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Accepted Manuscript

Phytochemicals content and antioxidant properties of sea buckthorn (Hippophae


rhamnoides L.) as affected by heat treatment – quantitative spectroscopic and
kinetic approaches

Florentina-Mihaela Ursache, Ioana Otilia Ghinea, Mihaela Turturică, Iuliana


Aprodu, Gabriela Râpeanu, Nicoleta Stănciuc

PII: S0308-8146(17)30669-6
DOI: http://dx.doi.org/10.1016/j.foodchem.2017.04.107
Reference: FOCH 20980

To appear in: Food Chemistry

Received Date: 4 February 2016


Revised Date: 7 April 2017
Accepted Date: 17 April 2017

Please cite this article as: Ursache, F-M., Ghinea, I.O., Turturică, M., Aprodu, I., Râpeanu, G., Stănciuc, N.,
Phytochemicals content and antioxidant properties of sea buckthorn (Hippophae rhamnoides L.) as affected by heat
treatment – quantitative spectroscopic and kinetic approaches, Food Chemistry (2017), doi: http://dx.doi.org/
10.1016/j.foodchem.2017.04.107

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1 Phytochemicals content and antioxidant properties of sea buckthorn (Hippophae

2 rhamnoides L.) as affected by heat treatment – quantitative spectroscopic and kinetic

3 approaches

4 Florentina-Mihaela Ursache1, Ioana Otilia Ghinea2 , Mihaela Turturică, Iuliana Aprodu, Gabriela

5 Râpeanu1, Nicoleta Stănciuc1*


1
6 Faculty of Food Science and Engineering, 2Faculty of Science, Dunarea de Jos University of

7 Galati

10

11

12

*Correspondence full address: NICOLETA STĂNCIUC, PhD, Professor

Dunărea de Jos University of Galati, Faculty of Food Science and Engineering, Domnească

Street 111, Building E, Room 304,

800201, Galati, Romania,

Telephone: 004 0336.130.177

Fax: 004 0236.460.165

E-mail: Nicoleta.Stanciuc@ugal.ro

www.funfood.ugal.ro, www.sia.ugal.ro, www.bioaliment.ugal.ro

1
13 Abstract

14 The effect of thermal processing (50-100°C) on the degradation of the phytochemicals in sea

15 buckthorn extract was investigated using chromatographic, fluorescence and FT-IR spectroscopy

16 techniques and degradation kinetics. Heating the sea buckthorn extract resulted in structural

17 changes that led to red- or blue-shifts in maximum emission, depending on temperature and

18 excitation wavelengths. The attenuated total reflectance analysis of the sea buckthorn extract

19 revealed a satisfactory thermostability of compounds at high temperatures. A fractional

20 conversion kinetic model was used to describe the mechanism of degradation in terms of rate and

21 activation energy. Activation energies for total carotenoids, polyphenolic, flavonoids, and

22 antioxidant activity were 8.45±0.93 kJ/mol, 2.50±0.66 kJ/mol, 22.50±7.26 kJ/mol and

23 15.22±2.75 kJ/mol, respectively. The kinetic parameters evidence a higher thermal stability of

24 carotenoids and polyphenols, suggesting higher degradation rates for flavonoids and antioxidant

25 activity. Our results demonstrate that industrial process optimization in terms of time-

26 temperature combinations demands product specific kinetic data.

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33 Key words: sea buckthorn, phytochemicals content, thermal treatment, kinetic, carotenoids,

34 polyphenols

35

2
36 1. Introduction

37 Sea buckthorn (Hippophae rhamnoides L., Elaeagnaceae) is native in Eurasia and has been

38 domesticated in several countries, including Romania. The berries are rich in vitamin C,

39 flavonoids, oils and oil-soluble compounds, as well as minerals. The nutritional attributes,

40 medicinal and therapeutic potential have been reviewed recently. For example, Kim, Kwon, Sa,

41 & Kim (2011) suggested that the butanol fraction, which contains the highest amount of phenolic

42 compounds, has a powerful α-glucosidase inhibitory effect. Maheshwari et al. (2011) reported

43 that, oral administration of phenolic rich fraction of sea buckthorn leaves significantly protected

44 from CCl(4) induced elevation in aspartate aminotransferase, alanine aminotransferase, γ-

45 glutamyl transpeptidase and bilirubin in serum of Sprague Dawley rats. The study performed by

46 Pang et al. (2008) demonstrated the antihypertensive effect of total flavones, extracted from sea

47 buckthorn, in chronic sucrose fed rats through regulation of insulin and angiotensin II levels.

48 Due to their unique taste, the food industry processes sea buckthorn berries to create a variety of

49 products, such as juice and marmalade, or flavorings for dairy products. Sea buckthorn are rich

50 in carotenoids, such as zeaxanthin, <beta>-carotene, <beta>-cryptoxanthin, lutein, lycopene and

51 <gamma>-carotene (Anderson, Olsson, Johansson, & Rumpunen, 2009). Some carotenoids have

52 an important role in the diet due to their vitamin A activity. The importance of carotenoids is

53 related not only to the public health, but also to agriculture, livestock and food, cosmetics and

54 pharmaceuticals.

55 Phenolic compounds widely distributed in the medicinal plants, pharmaceuticals, spices,

56 vegetables, fruits, grains and other seeds are an important group of natural antioxidants with

57 possible beneficial effects on human health (Pandey & Rizvi, 2009). They can participate in

58 protection against the harmful action of reactive oxygen species, mainly oxygen free radicals.

3
59 Free radicals are produced in higher amounts in a lot of pathological conditions and are involved

60 in the development of the most common chronic degenerative diseases, such as cardiovascular

61 disease and cancer. The major phenolic compounds detected in sea buckthorn juice (mg/L), as

62 reported by Rösch, Bergmann, Knorr, & Kroh (2003) are: gallic acid (1220), proanthocyanidins

63 (351), isorhamnetin 3-O-rutinoside (181), isorhanetin 3-O-glucoside-7-O-rhamnoside (75),

64 isorhamnetin 3-O-glucoside (75), catechin (19), quercetin 3-O-rutinoside (14.5), quercetin 3-O-

65 glucoside (9.0), epicatechin (2.8), protocatechuic acid (1.5), isorhamnetin (1.4), and isorhamnetin

66 7-O-rhamnoside (1.3). Regarding the content of phenolic acids, Zadernowski, Naczk, Czaplicki,

67 Rubinskiene, & Szalkiewicz (2005) suggested the presence of 2,5-dihydrobenzoic acid, gallic

68 acid, pyrocatechuic acid, protocatechuic acid, salicylic acid, syringic acid, vanillic acid,

69 veratricacid, caffeic acid, meta-, o- and p-coumaric acid, ferulic acid, hydroxycaffeic acid, p-

70 hydroxyphenyl-lactic and quinic acids.

