Accepted Manuscript

Effects of Portulaca oleracea L. extract on lipid oxidation and

color of pork meat during refrigerated storage

Xiao-Jing Fan, Shan-Zhi Liu, Huan-Huan Li, Jun He, Jun-Tao

Feng, Xing Zhang, He Yan

PII: S0309-1740(18)30349-8
DOI: doi:10.1016/j.meatsci.2018.08.022
Reference: MESC 7671
To appear in: Meat Science
Received date: 27 March 2018
Revised date: 23 August 2018
Accepted date: 25 August 2018

Please cite this article as: Xiao-Jing Fan, Shan-Zhi Liu, Huan-Huan Li, Jun He, Jun-Tao
Feng, Xing Zhang, He Yan , Effects of Portulaca oleracea L. extract on lipid oxidation
and color of pork meat during refrigerated storage. Mesc (2018), doi:10.1016/
j.meatsci.2018.08.022

This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.
 ACCEPTED MANUSCRIPT

1 Effects of Portulaca oleracea L. extract on lipid oxidation and color of pork meat

2 during refrigerated storage

3 Xiao-Jing Fan a, Shan-Zhi Liu a, Huan-Huan Li a, Jun He a, b, Jun-Tao Feng a, b

, Xing

a,b,*
4 Zhang , and He Yan a,b,*

a
5 Research & Development Center of Biorational Pesticide, Northwest A&F

PT
6 University, Yangling 712100, Shaanxi, China

RI
b
7 Engineering and Research Center of Biological Pesticide of Shaanxi Province,

SC
8 Yangling 712100, Shaanxi, China

9 * Correspondence: E-mail: zhxing1952@gmail.com (X.Z.); Tel.: +86-029-8709-2122

NU
10
MA

11 Abstract

12 The study explored the preservation effect of Portulaca oleracea L. extract (POE)
ED

13 on pork meat under refrigerated conditions for 9 days. POE was tested for antioxidant
T

14 activity and antibacterial activity in vitro and the results showed that POE has strong
EP

15 antioxidant activity and has antibacterial activity against Pseudomonas aeruginosa ,

16 Bacillus subtilis and Bacillus cereus to some extent. Effect of POE in different levels
C
AC

17 (0.25%, 0.50% and 1.0%) on quality and shelflife of pork meat storage were

18 evaluated. Results showed that the treatments of POE significantly inhibited microbial

19 growth，delayed lipids oxidation, reduced values of thiobarbituric acid reactive

20 substances (TBARS) and total volatile base- nitrogen (TVB-N), increased superoxide

21 dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and in a

22 dose-dependent manner (P < 0.05). Concomitantly, 1.0%POE and 0.50%POE

1
 ACCEPTED MANUSCRIPT

23 treatments had better appearance compared with control after 9 days storage. All

24 results confirmed that POE could effectively maintain the quality of chilled pork

25 compared to control.

26 Keywords: Portulaca oleracea L.; Lipid oxidation; Meat quality; Natural

27 antioxidant; Food preservative

PT
28 1. Introduction

RI
29 Chilled pork is the current meat consumption trend because of advantages of

SC
30 taste, color and nutritive value in China. The main factors that determine meat quality

31 loss and shelf- life reduction are bacterial contamination and lipid oxidation (Shahidi
NU
32 & Zhong, 2010; Tajkarimi, Ibrahim & Cliver, 2010). How to reduce the pollution
MA

33 level of cooled pork by spoilage bacteria was a problem for meat preservation.

34 Spoilage bacteria, such as Enterobacteriaceae spp., Pseudomonas spp.,

ED

35 Staphylococcus spp., and Bacillus spp., can cause discoloration, off- flavors, off-odors
T

36 and amine formation in meat (Nychas, Skandamis, Tassou & Koutsoumanis, 2008).
EP

37 Lipid oxidation is a key factor of the degradation of meat (Shahidi & Zhong, 2010),

38 especially in pork meat which contains more unsaturated fatty acids than other meat
C
AC

39 such as beef or lamb (Muzolf-Panek, Waskiewicz, Kowalski & Konieczny, 2016). The

40 harmful effects on the meat quality caused by lipid oxidation include rancid odour,

41 discolouration, nutrient losses, decrease in shelf life, and the accumulation of harmful

42 compounds (Falowo, Fayemi & Muchenje, 2014). Therefore, finding effective

43 exogenous preservatives to inhibit spoilage bacteria and to control the process of lipid

44 oxidation of meat are necessary in China.

2
 ACCEPTED MANUSCRIPT

45 Synthetic preservatives, such as buthlated hydroxyl ansisole (BHA), propyl

46 gallate (PG) and tert-butylhydroquinone (TBHQ), are widely and effectively used in

47 foods to extend the shelf- life (Aziz & Karboune, 2016). Although have a good effect,

48 an increasing number of studies have reported the synthetics may cause health risks to

49 man (Ahmad, Gokulakrishnan, Giriprasad & Yatoo, 2015; Brewer, 2011; Shahidi &

PT
50 Zhong, 2010). So, natural additives have become a significant consumer demand.

RI
51 Safe, efficient and broad spectrum natural additives have substituted for synthetic

SC
52 preservatives in more and more foods and attract more and more concern.

53 As a category of natural antimicrobials and antioxidants, p lant extracts have

NU
54 potential to improve the safety and enhance the overall quality of meat products
MA

55 (Jiang & Xiong, 2016; Tajkarimi, Ibrahim & Cliver, 2010; Zhang, Wu & Guo, 2016).

56 More and more plant extracts were found contain antimicrobial or antioxidant
ED

57 activities (blueberry, tea, rosemary, cranberry, pomegranate etc.). Many application

T

58 researches on the preservation effects of the extracts on meat were carried out
EP

59 (Contini et al., 2014; Li et al., 2015; Mhalla et al., 2017). The antimicrobial or

60 antioxidative effects of plant extracts are due largely to some bioactive compounds
C
AC

61 such as unsaturated aldehydes, alcohols, unsaturated aliphatic, acids, phenolics and

62 terpenes (Tiwari et al., 2009). Natural bioactive substances not only possess strong

63 antimicrobial and antioxidant activities but also have the potential to resist various

64 human diseases (Ahmad, Gokulakrishnan, Giriprasad & Yatoo, 2015). Compared to

65 synthetic chemicals, natural bioactive substances seem to be safer and better choices

66 for consumers. However, many plants also have certain toxicity, whether it can be

3
 ACCEPTED MANUSCRIPT

67 developed as a preservative is based on whether the plant is poisonous or not.

68 Portulaca oleracea L., belonging to the genus Portulaca of the family

69 Portulacaceae, is a cosmopolitan annual succulent herb and commonly used as a

70 edible vegetable (Li et al., 2014) . This plant is a potential vegetable crop and rich in

71 vitamins and minerals. It is versatile and can be fried or mixed into other foods and

PT
72 taste similar to spinach (Amirul Alam et al., 2014). Many studies have also

RI
73 demonstrated that this plant also have many biological activities as medicinal plant,

SC
74 including antioxidant (Liu et al., 2015), analgesic, anti- inflammatory (Zhou et al.,

75 2015), hepatoprotective (Al-Sheddi et al., 2015), antibacterial (Chan et al., 2015),

NU
76 antivirus (Dong, Hayashi, Lee & Hayashi, 2010), neuroprotective (E Abdel Moneim,
MA

77 2013), antitumor (Shen et al., 2013) and antidiabetic (Gong et al., 2009). P. oleracea

78 has been certified safe and associated with health benefits. It could be used for
ED

79 functional food.
T

80 There was no literature found on the impact of POE on preservation of meat or

EP

81 meat product. The aim of this study was to estimate the preservation activity and

82 investigate the preservation mechanism of POE on chilled pork by measuring some

C
AC

83 sensory, physiological and biochemical indexes.

