Preserving Apple (Malus domestica var. Anna) Fruit Bioactive Substances Using Olive Wastes Extract-Chitosan Film Coating Academic research paper on "Chemical sciences"

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Preserving Apple (Malus domestica var. Anna) Fruit Bioactive Substances Using Olive Wastes Extract-Chitosan Film Coating

Ibrahim Khalifa, Hassan Barakat, Hamdy A. El-Mansy, SolimanA. Soliman

PII: DOI:

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S2214-3173(16)30061-0 http://dx.doi.org/10.1016/j.inpa.2016.11.001 INPA 68

Information Processing in Agriculture

Received Date: Revised Date: Accepted Date:

26 June 2016 25 October 2016 10 November 2016

Please cite this article as: I. Khalifa, H. Barakat, H.A. El-Mansy, SolimanA. Soliman, Preserving Apple (Malus domestica var. Anna) Fruit Bioactive Substances Using Olive Wastes Extract-Chitosan Film Coating, Information Processing in Agriculture (2016), doi: http://dx.doi.org/10.1016/j.inpa.2016.11.001

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Preserving Apple (Malus domestica var. Anna) Fruit Bioactive Substances Using Olive Wastes Extract-Chitosan Film Coating

Ibrahim Khalifaa*, Hassan Barakatab, Hamdy A. El-Mansya, Soliman A. Soliman: aFood Technology Department, Faculty of Agriculture, Benha University, Moshtohor, 13736 Qaliuobia, Egypt.

Food Science and Human Nutrition Department, College of Agriculture and

7 Veterinary Medicine, Qassim University, Buraidah, Saudi Arabia.

E-mail: hassan.barakat@fagr.bu.edu.eg, Hamdy.elmansy@fagr.bu.edu.eg, Soliman.soliman@fagr.bu.e *Corresponding author: Mr. Ibrahim Khalifa Tel. +20122 Fax. +20132467786, E-mail: Ibrahiem.khalifa@fagr.bu.edu.eg

15 Address: Department of Food Technology, Faculty of Agriculture, Benha University,

16 Moshtohor, 13736 Qaluiobia, Egypt.

17 Abstract

19 Apple fruits have components of therapeutic nature. Such components, to a great

20 extent, decline or decompose during post-harvest that negatively affect fruit shelf-life.

21 Chitosan fruit-filming has proved useful in maintaining these compounds. This study

22 aims, therefore, at enhancing Chitosan coating-film performance by mixing with some

23 olive wastes extracts of leaf and pomace extracts. Apple fruits were sprayed with six

24 different coating formulas including chitosan-water wax coating, in addition to the

25 uncoated fruits. Then, the total phenolic, flavonoids, antioxidants, pigments, weight

26 loss, decay area, and microstructure were assayed. The bioactive substances

27 drastically changed in uncoated rather than coated fruits. Conversely, weight loss and

28 decay area significantly increased in uncoated fruits. Amazingly, the addition of olive

29 leaf extract to chitosan coating films meaningfully reduced the gradual decline in total

30 phenolic, flavonoids and antioxidants. Olive pomace extract recorded the lowest

31 influencing on anthocyanins during storage at 4±1 °C for 35 d. Also, both olive leaf

32 and pomace extracts enhanced the coating distribution, due to no pores were observed

33 in the fruits' surfaces. Decidedly, incorporation of olive leaf extracts with 2% into

34 chitosan coating solution was the best formula comparable with the others. Thus,

35 olive wastes extracts, incorporated into chitosan fruit coatings; relatively improve the 6 nutritional quality of apple fruits during post-harvest.

7 Keywords: Olive wastes extracts; chitosan; coating; nutritional quality;

38 microstructure analysis.

