each of the

255 experimental treatments, were placed in white plastic dishes and coded with

256 randomly chosen 3-digit numbers. Panellists were asked to assess the colour,

257 odour and general appearance acceptability of each treatment using a 9-point
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258 hedonic scale: 1 -dislike extremely; 2 -dislike very much; 3 -dislike moderately: 4 -

259 dislike slightly; 5 -neither like nor dislike; 6 -like slightly; 7 -like moderately; 8 -like

260 very much; and 9 -like extremely. The scores of means from 4 to 9 were

261 considered acceptable.

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262

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263 2.8. Statistical Analysis

264 Statistical analyses were carried out using the Statistica for Windows software

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265 (v.6.0; Statsoft Inc. 2001). The characterization of the extracts and the proximal

266 composition was analysed by a one-way analysis of variance (ANOVA), reporting

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the results as the mean ± standard error. In addition, a model that included
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268 treatment (CTL, MEI, MEE and NaASC) and storage period (0, 3, 6 and 9 days)

269 was used to evaluate TBARS values for meat, while for instrumental colour, pH,
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270 microbiological analysis and sensory analysis the same model was used, without
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271 considering NaASC treatment. Treatment and storage time were considered as
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272 main effects, and all their interactions. The differences among means were tested

273 for significance (P<0.05) by Duncan test.

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274

275 3. Results and discussion

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276 3.1. Identification of the epazote plant

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277 The epazote plant used in this research was identified as Chenopodium

278 ambrosioides L., with taxonomic synonymy with Disphania ambrosioides (L.)

279 Mosyakin and Clements, as well as Teloxys ambrosioides (L.) W.A. Weber.

280

281 3.2. Characterisation of the infusion and ethanolic extract of epazote

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282 EE showed a slightly more acidic pH than EI (P<0.05) (Table 1). These results can

283 be attributed to the presence of epazote-specific organic acids such as citric acid

284 identified by UHPLC-qTOF for both samples, as reported by Barros et al. (2013),

285 as well as the pH (5.34) of ethanol. On the other hand, EI showed higher TPC and

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286 TFC (P<0.05), though there were no significant differences in AA (P>0.05) when

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287 compared to EE, possibly due to the difference in the content of biocompounds

288 associated with the solvents and extraction methods used (Faraji & Jamei, 2016).

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289 The antioxidant activity reported for C. ambrosioides by Rodríguez, Tomassini,

290 Manca de Nadra, & Strasser (2010) was 21.5%, while for AbouZid, Elshahaat, Ali,

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& Choudhary (2008) it was less than 20%, similar to the values obtained in this
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292 study. On the other hand, there was a variation in the intensity percentage of the

293 compounds identified (Table 2), with a significant presence of p-coumaric acid,
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294 quercetin, kaempferol 3-O-rutinoside, kaempferol O-rhamnosyl-pentoside and

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295 quercetin dirhamnoside reported in EE compared to EI (Figure 1). This difference

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296 is clearly shown in the mass spectrum, with quercetin as an example ([302-H]-)

297 (Figure 2). Other phenolic compounds identified in C. ambrosioides L. were

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298 isohamnetin, rutin, luteolin, and some glycosides of quercetin and kaempferol, in

299 accordance with the findings of Kokanova-Nedialkova et al. (2010).

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300
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301 3.3. Characterisation of raw ground pork

302 3.3.1 Proximal composition

303 The results obtained on day 0 (Table 3), showed that the contents of fat, protein

304 and ash were similar (P>0.05) between treatments, while the moisture content

305 increased (P<0.05) in MEI and MEE. EI and EE were incorporated into the meat in
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306 liquid form, which explains these results. The values found for the chemical

307 composition in this research are in accordance with the range reported by other

308 authors for raw pork (Barbin, Elmasry, Sun & Allen, 2013; Kim et al., 2008).

309

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310 3.3.2. Lipid oxidation (TBARS) and instrumental colour

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311 TBARS analysis showed that the MEI and MEE treatments had significantly lower

312 values than the CTL (P<0.05) (Table 4), indicating that the epazote protected the

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313 raw ground pork from lipid oxidation. The rancidity starts at values of 0.4-0.6 mg of

314 malonaldehyde/kg sample (Feiner, 2006), and treatments with epazote showed

315
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values below 0.4. EE had the most effect, even when compared to NaASC, an
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316 additive commonly used in muscle foods (Erickson, 2002). The antioxidant effect of

317 EI and EE can be attributed to their flavonoids, as well as their organic acids such
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318 as citric acid (Barros et al., 2013; Embuscado, 2015). EE was the most efficient,
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319 possibly due to the quercetin content, which has been reported to be one of the
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320 flavonoids with the highest antioxidant activity (Brewer, 2011). The results

321 observed on lipid oxidation are comparable to those reported for pork treated with
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322 extracts obtained from different vegetable sources (0.14-1.00 mg of

323 malonaldehyde/kg sample) (Jia, Kong, Liu, Diao, & Xia, 2012; Lee et al., 2010;
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324 Shan, Cai, Brooks & Corke, 2009). On the other hand, TBARS values increased
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325 throughout the storage period as expected, with the highest values (P<0.05) on

326 days 6 and 9.