71 Although the benefits of sea buckthorn are widely established through numerous studies, limited

72 information about how these bioactive components and antioxidant capacity are affected by

73 processing under different time-temperature combination. Obviously, biologically active

74 compounds thermal degradation should be prevented to maintain biological activity, while the

75 carotenoids degradation and isomerization reactions might be of interest to either prevent or

76 promote depending on the carotenoid type, as suggested by Colle, Lemmens, Knockaert, Van

77 Buggenhout, Van Loey, & Hendrickx (2013). In order to design on optimized thermal process

78 leading to a maximized preservation of phytochemicals, kinetic modelling is necessary to derive

79 basic kinetic information for a system in order to describe the reaction rate as a function of

80 experimental variables and hence, to predict changes in a particular food during processing (Yu,

81 Wu, Hu, Cui, & Yu, 2011).

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82 To our knowledge no attempts have been made to combine these methods for examining the

83 thermal degradation of bioactive compounds from sea buckthorn extract. Therefore, this study

84 combines different methods with the aim to investigate the mechanisms of thermal degradation

85 of the biologically active compounds. Fluorescence microscopy is a noninvasive technique that

86 can be used to extend the possibilities of analyzing the changes of bioactive compounds in

87 different vegetables. Fourier transform infrared (FT-IR) spectroscopy is a simple, rapid and non-

88 destructive method, suitable for investigations which assure high reproducibility and specificity.

89 Therefore, in order to describe the effect of heat treatment on different chemical constituents

90 from sea buckthorn extract under thermal treatment, the changes in total carotenoid content

91 (TCC), total phenolic content (TPC), total flavonoid content (TFC), as well as antioxidant

92 capacities of sea buckthorn extracts were investigated in the temperature range of 50 to 100˚C,

93 for different times (0-25 min) on a quantitative kinetic basis. In addition, the fluorescent

94 properties of the sea buckthorn extracts in phosphate buffer at pH 4.0 were investigated at 250

95 nm, 340 nm, 410 nm and 448 nm (excitation wavelengths). Additionally, FT-IR spectroscopy

96 was used to gain insights into the compositional complexity of the extract and evaluate the

97 thermal behavior.

98 2. Materials and methods

99 2.1. Materials

100 Sea buckthorn berries (Hippophae rhamnoides L., ssp. Carpatica) were purchased from a local

101 market (Galați, România) in the first part of October of the year 2014. According to the trader

102 statement, the berries originated from Urzica field, Olt County, Romania. The initial moisture,

103 pH, °Brix, and titratable acidity were determined, as described by Araya-Farias, Makhlouf, &

104 Ratti (2011). The berries were kept in plastic pots, directly refrigerated at 4°C, kept for 8 h, and

5
105 subsequently frozen and stored at -20°C. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 3,4,5-

106 trihydroxybenzoic acid (gallic acid) and β-carotene were obtained from Sigma Chemical Co. (St.

107 Louis, MO, USA). 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), Folin-

108 Ciocalteu reagent, sodium carbonate, sodium hydroxide and ethanol (HPLC grade) were

109 obtained from Sigma Aldrich (Steinheim, Germany). All solvents/chemicals used were of

110 analytical grade.

111 2.2. Phytochemicals extraction

112 Frozen sea buckthorn berries were crushed in a mortar, and 5 g mixed with 35 ml ethanol:hexane

113 (4:3, v/v) containing 0.05 g magnesium carbonate for 1 h at room temperature on an orbital

114 shaker. After centrifugation at 7000 rpm for 10 min, the supernatant was collected and the

115 residue was further re-extracted with 70 ml ethanol:hexane (4:3, v/v). Finally, the residue was

116 washed with 25 mL ethanol and 12.5 mL hexane. All extracts obtained were combined and

117 washed with 100 mL of 10% sodium chloride and 150 mL of water. The carotenoids extract was

118 concentrated under reduced pressure at 40°C to dryness (AVC 2-18, Christ, UK), dissolved in 10

119 mL ethanol (70%) and passed through a 0.45 µm filter (Rotilabo®-syringe filters, Carl Roth,

120 Germany).

121 2.3. High-performance liquid chromatography (HPLC) analysis of carotenoids

122 To separate, identify and quantify the carotenoids from sea buckthorn extracts, chromatographic

123 analysis was performed using a Thermo Finnigan Surveyor HPLC system, controlled by

124 Xcalibur software (Finnigan Surveyor LC, Thermo Scientific, USA). The carotenoids were

125 analyzed at 450 nm on a Lichrosorb RP-18 (5 µm) Hibar RT 125-4 column, operated at 30°C.

126 The elution solvents were aqueous 90% acetonitrile (A) and 100% ethyl acetate (B). The

127 injection amount was 20 µL, and the flow was maintained at 0.500 mL/min. The elution profile

6
128 used was the following: 0-16 min, isocratic on 15% B; 16-54 min, linear gradient from 15% to

129 62% B, 54-56 min, isocratic on 62% B; 56–60 min, linear gradient from 62% to 15% B; 60–70

130 min, isocratic on 15% B (Pop et al. 2014). The calibration curve for the β-carotene standard was

131 prepared using six different concentrations (0.04–0.1 mg/mL), and the ethyl acetate was used as

132 solvent. The linear regression factor of the calibration curve was 0.988.

133 2.4. Heat treatment

134 Heating experiments were performed in triplicates by filling Eppendorf tubes (2 mL) with 100

135 µL of extract solutions in 70% ethanol. For the fluorescence and FT-IR spectroscopy

136 experiments, the samples were heated at temperatures ranging from 50 to 100 °C for 10 min. For

137 the thermal degradation kinetic studies, the samples were heated in the same temperature range

138 for different times (0-25 min). All heating experiments were conducted in a thermostatic bath

139 (Raypa Trade BBO-4, Barcelona, Spain). After the thermal treatment, the tubes were

140 immediately cooled in an ice-bath for 2 min to stop thermal degradation.

141 2.5. Spectroscopic measurements

142 Fluorescence measurements were carried out using a LS-55 luminescence spectrometer

143 (PerkinElmer Life Sciences, Shelton, CT, USA) in a 10 x 10 mm quartz cuvette, by diluting 50

144 µL of sample solutions in 2.5 mL of phosphate buffer at pH 4.0. The fluorescence spectra were

145 measured at excitation wavelengths of 250, 340, 410 nm and 448 nm. The scan speed was 500

146 nm/min and the slits had 10 nm.

147 2.6. FT-IR spectroscopy

148 The attenuated total reflectance (ATR) spectra were obtained using a Nicolet iS50 FT-IR

149 spectrometer (ThermoScientific, USA) equipped with a diamond crystal and were plotted

150 between 4000 and 400 cm-1. As reference, the background spectrum of air was collected. The

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151 TCC and TPC were measured without any preparation, directly on the ZnSe ATR crystal.

152 Between measurements, the ATR crystal was carefully cleaned using acetone.

153 2.7. Determination of total carotenoids content

154 TCC were analyzed using the colorimetric method described by Ranjith et al. (2006). Briefly, 1

155 ml of aliquot of extract in ethanol was added to 0.5 ml of 0.05 g/L NaCl, vortexed for 30 s, and

156 centrifuged at 1500 g for 10 min. The supernatant was diluted, and the absorbance at 460 nm was

157 measured. The amount of TCC was calculated by plotting a calibration curve with β-carotene as

158 standard (0-0.5 mg/mL).

