Development, Quantification, Method Validation, and Stability Study of

Publication Date (Web): July 25, 2016 ... The current method demonstrated good linearity over the range of 0.125–100 μg/mL fucoxanthin with R2 = 1...
0 downloads 0 Views 832KB Size
Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Development, Quantification, Method Validation and Stability Study of a Novel Fucoxanthin-Fortified Milk Il-Kyoon Mok, Jungro Yoon, Cheol-Ho Pan, and Sang Min Kim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02206 • Publication Date (Web): 25 Jul 2016 Downloaded from http://pubs.acs.org on July 26, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29

Journal of Agricultural and Food Chemistry

1

Development, Quantification, Method Validation and Stability Study of

2

a Novel Fucoxanthin-Fortified Milk

3

Il-Kyoon Mok†,§, Jung-Ro Yoon§, Cheol-Ho Pan†, Sang-Min Kim†, ‡, *

4 5 6



7

Gangneung, Gangwon-do 25451, Republic of Korea

8



9

Products, Gangneung, Gangwon-do 25451, Republic of Korea

Systems Biotechnology Research Center, KIST Gangneung Institute of Natural Products,

Convergence Research Center for Smart Farm Solution, KIST Gangneung Institute of Natural

10

§

11

Gangneung, Gangwon-do 210-702, Republic of Korea

Department of Food Processing and Distribution, Gangneung-Wonju National University,

12

13

14

15

16

17

*Corresponding author:

18

Sang-Min Kim, Tel:+82-33-650-3640, Fax: +82-33-650-3679

19

E-mail:

[email protected]

20 1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

21

Page 2 of 29

■ABSTRACT

22

In order to extend the scope of application of fucoxanthin, a marine carotenoid, whole milk (WM)

23

and skimmed milk (SM) were fortified with fucoxanthin isolated from microalga Phaeodactylum

24

tricornutum for a final 8 µg/mL milk solution concentration. Using these liquid systems, a

25

fucoxanthin analysis method implementing extraction and HPLC-DAD was developed and validated

26

by accuracy, precision, system suitability and robustness tests. The current method demonstrated

27

good linearity over the range of 0.125 ~ 100 µg/mL of fucoxanthin with R2 = 1.0000 and all

28

validation data supported the adequateness for the use in fucoxanthin analysis from milk solution. In

29

order to investigate fucoxanthin stability during milk production and distribution, fucoxanthin

30

content was examined during storage, pasteurization and drying processes under various conditions.

31

Fucoxanthin in milk solutions showed better stabilizing effect in one month of storage period.

32

Degradation rate constant (k) on fucoxanthin during this storage period suggested that fucoxanthin

33

stability might be negatively correlated with decrease of temperature and increase of protein content

34

such as casein and whey protein in milk matrix. In a comparison between SM and WM, fucoxantin

35

in SM always showed better stability than that in WM during storage and three kinds of drying

36

process. This effect was also deduced to relate with proteins content. In pasteurization step, more

37

than 91% of fucoxanthin was retained after three pasteurization processes even though above trend

38

was not found. This study demonstrated for the first time that milk products can be used as a basic

39

food matrix for fucoxanthin application and protein content in the milk is an important factor for

40

fucoxanthin stability.

41 42

KEYWORDS: fucoxanthin; carotenoid analysis; stability; whole milk; skimmed milk

43

2

ACS Paragon Plus Environment

Page 3 of 29

Journal of Agricultural and Food Chemistry

44

45

■INTRODUCTION

46

Fucoxanthin, one of the main marine carotenoids, contributes to more than 10% of the total

47

carotenoids in nature.1 This carotenoid has been reported to exhibit various beneficial biological

48

properties such as protective effects on the liver, as well as anti-oxidant, anticancer, anti-

49

inflammatory, anti-diabetic, anti-angiogenic, anti-malarial, neuro-protective, and anti-obesity

50

activities.2 Among them, the anti-obesity effect is the most prominent biological activity and a

51

number of reports have supported this fact.3 Industrially, fucoxanthin has been produced from