84 2. Materials and methods

85 2.1 Materials and chemicals

86 Pork tenderloin material was purchased form “Benxiang” store in Yangling,

87 Shaanxi, China and brought to laboratory under refrigeration (4±1ºC).

88 Portulaca oleracea L. samples were collected from the Northwest A&F

4
 ACCEPTED MANUSCRIPT

89 University surrounding farmland in Yangling, Shaanxi, China.

90 Microbial strains are obtained from China Center of Industrial Culture Collection

91 (CICC).

92 The chemicals used in this research were all analytical reagent.

93 2,2-diphenyl-1-picrylhydrazyl (DPPH), ascorbic acid (Vc), TBA and butylated

PT
94 hydroxyl anisole (BHA) were purchased from Sigma-Aldrich (USA). Trichloroacetic

RI
95 acid (TCA) and organic solvents were purchased from Sinopharm Grop Chemical

SC
96 Reagent Co., Ltd (Shanghai China).

97 2.2 Preparation of P. oleracea extract

NU
98 The above- ground of P. oleracea (2.0 kg) were crushed and then extracted with
MA

99 80% ethanol (3 × 20 L, 24 h each time) at room temperature to result in a P. oleracea

100 crude ethanol extract (POE, 197.4 g) after concentrated in vacuum at 45°C using
ED

101 rotary evaporator (R-SENCO, Shanghai, China).

T

102 2.3 Preparation meat samples and experimental design

EP

103 Pork tenderloin was sliced into square with approximately 5 cm side length and 5

104 mm thickness. Then, these meat slices was randomly divided into 6 treatments with
C
AC

105 three groups (replicates) in each treatment, 20 slices in each replicate.

106 Each treatment was sprayed with 10ml of solution according to the following

107 formulation: (1) Control (deionized water); (2) TP (0.05% tea polyphenols); (3) BHA

108 (0.02% butylated hydroxyl anisole) (Ahn,Grun & Mustapha, 2004); (4) 0.25% POE;

109 (5) 0.50% POE; (6) 1.0% POE. The treatments were transferred to the enamel trays

110 and sealed with plastic wrap with bleeder vents in the 4o C cold storage. When

5
 ACCEPTED MANUSCRIPT

111 sampling (day 1, 3, 5, 7, 9 ), four slices per group were taken out and analyzed for

112 microbiological analyses, pH, lipid oxidation, total volatile base- nitrogen, color and

113 two enzyme activities.

114 2.4 Evaluation of the antibacterial activity (Inhibitory zone assay) of P. oleracea

115 extract

PT
116 Three Gram positive pathogens: Bacillus subtilis (ATCC 21216), Bacillus cereus

RI
117 (ATCC 14579), and Staphylococcus aureus (ATCC 6538); Three Gram negative

SC
118 pathogen bacteria: Salmonella enteritidis (ATCC 13311), Escherichia coli (ATCC

119 25922), and Pseudomonas aeruginosa (ATCC 13525) were used as the test
NU
120 organisms.
MA

121 The POE were firstly dissolved in alcohol, and then diluted with sterile water to

122 the concentration of 50 mg/ml. The extract sample was sterilized by filtrating through
ED

123 0.22 μm Millipore filters.

T

124 In vitro antibacterial activity of POE was detected by Oxford cup method as
EP

125 follows (Beverlya, Janes, Prinyawlwatkula & No, 2008). The organisms were grown

126 in Luria-Bertani at 30o C for 24 h with shaking (200 rpm), then diluted with LB liquid
C
AC

127 culture to 106 CFU/ml. Mueller-Hinton agar (16 ml) was inoculated with 200 μL

128 bacterial suspension and solidified in culture dish. The Oxford cups (inner diameter 6

129 mm) were put on the inoculated LB plates and impregnated with 200 μL of POE

130 samples. Streptomycin sulfate and penicillin (both 100 μg/ml) were used as positive

131 controls and the solvent was used as a negative control. All plates were cultured at

132 30o C for 24 h. The antibacterial activity in vitro was estimated by measuring the

6
 ACCEPTED MANUSCRIPT

133 diameter of inhibition zones against the tested microorganisms. Each assay was

134 replicated three times.

135 2.5 Antioxidant activity of P. oleracea extract in vitro

136 2.5.1 Scavenging ability of P. oleracea extract towards DPPH free radical

137 The capability to scavenge DPPH was determined with the method described by

PT
138 Yang, et al.(2013). Equal volume of POE methanolic solution was added to 2 ml of

RI
139 methanolic DPPH solution (25μg/ml). The mixture was shaken and left to stand at

SC
140 room temperature and protected from light for 30 min. 2ml of methanolic solution and

141 2 ml of methanolic DPPH solution (25μg/ml) served as the control. The absorbance of
NU
142 the solution was measured at 517 nm using a spectrophotometer (HITACHI U-3310).
MA

143 The result was expressed as EC50 which represents the concentration of sample

144 required to scavenge 50% of DPPH. Each analysis was performed in triplicate.
ED

145 2.5.2 Total antioxidant capacity (TAC)

T

146 TAC assay was implemented using ammonium molybdate method (Prieto,
EP

147 Pineda & Aguilar, 1999) , with slight modifications. 0.3 ml extract solution (200, 300,

148 500, 700, 800, 900 μg/ml) mixed into 3 ml of reaction solution (4 mM Ammonium
C
AC

149 molybdate, 28 mM sodium phosphate, and 600 mM H2 SO 4 ) was heated at 95o C for 90

150 minutes. After decreased to room temperature, the absorbancy of blank (ethyl replaced

151 extract solution) and samples were determined at 695 nm. Each analysis was

152 conducted in triplicate. Ascorbic acid solution (20-200 μg/ml) was used to construct

153 the standard curve. The result expressed as mg equivalent to ascorbic acid /g extract.

154 2.6 Meat samples measurements of different treatments during shelflife

7
 ACCEPTED MANUSCRIPT

155 2.6.1 Microbial analysis

156 According to the methods of Chinese standard GB/4789-2016 (Microbiology test

157 standard for food hygiene), samples (25 g meat) were diluted with 225 ml sterilized

158 0.85% NaCl solution and homogenized using an Ultra Turrax T18 (IKA. Co.,

159 Germany) for 2 min, which were prepared as the initial concentration (100 mg/mL).

PT
160 Serial dilutions (1:10) were made with 0.1% saline and spread on the surface of

RI
161 different, selective agars. Dilutions of 1 ml were spread. Plate count agar was used for

SC
162 total viable counts (TVC). Violet red bile glucose agar (VRBGA) was used for

163 coliform counts. Manitol salt agar (MSA) was used for total staphylococcus counts.
NU
164 TVC, coliform counts and total staphylococcus counts were determined by counting
MA

165 the colonies formed on the plates after incubating at 37o C for 48h. Pseudomonas spp.

166 were counted on Pseudomonads selective agar and incubated at 25 °C for 48 h. Lactic
ED

167 acid bacterial (LAB) counts were counted on MRS agar and incubated at 30o C for 72
T

168 h. All treatments were repeated twice with three replications per experiment. The
EP

169 average of the results was reported as a logarithm of colony forming units (log CFU/g)

170 per gram of meat.

C
AC

171 2.6.2 pH

172 The pH values were measured by a pH- meter (Hanna Instrument HI-9024,

173 Portugal) previously calibrated in buffers at pH 4.01 and 7.01 at ambient temperature.

174 5 g of minced meat samples were homogenized with 45 ml deionized water, then a

175 glass electrode was immersed directly into the sample. The pH was measured in

176 triplicate.