159 2.8. Determination of total phenolic, flavonoid content and free radical scavenging

160 activity

161 TPC, TFC and DPPH RSA of the extract were estimated as reported earlier (Turturică, Stănciuc,

162 Bahrim, & Râpeanu, 2016).

163 2.9. Kinetic data analysis

164 The degradation kinetics of TCC, TPC, TFC and DPPH RSA were described by fitting the

165 experimental results to the first-order fractional conversion kinetic model. In this model, the

166 changes in phytochemicals content and DPPH RSA (Ct) as a function of heating time were

167 described by Eq. 1:

168 (1)

169 where C∞ is the equilibrium value at infinite heating time (the value after which longer heating

170 time does not result in changes in C value), and Ci is the phytochemicals content and DPPH RSA

171 values of the samples at time 0 of thermal treatment (initial content). The kinetic parameters

172 were estimated using SAS software package (SAS Institute Inc., Cary, NC, USA).

8
173 The Arrhenius model was used to describe the temperature dependence of degradation rate

174 constants as described by Turturică et al. (2016).

175 2.10. Statistical analysis of data

176 All experiments were performed in triplicates. Results are expressed as mean ± SD. Statistical

177 comparisons were made by one-way analysis of variance (ANOVA). Differences were

178 considered to be significant when the p values were <0.05.

179 3. Results and discussion

180 3.1. Physical characteristics and phytochemicals content of the extract

181 The physical characteristics of the sea buckthorn berries were 86.37±0.89% moisture, pH of

182 2.53±0.03 and 1.69±0.05 titratable acidity expressed in % malic acid. Further, HPLC analysis of

183 extract was used to identify free carotenoids and carotenoid esters. Figure 1 shows a typical

184 HPLC chromatogram for carotenoids in sea buckthorn extracts. Carotenoids identification was

185 performed based on their retention times and comparisons with data from the literature. Twelve

186 compounds were identified as follow: astaxanthin (peak 1), zeaxanthin (peak 2), zeaxanthin-

187 palmitate (peak 3), γ-carotene (peak 4), cis β-carotene (peak 5), β-cryptoxanthin (peak 6),

188 lycopene (peak 7), lutein-palmitate-myristate (peak 8), lutein di-palmitate (peak 9), β-carotene

189 (peak 10), α-carotene (peak 11), and zeaxanthin di-palmitate (peak 12). Among the carotenoids,

190 β-carotene displayed a content of 15.19 mg/g DW, followed by astaxanthin with 11.94 mg/g

191 DW, β-cryptoxanthin with 8.93 mg/g DW and lycopene with 2.24 mg/g DW, whereas zeaxanthin

192 was found in the highest concentration of 81.29 mg/g DW, respectively.

193 The results are in line with those reported by Pop et al. (2014) who found a content of β-carotene

194 ranging from 1.9 to 7.4 mg/100 g DW, β-cryptoxanthin (1.3-1.6 mg/100g DW), lycopene (1.4-

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195 2.3 mg/100g DW) and zeaxanthin (1.8-2.5 mg/100g DW) in six Romanian sea buckthorn

196 varieties.

197 The relative mean amounts of different carotenoids in sea buckthorn berries were reported as:

198 1% lutein, 8% zeaxanthin, 0.3% β-cryptoxanthin, 8% lycopene, 4% γ-carotene, 14% β-carotene

199 and 10 % minor carotenoids (Andersson, 2009).

200 Sea buckthorn extract revealed a TCC of 38.34±5.71 mg β-carotene/g DW. Korekar et al. (2014)

201 reported a variation in TCC from 0.1 to 14.4 mg/100 g FW in seventeen natural population of sea

202 buckthorn from trans-Himalaya. Andersson et al. (2009) studied the carotenoids in sea buckthorn

203 berries during ripening and suggested a variation between 1.5 and 18.5 mg/100 g FW. These

204 authors suggested large variations in carotenoid content, as a function of sea buckthorn origin,

205 growth condition and subspecies (e.g. 0.27 mg/g DW in Hippophae neurocarpa from China, and

206 1.05 mg/g DW in ssp. turkestanica from Kyrgyzstan). Pop et al. (2014) identified twenty-seven

207 carotenoids in sea buckthorn berries, as follow: free carotenoids represented by lutein,

208 zeaxanthin, β-cryptoxanthin, α-carotene, γ-carotene, β-carotene and lycopene; the carotenoid

209 esters fraction was mainly represented by the zeaxanthin esters, followed by the lutein esters and

210 cryptoxanthin esters. From a quantitative point a view, variations in TCC between 0.53 and 0.97

211 mg/g DW in berries, and between 0.035 and 0.042 mg/g DW in leaves were reported.

212 In comparison with other fruits and berries, sea buckthorn berries have a relatively high content

213 of carotenoids. The esterified carotenoids corresponded to 55% of the total carotenoids content

214 (Andersson, 2009).

215 Phenolics are the major plant compounds that exhibit antioxidant activity. This activity is

216 believed to be mainly due to redox properties, which play important role in adsorbing and

217 neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides. Sea

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218 buckthorn extract revealed TPC of 140.14±6.64 mg GAE/g DW. Korekar et al. (2014) reported

219 TPC ranging from 9.64 to 107.04 mg GAE/g DW. Kumar, Dutta, Prasad, & Misra (2011) tested

220 different methods for TPC extraction from sea buckthorn and reported values varying between

221 28.35 ± 1.31 (mg/g) at maceration, to 93.72 ± 2.83 (mg/g) at subcritical water extraction at

222 100°C.

223 Flavonoids represent a class of benzo-γ-pyrone derivatives including flavones, flavanes,

224 flavonols, anthocyanidines, and catechins. It has been reported that sea buckthorn is a natural

225 source of flavonoids, which have been claimed to lower cholesterol, platelet aggregation, blood

226 pressure and blood sugar. They possess a wide spectrum of biological activities such as

227 anticancer, antibacterial, antifungal, antiviral, spasmolytic, hypoglycemic, antihistaminic and

228 radioprotective potential (Jagtap et al., 2009).

229 The TFC in sea buckthorn extract was 5.04±0.05 mg CE/g DW, which is close to the TFC of

230 6.79 ± 0.30 mg rutin/g in 70% ethanol reported by Chauhan & Varshneya (2012). These authors

231 identified isorhamnetin-rutinoside (355 mg/L) and quercetin-glycoside (35 mg/L) as the main

232 flavonoids. Kumar et al. (2011) reported higher TFC values, depending on the method of

233 extraction. Thus, the TFC of 14.14±1.12 mg rutin equivalents/g DW was found in the buckthorn

234 leaves extract resulting from maceration, and of 66.40±2.36 mg rutin equivalents/g DW in case

235 of the subcritical water extraction method at 100°C.

236 The reducing ability of a compound generally depends on the presence of reductants which have

237 exhibited antioxidant potential by breaking the free radical chain and donating a hydrogen atom

238 (Turturică et al. 2016). In our study, the extract showed an initial DPPH RSA of 33.70±0.29%,

239 corresponding to 2.50±0.02 µM Trolox/g DW. Kumar et al. (2011) reported values ranging from

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240 86.35±2.93 mg Trolox eqt./g for maceration method, to 343.86±7.71 mg Trolox eqt./g for the

241 subcritical water extraction at 200°C.