52

macroalgae such as Laminaria japonica and Undaria pinnatifida.4 Recently, microalga

53

Phaeodactylum tricornutum has been suggested as another source for fucoxanthin production.5

54

Although fucoxanthin can be produced industrially, its application in the food industry is limited

55

for several reasons. On one hand, the unique molecular structure of fucoxanthin—including the

56

unusual allenic bond, a 5,6-monoepoxide, and 9 conjugated double bonds—is susceptible to

57

oxidation and isomerization. Thus, fucoxanthin would be unstable when exposed to heat, light,

58

oxygen, metals, enzymes, unsaturated lipids and other pro-oxidant molecules.6, 7 On the other hand,

59

the low pH and water-based environment in the digestive system often make it difficult for

60

fucoxanthin to be absorbed in the small intestine.8 In order to overcome this drawback, several

61

processing methods for fucoxanthin have been attempted using canola oil, biodegradable chitosan-

62

glycolipid, and cetyl palmitate-canola oil mixed with a solid lipid core of fish gelatin..7, 9, 10 The

63

stability and bioavailability of fucoxanthin was significantly improved by these functional

64

encapsulation materials even though industrial process of production should be more discussed.

65

Milk is an effective vehicle for micronutrients and is a good food system model for carotenoid 3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 4 of 29

66

application. Whey protein, β-lactoglobulin, and casein isolated from milk have been widely used as

67

encapsulation materials for a number of bioactive substances including carotenoids.

68

combination of sodium caseinate and whey protein isolate was used as a stabilizer for β-carotene in

69

an oil-in-water emulsion system, demonstrating the highly increased stability of the pigment under

70

these conditions.11 β-Lactoglobulin was also used as a vector for β-carotene, and its absorption was

71

highly increased in in-vivo.12 Astaxanthin incorporation in different types of milk—whole milk

72

(WM), semi-skimmed milk (SSM), and skimmed milk (SM)—showed that the retention of

73

astaxanthin during storage decreased according to the increase of the fat content.13

11–13

A

74

The fact that milk constituents bind strongly to carotenoid, as demonstrated by milk being a

75

suitable vehicle for the pigment, can be a source of difficulty in carotenoid analysis. Some

76

carotenoids such as lutein and zeaxanthin naturally occurring in milk and formulas are treated with a

77

saponification or an enzymatic hydrolysis step because of the high fat content of the matrices.14

78

However, these processes cause carotenoid losses of up to 40%. Thus, several modification methods

79

have been developed for a more precise and accurate measurement of carotenoids in milk products.

80

For instance, Yuhas et al.15 changed the order of extraction and saponification steps to recover more

81

lipophilic carotenoids and used echinenone as an internal standard for the correction of losses.

82

The aim of this study is to prepare value-added milk products by fucoxanthin fortification of WM

83

and SM. Milk is generally considered the ideal food that contains the most nutrients for human

84

health. Until now, a range of vitamins (vitamins A and D), minerals (iron, calcium, copper and zinc),

85

and other nutrients such as polyunsaturated fatty acids have been used to fortify milk and provide

86

various biological advantages.16 However, in fortification of milk by such function-inducing

87

materials, several points should be confirmed prior to commercial production. The added nutrients

88

should be stable during the storage period under the appropriate conditions and during industrial 4

ACS Paragon Plus Environment

Page 5 of 29

Journal of Agricultural and Food Chemistry

89

processing steps such as drying, pasteurization, and fermentation adopted to prepare dried milk

90

powder or yogurt.

91

Thus, in this study, we developed and validated fucoxanthin analysis methods for WM and SM

92

based on a previously reported carotenoid analysis method.17 Additionally, the stability of

93

fucoxanthin within the WM and SM matrices was investigated under the appropriate storage,

94

pasteurization, and drying conditions.