39 Chemical compounds:

40 DPPH* (PubChem CID: 2735032), Trolox (PubChem CID: 40634), Gallic acid

41 (PubChem CID: 370), Quercetin (PubChem CID: 5280343), Chitosan (PubChem

42 CID: 3086191) and Thiabendazole (PubChem CID: 5430).

44 1. Introduction

45 Apple (Malus domestica var. Anna) fruits greatly vary in the bioactive substances like

46 polyphenols, flavonoids, vitamins, pigments and others [1, 2] which provide high

47 nutritional values. Unfortunately, these components dramatically decline during post-

48 harvest times [3, 4-5]. This might refer to the activity of some microorganisms which

49 grow on the fruit surfaces' during post-harvest time. They produce mycotoxins and

50 degrade phytochemicals [6]. Additionally, other factors may responsible for

51 phytochemicals degradation includes cultivar type, environmental and agronomic

52 conditions, harvest and food processing operations, and storage factors [7].

53 Commonly, these challenges might be fixed using coating fruits with commercial

54 waxes such as water wax incorporated with artificial additives like thiabendazole

55 (WW-TBZ). But, such materials might cause some dangerous side effects related to

56 bladder cancer [8]. Switching to using some natural polymers, such as chitosan (CH)

57 (poly #-(1,4) N-acetyl-D-glucosamine) incorporated with natural additives like food

58 processing wastes, has recently been applied to fruit coating techniques [9, 10-11].

59 The CH, following cellulose, is the second most abundant polysaccharide found in

60 nature [12]. In addition to being environmentally safe, it has good film-forming

61 properties, antimicrobial activity, and has been recommended as GRAS food additive

62 [13, 14,15-16].

63 Olive (Olea europaea var. Kronakii) plant extracts has been one of these natural

64 additives which play a functional role in fruit film-coating components. Olive oil

65 processing wastes (OOW) contain considerable amounts of bioactive substances,

66 although it causes some economic losses and some environmental problems. [17, 18,

67 19-20]. However, OOW are promoting by-products for functional food and/or

68 nutraceuticals. They can be used as antioxidants, antifungal and antibacterial agents

69 [20, 21]. Thus, OOW has been valued before in edible-film approach as in the cases

70 of polyesters and polylactic films [22, 23]. Incorporating OOW extracts into chitosan

71 coating materials may be one safe approach to maintaining quality of cold-stored

72 apple fruits. The objective of this study was to infer how both olive wastes extracts,

73 when incorporated into Chitosan film coating, enhance apple fruits shelf-life during

74 post-harvest cold storage time by maintaining fruit nutritional and keeping quality.

ruit nutrit

Materials and methods

77 2.1. Reagents and solutions

78 1, 1-diphenyl-2 picrylhydrazyl radical (DPPH*), 2-(3, 4-dihydroxyphenyl)-3, 5, 7-

79 trihydroxy-4H-chro

men-4-c

(Quercetin)

6-hydroxy-2,5,7,8-

80 tetramethylchroman carboxylic acid (Trolox) were obtained from Sigma Aldrich, Co.,

81 Germany. Chitosan 95% deacetylation, high molecular weight (viscosity 500-2000

82 cps) was procured from Oxford Co., India. Folin-Ciocalteu reagent was purchased

83 from Fluka Biochemical, Co., Switzerland. Gallic acid Serva was obtained from iochemical, Co., New York. Thiabendazole and water wax® WW-TBZ were

obtained from Fomesa Fruitech, Co., Spain. All reagents and indicators are pure and

86 analytical grade.

87 2.2. Microbial strain and media

maturity form

88 Penicillium expansum ATCC 7861 was obtained from Cairo Microbiological

89 Resource Center (MIRCEN), Faculty of Agriculture, Ain Shams University, Cairo,

90 Egypt. Sabouraud agar No. 402005 was obtained from Biolife, Co., Italy.

91 2.3. Raw materials

92 a. Olive (Olea europaea var. Kronakii) wastes including olive leaves and olive

93 pomace were obtained from Cairo for Oil Industry, Co., Industrial Zone, 6th October

94 City, Egypt.

95 b. Apple fruits (Malus domestica var. Anna) eatable maturity form was obtained

96 from the Alexandria Agriculture Farm, Co., Egypt.