242 3.2. Fluorescence spectra

243 The spectra given in Figure 2 illustrate the fluorescence properties of the sea buckthorn extracts.

244 The maximum of fluorescence emission in these spectra were located at 369 nm, 527 nm, 550

245 nm and 600 nm, suggesting the presence of at least four fluorescent species in the extract. Our

246 observations comply with those of Sikorska et al. (2004), who showed that when solutions were

247 excited at 250 nm and 340 nm, spectra with specific structure were obtained, the fluorescence

248 maximum positioned at 369 nm and 527 nm, being specific to phenols and tocopherols and

249 tocotrienols, respectively. Only few polyphenols exhibit natural fluorescence, including

250 isoflavones without an OH group at position 5, and flavonoids with an OH group at position 3,

251 such as catechin and methoxylated flavones (Lamuela-Raventos, Vallverdu-Queralt, Jaurequi,

252 Martinez-Huelamo, & Quifer-Rada, 2014). Due to their structural similarity, all these

253 compounds may exhibit very similar UV-absorption spectra and have similar fluorescence

254 properties. Sea buckthorn contains a considerable number of minor components belonging to

255 different classes of phenolic compounds, such as phenols, phenolic acids and flavonoids. Since

256 most of polyphenols are fluorescent, it can be concluded that absorption in the range 260-310 nm

257 and emission in the near-UV range, with bands centered at 310-370 nm, are specific for this class

258 of compounds (Zandomeneghi, Carbonaro, & Caffarata, 2005). Similar conclusions regarding

259 the spectral contribution of both tocopherols and phenolic compounds were reported by

260 Sikorska, Khmelinskii, & Sikorski (2012) when studying the fluorescence properties of extra

261 virgin olive oil.

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262 Kyriakidis & Skarkalis (2000) suggested that the bands in the emission spectrum (λex=365 nm)

263 with the maximum at 525 nm may partly originate from compounds belonging to the vitamin E

264 group, or their derivatives formed upon oxidation.

265 The third class of fluorescent compounds, which exhibited a specific emission spectrum at

266 excitation wavelength of 410 nm and emission maximum at 550 nm, belongs to flavonoids.

267 Baran et al. (2011) used spectrofluorimetric measurements to evaluate the emission/excitation

268 spectra of intracellular quercetin, who displayed a dominant emission maximum at ~540 nm and

269 two different excitation maxima at ~380 nm and ~440 nm, respectively.

270 The fourth emission spectrum obtained at excitation wavelength of 448 nm with a maximum at

271 600 nm (Figure 2), denotes the carotenoids contribution to the fluorescent properties of sea

272 buckthorn extracts. Similar observations are reported by Pawlak, Szurkowski, Skrzypczak, &

273 Bialek-Bylka (2013) who investigated the fluorescence emission of all-trans and 15-cis β-

274 carotene in imidazolium based room temperature ionic liquids. These authors suggested that

275 monomeric form of β-carotene have a maximum emission at 525 nm, whereas the crystal and

276 aggregated forms have maximum emission at 564 nm and 608 nm, respectively, when the sample

277 was excited at 413 nm.

278 Heating of sea buckthorn extracts resulted in structural changes that led to a slight increase in

279 fluorescence intensity, except for the sample treated at 80ºC. Small red- and blue-shifts were

280 registered in the maximum fluorescence intensity wavelengths (λmax) due to heating. For

281 example, a small 3 nm red shift was obtained at 100ºC when excited at 250 nm. A blue-shift of 5

282 nm was registered in case of the sample heated at temperature of 100ºC, when the extract

283 solutions were excited at 340 nm. When exciting at 410 nm, the increase of the temperature up to

284 90 and 100ºC resulted in red-shifts in λmax of 3 nm and 6 nm, respectively. No significant heat-
13
285 induced changes were recorded in the maximum emission wavelength, when exciting at 448 nm.

286 Based on λmax variation, it can be concluded that thermal treatment induced sequential structural

287 changes in phytochemicals structure.

288 3.3. The IR spectrum

289 The IR spectrum of the sea buckthorn extract is predictably complex. Characteristic peaks

290 include ethers and lactones (880 cm-1, 1045 cm-1, 1087 cm-1, 2880 cm-1), H bonded O-H and N-H

291 stretching (3320 cm-1), phenols (1274 cm-1), C=O in esters, dicarboxylic acids and α-amino acids

292 (1745 cm-1), C-H and CH2 stretching and deformation vibration (1454 cm-1, 2880 cm-1) (Pretsch,

293 Bühlmann, & Badertscher, 2009). The ATR of the samples revealed a generally high thermal

294 stability. Therefore, heating at 50°C produced an overall weakening of peaks (Figure 3) and a

295 slight increase at 1160 cm-1, that could be attributed to C-O ester group vibration (Trif, Ansorge-

296 Schumacher, & Socaciu, 2001). A weak band was registered in the fingerprint region of the

297 samples heated at 60-100°C, at 1419 cm-1, becoming stronger with the temperature increase.

298 Keto-enolic tautomerism could also be observed, with the significant reduction of the carbonyl

299 peak (1745 cm-1) at 70°C and 80°C. This characteristic band appeared again in the samples

300 exposed to higher temperatures (90°C and 100°C), as the keto form could be favored by

301 temperature increases (Laurella, Sierra, Furlong, & Allegretti, 2013). Another effect of the

302 thermal treatment was observed in the narrowing and weakening of the 3320 cm-1 band, possibly

303 caused by the breaking of hydrogen bonds. Due to the complexity of the spectrum, the expected

304 oxidation of ascorbic acid in the samples heated at lower temperatures (Munyaka et al. 2010)

305 could not be observed.

306

307

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308 3.4. The effect of heating on phytochemicals content

309 Current knowledge indicates that heat processing may affect phytochemicals levels in fruit

310 products, thus it is important to consider the heating impact and to evaluate the potential loss in

311 bioactive compounds and, therefore in bioavailability. Figure 4 shows the correlation between

312 thermal degradation of DPPH RSA and TCC, TPC or TFC at 60°C, which resulted to be highly

313 correlated (R2 = 0.97, P < 0.01 for TCC, R2 = 0.96, P < 0.01 for TPC, R2 = 0.93, P < 0.01 for

314 TFC). At each tested temperature, correlation analysis data revealed that DPPH RSA of the

315 extracts is strongly dependent on the phytochemical content (data not shown).

316 For all studied temperatures, heating resulted in significant loss of phytochemical compounds.

317 For example, a reduction between 22% and 42% was observed in TCC in the whole temperature-

318 time combinations studied (Figure 5, a). Ahmed, Shivhare, & Sandhu (2013) reported a decrease

319 in the carotenoids content during thermal treatment of fresh papaya puree, and the corresponding

320 values at 70, 80, 90, and 105°C after 3 h were 34.73, 27.11, 20.45, and 16.54 mg/L, respectively.

321 Benlloch-Tinoco et al. (2015) suggested a TCC reduction by 67±7% in case of pasteurized kiwi

322 puree during storage, highlighting that neoxanthin (loss of 91%) and lutein (loss of 62%) were

323 the most and the least thermolabile compounds. Mertz, Brat, Caris-Veyrat, & Gunata (2010)

324 reported that increasing temperatures led to significant losses in some carotenoids of tamarillo

325 fruit, such as zeaxanthin and β-carotene. Esters of zeaxanthin and β-cryptoxanthin were less

326 affected than the corresponding free xanthophylls. Von Doering, Sotiriou-Leventis, & Roth

327 (1995) suggested that bellow 100°C, the heat treatment of all trans β-carotene induced the

328 formation of 13- and 15-cis-β-carotene, whereas 9-cis-β-carotene was formed above 100°C. 13-

329 cis-β-carotene was the only isomer detected by Mertz et al. (2010) in tamarillo fruit. A

330 mechanism for thermal carotenoid degradation was proposed by Zepka et al. (2009), involving

15
331 parallel irreversible and reversible coupled reactions of the initial all-trans-β-cryptoxanthin and

332 all-trans-β-carotene to yield, respectively, degradation compounds and mono-cis isomers.