8
 ACCEPTED MANUSCRIPT

177 2.6.3 Lipid oxidation

178 Lipid oxidation was determined by measuring the thiobarbituric acid reactive

179 substances (TBARS) as previously reported (Erkan & Oezden, 2008). 10 g of minced

180 meat were homogenized in 50 ml of 7.5% cold trichloroacetic acid (TCA) containing

181 0.1% EDTA, shaking for 30 min and filtered. 5 ml filtrate was then transferred into a

PT
182 test tube and added equal volume of freshly prepared chilled 2-thiobarbituric acid

RI
183 (TBA) (0.02 mol/L). The test tube was heated at 100 o C for 40 min. The mixture was

SC
184 centrifuged at 1600rpm for 5 min after cooling for an hour. The supernatant was

185 added 5 ml chloroform and shook well. After stratifying, the absorbancy of the
NU
186 supernatant was determined at 532 nm and 600 nm. The blank control included 5 ml
MA

187 TBA and 5 ml TCA. The TBARS content was expressed as mg malonaldehyde (MDA)

188 per kg meat and calculated by the following formula: TBARS (mg/kg) = (A532 -
ED

189 A600) / 155 × (1/10) × 72.6 × 1000. The analysis was performed in triplicate.
T

190 2.6.4 Total volatile base-nitrogen (TVB-N)

EP

191 The determination of TVB-N was performed according to Liu, et al. (2013) with

192 slight modifications. First, 10 g of meat were minced and homogenized with 100 ml
C
AC

193 deionized water, filtered after shaking the bottles for 30 min. Equal volume of MgO

194 (10 g/L) as catalyst was added to 10 ml of the filtrate. Then, the mixture was distilled

195 using a KDN-103F kjeldahl apparatus (Shanghai Qianjian Instrument Co., Ltd. China).

196 The distillate was absorbed into 25 g/L H3 BO4 solution containing mixed indicator of

197 methyl red and bromocresol green and then titrated with 0.01 M HCl. The results

198 were expressed as mg of TVB-N per 100 g of meat.

9
 ACCEPTED MANUSCRIPT

199 2.6.5 Color measurement

200 The color of the samples was measured according to the method of Cheng et al.,

201 (2017) by Cr-310 colourimeter (Minolta Co., Ltd. Shanghai. China) with an aperture

202 of 8 mm diameter measuring area, 0° standard observer and D65 illuminant, after

203 calibration using a white tile. After 30 min of blooming time at 4o C, each slice was

PT
204 performed 6 measurements in different areas. The results were expressed in CIE LAB

RI
205 (L* a* b* parameters) color scale, where L*, a*, and b* indicated lightness, redness,

SC
206 yellowness, respectively.

207 2.6.6 The changes of the Superoxide dismutase (SOD) and Glutathione peroxidase
NU
208 (GSH-Px) activity
MA

209 The indexes of SOD and GSH-Px were measured by Reagent Kits method. The

210 Reagent Kits purchased from Suzhou Comin Biotechnology co., Ltd.
ED

211 A pre-experiment was carried out to determine the optimum concentration of

T

212 supernatant. For the determination of SOD activity, the absorbance at 560 nm was
EP

213 recorded. According to the kit instructions, one unit (U) was taken as the activity that

214 inhibits the xanthine oxidase coupled reaction by 50%. The results were expressed as
C
AC

215 U/g fresh meat.

216 The activity of GSH-Px was measured by the decrease in absorbance at 340 nm

217 during 3 min. The GSH-Px activity unit was expressed as nmol of oxidized

218 NADPH/min/g of fresh meat. The results were expressed as nmol/min/g fresh meat.

219 2.6.7 Sensory evaluation

220 To evaluate the effect of addition POE on sensory characteristics of pork meat,

10
 ACCEPTED MANUSCRIPT

221 the meat samples were sliced to pieces 0.8-1.0 cm wide and cooked at 180 o C for 2

222 min, until the internal temperature reached 70 o C (Fernandes, Trindade, Lorenzo, &

223 De, 2017). After cooking, the meat slices were allowed to cool to room temperature.

224 The samples were labeled individually with random numbers and served to panelists

225 in separate booths. Warm water was provided for cleansing the mouth between

PT
226 samples.

RI
227 Panel members were recruited from students in the Collage of Food Science and

SC
228 Technology at Northwest Agriculture and Forestry University of China. 10 Panelists

229 were 20-35 years old (5 female, 5 male). Panel members were given verbal
NU
230 instructions before they evaluated the products. All requirements for the achievement
MA

231 of sensory tests were complied with according to the Chinese Standard

232 GB/T22210-2008 (Criterion for sensory evaluation of meat and meat pro-ducts),
ED

233 following the general guidelines intended for sensory analysis of meat products.
T

234 The sensory attributes of meat samples including oder, taste, texture and overall
EP

235 acceptability were evaluated on day 0. A 9-point hedonic scale was used to determine

236 the four sensory attributes (Stone & Sidel, 2004)：1, dislike extremely; 2, dislike very
C
AC

237 much; 3, dislike moderately; 4, dislike slightly; 5, neither like nor dislike; 6, like

238 slightly; 7, like moderately; 8, like very much; 9,like extremely.

239 2.7 Statistical analysis

240 SPSS version 16.0 (IBM, USA) were used for data analysis. Differences between

241 treatments were analyzed using a one-way ANOVA, with treatments as fixed factor

242 and replicates as random factor to examine the effect of POE on meat characteristics.

11
 ACCEPTED MANUSCRIPT

243 The data of each treatment was assessed using another one-way analysis of variance

244 (ANOVA) to examine the effect of time on meat characteristics. The results were

245 expressed by mean ± standard error of three repetitions. Duncan’s Multiple Range

246 Test to identify significant differences with a confidence level set at P < 0.05.

247 3. Results and discussion

PT
248 3.1 In vitro antibacterial activity

RI
249 The antibacterial activities in vitro of P. oleracea extract against the tested

SC
250 microorganisms were estimated by inhibition zones qualitatively. The results were

251 shown in Table.1. Diameters of bacteriostatic rings of POE range from 0 to 9.05 mm.
NU
252 The similar results showed antibacterial activity against the enteropathogenic bacteria
MA

253 with the inhibition zones of 13-15mm (Zhou et al., 2015). The solvent (negative

254 control) had no inhibitory effect on the tested microorganisms under the current
ED

255 experimental conditions. The results were similar to those reported by Al-Moghazy,
T

256 Ammar, Sief & Mohamed (2017) who found Portulaca extract had antimicrobial
EP

257 activity against Staphylococcus aureus but had no antimicrobial activity against

258 Pseudomonas aeruginosa, Salmonella typhimurium, Listeria monocytogenes and

C
AC

259 E.coli O157:H7. Additionally, the minimum inhibitory concentration (MIC) and the

260 minimum bactericidal concentration (MBC) of POE on 6 kinds of bacteria were also

261 measured. However, when the concentration was 5mg/ml, the growths of all tested

262 bacteria were still visible. In general, the results of in vitro antibacterial activity

263 showed that POE had weak antibacterial activity.

264 3.2 DPPH free radical scavenging activity and total antioxidant capacity of P.

12
 ACCEPTED MANUSCRIPT

265 oleracea extract

266 The results of the DPPH radical scavenging activity and the total antioxidant

267 capacity test of the extract were shown in Table.2. The half- maximal effective

268 concentration of POE for scavenging DPPH radical was 42.87 μg/ml. The DPPH

269 radical scavenging activity of POE was approximately 14 times and 8 times lower

PT
270 than TP and BHA, respectively. However, TP is a class of chemical compounds and

RI
271 BHA is a pure compound. POE is just a plant crude extraction contains many

SC
272 impurities. So, with this in mind, the activity of POE for scavenging DPPH radical

273 was significant.