97 2.4. Analytical techniques

98 2.4.1. Olive oil processing wastes preparation and extraction

99 Olive leaves and olive pomace were oven-dried (Tit Axon S.R.L via Canova, Italy) at

100 40-50 °C gradually till the weight constant (4.37 and 7.60% moisture, respectively).

101 Subsequently, they were milled by grinder (Severin, type 3871, Germany). They were

102 passed through a 60 mesh sieve to obtain a fine homogenous powder, then packed in

103 dark glass jars then kept at -20±1 °C until use. Both olive leaves and pomace were

104 mixed with ethanol 80% (1:20, w/v) in dark bottles, and shacked at 120 rpm for 86400

105 s (Centrifuge (MLM Zentrifugenbau.TS21, Germany). The mixtures were filtered

106 through filter paper Whatman No.1. The filtrates were collected, then solvents were emoved by rotary evaporator (NE-1-Rikakikai Co., LTD, Japan) at 40 °C according

8 to [24]. The residue was collected and kept at -18+1 °C.

109 2.4.2. Film forming solution

110 The incorporated CH solutions with both ethanolic olive leaf and olive pomace

111 extracts were prepared according to [25] with some modifications. Chitosan 2% was

112 dispersed in an aqueous solution of glacial acetic acid (0.5%, v/v) at 40 °C. The

107 r 10

113 solution was heated and agitated constantly for 43200 s. Then the pH was adjusted to

114 5.6 with 1 M NaOH. Subsequently, glycerol 1.6% was added as a plasticizer [26]. The

115 solution was stirred overnight at room temperature. Finally, olive leaves and olive

116 pomace extracts at 1 and 2% were added and mixed to achieve the complete

117 dispersion.

118 2.4.3. Apple fruits coating applying

119 Apple fruits were sorted for uniform size, full color, % maturities, and for being free

120 of visible defect and of decay. Then, they were sanitized by inundation on sodium

121 hypochlorite solution 250 ppm for 2 min and washed with distilled water to eliminate

122 chlorine traces. Subsequently, cross-shaped wounds (2, 0.1 and 0.5 mm for length,

123 width and depth, respectively) were made to the fruits using sterilized Spatula

124 (Dynalon 1212W16CS 391905) and inoculated by 10 |jL P. expansum spore

125 suspension (105 CFU/ mL). The coating solutions (as described above section 2.4.2)

126 were sprayed twice on the whole fruit surface using a multi-function hand 2L pressure

127 sprayer (Ningbo Synkemi Co., type SK-2B, China) and allowed to be air-dried at

128 ambient temperature for 7200 s. Seven groups of samples were prepared in total:

129 uncoated (control), CH (2% w/v), Chitosan-Olive leaves extracts CH-OLE (1 and 2%

130 w/v), Chitosan-Olive pomace extracts CH-OPE (1 and 2% w/v), and WW-TBZ 0.1%

131 -coated fruits as a positive control, according to [27]. Fruits were packed in boxes

32 (~3 fruit per box) as a three replicate and wrapped with polyethylene sheets, then

33 stored at 4±1 °C for 35 d. The nutritional characteristics, keeping parameters and

134 weight loss of fruits were evaluated at the beginning of the experiment (i.e, 0 d) then

135 at a 7 d interval up to 35 d.

136 2.4.4. Bioactive substances of coated apple fruits

137 2.4.4.1. Anthocyanins content

138 The anthocyanins content of apple fruits was determined according to [28]. A 0.005

139 kg apple samples were extracted with 45 mL of acidified ethanol (95% ethanol: HCl

140 1.5 N 85:15) for 7200 s at room temperature in the dark, filtered and measured at 535

141 nm using spectrophotometer (CE599- Automatic Scanning Spectrophotometer,

142 GECIL, England) .

143 2.4.4.2. Carotenoids and chlorophylls content

144 A 0.01 kg apple sample was mixed with 50 mL acetone 85% in dark bottle and left to

145 stand for 54000 s at room temperature. The mixture was filtered through glass wool

dark bottle and ] d throu

146 into a 100 mL volumetric flask and made up to appropriate volume by the same

147 solvent. The chlorophyll a, b and carotenoids wert immediately measured at 440, 644

148 and 662 nm using the same spectrophotometer according to [29].