333 Therefore, thermal lability seems to be influenced by both experimental conditions and nature of

334 food matrices.

335 The antioxidant activity of plant foods is mainly related to their content of polyphenols. Heating

336 the extract solutions at 60°C for 5 to 20 minutes resulted in TPC reduction ranging from 85% to

337 89% (Figure 5, b). Increasing the temperature at 100°C up to 25 minutes resulted in a more

338 severe reduction of 95%, when compared with untreated extracts. Our results are in good

339 agreement with those suggested by Jaiswal, Gupta, Abu-Ghannam (2012), who studied the effect

340 of blanching on the TPC content of Irish York cabbage. These authors reported that for all

341 studied temperatures, blanching for up to 2 min resulted in a severe loss of phenolic compounds.

342 Therefore, a reduction of 43.5–45.4% was observed at lower blanching temperatures (80, 85 and

343 90ºC), whereas 47.3–50.4% reduction was observed at 95 and 100ºC after 2 min. The

344 degradation continued up to 6 min of blanching resulting in a reduction of 63.9–65.3% at 80-

345 90ºC and 69.1–76.5% at 95 and 100ºC, as compared to fresh cabbage.

346 The changes in the TFC of sea buckthorn extract after heat treatment from 60°C to 100°C as a

347 function of heating time are presented in Figure 5 (c). TFC showed similar trends in the whole

348 temperature range studied. TFC reduction of 77.91%, 79.46%, 81.52%, 86.33% and 90.04% was

349 observed as a result of heating at 60, 70, 80, 90 and 100°C, respectively after 25 min. Ahmed &

350 Ali (2013) noted the highest reduction in TFC of white cauliflower after water boiling (56.39%)

351 followed by water blanching (43.42%) and stir-frying (30.23%).

352 The effect of heating on DPPH RSA of extract is presented in Figure 5 (d). Similar to the

353 phytochemicals content, heating up to 5 min in the temperature range of 70-90°C, caused a loss

16
354 ranging from 5 to 29% in DPPH RSA. The rate of loss gradually increased with the increase of

355 heating time, reaching a maximum of 44% after 25 min at 100°C. The decrease could be due to

356 the degradation of phenolic compounds during heating (Turturică et al. 2016). In particular

357 flavonoids are easily available to leach into processing water, while hydroxycinnamic acid

358 derivatives (caffeic, p-coumaric and ferulic acids) require higher temperatures in order to be

359 released. Jaiswal et al. (2012) reported a reduction of 60-65% after 6 min of blanching in the

360 temperature range of 80-90°C. Ahmed & Ali (2009) suggested a reduction of approximately 49%

361 in DPPH RSA for the methanolic extract of fresh cauliflower heat treated in water at temperature

362 of 100ºC for 3 min.

363

364 3.5. Kinetic analysis of phytochemicals content

365 In order to design new food processing techniques and safe food products with high nutritional

366 and quality attributes, the knowledge of thermal degradation mechanisms of the phytochemicals

367 in food is essential. Patras, Brunton, O′Donnell, & Tiwari (2010) emphasized the importance of

368 knowing the degradation kinetics, including reaction order, rate constant and activation energy,

369 with great impact in predicting food quality loss during thermal process treatments. Most

370 previous studies have used first-order kinetic model to describe thermal degradation mechanisms

371 of phytochemicals in general. However, Borsarelli & Mercande (2010) reported that the kinetic

372 profile for the degradation of carotenoid is not fitted well by a first-order equation. These authors

373 suggested the use of a bi-exponential equation, which led to an adequate estimation of kinetic

374 parameters. In our study, degradation kinetics of the phytochemicals content in heat-treated sea

375 buckthorn extracts was modeled by fractional conversion kinetic model. To verify the validity of

376 the model and to measure the linearity, coefficients of determination (R2) were calculated and

17
377 residual plots checked for the absence of trends or correlations. The model was appropriate for

378 the data, since residuals represented only the experimental errors and were randomly distributed

379 when plotted. In all cases, there was a good correlation between experimental and predicted

380 values (Figure 6).

381 The degradation rate constants and activation energies are presented in Table 1. In case of TCC,

382 the degradation rate constant increased from 17.83 ± 2.85·10-2 1/min at 60°C to 24.02 ± 3.67·10-
2
383 1/min at 100°C. For TPC, the estimated k value showed values ranging from 31.27 ± 3.46·10-2

384 1/min to 34.27 ± 6.19·10-2 1/min, as a result of temperature increase from 60 to 100°C. The

385 degradation rate of TFC and DPPH RSA increased from 14.13 ± 1.89·10-2 1/min and 16.30 ±

386 3.09·10-2 1/min at 60°C to 38.47 ± 13.30·10-2 1/min and 30.52 ± 6.40·10-2 1/min at 100 °C,

387 respectively (Table 1).

388 Thermal degradation of carotenoids was assumed to follow a 1st-order rate indicating a

389 logarithmic order of inactivation. Aparicio-Ruiz, Mínguez-Mosquera, & Gandul-Rojasa (2011)

390 demonstrated that the first-order mechanism is appropriate for describing the thermal

391 degradation of β-carotene, β-cryptoxanthin and lutein in virgin olive oils, with k values ranging

392 from 17.16±0.33 1/min at 60ºC to 188.66±5.66 1/min at 100ºC for β-carotene degradation.

393 Ahmed et al. (2002) used the first order kinetic model to describe the thermal degradation of

394 carotenoids in papaya puree and reported k values of 13.1·10-2 1/min at 70ºC and of 37.80· 10-2

395 1/min at 105ºC. Pérez-Gálvez, Jarén-Galán, & Mınguez-Mosquera (2005) suggested that the

396 cyclization of polyolefins could be considered as the general reaction pathway in thermally

397 induced reactions, yielding xylene as byproduct and the corresponding nor-carotenoids. The

398 thermolability of carotenoids to heat is due to their conjugated double bonds. When intense heat

399 is applied, the tiny structures are cleaved and molecular reactions occur, involving the double

18
400 bonds. Ty & Cho (1999) suggested that two types of thermal degradation products are formed: a

401 volatile fraction of low molecular weight molecules, which is vaporized, and a non-volatile

402 fraction from the larger fragments of the carotene molecules after cleaving off the volatile

403 fraction.

404 Degradation kinetics of the TPC in Irish York cabbage after blanching treatment was modeled by

405 Jaiswal et al. (2012) using first-order and fractional conversion first-order kinetics model. These

406 authors reported an increase in the first order degradation rate constant from 14.90±0.00·10-2

407 1/min to 20.30±2.00·10-2 1/min, when the temperature increased from 80°C to 100°C. The

408 corresponding fractional conversion degradation rate constants were 37.90±0.00·10-2 1/min and

409 48.40±4.00·10-2 1/min. Turturică et al. (2016) also suggested a first order kinetic model for the

410 thermal degradation of TPC in plum extract, with an increase in k values from 0.7±0.1·10-2 1/min

411 at 70º C to 2.90 ± 0.7·10-2 1/min at 110ºC.