NU
274 By the TAC assay, the total antioxidant capacity was expressed by the average of
MA

275 the six different concentrations. The antioxidant activity of POE was estimated at

276 149.07 mg ascorbic acid /g extract, TP and BHA was approximately 2 times and 1.5
ED

277 times higher, respectively.

T

278 The high antioxidant activity of POE would be due to there are some antioxidant
EP

279 components in it, such as ω-3 unsaturated fatty acid, coumarins and phenolic acids. P.

280 oleracea has been known as the richest source of β-carotene, α-tocopherol,
C
AC

281 glutathione, and essential unsaturated fatty acids (Simopoulos & Salem, 1986; Wenzel,

282 Fontana & Correa, 1990). Meanwhile, phenolic alkaloids also served as a class of

283 antioxidant agent (Xiang et al., 2005; Yang, Liu, Xiang & Zheng, 2009).

284 3.3 The protective effect of POE on the storage qualities of chilled pork

285 3.3.1 Microbial analysis

286 The total viable count TVC in the chilled pork was monitored during refrigerated

13
 ACCEPTED MANUSCRIPT

287 storage for 9 d (Fig.1.A). The initial bacterial count of the control sample was 3.68

288 log CFU/g, the result was close to 3.82 log CFU/g and 4 log CFU/g which in the

289 researches of (Pogorzelska, Godziszewska, Brodowska, & Wierzbicka, 2018) and

290 (Lorenzo, Sineiro, Amado, & Franco, 2014), respectively. From Fig.1.A, the addition

291 of POE led to a significantly (P < 0.05) decrease in growth rate of TVC. The TVC of

PT
292 control increased with storage time and reached to 6.26 log CFU/g meat at day 5.

RI
293 However, by the end of 7 days, TVC in the samples treated with 0.5% and 1.0%

SC
294 concentration of POE were 5.93 log CFU/g and 5.51 log CFU/g, respectively.

295 According to Chinese Standard GB/T9959.2-2008 (Fresh and frozen pork lean, cuts),
NU
296 TVC of 106 CFU/g was considered as spoilage for fresh pork. Therefore, the shelf- life
MA

297 of control sample would be 4 days, while meat samples treated with 0.5% and 1.0%

298 concentration of POE could prolong this life to 7 days.

ED

299 The coliform growth counts were also inhibited by the addition of POE, as
T

300 shown in Fig.1.B. At the beginning of storage, lower coliform counts were observed
EP

301 in 0.5% and 1.0% concentration of POE treated samples (below 3 log CFU/g) than

302 other treatments. And, coliform counts increased significantly (P < 0.05), reaching
C
AC

303 higher values in TP samples than in 0.5% and 1.0% POE samples during the whole

304 storage period. Thus addition of POE reduced coliform counts growth in meat

305 effectively.

306 The antimicrobial effect of POE was also investigated on Pseudomonas. As

307 shown in Fig.1.C, Pseudomonas counts of control meat sample showed a significant

308 increase from 2.30 log CFU/g to 6.22 log CFU/g during storage (P < 0.05). After 9

14
 ACCEPTED MANUSCRIPT

309 days storage, Pseudomonas counts were significantly reduced, reached about 4.15 log

310 CFU/g and 3.61 log CFU/g for 0.5% and 1.0% POE treated samples, respectively (P <

311 0.05).

312 Staphylococcus counts of each treatment were increased with the extension of

313 storage time. However, the addition of POE significantly affected the Staphylococcus

PT
314 growth counts (P < 0.05) throughout the storage period, and showed a similar

RI
315 tendency with Pseudomonas counts (Fig.1.D).

SC
316 From Fig.1.E, Lactic acid bacteria (LAB) counts of control sample showed a

317 significant increase from 3.25 log CFU/g to 6.24 log CFU/g than other treatments (P <
NU
318 0.05). At the end of storage, LAB counts showed lower values in TP and 1.0% POE
MA

319 treated groups (below 5.28 log CFU/g) than in control (above 6.24 log CFU/g),

320 followed by 0.5% POE treated samples (5.62 log CFU/g). The results indicated an
ED

321 inhibitory effect of POE on the growth of LAB.

T

322 In general, the results indicated that all counts for all treatments became higher
EP

323 with storage time. However, POE inhibited the growth of all targets in a dose related

324 manner, with a stronger effect at 1.0% than at 0.5% and 0.25%. This implied POE
C
AC

325 played a role in inhibiting the growth of bacteria. The antibacterial activities of POE

326 have been reported in some studies (Chan et al., 2015; Elkhayat, Ibrahim, & Aziz,

327 2008). Four amphipathic molecules compounds with antibacterial activity,

328 portulacerebroside A-D, were separated and identified from P. oleracea.(Lei, Li, Liu,

329 Zhang, & Liu, 2015). However, the microbiological changes of meat samples during

330 storage at 4o C were affected by many aspects, and the mechanism has not yet been

15
 ACCEPTED MANUSCRIPT

331 identified.

332 3.3.2 pH

333 The values of the pH were shown in Fig.2.A. The initial values were in a range

334 from 5.38 to 5.46, and similar values for pork meat are reported in the literature (Suo

335 et al., 2017). Statistical analysis indicated that pH values of different treatments were

PT
336 not significant at the beginning of storage (P > 0.05). Until the fifth day, pH value of

RI
337 control groups (5.89) was significantly higher than other treatments (P < 0.05). After

SC
338 9 days of cold storage, the lowest pH value was observed in 1.0% POE (5.86)

339 followed by 0.5% POE (5.91) and TP (5.97). Comparatively, the pH values of 0.25%
NU
340 POE and BHA increased to 6.02 and 6.16, respectively. These results showed that
MA

341 POE delayed the increase of pH values in pork meat during cold storage. The pH

342 values showed increasing trends and were similar to other studies (Hu, Wang, Xiao &
ED

343 Bi, 2015; Suo et al., 2017). And, correlation analysis revealed a strong relationship (P
T

344 < 0.001) between pH levels and the microbial counts, which probably due to the
EP

345 accumulation of alkaline substances produced by the microbiological degradation of

346 protein.
C
AC

347 3.3.3 Lipid oxidation

348 Lipid oxidation is an important factor concerned with the deterioration in food

349 quality. The measurement of TBARS is a commonly used method for evaluating the

350 oxidation changes in food during storage. The effects of POE on the TBARS values of

351 cooled pork were shown in Fig.2.B.

352 Initially, the TBARS values of all samples were about 0.2 mg MDA/Kg of the

16
 ACCEPTED MANUSCRIPT

353 meat. Then, the values increased rapidly beyond day 5, especially in control. The

354 TBARS in control sample were significantly increased to be 0.66 mg MDA/Kg,

355 whereas the samples treated with 0.50% POE was 0.42 mg MDA/Kg. During the

356 whole storage, the TBARS values of 0.5% POE and 1.0% POE were always

357 significantly lower than other treatments (P < 0.05). At the 9th day, the TBARS value

PT
358 of BHA (1.10 mg MDA/Kg) and TP (1.00 mg MDA/Kg) was 1.47-, 1.33-fold higher

RI
359 than 1.0% POE (0.75 mg MDA/Kg) treatment respectively. The value of 0.25% POE

SC
360 (0.98 mg MDA/Kg) was also significant lower than BHA and control (1.24 mg

361 MDA/Kg) (P < 0.05). The results indicated that POE could decrease the TBARS
NU
362 values in meat effectively compared to other treatments throughout storage.
MA

363 The reduction in lipid oxidation of meat would be due to the antioxidant activity

364 of POE. Several studies have showed that the polysaccharides isolated from P.
ED

365 oleracea possess a strong antioxidant ability (Chen et al., 2012; Dong, Hayashi, Lee
T

366 & Hayashi, 2010; Uddin, Juraimi, Ali & Ismail, 2012) and could scavenge DPPH,
EP

367 nitric oxide, superoxide anion, and hydroxyl radicals,. What's more, many chemical

368 constituents with high antioxidant also have been found in P. oleracea including
C
AC

369 alkaloids (Yang, Liu, Xiang & Zheng, 2009), polysaccharides (Dong, Hayashi, Lee &

370 Hayashi, 2010), phenolic (Uddin, Juraimi, Ali & Ismail, 2012), flavonoids (Zhou et

371 al., 2015) and portulacerebroside (Lei, Li, Liu, Zhang & Liu, 2015).