149 2.4.4.3. Total phenolic compounds content

150 The total phenolic compounds (TPC) for acetone extracts of apple were determined

151 according to [30]. In brief, 200 ^L of each sample was mixed with 1 mL of 10-fold

152 diluted Folin-Ciocalteu reagent; the reaction was terminated after 300 s by 1 mL of

153 Na2CO3 7.5%, then 1.5 mL distilled water was added. The mixtures were incubated in

154 dark for 3600 s then the absorbance at 760 nm was measured using the same

155 spectrophotometer. The TPC was expressed as gallic acid equivalents (mg GAE 100 g-1)

156 using the following straight-line linear regression equation based on the calibration

157 curve:

58 Y= 0.0201 x + 0.0538 (R2= 0.99)....................................(1)

159 Where, Y is the concentration and X is the absorbance.

160 2.4.4.4. Total flavonoids content

161 The total flavonoids content (TF), for acetone extracts of apple, was determined

162 according to [31]. A 0.5 mL aliquot of AICI3 2% ethanolic solution was added to 0.5

163 mL of extracts and mixed well. Then it was kept for 3600 s at room temperature and

164 the absorbance at 420 nm was measured using the same spectrophotometer. The final

165 concentration of TF was expressed as quercetin equivalent (mg QE g-1) which was

166 calculated using the following straight line linear regression equation based on the

167 calibration curve:

168 Y = 0.037x+0.1363 (R2= 0.98) ...................................(2)

169 Where, Y is the concentration and X is the absorbance.

170 2.4.4.5. Antioxidant activity

171 The antioxidant activity (AOA) of apple acetonic extracts was evaluated according to

172 [32]. A 0.1 mL extract was added to 3.9 mL of DPPH* methanolic solution, then the

173 absorbance at 517 nm was measured after the solution had been allowed to stand in

174 the dark for 600 s at 517 nm using the same spectrophotometer. The final results were

g the same spectr g the same spectr

(RmoL TE g

175 expressed as Trolox equivalents (^moL TE g-1).

176 2.4.4.6. Decay area determination

177 Mold growth area of inoculated apple was checked by measuring in terms of the

178 decay area every seven days using micrometric ruler according to [33].

179 2.4.4.7. ^M icrostructure analysis

180 The surface and cross-section microstructures of apple skins, which had been coated

181 by selected formulas such as CH 2%, CH-OLE 2%, CH-OPE 2%, and uncoated fruits,

182 were examined and scanned using an electron microscope. Tissues from different 3 treatments were fixed in 4% glutaradehyde in 0.2 M sodium cocodylate buffer (pH

184 4.1) for 14400 s, formerly fixated later in osmium tetraoxide for 7200 s. Fixed tissues

185 were rinsed in the same buffer three times and dehydrated through a graded ethanol

186 series 10 to 100% for 600 s up to 1800 s in final concentration. The specimens were

187 transferred on cupper slide and dehydrated using critical point dryer with liquid

188 carbon dioxide, then coated with gold using (S150A Sputter coater-Edwards-

189 England). The specimens were examined and photographed using scanning electron

190 microscope with proper magnification (JXA-840A, Electron Probe Micro analyzer

191 JEOL, Japan).

192 2.4.5. Statistical analysis

193 The statistical analysis was carried out using SPSS program with multi-function

194 utility regarding to the experimental design and multiple comparisons were carried

195 out applying LSD according to [34].

196 3. Results and discussions

197 3.1. Weight loss

198 The effect of CH-incorporated films on weight loss of apple fruits during cold storage » „ ^ in T* we^, 10SS Ste.„y ^ «ng a Prol0„8ed ^

200 period either coated or uncoated fruits. Significant difference (p<0.05) was observed

201 between uncoated and coated fruits with the progression of storage period. Uncoated

202 apples fruits showed weight loss was as high during storage period. Significant

203 difference (p<0.05) was found between CH incorporated film and WW-TBZ.