412 Zorić et al. (2014) reported a first-order degradation for quercetin-3-glucoside in sour cherry

413 marasca paste, with k values ranging from 1.5 to 2.6·10–2 1/min, as a result of temperature

414 increase from 80 to 120°C. Kinetic analysis of DPPH RSA using the first-order model was also

415 performed by Jaiswal et al. (2012), who reported values for k ranging from 15.90±3.00·10-2

416 1/min at 80ºC to 18.60±4.00·10-2 1/min at 100ºC. The degradation constant rate values were

417 higher when the fractional conversion model was used to describe the thermal degradation of

418 DPPH RSA after blanching (26.90±3.00·10-2 1/min at 80ºC and 41.40 ± 4.00·10-2 1/min at

419 100ºC). Significantly lower values were reported by Turturică et al. (2016), who found an

420 increase in k values from 0.4 ± 0.2·10-2 1/min at 70ºC to 2.1 ± 0.1·10-2 1/min at 110ºC.

421 The temperature dependence of rate constants was expressed by the Arrhenius equation. The

422 activation energies are presented in Table 1. Ea values were 8.45±0.93 kJ/mol, 2.50±0.66

19
423 kJ/mol, 22.50±7.26 kJ/mol and 15.22±2.75 kJ/mol for TCC, TPC, TFC, and DPPH RSA,

424 respectively. The obtained activation energies are rather low compared to the typical activation

425 energy (about 100 kJ/mol) of a chemical reaction. Boon, Mc Clements, Weiss, & Decker (2010)

426 explained that radical intermediates play an important role during carotenoid degradation,

427 whereas radical reactions are only slightly temperature dependent.

428 The activation energy value for degradation of TCC is lower compared with the results reported

429 by Ahmed et al. (2002) for the thermal degradation of carotenoids in papaya puree (20.56

430 kJ/mol). Aparicio-Ruiz et al. (2011) reported Ea value of 14.77±0.06 kJ/mol for thermal

431 degradation of β-carotene in virgin olive oils in the temperature range of 60-120ºC. For the

432 thermal degradation of TPC, TFC and DPPH RSA in Irish York cabbage, Jaiswal et al. (2012)

433 reported Ea values of 11.54, 9.22 and 22.37 kJ/mol, respectively, whereas Turturică et al. (2016)

434 suggested values of 36.0 ± 8.0 kJ/mol, 18.0 ± 2.0 and 48.0 ± 6.0 kJ/mol in plum extract.

435

436 Conclusions

437 The influence of thermal treatment was used to quantitatively describe the impact of processing

438 on bioactive compounds in sea buckthorn fruits extract. The structural complexity of the sea

439 buckthorn extract was emphasized by fluorescence and FT-IR spectroscopy techniques. Four

440 major classes of compounds were identified as follow: phenols, flavonoids, tocopherols and

441 carotenoids. The IR spectrum revealed also the complexity of the sea buckthorn extract,

442 involving characteristic peaks for ethers and lactones, H bonded O-H and N-H stretching,

443 phenols, C=O in esters, dicarboxilic acids and α-aminoacids, C-H and CH2 stretching and

444 deformation vibrations. Heat treatment caused changes in maximum emission, indicating the

20
445 sequential character of structural changes of different phytochemicals, whereas the ATR of the

446 samples revealed a good thermostability of the compounds at high temperatures.

447 Degradation kinetic of the phytochemicals content in heat-treated sea buckthorn extract was

448 modeled by using fractional conversion model. Variation of degradation rate constants with

449 temperature was described by the Arrhenius equation. The calculated values of the activation

450 energy for different phytochemical content revealed that, the carotenoids and polyphenols, are

451 more heat stable, whereas the flavonoids presented the lowest thermal stability. Our study

452 provides important knowledge regarding the kinetics of phytochemicals degradation, which will

453 facilitate the optimization of the thermal treatment in food industry in terms of food safety and

454 functionality.

455

456 Acknowledgements

457 This work was supported by a grant of the Romanian National Authority for Scientific Research

458 and Innovation, CNCS-UEFISCDI, project number PN-II-RU-TE-2014-4-0115.

459

460 References

461 1. Ahmed, F.A., & Ali, R.F.M. (2013). Bioactive compounds and antioxidant activity of

462 fresh and processed white cauliflower. BioMed Research International, Article ID

463 367819. http://dx.doi.org/10.1155/2013/367819.

464 2. Ahmed, J., Shivhare, U.S., & Sandhu, K.S. (2002). Thermal degradation kinetics of

465 carotenoids and visual color of papaya puree. Journal of Food Science, 67, 2692-2695.

466 3. Andersson, S.T. (2009). Carotenoids, tocochromanols and chlorophylls in sea buckthorn

467 berries (Hippophae rhamnoides) and rose hips (Rosa sp.). Variation during ripening, and

21
468 among cultivars/Species and Years. Doctoral Thesis, Swedish University of Agricultural

469 Sciences, Alnarp.

470 4. Andersson, S.C., Olsson, M.E., Johansson, E., & Rumpunen, K. (2009). Carotenoids in

471 sea buckthorn (Hippophae rhamnoides L.) berries during ripening and use of pheophytin

472 a as a maturity marker. Journal of Agricultural and Food Chemistry, 14, 250-258.

473 5. Araya-Farias, M., Makhlouf, J., & Ratti, C., (2011). Drying of sea buckthorn (Hippophae

474 rhamnoides L.) berry: Impact of dehydration methods on kinetics and quality. Drying

475 Technology, 29, 351–359.

476 6. Association of Official Analytical Chemists. (1984). Official Methods of Analysis of the

477 Association of Official Analytical Chemists. Section 43.014-43.017 Carotenes in Fresh

478 Plant Materials and Silages Spectrophotometric Method Final Action, 14th Edn. The

479 Association of Official Analytical Chemists, Inc., Arlington, VA.

480 7. Aparicio-Ruiz, R., Mínguez-Mosquera, M.I., & Gandul-Rojasa, B. (2011). Thermal

481 degradation kinetics of lutein, β-carotene and β-cryptoxanthin in virgin olive oils. Journal

482 of Food Composition and Analysis, 24, 811–820.

483 8. Baran, I., Ganea, C., Ursu, I., Baran, V., Calinescu, O., Iftimie, A., Ungureanu, R., &

484 Tooloeanu, I.T. (2011). Fluorescence properties of quercetin in human leukemia jurkat t-

485 cells. Romanian Journal of Physics, 56, 388-398.

486 9. Benlloch-Tinoco, M., Kaulmann, A., Corte-Real, J., Rodrigo, D., Martínez-Navarrete, N.,

487 & Bohn, T. (2015). Chlorophylls and carotenoids of kiwifruit puree are affected similarly

488 or less by microwave than by conventional heat processing and storage. Food Chemistry,

489 187, 254–262.

22
490 10. Boon, C.S., Mc Clements, D.J., Weiss, J., & Decker, E.A. (2010). Factors influencing the

491 chemical stability of carotenoids in foods. Critical Reviews in Food Science and

492 Nutrition, 50, 515-532.

493 11. Borsarelli, C.D., & Mercande, A.Z. (2010). Thermal and photochemical degradation of

494 carotenoids. In Carotenoids: Physical, Chemical and Biological Functions and Properties,

495 Landrum, J.T. (Ed.), CRC Press, Taylor and Francis, pg. 249.

496 12. Chauhan, S., & Varshneya, C. (2012). The Profile of Bioactive Compounds in

497 Seabuckthorn: Berries and Seed oil. International Journal of Theoretical and Applied

498 Sciences, 4, 216-220.

499 13. Jagtap, S., Meganathan, K., Wagh, V., Winkler, J., Hescheler, J., & Sachinidis, A.