95

96

■ MATERIALS AND METHODS

97

Materials. Fucoxanthin was purified from ethanol extract of microalgae Phaeodactylum tricornutum

98

by silica-gel chromatography as described by Kim et al.5 and its purity was identified to be over 95%

99

by NMR. Astaxanthin (purity ≥ 97%, HPLC grade) was purchased from Sigma-Aldrich. Two kinds

100

of milk powders (WM, SM from Seoul Daily Cooperative, Korea) used as a model food matrix were

101

purchased from the local market (Gangneung, Korea). The analytical grade extraction solvents

102

including tert-butyl methyl ether (TBME) and petroleum ether were purchased from Sigma-Aldrich.

103

Water and methanol with HPLC grade from Fisher Scientific Korea Ltd. were used for HPLC

104

analysis.

105

Fucoxanthin Fortification of Milk. In order to fortify fucoxanthin, fucoxanthin stock solution (4

106

mg/mL in ethanol) was prepared with purified fucoxanthin. Milk solutions from WM and SM were

107

prepared by mixing each powder with distilled water according to manufacturer’s suggestion (12.15%

108

for WM powder, 12.27% for SM powder in water (w/w)). Fucoxanthin stock solutions were added to

109

each milk solution while stirring the milk solution with a magnetic bar to make a final concentration 5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 6 of 29

110

of 8 µg/mL, corresponding to the recommended daily intake of fucoxanthin (2 mg in 250 mL milk

111

solution). This concentration is also the minimum level of fucoxanthin demonstrated to have an anti-

112

obesity effect in humans.18 Distilled pure water was used as a negative control for the matrix.

113

Development of Fucoxanthin Extraction from Milk Solution. Fucoxanthin extraction from a milk

114

solution was developed based on carotenoid analysis methods for foods.17 Briefly, the milk solution

115

was deproteinated with ethanol, and then, fucoxanthin was extracted with petroleum ether and

116

TBME solvents. In WM solution, an additional extraction step with hexane and 90% aqueous ethanol

117

was added to remove milk fat. The steps are details as follows: 2 mL of fucoxanthin-fortified milk

118

solution was mixed with the same volume of ethanol for deproteination and then, 1 mL of petroleum

119

ether and TBME solvents was serially added to sample tube. The tube was vortexed for 30 seconds

120

and centrifuged at 3,500 rpm for 5 min. to extract fucoxanthin from the food matrix. The supernatant

121

was collected in an 8 mL glass vial. This extraction step was repeated three times and the collected

122

extract solution was dried under N2 gas. The sample was dissolved in 1 mL of 90% aqueous ethanol

123

and filtrated with 0.45 µm nylon membrane filter for HPLC analysis. In case of WM solution, milk

124

fat was further removed from sample solution by extraction with 1 mL hexane solvent. The solution

125

in ethanol phase was directly used in HPLC analysis following 0.45 µm nylon membrane filtration.

126

HPLC Analysis. Quantification of fucoxanthin was performed by Agilent HPLC-DAD system

127

(Agilent 1200 series, USA). YMC C-30 carotenoid column (250 × 4.6 mm ID, 3 µm particle size,

128

Waters, Ireland) was used for the separation. Methanol and water solvent system was used for mobile

129

phase at a flow rate of 0.7 mL/min with a column temperature of 35 ℃. The solvent gradient

130

program was as follows: methanol/water ratio was increased from 90:10 to 100:0 over 20 min, and

131

then, 100% methanol was held for the next 5 min. The chromatogram obtained at 450 nm was used

132

for quantitative analysis of fucoxanthin (Fig. 1A). 6

ACS Paragon Plus Environment

Page 7 of 29

Journal of Agricultural and Food Chemistry

133

Validation of the Fucoxanthin Analysis Method. All validation tests except for accuracy test were

134

performed with fucoxanthin stock solution dissolved in ethanol, but the accuracy test was assayed

135

using fucoxanthin dispersed into milk solutions. The limit of detection (LOD) and the limit of

136

quantification (LOQ) of fucoxanthin were measured with HPLC quantification at 10 concentration

137

points from 0.125 to 100 µg/mL in ethanol. LOD and LOQ were carried out in triplicate. Precision

138

data were obtained from the three stock solutions of the following fucoxanthin concentrations: 10, 20,

139

and 50 µg/mL. Accuracy tests were carried out with two milk products fortified with fucoxanthin in

140

two different concentrations (20 and 50 µg/mL) by calculating the fucoxanthin recovery value in

141

terms of intra-day and inter-day variation. The precision and accuracy experiments were repeated 5

142

times.19 The system suitability test was performed by analyzing the mixed stock solution of

143

fucoxanthin and astaxanthin (as an internal standard) with each concentration of 20 µg/mL in ethanol.