204 Statistically, uncoated apples evident the highest weight loss to be 3.03% during the

205 whole storage period and 8.50% after 35 days. Conversely, the lowest observed loss

206 was 2.66 % using CH-OLE 2%. The formed CH film on surface of coated fruits 07 delayed the migration of moisture. These CH- based coating strategies to reduce the

8 weight loss during storage and its concept were used before as mentioned [10].

209 3.2. Anthocyanins content

210 The initial anthocyanins of coated apple with CH-OPE 2% and uncoated apple were

211 20.83 and 19.00 mg 100g-1, respectively (Fig.2). Indeed, during the preliminary stage

212 of cold storage the uncoated and coated fruits show a significant increase in

parisons

eight loss of ap

loss steadily incr fruits. Sign

213 anthocyanins content. [35] reported that fruits become darker during storage due to

214 releasing cell anthocyanins' after its decomposition. Following 14 d of storage, there

215 were, generally, plodding declines of anthocyanins. At 35 d, CH-OPE 2% film had

216 relatively higher anthocyanins content. Based on Fig. 2, the control had about 41%

217 less anthocyanin relative to both CH-OPE 2% and CH-OLE 2% at 35 d. Applying

218 CH-OOW films maintained the anthocyanins better than WW-TBZ films. Coating

219 acts as a gas barrier, thus lowering internal O2 levels and increasing CO2. Under these

220 conditions, metabolism and catabolism are slowed down which contribute to

221 preserving nutraceuticals for longer periods [36]. Also, olive pomace extracts contains

222 a lot of AOA that prevent cell wall oxidation [37].

223 3.3. Carotenoids contents

224 Over the 35 d period, a decremental rate has been observed during cold storage in

225 either uncoated or coated fruits (Fig. 3). Between both CH-OLE 2% and CH-OPE 2%

226 on one side and the rest of the other films, there were marginal differences (p<0.05).

227 The coated apple with CH-OPE 2% was the lowest decreases in carotenoids contents

228 (1.59 mg g-1) compared with uncoated apple (0.52 mg g-1) at the end of storage

229 period. So far, there are no studies on the effect of the coating materials on the content

230 of carotenoids in fruits. However, [38] found that cold storage was decreased the

231 carotenoids in some vegetable and fruits. 32^3.4. Chlorophylls contents

33 The differences among coatings were quite trivial up to 21 d; however, a sharp

234 upward content of Chlorophyll a (~2.5 to ~4.5 mg g-1), and for Chloroyphyl b (~4.5 to

235 ~ 9.0 mg g-1) occurred towards the 28 d for the uncoated fruits (Fig.4 A, B). However,

236 the uncoated apple scored the highest chlorophyll a and b regardless the storage

237 periods. Then it was followed by CH 2% for chlorophyll a and WW-TBZ for

238 chlorophyll b. In contrarily, CH-OOW led to decrease the increasing of chlorophylls

239 releasing during storage. These changes may occur because so-called weight losses in

240 fruits during post-harvest related to the result mentioned before (3.1). There are no

241 studies on the effect of the coating materials on apples' chlorophylls content.

242 3.5. Total phenolic compounds

243 The initial TPC content was varied, for instance the mean value of TPC in uncoated

244 and CH-OLE 2% for apple fruits were (1.62 and 1.71) mg GAE g-1, respectively.

245 These differences owing to the phenolic were involved in the composition of OOW

246 which used in film formation and fruits coating. However, it was 0.43 mg GAE g-1 at

247 the end of storage. Over the 35 d trial period, all coating films had varying positive

248 impacts (p<0.05) on slowing down apple fruit total TPC degradation rate relative to

249 that of the Control (Fig. 5). All four CH-OPE/OLE films caused TPC degradation

250 rates steadily drop towards the 35 d time; yet CH 2%, TBZ 0.1%, and control, all

251 exhibited relatively faster downward degradation rates. Both olive extracts, as basic

252 components of CH coating thereby had potentials in maintaining fruit TPC as intact as

253 possible in relation to CH coating impact by itself. The lowest decreases in TPC were

254 showed in coated apple with CH-OLE 2% to be 1.24 mg GAE g-1. Otherwise the

255 highest decreases in TPC were observed in uncoated fruits reached to 028 mg GAE g-1 at

256 end of storage period. [39] explained how cell breakdown releases phenolic

57 compounds that are exposed to enzymatic oxidation. CH-OOW compounds might

58 function as protective barriers on the fruits surface to reduce oxygen supply. This

259 finding was similar to what [40] had found in apple fruits.