327 MEI and MEE showed higher values in L*, a*, b*, a*/b*, Chroma and R630/R580

328 compared to CTL (Table 4), indicating greater redness and lower discoloration of

329 meat (AMSA, 2012; Mancini, 2013). Both epazote samples, therefore, provided the
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330 meat with a clearer appearance with a more striking red colour. The value of a* can

331 be associated with an increase in the concentration of OxyMb (Warner, 2014).

332 During storage (Table 4), the a*, a*/b* and R630/R580 values decreased significantly

333 (P<0.05), which revealed a loss in meat redness. The discoloration of the meat is

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334 strongly associated with the conversion of OxyMb (bright red colour) to

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335 metmyoglobin (MetMb), leading to a brown coloration. The accumulation of MetMb

336 on the surface of the meat and subsequent discoloration depends on lipid oxidation

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337 (Faustman & Cassens 1990; Faustman, Sun, Mancini & Suman, 2010; Mancini &

338 Hunt, 2005).

339
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On the other hand, the treatment-time interaction showed significant effects for L*
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340 (P<0.01) and R630/R580 (P<0.05) values. Both interactions showed that the

341 oxidation of the pigment in the meat decreased with the incorporation of EI and EE
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342 considering that these treatments had L* values close to 50 (Murray, 1995), and
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343 above 1.0 for R630/R580 (AMSA, 2012) (Figures 3-4), which is indicative of a light
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344 red colour. Biswas et al. (2012) reported that the incorporation of curry extracts

345 (Murraya koeniggi L.) and mint (Mentha spicata) improved colour stability in raw
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346 ground pork stored in refrigeration for 12 days.

347
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348 3.3.3. pH and microbiological status

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349 MEE showed lower pH (P<0.1) than the CTL (Table 5), values that comply with

350 regulations established by Mexican Legislation (Secretaría de Salud, 1993), which

351 indicates a maximum limit of 6.5-6.8 for fresh ground meat. As expected, pH

352 values increased significantly (P<0.05) during storage. An increase in pH is

353 associated with the accumulation of ammonia caused by the degradation of

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354 proteins and amino acids (Gill & Gill, 2010). The total bacterial count was lower in

355 MEE than in MEI and CTL (P<0.05) (Table 5), which implies a higher antimicrobial

356 activity of the EE, perhaps due to the higher content of quercetin, rutin and p-

357 coumaric acid (Cushine & Lamb, 2005; Gyawali & Ibrahim, 2014). It has been

PT
358 reported that rutin and p-coumaric acid showed antimicrobial activity in chicken

RI
359 soup (Stojkovićet al., 2013), while Lee et al. (2010) found that the total bacterial

360 count decreased in raw ground pork by increasing the concentration of ethanolic

SC
361 extracts of mustard leaf kimchi (Brassica juncea). The total bacterial count

362 increased significantly (P<0.05) throughout the storage period.

363
U
AN
364 3.3.4. Sensory evaluation

365 From day 3, MEI scored the highest for colour and appearance (P<0.05) with
M

366 respect to CTL and MEE (Table 6). These results reveal that EI contributed to the
D

367 decrease in colour loss more so than EE, which helped to prevent the formation of
TE

368 MetMb, which consumers associate with a lack freshness and unacceptability

369 (Troy & Kerry, 2010). This can be attributed to a higher percentage of citric acid in
EP

370 EI (1.45%) compared with EE (0.03%). Colour and appearance are among the

371 most important sensorial properties by which consumers judge the quality of meat
C

372 and influence them when they decide to purchase (Brewer & McKeith, 1999;
AC

373 Faustman & Cassens, 1990). MEI and MEE were evaluated as having an

374 acceptable odour (scores above 4) until day 6, with no significant differences

375 between both treatments (P>0.05). As shown in Table 6, all sensory traits of raw

376 ground pork were significantly reduced (P<0.05) for the three treatments

377 throughout the storage period.