500 (2009). Chemoprotective mechanism of the natural compounds, epigallocatechin-3-

501 Ogallate, quercetin and curcumin against cancer and cardiovascular diseases. Current

502 Medicinal Chemistry, 16, 1451–1462.

503 14. Jaiswal, A.K., Gupta, S., & Abu-Ghannam, N. (2012). Kinetic evaluation of colour,

504 texture, polyphenols and antioxidant capacity of Irish York cabbage after blanching

505 treatment. Food Chemistry, 131, 63-72.

506 15. Kim, J.S., Kwon, Y.S., Sa, Y.J., & Kim, M.J. (2011). Isolation and identification of sea

507 buckthorn (Hippophae rhamnoides) phenolics with antioxidant activity and - glucosidase

508 inhibitory effect. Journal of Agricultural and Food Chemistry, 59, 138-144.

509 16. Korekar, G., Dolkar, P., Singh, H., Srivastava, R.B., & Stobdan, T. (2014). Variability

510 and the genotypic effect on antioxidant activity, total phenolics, carotenoids and ascorbic

511 acid content in seventeen natural population of sea buckthorn (Hippophae rhamnoides L.)

512 from trans-Himalaya. LWT - Food Science and Technology, 55, 157-162.

23
513 17. Kumar, Y.M.S., Dutta, R., Prasad, D., & Misra, K. (2011). Subcritical water extraction of

514 antioxidant compounds from sea buckthorn (Hippophae rhamnoides) leaves for the

515 comparative evaluation of antioxidant activity. Food Chemistry, 127, 1309-1316.

516 18. Kyriakidis, N. B., & Skarkalis, P. (2000). Fluorescence Spectra Measurement of Olive

517 Oil and Other Vegetable Oils. Journal of AOAC International, 83, 1435-1439.

518 19. Lamuela-Raventos, R.M., Vallverdu-Queralt, A., Jaurequi, O., Martinez-Huelamo, M., &

519 Quifer-Rada, P. (2014). Improved characterization of polyphenols using liquid

520 chromatography. In Watson, R.R. (Ed.) Polyphenols in Plants: Isolation, purification and

521 extract preparation, Academic Press, London, UK.

522 20. Laurella, S.L., Sierra, M.G., Furlong, J.J.P., & Allegretti, P.E. (2013). Substituent,

523 Temperature and Solvent Effects on the Keto-Enol Equilibrium in β-Ketoamides: A

524 Nuclear Magnetic Resonance Study. Open Journal of Physical Chemistry, 3, 138-149.

525 21. Maheshwari, D.T., Kumar, Y.M.S., Verma, S.K., Singh, V.K., & Singh, S.N. (2011).

526 Antioxidant and hepatoprotective activities of phenolic rich fraction of sea buckthorn

527 (Hippophae rhamnoides L.) leaves. Food and Chemical Toxicology, 49, 2422-2428.

528 22. Mertz, C., Brat, P., Caris-Veyrat, C., & Gunata, Z. (2010). Characterization and thermal

529 lability of carotenoids and vitamin C of tamarillo fruit (Solanum betaceum Cav.). Food

530 Chemistry, 119, 653-659.

531 23. Munyaka, A.W., Makule, E.E., Oey, I., Van Loey, A., & Hendrickx, M. (2010). Thermal

532 Stability of L-ascorbic acid and ascorbic acid oxidase in broccoli (Brassica oleracea var

533 italica), Journal of Food Science, 75(4), 36-40.

534 24. Pandey, K.B., & Rizvi, S.I., (2009). Plant polyphenols as dietary antioxidants in human

535 health and disease. Oxidative Medicine and Cellular Longevity, 2, 270-278.

24
536 25. Pang, X., Zhao, J., Zhang, W., Zhuang, X., Wang, J., Xu, R. et al. (2008).

537 Antihypertensive effect of total flavones extracted from seed residues of Hippophae

538 rhamnoides L. in sucrose-fed rats. Journal of Ethnopharmacology, 117, 325-331.

539 26. Patras, A., Brunton, N.P., O′Donnell, C., & Tiwari, B.K. (2010). Effect of thermal

540 processing on anthocyanin stability in foods; mechanisms and kinetics of degradation.

541 Trends in Food Science & Technology, 21, 3-11.

542 27. Pawlak, K., Szurkowski, J., Skrzypczak, A., & Bialek-Bylka, G.E. (2015). The thermal

543 deactivation of all-trans and 15-cis beta-carotene excited states in the ionic liquids

544 without and with methylenoxy group. Journal of Thermal Analysis and Calorimetry, 120,

545 627-632.

546 28. Pérez-Gálvez, A., Rios, J.J., & Mínguez-Mosquera, M.I. (2005). Thermal degradation

547 products formed from carotenoids during a heat-induced degradation process of paprika

548 oleoresins (Capsicum annuum L.). Journal of Agricultural and Food Chemistry, 53,

549 4820-6.

550 29. Pop, R.M., Weesepoel, Y., Socaciu, C., Pintea, A., Vincken, J.P., & Gruppen, H. (2014).

551 Carotenoid composition of berries and leaves from six Romanian sea buckthorn

552 (Hippophae rhamnoides L.) varieties. Food Chemistry, 147, 1-9.

553 30. Pretsch, E., Bühlmann, P., & Badertscher, M. (2009). Structure Determination of Organic

554 Compounds, Springer-Verlag Berlin Heidelberg, p. 269-332.

555 31. Ranjith, A., Kumar, K.S., Venogupalan, V.V., Arumughan, V., Shawney, R.S., & Singh,

556 V. (2006). Fatty acids, tocols, and carotenoids in pulp oil of three sea buckthorn species

557 (Hippophae rhamnoides, H. salicifolia, and H. tibetana) grown in the Indian Himalayas.

558 Journal of American Oil Chemists Society, 83, 359-364.

25
559 32. Rösch, D., Bergmann, M., Knorr, D., & Kroh, L.W. (2003). Structure-antioxidant

560 efficiency relationships of phenolic compounds and their contribution to the antioxidant

561 activity of sea buckthorn juice. Journal of Agricultural and Food Chemistry, 51, 4233-

562 4239.

563 33. Sikorska, E., Khmelinskii I., & Sikorski, M. (2012). Analysis of Olive Oils by

564 Fluorescence Spectroscopy: Methods and Applications, Olive Oil - Constituents, Quality,

565 Health Properties and Bioconversions, Dr. Dimitrios Boskou (Ed.), InTech.

566 34. Sikorska, E., Romaniuk, A., Khmelinskii, I.V., Herance, R., Bourdelande, J.L., Sikorski,

567 M., & Koziol, J. (2004). Characterization of Edible Oils Using Total Luminescence

568 Spectroscopy. Journal of Fluorescence, 14, 25-35.

569 35. Trif, M., Ansorge-Schumacher, M., & Socaciu, C. (2001). Application of FTIR

570 Spectroscopy for determination of oxidation of encapsulated sea buckthorn oil, XVth

571 International Workshop on Bioencapsulation, Vienna, 6-8, P3-07, p. 2.

572 36. Ty, B.P., Y., M., & Choo, Y. M. (1999). Oxidation and thermal degradation of

573 carotenoids. Journal of Oil Palm Research, 1, 62-78.