260 3.6. Total flavonoids content

261 The variation in TF content during cold storage of uncoated and coated fruits is

262 exhibited in Fig.6. The TF content progressively decreased during storage period,

263 recording a relatively greater reduction in uncoated fruits. The loss in TF in uncoated

264 fruits was extremely rapid compared with the CH-OLE 2% coated fruits to be 0.02 vs

265 0.93 mg QE 100g-1, respectively at the end of storage period. Indeed, significant

266 difference (P<0.05) in TF content was noticed between all coated and uncoated fruits.

267 Otherwise, no significant difference (p>0.05) was found between both CH-OLE 1%

268 and CH-OPE 1%, or between CH-OLE 2% and CH-OPE 2%. Yet there are rarely

269 available studies have examined the effect of coating on TF in apple fruits, whereas,

270 [40] mentioned that the TF was decreased during cold storage.

271 3.7. Antioxidant activity

272 Similarly, during the storage period the AOA sharply decreased especially in

273 uncoated fruits compared to the coated ones (Fig. 7). The AOA significantly

274 decreased (P<0.05) from 13.80 to 6.90 ¡moL TE g-1 in uncoated apple after 14 d.

to 6.90 ¡imoL TE was observ

275 However, low decremental rate was observed in coated fruits. Coating apple fruits

276 with CH-OLE 2% or CH-OPE 2% exhibit the lowest decreases rather than WW-TBZ

277 0.1%. [3, 39] stated valuable finding about the preventing of AOA losses in apple

278 fruits during cold storage using edible coating.

279 3.8. Decay area

280 Generally, the infected area gradually increased by extending storage periods (Fig.8

281 A, B). Regardless the coating treatments the initial and the final infected area was 3 82 iand 13.28 mm2 13.28, respectively. The decayed area of coated apple was smaller

3 significantly reduced compared to the uncoated fruits. For example the early signs of

284 mold development in uncoated apple appeared after 7 d of storage and it was delayed

285 in the others fruits. CH-OLE 2% had a relatively low decay area compared to each of

286 CH, WW-TBZ, and uncoated fruits. Mean decayed area on the control vs CH-OLE2%

287 fruits was about 3-folds as much. Obviously, the highest observed areas were 25.33

288 mm2 in uncoated apple at the end of storage. While, the lowest observed areas were

289 7.33 mm2 in coated apple with CH-OLE 2%. Both CH and CH- OOW were more

290 effective than the commercial coating material of WW-TBZ regarding growth of

291 fungal strains Fig.8. This is due to its antifungal activity [41]. [42] suggested that CH

292 induces chitinase, as defense enzyme catalyzes the hydrolysis of chitin, a common

293 component of fungal cell walls, preventing or delayed the growth of fungi on the fruit.

294 Also, these results are complimentary to those of [43] who reported that significant

295 antimycotic activity of methylcellulose and CH composite films incorporated with 4%

296 of sodium benzoate or potassium sorbate. JO

297 3.9. Microstructure examination

298 The homogeneity of both CH and CH-OOW coatings, micrographs of both the

299 surface and the cross-section areas are shown in Fig.9. Coated apples, especially with

300 CH-OOW, showed uniform coating distribution, and pores were not observed

301 compared with uncoated fruits Fig.9 A, B. The higher percentage of covered surface

302 relates to the higher water vapor resistance which slowed respiration process and

303 water loss as observed in coated apple with CH-OLE 2% and CH-OPE 2% Fig. 9 E to

304 H. In addition, both CH Fig .9 C, D and CH-OOW coatings covered all irregularities

305 in the fruits skin. [44] argued that the extensibility of the liquid dispersion on the

306 covered fruit surface plays an important role in limiting water migration from the ruits.