372 3.3.4 Total volatile base-nitrogen (TVB-N)

373 TVB-N is a commonly used index of meat quality and shelflife. TVB-N values

374 for six treatments were shown in Fig.2.C. At the beginning of the storage, The TVB-N

17
 ACCEPTED MANUSCRIPT

375 values of meat ranged from 6.61 to 8.12 mg/100g and there were no significant

376 changes between different treated samples (P < 0.05). The TVB-N values showed

377 increasing trends during storage time and rapidly for control sample. The reason

378 would be for the increase of spoilage bacteria and the activity of endogenous enzymes.

379 The TVB-N value of control sample was significantly reached to 16.6 mg/100g at day

PT
380 5. However, at the end of storage, the TVB-N values of samples from 1.0% POE and

RI
381 0.5% POE had lower levels of 11.90 mg/100g and 12.73 mg/100g, respectively,

SC
382 followed by 0.25% POE samples (14.03 mg/100g). According to Chinese Standard

383 GB/T9959.2-2008 (Fresh and frozen pork lean, cuts), chilled pork is considered as
NU
384 spoilage if its TVB-N exceed the upper limit 15 mg/100g. So, the shelf life of meat
MA

385 treated with POE extended for 4 days compared to control. These results suggest that

386 POE could delay the increase of pork meat TVB-N values during cold storage.
ED

387 3.3.5 Measurement of color

T

388 Color values (L*, a*, b*) of the different treat samples were presented in Table 3.
EP

389 It indicated that the samples treated with different levels of POE could not cause color

390 alteration first in the chilled pork. The L*（lightness）values showed continued decline
C
AC

391 during whole storage time for all samples, especially significantly in the control

392 groups. At the later stage of storage, L* values for two higher levels of POE were

393 significantly higher compared to TP and BHA treatments. The variations tendency of

394 L* values were similar to a previous study in chilled pork treated with blueberry

395 extract (Muzolf-Panek, Waskiewicz, Kowalski & Konieczny, 2016).

396 The a* (redness) values showed a decrease in redness during storage from 9.34

18
 ACCEPTED MANUSCRIPT

397 to 4.28 in control, with significantly lower than other treatments on day 9. The a*

398 values of 1.0% POE samples were significantly higher compared to TP, BHA and

399 control at later storage period. It indicated that POE was effective in maintaining the

400 redness of meat. A rapid reduction of meat redness is mainly due to the lipidic

401 oxidation, while the antioxidants could retard such a decrease (Yu, Scanlin, Wilson &

PT
402 Schmidt, 2002).

RI
403 The b* (yellowness) values of meat showed a progressive increase trend with

SC
404 storage time, which was opposite to that of the a* values. The b* values of all samples

405 had no big changes until day 5. Only 0.5% POE and 1.0% POE treatments were
NU
406 significantly lower than other treatments and these two treatments had no significant
MA

407 differences during whole storage period. The results showed that the original color of

408 POE had little effect on b* value of meat. In addition, there was no significant
ED

409 difference between control and samples containing Portulaca extract had also been
T

410 described in beef burgers (Al-Moghazy, Ammar, Sief & Mohamed, 2017). However,
EP

411 there were studies showed that some plant such as citrus extract (Sammel & Claus,

412 2006) and red grape extract (Selani et al., 2011) could be responsible for an increase
C
AC

413 in the b* value of meat. This was an advantage of POE.

414 The acceptability of meat was firstly dependent on the change of sensory

415 attributes, in which color is the first criterion. So, the photos of samples on day 9 were

416 shown in Fig.3. After 8 days storage, there was a large difference in the appearance of

417 different chilled meat treatments. The redness of control meat samples had been gone

418 off obviously and turned gray, whereas TP, 0.5%POE and 1.0%POE samples

19
 ACCEPTED MANUSCRIPT

419 remained in the acceptable range. Compared with POE samples, the color of TP were

420 a bit darker like red purple. The color of 0.5%POE samples turned a little yellowish,

421 while 1.0%POE samples still bright red and maintained in a better quality.

422 1.0%POE was more effective in keeping meat color than others.

423 The POE groups significantly improved the color of chilled pork compared to

PT
424 BHA, TP and control. This inspiring result suggested that POE was a potential natural

RI
425 preservation inhibited the discoloration of chilled pork. This is greatly beneficial

SC
426 because color is an important quality attributes determining consumers’ purchase

427 decisions.
NU
428 3.3.6 Antioxidant enzyme activity
MA

429 SOD and GSH-Px enzymes in animal muscles have scavenging action on free

430 radical. The higher enzymes activities were, the better meat quality was. The activity
ED

431 levels of SOD and GSH-Px were observed in vitro.

T

432 There were significant differences in the SOD activities of the different
EP

433 treatments from Fig.4.A (P < 0.05). The activities of SOD kept a decrease continually

434 and control groups dropped more quickly than other treatments. At the end of storage,
C
AC

435 the activity of 1.0% POE was 74.48 U/g, and which significantly higher than sample

436 of TP (30.09), BHA (20.83) and control (4.14). The SOD activities of POE samples

437 decreased slightly and maintained in higher levels during the whole storage period.

438 GSH-Px activities of different treatments were showed in Fig.4.B. The results

439 showed that control sample had lower GSH-Px activity than other treatments during

440 the whole storage. GSH-Px showed higher activities in samples treated with different

20
 ACCEPTED MANUSCRIPT

441 concentrations of POE and presented a dose-dependent effect. At day 7, the GSH-Px

442 activities of TP and BHA were dropped to 0.39 Umol/min/g and 0.36 Umol/min/g,

443 respectively. However, 1.0% POE had equivalent activity at day 9 (0.39 Umol/min/g).

444 These results showed that the addition of POE was effective in delaying

445 antioxidant enzyme activities SOD and GSH-Px decrease in meat. Combining with

PT
446 the front result of In vitro antibacterial activity of POE, it revealed that the main

RI
447 preservation mechanism of P. oleracea may be the antioxidant properties.

SC
448 3.3.7 Sensory evaluation

449 The sensory properties of meat slices, including oder, taste, texture and overall
NU
450 acceptability, with different concentrations of POE after cooking, were shown in
MA

451 Table 4. There was no significant difference (P < 0.05) between control and samples

452 containing POE in all sensory properties of meat, 1.0% POE could even enhance the
ED

453 oder, taste, texture and overall acceptability of meat slices. The similar discovery had
T

454 been reported previously (Al-Moghazy, Ammar, Sief & Mohamed, 2017).
EP

455 4. Conclusions

456 In this study, it was found that the preservation effect of POE was superior to
C
AC

457 BHA and TP which are recognized as good fresh-keeping agents. According to the

458 results, POE inhibited the growth of microorganisms in chilled pork during storage

459 for 9 d at 4 o C. The pH levels, TBARS and TVB-N values decreased with increasing

460 amounts of POE. In addition, POE also displayed a protective effect against the

461 decreases of SOD and GSH-Px activity and the degradation of pork color. Therefore,

462 as a source of natural antioxidants, P. oleracea extract has a good potential to improve

21
 ACCEPTED MANUSCRIPT

463 shelflife and sensory quality of chilled pork. It could be used in many

464 biotechnological fields as a natural preservative ingredient of food industry.