144

The developed HPLC-DAD method was used for 8 times repetition analysis and the RSD % of peak

145

area and retention time, tailing factor, plate number and resolution were calculated with the analyzed

146

HPLC chromatograms. The system suitability data was evaluated by the criteria of United States

147

Pharmacopeia.20, 21 The robustness test was performed by changing the chromatographic conditions

148

with ± 5% modifications each time in three HPLC analytical parameters (flow-rate (0.6 ~ 0.8

149

mL/min), mobile phase % (methanol : water = 88 : 12 ~ 92 : 8) and oven temperature (33 ~ 37 ℃)).

150

Three fucoxanthin stock concentrations (5, 10 and 20 µg/mL) were analyzed by HPLC and the result

151

was evaluated by one-way ANOVA analysis of the fucoxanthin recovery percentage. 22, 23

152

153

Stability Tests of Fucoxanthin in Milk Matrices. Fucoxanthin stability under storage conditions,

154

pasteurization, and drying methods was investigated with fucoxanthin-fortified milk solutions. Three

155

different temperatures (2, 10, and 26 ℃) were selected as the storage temperatures and milk 7

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 8 of 29

156

solutions were stored in the dark. Samples were harvested weekly for fucoxanthin analysis for 2-4

157

weeks. For pasteurization of milk solution, three different heat treatment methods were applied: Low

158

Temperature Long Time (LTLT), 65 ℃/30 min; Hot Temperature Short Time (HTST), 75 ℃/15 sec;

159

Ultra-High Temperature (UHT), 135 ℃/2 sec. Sample solutions were packaged into 2 mL glass vial

160

and thermal treatment was carried out in an oil-bath. Thermal treatment time started when the

161

solution reached a target temperature. For drying processes, two milk solutions were treated by hot-

162

air drying, spray-drying, and freeze-drying methods. The equipment model and drying conditions

163

were as follows: hot-air dryer (EYELA, WFO-400, temperature = 45 ℃, drying time = 48 hr), spray-

164

dryer (OHKAWARA KAKOHKI, L-8, Inlet temperature = 180 ℃, Outlet temperature = 105 ℃,

165

DISC rpm = 25,000), freeze-dryer (Operon, FDCF-12003, Coil temperature = –120 ℃, drying time =

166

72 hr). After drying, each powder was re-dissolved to original ratio with water and fucoxanthin

167

amount was quantified by HPLC.

168

Kinetics Study of Fucoxanthin Degradation and Statistical Analysis. From the result of

169

fucoxanthin storage in milk solutions (Fig. 2), fucoxanthin degradation curves were kinetically

170

analyzed applying three equations of kinetic order (zero order: C = C0 – kt,

171

kt, second order: 1/C = 1/C0 + kt), where C is fucoxanthin content (µg/mL) at storage time t; C0 is

172

fucoxanthin content (µg/mL) at initial time; t is storage time (day); and k is degradation constant rate.

173

Finally, the best equation was determined by the highest k-value and correlation coefficient (R2). The

174

selected kinetic equation was used to evaluate the degradation rate (k) of fucoxanthin in milk

175

solutions under the selected storage conditions.

first order: lnC = lnC0 –

176

All experiments were performed in triplicate. All data are expressed as mean ± SD. Data was

177

analyzed by t-test and one-way or two-way ANOVA (P < 0.05). The significant differences among 8

ACS Paragon Plus Environment

Page 9 of 29

Journal of Agricultural and Food Chemistry

178

means were separated by Duncan’s multiple range tests. All statistical analysis was processed using

179

IBM SPSS 23.0 package and Microsoft office professional plus 2010 Excel.