8 4. Conclusion

309 The results of the present study asserted that the incorporation of OOW into CH

310 improved the nutritional quality for cold-stored apple fruits. Also, the coatings of CH

311 or CH-OOW have beneficial impacts on the quality retention of cold storage apple

307 f]

312 fruits especially CH-OLE 2%. The use of OOW also maintained lower weight loss.

313 Likewise, CH-OOW resulted in effectively delaying anthocyanins, total phenolic,

314 flavonoids, carotenoids, chlorophylls and antioxidants activities. Moreover, CH-OOW

315 fully covered the whole surface of apple fruits in term of skin irregularities and/or

316 pores. Hence, coatings of apple fruit with CH-OOW may be relatively more useful for

317 improving apple post-harvest quality and shelf-life stability compared to both only

318 CH and WW-TBZ coatings. These motivated results may encourage the food handlers

319 to replace the chemical coating materials with the presented coatings formulas of the

320 current study. Moreover, the applicability of such formulas on different fruits and

321 vegetables surfaces' has to further reviewing.

322 Acknowledgments

323 The authors gratefully acknowledge the financial support from the Benha university

324 project [Utilization of some agricultural wastes in food and feed processing - A/1/2]

325 2013-2015.

326 Conflict of interest

327 Authors have declared that there are no conflict of interest.

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Figure Captions:

Fijgp!. Effect of CH, CH-OOW and WW-TBZ coating solution on weight loss in apple fruits during cold storage at 4±1 °C, (Mean±SD, «=3).

Fig6>4E. Monitoring of anthocyanins for coated and uncoated apple fruits with different chifösan based coating formulas during cold storage at 4±1 °C, (Mean±SD, n=3). Figi3. Changing of carotenoids in coated and uncoated apple fruits with different chitosan based coating formulas during cold storage at 4±1 °C, (Mean±SD, n=3). Fig>4. Changing of chlorophyll a (A) and chlorophyll b (B) in coated and uncoated apple frftte with different chitosan based coating formulas during cold storage at 4±1 °C, (Mean+SD, n=3).

Fig.5. Monitoring of TPC for coated and uncoated apple fruits with different chitosan based coating formulas during cold storage at 4+1 °C, (Mean±SD, n=3).

pple fruits

ncoated

Fig.6. Monitoring of TF for coated and uncoated apple fruits with different chitosan based coating formulas during cold storage at 4+1 °C, (Mean+SD, n=3).

Fig.7. Monitoring of AOA for coated and uncoated apple fruits with different chitosan based coating formulas during cold storage at 4+1 °C, (Mean+SD, n=3).

Fig78. Decay area in uncoated apple and coated with CH and WW-TBZ (A), CH-OOW (B) in478ted with R. stolonifer during cold storage at 4+1 °C, (Mean+SD, n=3). Fig.9. Scanning electron micrographs of surface and cross-section of uncoated; (A, B) and coated apple fruits with CH; (C, D), CH-OLE 2%; (E, F) and CH-OPE 2%; (G, H) formulas, 1).

Surface Cross section

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Storage period (day)

7 14 21

Storage period (day)

■Uncoated rTBZ 0.1% <H-OPEl%

CH-OLEl%

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Storage period (day)

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CH-OLE1 % CH-OPEl% CH-OLE2% CH-OPE2%

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Storage period (day)

OOR + CH CH-OOR Spray coating

Highlights: 518

519 Revalorization of olive leaves and pomace has been concerned.

520 A novel edible coating solution of chitosan based had been developed.

521 CH-OLE 2% was the best emulsion coating rather than other formulas.

522 CH-OOW was significantly preserving the nutritional compounds of apple fruits. 528 CH-OOW was gradually kept bioactive substances in apple compared to WW-TBZ.