465 Conflict interest

466 The authors declare no conflict of interest.

467 Acknowledgments

PT
468 This work was supported by the National Key Technology R&D Program of

RI
469 China (2015BAD16B00).

SC
470 References

471 Ahmad, S. R., Gokulakrishnan, P., Giriprasad, R., & Yatoo, M. A. (2015). Fruit-based
NU
472 Natural Antioxidants in Meat and Meat Products: A Review. Critical Reviews
473 in Food Science and Nutrition, 55, 1503-1513.
MA

474 Al-Moghazy, M., Ammar, M., Sief, M. M., & Mohamed, S. R. (2017). Evaluation the

475 Antimicrobial Activity of Artemisia and Portulaca Plant Extracts in Beef

ED

476 Burger. Food Science and Nutrition Studies, 1, 31.

477 Al-Sheddi, E. S., Farshori, N. N., Al-Oqail, M. M., Musarrat, J., Al-Khedhairy, A. A.,
T

478 & Siddiqui, M. A. (2015). Portulaca oleracea seed oil exerts cytotoxic effects
EP

479 on human liver cancer (HepG2) and human lung cancer (A-549) cell lines.

480 Asian Pac J Cancer Prev, 16, 3383-3387.

C

481 Amirul Alam, M., Juraimi, A., Rafii, M., Hamid, A., Kamal Uddin, M., Alam, M., &
AC

482 Latif, M. (2014). Genetic improvement of purslane (Portulaca oleracea L.)

483 and its future prospects. Mol. Biol. Rep., 41, 7395-7411.
484 Aziz, M., & Karboune, S. (2016). Natural antimicrobial/antioxidant agents in meat

485 and poultry products as well as fruits and vegetables: A review. Critical
486 reviews in food science and nutrition, 1-26.
487 Beverlya, R. L., Janes, M. E., Prinyawlwatkula, W., & No, H. K. (2008). Edible
488 chitosan films on ready-to-eat roast beef for the control of Listeria
489 monocytogenes. Food Microbiology, 25, 534-537.
22
 ACCEPTED MANUSCRIPT

490 Brewer, M. S. (2011). Natural Antioxidants: Sources, Compounds, Mechanisms of

491 Action, and Potential Applications. Comprehensive Reviews in Food Science
492 and Food Safety, 10, 221-247.

493 Chan, B. C. L., Han, X. Q., Lui, S. L., Wong, C. W., Wang, T. B. Y., Cheung, D. W. S.,
494 Cheng, S. W., Ip, M., Han, S. Q. B., Yang, X.-S., Jolivalt, C., Lau, C. B. S.,
495 Leung, P. C., & Fung, K. P. (2015). Combating against methicillin-resistant

PT
496 Staphylococcus aureus - two fatty acids from Purslane (Portulaca oleracea L.)
497 exhibit synergistic effects with erythromycin. Journal of Pharmacy and

RI
498 Pharmacology, 67, 107-116.
499 Chen, B., Zhou, H., Zhao, W., Zhou, W., Yuan, Q., & Yang, G. (2012). Effects of

SC
500 aqueous extract of Portulaca oleracea L. on oxidative stress and liver, spleen
501 leptin, PARα and FAS mRNA expression in high- fat diet induced mice.
NU
502 Molecular biology reports, 39, 7981-7988.

503 Cheng, J. R., Liu, X. M., Zhang, Y. S., Zhang, Y. H., Chen, Z. Y., Tang, D. B., &
MA

504 Wang, J. Y. (2017). Protective effects of Momordica grosvenori extract against

505 lipid and protein oxidation-induced damage in dried minced pork slices. Meat
ED

506 Science, 133, 26-35.

507 Contini, C., Álvarez, R., O'Sullivan, M., Dowling, D. P., Gargan, S. Ó., & Monahan, F.
T

508 J. (2014). Effect of an active packaging with citrus extract on lipid oxidation
EP

509 and sensory quality of cooked turkey meat. Meat science, 96, 1171-1176.
510 Dong, C.-X., Hayashi, K., Lee, J.-B., & Hayashi, T. (2010). Characterization of
C

511 Structures and Antiviral Effects of Polysaccharides from Portulaca oleracea L.

AC

512 Chemical & Pharmaceutical Bulletin, 58, 507-510.

513 E Abdel Moneim, A. (2013). The neuroprotective effects of purslane (Portulaca

514 oleracea) on rotenone- induced biochemical changes and apoptosis in brain of
515 rat. CNS & Neurological Disorders-Drug Targets (Formerly Current Drug
516 Targets-CNS & Neurological Disorders), 12, 830-841.
517 Elkhayat, E. S., Ibrahim, S. R. M., & Aziz, M. A. (2008). Portulene, a new diterpene

518 from Portulaca oleracea L. Journal of Asian Natural Products Research, 10,
519 1039-1043.
23
 ACCEPTED MANUSCRIPT

520 Erkan, N., & Oezden, O. (2008). Quality assessment of whole and gutted sardines
521 (Sardina pilchardus) stored in ice. International Journal of Food Science and
522 Technology, 43, 1549-1559.

523 Falowo, A. B., Fayemi, P. O., & Muchenje, V. (2014). Natural antioxidants against
524 lipid–protein oxidative deterioration in meat and meat products: A review.
525 Food Research International, 64, 171-181.

PT
526 Fernandes, R., Trindade, M. A., Lorenzo, J. M., & De, M. M. (2017). Assessme nt of
527 the stability of sheep sausages with the addition of different concentrations of

RI
528 Origanum vulgare extract during storage. Meat Science, 137.
529 Gong, F., Li, F., Zhang, L., Li, J., Zhang, Z., & Wang, G. (2009). Hypoglycemic

SC
530 effects of crude polysaccharide from purslane. International journal of
531 molecular sciences, 10, 880-888.
NU
532 Hu, J., Wang, X., Xiao, Z., & Bi, W. (2015). Effect of chitosan nanoparticles loaded

533 with cinnamon essential oil on the quality of chilled pork. Lwt-Food Science
MA

534 and Technology, 63, 519-526.

535 Jiang, J., & Xiong, Y. L. L. (2016). Natural antioxidants as food and feed additives to
ED

536 promote health benefits and quality of meat products: A review. Meat Science,
537 120, 107-117.
T

538 Lei, X., Li, J., Liu, B., Zhang, N., & Liu, H. (2015). Separation and Identification of
EP

539 Four New Compounds with Antibacterial Activity from Portulaca oleracea L.
540 Molecules, 20, 16375-16387.
C

541 Li, J., Hui, T., Wang, F., Li, S., Cui, B., Cui, Y., & Peng, Z. (2015). Chinese red
AC

542 pepper (Zanthoxylum bungeanum Maxim.) leaf extract as natural antioxidants

543 in salted silver carp (Hypophthalmichthys molitrix) in dorsal and ventral

544 muscles during processing. Food Control, 56, 9-17.
545 Li, Y. P., Yao, L. H., Wu, G. J., Pi, X. F., Gong, Y. C., Ye, R. S., & Wang, C. X. (2014).
546 Antioxidant activities of novel small- molecule polysaccharide fractions
547 purified from Portulaca oleracea L. Food Science and Biotechnology, 23,

548 2045-2052.
549 Liu, D., Liang, L., Xia, W., Regenstein, J. M., & Zhou, P. (2013). Biochemical and
24
 ACCEPTED MANUSCRIPT

550 physical changes of grass carp (Ctenopharyngodon idella) fillets stored at-3
551 and 0 degrees C. Food Chemistry, 140, 105-114.
552 Liu, X.-F., Zheng, C.-G., Shi, H.-G., Tang, G.-S., Wang, W.-Y., Zhou, J., & Dong,

553 L.-W. (2015). Ethanol extract from Portulaca oleracea L. attenuated

554 acetaminophen- induced mice liver injury. American journal of translational
555 research, 7, 309.