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378 Therefore, of the three attributes evaluated, odour was most affected during the

379 storage period, with CTL scoring the lowest (2.57) probably because of lipid

380 oxidation and ammonia production from protein breakdown. In contrast, the

381 stability of odour in MEI and MEE could be attributed to the protective role of

PT
382 epazote against the deteriorating processes due to its antioxidant and antimicrobial

RI
383 activity according to the results obtained from this research.

384

SC
385 4. Conclusions

U
386 Research has been carried out for the first time on the use of epazote
AN
387 (Chenopodium ambrosioides L.) as a source of natural antioxidants in meat.

388 Flavonoids and citric acid present in the epazote plant may play an important role
M

389 in the lipid and myoglobin stability of the raw ground pork during storage in

390 refrigeration. Therefore, epazote extracts could be incorporated as natural

D

391 antioxidants into raw ground meat to prolong its shelf-life.

TE

392

393 Acknowledgements
EP

394 The authors would like to thank to the financial support received by “Programa
C

395 para el Desarrollo Profesional Docente” (PRODEP; Project UTMIX-EXB-036) and

AC

396 CONACyT (Consejo Nacional de Ciencia y Tecnología; Project 254019). The

397 authors express their gratitude to Dr. Emma Cristina Mapes Sánchez from the

398 Institute of Biology at the National Autonomous University of Mexico for the

399 taxonomic identification of the epazote plant. Finally, the authors acknowledge the

400 language support provided by M.A. Christopher Shackley.

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401

402 References

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Figure 2. Mass spectrometry of quercetin. A: Ethanolic extract of epazote, B:

Epazote infusion. ESI negative mode. 100-1200 m/z.

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U SC
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D
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C EP
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Table 1
pH values, total phenolic content (TPC), total flavonoid content (TFC) and
antioxidant activity (AA) of the infusion and the ethanolic extract of Chenopodium
ambrosioides L.

PT
pH TPCa TFCb AAc

EI 7.34±0.03a 193.50±6.68ª 380.87±18.22ª 13.63±1.20ª

RI
EE 6.90±0.02b 126.3±4.91b 147.26±19.68b 16.65±4.44ª
All values are mean ± SEM (Standard Error of the Mean) of the three replicates.

SC
EI: epazote infusion; EE: ethanolic extract of epazote.
a
Milligrams of gallic acid equivalents (mg EAG/100g dry weight).

U
b
Milligrams of quercetin equivalents (mg EQ/100g dry weight).
AN
c
Inhibition Percentage (% inhibition)
a,b:
Means within the same column with different letters are significantly different
(P<0.05; Duncan´s test).
M
D
TE
C EP
AC
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Table 2
Identification of phenolic compounds in epazote infusion (EI) and ethanolic extract
of epazote (EE).
Compound [M-H]- tR EI EE Formula
(m/z) (min) %Intensity

PT
1 Isorhamnetin 315 4.510 1.85 0.42 C16H12O7
2 Quercetin O- 579 6.808 0.3 ND C33H23O10
rhamnosyl-

RI
pentoside
3 Kaempferol 739 7.743 3.58 9.04 C33H40O19

SC
dirhamnoside-
O-hexoside

U
4 Kaempferol 3- 593 8.103 39.04 32.48 C27H30O15
AN
O-rutinoside
5 p-Coumaric 163 8.583 26.12 100 C9H8O3
acid
M

6 Quercetin 593 8.583 13.55 29.35 C27H30O15

dirhamnoside
D

7 Quercetin-3- 609 8.84 0.18 9.01 C27H30O16

TE

O-rutinoside
(Rutin)
8 Kaempferol 739 8.926 1.58 2.67 C33H40O19
EP

dirhamnoside-
O-hexoside
C

9 Luteolin 285 9.366 0.89 1.12 C15H10O6

AC

10 Kaempferol 285 9.492 4.79 2.99 C15H10O6

11 Quercetin-3- 463 11.404 0.14 0.24 C21H20O12
O-glucoside
12 Quercetin 301 13.213 2.37 68.28 C15H10O7
13 Kaempferol 563 9.492 15.71 31.95 C26H28O14
O-rhamnosyl-
pentoside
tR: time retention.
 ACCEPTED MANUSCRIPT

Table 3
Effect of the incorporation of epazote infusion (MEI) and ethanolic extract of
epazote (MEE) on the proximal composition of raw ground pork.
Treatment

PT
CTL MEI MEE
Moisture (%) 74.05±0.54b 75.21±0.30a 75.37±0.14a

RI
Fat (%) 3.24±0.67a 3.05±0.36a 2.43±0.40a
Protein (%) 20.83±0.73a 20.15±0.44a 20.05±0.41a

SC
Ash (%) 1.13±0.04a 1.07±0.04a 1.05±0.04a
All values are mean ± SEM (Standard Error of the Mean) of the three replicates.