574 37. Turturică, M., Stănciuc, N., Bahrim, G., & Râpeanu, R. (2016). Effect of thermal

575 treatment on phenolic compounds from plum (Prunus domestica) extracts – a kinetic

576 study. Journal of Food Engineering, 171, 200-207.

577 38. Zadernowski, R., Naczk, M., Czaplicki, S., Rubinskiene, M., & Szalkiewicz, M. (2005).

578 Composition of phenolic acids in sea buckthorn (Hippophaë rhamnoides L.) berries.

579 Journal of the American Oil Chemists' Society, 82, 175-179.

580 39. Zandomeneghi, M., Carbonaro, L., & Caffarata, C. (2005). Fluorescence of vegetable

581 oils: olive oils. Journal of Agricultural and Food Chemistry, 53, 759-766.

26
582 40. Zepka, Q., Borsarelli, L., Aparecida Azevedo Pereira da Silva, C.D., Zerlotti M., &

583 Mercadante, M. (2009). Thermal degradation kinetics of carotenoids in a cashew apple

584 juice model and its impact on the system color. Journal of Agricultural and Food

585 Chemistry, 57, 7841-7845.

586 41. Zorić, Z., Dragović-Uzelac, V., Pedisić, S., Kurtanjek, Z., & Garofulić, I.E. (2014).

587 Kinetics of the degradation of anthocyanins, phenolic acids and flavonols during heat

588 treatments of freeze-dried sour cherry marasca paste. Food Technology and

589 Biotechnology, 52, 101-108.

590 42. Von Doering, W. E., Sotiriou-Leventis, C., & Roth, W.R. (1995). Thermal

591 interconversions among 15-cis-, 13-cis-, and all-trans-β-carotene: kinetics, Arrhenius

592 parameters, thermochemistry, and potential relevance to anticarcinogenity of all-trans-β-

593 carotene. Journal of the American Chemical Society, 117, 2747-2757.

594 43. Yu, K., Wu, Y., Hu, Z., Cui, S., & Yu, X. (2011) Modeling thermal degradation of litchi

595 texture: Comparison of WeLL model and conventional methods. Food Research

596 International, 44, 1970-1976.

597

27
598 Figure 1. Representative HPLC fingerprint of carotenoids from sea buckthorn extracts recorded

599 at 450 nm

600

480000
2
430000
380000
330000
Absorbance (a.u.)

280000
230000 11
180000
130000 9
80000 1 5 10
6 8
30000
3 4 7 12
-20000
0 10 20 30 40 50 60
Retention time (min)
601

602

603

604

605

606

607

608

609

610

611

612

613
28
614 Figure 2. Fluorescence spectra of sea buckthorn extract at different excitation wavelengths.

615 Three independent tests were carried out in each case and SD was lower than 3.5%.

600 250 nm
Fluorescence intensity (a.u.)
500
340 nm
400

300
410 nm
200 448 nm

100

0
300 350 400 450 500 550 600 650 700
Wavelengths (nm)
616

617

618

619

620

621

622

623

624

625

626

627

628

629

630

29
631

632 Figure 3. Superimposed ATR spectra of samples after thermal treatment. a – 50°C, b – 100°C

633

634

635

636

637

638

639

640

641

642

643

644

645

646
30
647

648 Figure 4. Correlation between the thermal degradation at 60˚C in DPPH RSA and TCC

649 (triangles), TPC (squares), and TFC (diamonds)

1
0,9
Phytochemicals content

0,8
(expressed as C/C0)

0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
0,85 0,87 0,89 0,91 0,93 0,95 0,97 0,99

DPPH RSA (expressed as C/C0)


650

651

652

653

654

655

656

657

658

659

660

661

662

663

31
664 Figure 5. Isothermal degradation of TCC (a), TPC (b), TFC (c) and DPPH RSC (d) in sea

665 buckthorn extract, treated at different temperatures ( 60°C,  70,  80°C,  90°C and ∆

666 100°C). The solid lines represent fractional conversion model fits to experimental data (Eq. 1).

667

668 10
9,5
TCC (mg β-carotene/g FW)

669 9
8,5
670 a)
8
671 7,5
7
672 6,5
6
673
5,5
674 0 5 10 15 20 25
Heating time (min)
675

676
30
677 25
TPC (mg GAE/ g FW)

678 20

679 b) 15

680 10

681 5

0
682
0 5 10 15 20 25
683 Heating time (min)

684

685

686

687

688
32
689
0,1
690 0,09
0,08

TFC (mg CE/g FW)


691 0,07
0,06
692 c) 0,05
0,04
693 0,03
0,02
694 0,01
0
695
0 5 10 15 20 25
696 Heating time (min)

697

698
35
699 d) 33
31
700 29
DPPH-RSA (%)

27
701 25
23
702 21
19
703
17
704 15
0 5 10 15 20 25
705 Heating time (min)

706

707

708

709

710

711

712

33
713 Figure 6. Correlation between the predicted and experimental C/C0 values of sea buckthorn

714 extract simulated using eq. (1).

715
1
Predicted values (expressed as C/C0)

0,9
716
0,8
717 0,7
0,6
718 0,5
0,4
719 0,3
0,2
720 0,1
0
721 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1
Experimental values (expressed as C/C0 )
722 TCC TPC TFC DPPH-RSA

723

724

725

726

727

728

729

34
730 Table 1. Estimated kinetic parameters (k) and activation energy of the sea buckthorn phytochemicals

731 content

Temperature TCC TPC TFC DPPH RSA

°C k·10-2 (min- k·10-2 (min- k·10-2 (min-1) k·10-2 (min-


1
R2 1
R2 R2 1
R2
) ) )

17.83 ± 0.978 31.27 ± 0.989 0.988 16.30 ± 0.989


60 a
14.13 ± 1.89
2.85 3.46 3.09

0.985 31.40 ± 0.989 0.979 23.18 ± 0.979


70 18.83 ± 2.78 15.47 ± 3.01
3.43 7.26

0.976 31.51 ± 0.990 0.992 24.21 ± 0.932


80 21.42 ± 3.18 18.32 ± 1.56
3.17 9.37

0.969 33.42 ± 0.986 0.993 28.53 ± 0.961


90 23.47 ± 4.48 19.04 ± 1.57
4.18 5.84

0.978 34.27 ± 0.985 0.983 30.52 ± 0.928


100 24.02 ± 3.67 38.47 ± 13.30
6.19 6.40

Ea (kJ·mol-1) 8.45 ± 0.93 0.964 2.50 ± 0.66 0.827 22.50 ± 7.26 0.761 15.22 ± 0.910
2.75

a
732 Standard error

733

35
734 Research highlights

735 Impact of thermal treatment on phytochemicals from sea buckthorn was investigated.

736 Fluorescence spectra of extracts suggested the presence of at least four species.

737 The ATR revealed a generally high thermal stability.

738 During heating, phytochemicals degradation followed a fractional conversion model.

739 The carotenoids and polyphenols were more heat stable.

740

741

36