PT
556 Lorenzo, J. M., Sineiro, J., Amado, I. R., & Franco, D. (2014). Influence of natural
557 extracts on the shelf life of modified atmosphere-packaged pork patties. Meat

RI
558 science, 96, 526-534.
559 Mhalla, D., Bouaziz, A., Ennouri, K., Chawech, R., Smaoui, S., Jarraya, R., Tounsi, S.,

SC
560 & Trigui, M. (2017). Antimicrobial activity and bioguided fractionation of
561 Rumex tingitanus extracts for meat preservation. Meat science, 125, 22-29.
NU
562 Muzolf-Panek, M., Waskiewicz, A., Kowalski, R., & Konieczny, P. (2016). The Effect

563 of Blueberries on the Oxidative Stability of Pork Meatloaf During Chilled

MA

564 Storage. Journal of Food Processing and Preservation, 40, 899-909.

565 Nychas, G. J. E., Skandamis, P. N., Tassou, C. C., & Koutsoumanis, K. P. (2008).
ED

566 Meat spoilage during distribution. Meat Science, 78, 77-89.

567 Pogorzelska, E., Godziszewska, J., Brodowska, M., & Wierzbicka, A. (2018).
T

568 Antioxidant potential of Haematococcus pluvialis extract rich in astaxanthin

EP

569 on colour and oxidative stability of raw ground pork meat during refrigerated
570 storage. Meat Science, 135, 54-61
C

571 Prieto, P., Pineda, M., & Aguilar, M. (1999). Spectrophotometric quantitation of
AC

572 antioxidant capacity through the formation of a phosphomolybdenum complex:

573 Specific application to the determination of vitamin E. Analytical Biochemistry,

574 269, 337-341.
575 Sammel, L. M., & Claus, J. R. (2006). Citric acid and sodium citrate effects on pink
576 color development of cooked ground turkey irradiated pre- and post-cooking.
577 Meat Science, 72, 567-573.

578 Selani, M. M., Contreras-Castillo, C. J., Shirahigue, L. D., Gallo, C. R., Plata-Oviedo,
579 M., & Montes-Villanueva, N. D. (2011). Wine industry residues extracts as
25
 ACCEPTED MANUSCRIPT

580 natural antioxidants in raw and cooked chicken meat during frozen storage.
581 Meat Science, 88, 397-403.
582 Shahidi, F., & Zhong, Y. (2010). Novel antioxidants in food quality preservation and

583 health promotion. European Journal of Lipid Science and Technology, 112,
584 930-940.
585 Shen, H., Tang, G., Zeng, G., Yang, Y., Cai, X., Li, D., Liu, H., & Zhou, N. (2013).

PT
586 Purification and characterization of an antitumor polysaccharide from
587 Portulaca oleracea L. Carbohydrate polymers, 93, 395-400.

RI
588 Simopoulos, A. P., & Salem, N., Jr. (1986). Purslane: a terrestrial source of omega-3
589 fatty acids. The New England journal of medicine, 315, 833-833.

SC
590 Stone, H., & Sidel, J. L. (2004). Sensory Evaluation Practices. Sensory Evaluation
591 Practices, 296–304.
NU
592 Suo, B., Li, H., Wang, Y., Li, Z., Pan, Z., & Ai, Z. (2017). Effects of ZnO

593 nanoparticle-coated packaging film on pork meat quality during cold storage.
MA

594 Journal of the Science of Food and Agriculture, 97, 2023-2029.

595 Tajkarimi, M. M., Ibrahim, S. A., & Cliver, D. O. (2010). Antimicrobial herb and
ED

596 spice compounds in food. Food Control, 21, 1199-1218.

597 Tiwari, B. K., Valdramidis, V. P., O'Donnell, C. P., Muthukumarappan, K., Bourke, P.,
T

598 & Cullen, P. J. (2009). Application of Natural Antimicrobials for Food

EP

599 Preservation. Journal of Agricultural and Food Chemistry, 57, 5987-6000.

600 Uddin, M. K., Juraimi, A. S., Ali, M. E., & Ismail, M. R. (2012). Evaluation of
C

601 Antioxidant Properties and Mineral Composition of Purslane (Portulaca

AC

602 oleracea L.) at Different Growth Stages. International Journal of Molecular

603 Sciences, 13, 10257-10267.

604 Wenzel, G. E., Fontana, J., & Correa, J. B. (1990). The viscous mucilage from the
605 weed Portulaca oleracea L. Applied biochemistry and biotechnology, 24,
606 341-353.
607 Xiang, L., Xing, D., Wang, W., Wang, R., Ding, Y., & Du, L. (2005). Alkaloids from

608 Portulaca oleracea L. Phytochemistry, 66, 2595-2601.

609 Yang, L.-C., Li, R., Tan, J., & Jiang, Z.-T. (2013). Polyphenolics Composition of the
26
 ACCEPTED MANUSCRIPT

610 Leaves of Zanthoxylum bungeanum Maxim. Grown in Hebei, China, and

611 Their Radical Scavenging Activities. Journal of Agricultural and Food
612 Chemistry, 61, 1772-1778.

613 Yang, Z., Liu, C., Xiang, L., & Zheng, Y. (2009). Phenolic alkaloids as a new class of
614 antioxidants in Portulaca oleracea. Phytotherapy research, 23, 1032-1035.
615 Yu, L., Scanlin, L., Wilson, J., & Schmidt, G. (2002). Rosemary extracts as inhibitors

PT
616 of lipid oxidation and color change in cooked turkey products during
617 refrigerated storage. Journal of Food Science, 67, 582-585.

RI
618 Zhang, H., Wu, J., & Guo, X. (2016). Effects of antimicrobial and antioxidant
619 activities of spice extracts on raw chicken meat quality. Food Science and

SC
620 Human Wellness, 5, 39-48.
621 Zhou, Y.-X., Xin, H.-L., Rahman, K., Wang, S.-J., Peng, C., & Zhang, H. (2015).
NU
622 Portulaca oleracea L.: A Review of Phytochemistry and Pharmacological

623 Effects. Biomed Research International.

MA

624
T ED
C EP
AC

27
 ACCEPTED MANUSCRIPT

Figure legends

Fig.1. Changes of (A) TVC (total viable counts), (B) Coliform counts, (C)
Pseudomonads counts, (D) Total staphylococcus counts, (E) LAB (Lactic acid
bacterial) counts in the chilled pork of different treatments. Error bars represent the
standard errors of three replications. Control: Deionized water treated group, TP: 0.05%
tea polyphenol treated group, BHA: 0.02% butylated hydroxyl anisole treated group,

PT
POE: P. oleracea crude ethanol extract treated group

RI
Fig.2. Changes of (A) pH, (B) TBARS, (C) TVB-N values in the chilled pork of

SC
different treatments. Error bars represent the standard errors of three replications.
Control: Deionized water treated group, TP: 0.05% tea polyphenol treated group,
NU
BHA: 0.02% butylated hydroxyl anisole treated group, POE: P. oleracea crude
ethanol extract treated group
MA

Fig.3. Appearance differences on day 9 in the chilled pork of different treatments. (A)
Control sample. (B) 0.05% tea polyphenol sample. (C) 0.50% POE sample. (D) 1.0%
ED