U
CTL: without adding the extract; MEI: with the addition of the infusion; MEE: with
the addition of the ethanolic extract.
AN
a,b:
Means within the same row with different letters are significantly different
(P<0.05; Duncan´s test).
M
D
TE
C EP
AC
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Table 4
Effect of the incorporation of epazote (MEI and MEE) and storage time on thiobarbituric acid reactive substances (TBARS,

PT
mg of malondialdehyde/kg of meat) and the characteristics of instrumental colour of raw ground pork during refrigerated
storage.

RI
Treatment Storage Day SEM Significance
CTL MEI MEE NaASC 0 3 6 9 T S TxS

SC
TBARS 0.54a 0.37ab 0.19b 0.19b 0.21b 0.14b 0.49a 0.45a 0.045 ** ** NS

U
Colour

AN
L* 40.20b 44.12a 45.17a _ 44.10a 44.48a 42.45a 41.49a 0.656 ** NS **
b a a a a b b
a* 1.85 3.34 3.35 _ 3.26 4.01 2.08 1.98 0.228 ** ** NS

M
b* 14.16b 15.35a 15.56a _ 14.81a 15.15a 15.10a 15.01a 0.175 ** NS NS
b a a a a b b
a*/b* 0.12 0.21 0.21 _ 0.22 0.26 0.13 0.12 0.015 ** *** NS

D
Chroma 14.57b 15.82a 15.96a _ 15.20a 15.75a 15.41a 15.43a 0.191 ** NS NS

TE
a a b a a b ab
R630/R580 1.96 2.01 1.64 _ 2.03 1.91 1.69 1.87 0.040 *** ** *
CTL: without adding extract; MEI: with the addition of epazote infusion; MEE: with the addition of alcoholic extract;
EP
NaASC: with the addition of sodium ascorbate (positive control).
SEM: standard error of the mean.
C

Significance: T, treatment; S, storage day: T x S: treatment x storage day interaction.

AC

a,b:
Means in the same row with different letters are significantly different (P<0.05; Duncan’s test). NS: not significant; *:
P<0.05; **: P<0.01; ***: P<0.001.
 ACCEPTED MANUSCRIPT

Table 5

PT
Effect of the incorporation of epazote (MEI and MEE) and storage time on pH and total bacterial count (TBC) (log CFU / g)
on raw ground pork during refrigerated storage.

RI
Treatment Storage Day SEM Significance

SC
CTL MEI MEE 0 3 6 9 T S TxS

U
pH 6.92a 6.83ab 6.40b 6.26b 6.75ab 6.79ab 7.08a 0.104 + * NS

AN
TBC 7.66a 7.50a 6.56b 5.92c 7.27b 7.77ab 8.02a 0.180 *** *** NS
CFU: Colony-Forming Units.

M
CTL: without adding extract; MEI: with the addition of epazote infusion; MEE: with the addition of ethanolic extract of
epazote.

D
SEM: standard error of the mean.

a,b: TE
Significance: T, treatment; S, storage day: T x S: treatment x storage day interaction.
Means in the same row with different letters are significantly different (P<0.05; Duncan’s test). NS: not significant; +:
EP
P<0.1; *: P<0.05; ***: P<0.001.
C
AC
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Table 6
Effect of the incorporation of epazote (MEI and MEE) on sensory analysis (colour, odour and appearance) of raw ground

PT
pork meat during refrigerated storage.

RI
Attribute Days of storage Treatment
CTL MEI MEE
Colour 0 7.07±0.13a,v 6.22±0.15b,v 5.32±0.14c,v

SC
3 5.04±0.16b,x 5.78±0.18a,vx 4.34±0.18c,x
6 4.23±0.22b,y 5.60±0.22a,x 3.53±1.62c,y
3.42±0.22b,z 4.62±0.25a,y 3.22±0.19b,y

U
9

AN
Odour 0 6.14±0.14a,v 5.92±0.16a,v 4.81±0.19b,v
3 4.31±2.10a,x 4.66±0.21a,x 4.53±0.16a,vx
6 3.67±0.21b,y 4.33±0.21a,x 4.11±0.18ab,x

M
9 2.57±0.19b,z 3.10±0.21a,y 3.61±0.18a,y

D
Appearance 0 6.84±0.14a,v 6.20±0.15b,v 5.25±0.15c,v
3 4.66±0.19b,x 5.21±0.19a,x 4.33±0.19b,x

TE
6 3.90±0.22b,y 5.25±0.23a,x 3.70±0.17b,y
9 3.06±0.21b,z 3.98±0.22a,y 3.29±0.20b,y
EP
All values are mean ± SEM (Standard Error of the Mean) of the three replicates.
CTL: without adding extract; MEI: with the addition of epazote infusion; MEE: with the addition of alcoholic extract;
C

a,b,c:
Means in the same row with different letters are significantly different (P<0.05; Duncan’s test).
AC

v,x,y,z:
Means in the same column with different letters are significantly different (P<0.05; Duncan’s test).
 ACCEPTED MANUSCRIPT

100
EI
EE

PT
80

RI
% Intensity

60

SC
40

20
U
AN
0
M

--
L
QRP

CA

KRP
QG

Q
IH

KDRH

KR

QDH

KDH

Compounds identified in Chenopodium ambrosioides L.

D
TE

Fig. 1. Percentage of intensity of phenolic compounds in infusion (EI) and ethanolic

EP

extract (EE) of C. ambrosioides L. IH: Isorhamnetin; QRP: Quercetin O-rhamnosyl-

pentoside; KDRH: Kaempferol dirhamnoside-O-hexoside; KR: Kaempferol 3-O-

C

rutinoside; CA: p-Coumaric acid; QDH: Quercetin dirhamnoside; R: Rutin; KDH:

AC

Kaempferol dirhamnoside-O-hexoside; L: Luteolin; K: Kaempferol; QG: Quercetin-

3-O-glucoside; Q: Quercetin; KRP: Kaempferol O-rhamnosyl-pentoside.

 ACCEPTED MANUSCRIPT TOF MS ES-
ETHANOLIC EXTRACT 1090 (9.363) Cm (99:1663)
100
301.0387 9.81e7

291.9896

PT
%

RI
307.1242
299.0166

U SC
302.0381 314.9803
292.9914

AN
308.1264 321.9096 323.1344
313.1656 317.0301

0 m/z
292 294 296 298 300 302 304 306 308 310 312 314 316 318 320 322

M
INFUSION 1091 (9.372) Cm (92:1663) TOF MS ES-

D
100
291.9896 7.06e7

299.1867
TE B
C EP
AC
%

299.0060

311.0561
297.1708 314.9803
292.9879 301.0352 304.9101
323.1859
303.8989 305.0246 310.9875 321.9096
313.1656
316.7384 321.0283
309.2059

0 m/z
292 294 296 298 300 302 304 306 308 310 312 314 316 318 320 322
 ACCEPTED MANUSCRIPT

60

a a

PT
50 a a a
a a a a
ab
40 b
b

RI
L* value

30 CTL

SC
MEI
20 MEE

U
10
AN
0
Day 1 Day 3 Day 6 Day 9
M

Storage Period
D

Fig. 3. Effect of the incorporation of epazote (MEI and MEE) on L* values in raw
TE

ground pork during refrigerated storage.

CTL: without adding extract; MEI: with the addition of infusion; MEE: with the
addition of ethanolic extract.
EP

a,b:
Means between treatments at the same storage time with different letters are
significantly different (P<0.05; Duncan's test).
C
AC
 ACCEPTED MANUSCRIPT

2.5
a
a ab a

PT
a ab
b a
2 a
b

RI
b b
R630/R580

1.5
CTL

SC
1 MEI
MEE

U
0.5
AN
0
Day 1 Day 3 Day 6 Day 9
M

Storage Period
D
TE

Fig. 4. Effect of the incorporation of epazote (MEI and MEE) on the ratio of
reflectance at R630/R580 on raw ground pork during refrigerated storage.
CTL: without adding extract; MEI: with the addition of infusion; MEE: with the
EP

addition of ethanolic extract.

a,b:
Means between treatments at the same storage time with different letters are
C

significantly different (P<0.05; Duncan's test).

AC
 ACCEPTED MANUSCRIPT

Highlights

• Chenopodium ambrosioides L. as natural antioxidant in raw ground pork

was evaluated.

PT
RI
• Oxidative stability of raw ground pork was improved by the addition of

Chenopodium ambrosioides L.

SC
• Raw ground pork meat contained more redness with Chenopodium

ambrosioides L.
U
AN
•
M

Chenopodium ambrosioides L. modified the sensorial attributes of the raw

ground pork.
D
TE
C EP
AC