POE sample.
T

Fig.4. Changes of (A) SOD, (B) GSH-Px activities in the chilled pork of different
EP

treatments. Error bars represent the standard errors of three replications. Control:
C

Deionized water treated group, TP: 0.05% tea polyphenol treated group, BHA: 0.02%
AC

butylated hydroxyl anisole treated group, POE: P. oleracea crude ethanol extract
treated group

28
 ACCEPTED MANUSCRIPT

Table.1. Experiment arrangement and growth of tested bacteria

Inhibition zone (mm)
Microorganisms Strain a Negative Streptomycin
POE d
Penicillin
control b sulfate c
Gram negative
ATCC
E.coli Nil f Nil 10.87 ± 0.36 Nil
25922
ATCC
S.enteritidis Nil Nil 8.85 ± 0.47 18.23 ± 0.52

PT
13311
ATCC
P.aeruginosa 9.05 ± 0.34 e Nil 8.13 ± 1.09 Nil
13525

RI
Gram positive
ATCC

SC
S.aureus Nil Nil 15.50 ± 0.77 25.10 ± 1.75
6538
ATCC
B.subtilis 8.37 ± 1.25 Nil 22.05 ± 1.58 18.13 ± 2.40
21216
NU
ATCC
B.cereus 7.68 ± 0.33 Nil 18.63± 0.45 15.35 ± 0.65
14579
a
P. oleracea crude ethanol extract (2mg/disc).
MA

b
Sterile water plus equivalent solvent (0.2 ml/disc ) was used as black control.
c
Streptomycin sulfate (0.02mg/disc)was used as the first positive control.
d
Penicillin (0.02mg/disc)was used as the second positive control.
ED

e
Each value is expressed as mean ± SE (n=3).
f
Nil represent no zone
T
C EP
AC

29
 ACCEPTED MANUSCRIPT

Table.2. Scavenging activity (%) on DPPH radicals and total antioxidant capacity of P.
oleracea extracts.
DPPH radical scavenging Total antioxidant capacity
Extracts activity (mg equivalent to ascorbic
a
EC50 (μg/ml) acid/g extract)b
POE 42.87±2.87 a 149.07±4.24 c
TP 3.91±0.41b 301.13±6.98 a
BHA 5.26±0.35 b 217.13±6.87 b
Vc 1.93±0.14 c --

PT
Values are expressed as means ± SE of three replicates. Different letters in the same column
indicate significant differences within the different treatments according to Duncan’s test (P <
0.05).

RI
a
EC50 : concentration suppressing 50% DPPH free radical
b
: Antioxidant capacity monitored by the phosphomolybdenum method

SC
PEE: P. oleracea crude ethanol extract, TP: Tea polyphenol, BHA: butylated hydroxyl anisole ,
Vc: Ascorbic acid NU
MA
T ED
C EP
AC

30
 ACCEPTED MANUSCRIPT

Table.3. Colour variables (L*, a*,b*) in chilled pork with various treatments during 9 days of
storage at 4o C.
Days of storage
Parameters Samples
1 3 5 7 9
control 51.93±0.87abA 48.78±0.62cA 43.51±1.76dB 39.35±0.91dC 33.94±0.33cD
aA bcB bcB abcB cC
TP 52.81±0.39 49.23±0.32 46.83±0.93 46.62±1.16 36.02±1.18
BHA 52.68±0.83aA 51.27±0.55abAB 49.35±0.77abcB 43.81±0.97bcC 36.83±1.24bcD
L* bA abcA cdAB cdBC cC
0.25% POE 49.93±0.45 49.90±1.44 46.34±0.42 42.70±2.07 39.77±1.99
abA abc B abB abC aC
0.50% POE 52.15±0.32 49.67±0.56 49.58±0.18 47.58±0.98 46.38±0.78

PT
1.0% POE 54.19±1.37aA 52.07±0.28aAB 50.48±0.80aBC 49.72±0.79aBC 48.39±0.36aC

control 9.34±0.34aA 8.58±0.48aA 7.94±0.74bA 6.27±0.18bB 4.28±0.29eC

RI
TP 8.86±0.40aAB 9.87±0.44aA 8.28±0.63bAB 7.34±0.66abB 5.62±0.27cdC
* BHA 9.72±0.40aA 6.50±0.67bB 7.69±0.36bB 6.89±0.54bB 4.48±0.73deC
a

SC
0.25% POE 9.37±0.37aA 8.62±0.38aA 8.06±0.49bA 8.14±0.70abA 6.52±0.26bcB
aAB aABC abA abBC abC
0.50% POE 8.70±0.16 8.40±0.18 8.91±0.23 7.53±0.74 7.36±0.34
1.0% POE 9.54±0.37aAB 9.83±0.44aA 10.21±0.42aA 9.02±0.36aAB 8.41±0.34a B
NU
control 4.51±0.46aC 5.53±0.67abBC 6.99±0.17aAB 7.58±0.46aA 8.73±0.51aA
aB abB bB bcA abA
TP 3.54±0.16 4.52±0.92 4.04±0.13 6.18±0.34 7.41±0.47
aB bB aA abA abA
MA

*
BHA 4.40±0.43 4.32±0.24 6.93±0.26 6.82±0.41 7.82±0.31
b
0.25% POE 4.35±0.49aB 5.19±0.22abB 4.77±0.46bB 6.57±0.43abcA 6.61±0.33bcA
0.50% POE 4.81±0.54aAB 4.11±0.25bB 4.76±0.54bAB 5.36±0.24cdAB 5.81±0.36cA
1.0% POE 4.88±0.27aB 6.18±0.21aB 4.71±0.55bB 5.12±0.45dAB 5.94±0.31cAB
ED

Values are expressed as means ± SE of three replicates. a,b Within each storage time, values
with different letters are significantly different due to the treatment (P<0.05). A,B Within each
treatment, values with different letters are significantly different due to the storage time
T

(P<0.05). Control: Deionized water treated group; TP: 0.05% tea polyphenol treated group;
EP

BHA: 0.02% butylated hydroxyl anisole; POE: P. oleracea crude ethanol extract treated
group.
C
AC

31
 ACCEPTED MANUSCRIPT

Table.4. Effect of addition of POE on sensory characteristics to pork meat

Dosage of POE
Sensory characteristics
0 0.25% 0.50% 1.00%
oder 8.00 ± 0.29 a 7.97 ± 0.29 a 8.40 ± 0.21 a 8.53 ± 0.11 a
taste 8.33 ± 0.22 a 8.30 ± 0.17 a 8.60 ± 0.08 a 0.62 ± 0.09 a
texture 8.30 ± 0.17 a 8.23 ± 0.15 a 8.20 ± 0.35 a 8.40 ± 0.15 a
Overall acceptability 8.23 ± 0.14 b 8.30 ± 0.10 ab 8.46 ± 0.09 ab 8.63 ± 0.07 a
a-b
Each value is expressed as mean ± SE (n=30). Different letters within a row are

PT
significantly different (P < 0.05). POE: P. oleracea crude ethanol extract

RI
SC
NU
MA
T ED
C EP
AC

32
 ACCEPTED MANUSCRIPT

Highlights

① Portulaca oleracea L. extract was added to chilled pork meat in comparison to

BHA and TP (tea polyphenol).

② Shelflife of chilled pork treated with Purslane extract increased 2 days.

③ Chilled meat with Purslane extract presented better oxidative stability and sensory

index.

PT
④ As a potherb, Purslane not only contains rich nutrients which are edible and

RI
officinal value, but also process high antioxidant activity and better preservation
effect.

SC
⑤ Portulaca oleracea L. as natural food antioxidant have broad application and
development value prospects
NU
MA
T ED
C EP
AC

33
Figure 1
Figure 2
Figure 3
Figure 4