180 181

■ RESULTS AND DISCUSSION

182 183

Optimization of Fucoxanthin Extraction Method from Milk Solution. There have been various

184

carotenoid extraction methods from food products and most extraction methods generally follow

185

three common steps: the release of carotenoid from food matrices by disrupting tissue, removal of

186

unwanted components, and carotenoid concentration by liquid-liquid or liquid-solid extraction.17

187

Milk also contains small amount of carotenoids such as lutein and zeaxanthin and several studies

188

have developed analytical methods of carotenoids naturally found in milk matrices.14,15 In addition,

189

the stability of astaxanthin is also used in milk products as a natural colorant was investigated under

190

various conditions. As milk and related dairy products have more fat and protein content relative to

191

common fruits and vegetables, carotenoid analysis methods based on the latter food models are

192

different in terms of removing unwanted components. In this study, fucoxanthin was used to fortify

193

two milk products (WM and SM)

194

recommended daily intake amount of fucoxanthin (as 2 mg in 250 mL milk solution) and its

195

analytical method was optimized for these food systems using previously reported methods.18, 24, 25

196

Even though there have been several reports on fucoxanthin extraction from macro- or microalgaes,

197

the extraction properties of fucoxanthin from the reported matrices are quite different from that of

198

milk products.26, 27 As the first step, the volume ratio between ethanol and aqueous milk solution was

199

optimized in a deproteination procedure. This step is necessary to separate the extraction phase

200

(organic solvent) from the aqueous phase of the milk solution without gelation, which is an issue in

with final concentrations of 8 µg/mL corresponding to the

9

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 10 of 29

201

the carotenoid analysis process using food systems containing high protein content such as milk

202

products.17 Among the four tested volume ratios of ethanol (0.5, 1, 1.5 and 2) to the aqueous phase,

203

the 1:1 ratio of ethanol and milk solution showed the best recovery yield for fucoxanthin (The

204

fucoxanthin recovery % from milk solution (volume ratio of ethanol to milk solution) : 68.77% (0.5),

205

95.51% (1), 80.13% (1.5), 56.53% (2)). As the next step, various organic solvents were investigated

206

for the extraction efficiency of fucoxanthin from milk matrices. A wide variety of solvent

207

combinations such as acetone/dichloromethane, acetone/ethanol, acetone/hexane, acetone/petroleum

208

ether, and n-hexane/diethyl ether have been used as carotenoid extraction solvents for food

209

systems.28-30 In this study, TBME, petroleum ether, dichloromethane, diethyl ether, n-hexane, and

210

ethyl acetate were selected as the extraction solvents and their various combinations were tested in

211

respect to the recovery yield of fucoxanthin from the milk solution. The mixed solvent of petroleum

212

ether and TBME in equal parts was selected as the most appropriate extraction solvent system for

213

fucoxanthin from milk products (data not shown).

214

Among three model systems (WM, SM and water) fortified with fucoxanthin, SM and water

215

showed high recovery yields of fucoxanthin (over 95% of the initial added amount) after three

216

extraction steps. However, the recovery was significantly low in WM (45.29%) indicating that the

217

extraction step with petroleum ether and TBME was not sufficient for the higher fat content solution.

218

Following the drying process with N2 gas, the vial sample from WM exhibited a sticky property,

219

which was not found in SM and water. Re-suspending the residue in the vial with 90% aqueous

220

ethanol for HPLC analysis was not successful and led to a low recovery yield. As this phenomenon

221

was attributed to the milk fat (milk fat content 27 g/100 g powder for WM, 1 g/100 g powder for

222

SM), an additional solvent partition step with n-hexane was applied after the extraction step. Fig. 1B

223

shows the effect of the n-hexane partition step in fucoxanthin extraction from the three systems. The

224

recovery of fucoxanthin from WM was increased up to 89.83% from 45.29%. This result 10

ACS Paragon Plus Environment

Page 11 of 29

Journal of Agricultural and Food Chemistry

225

demonstrates that non-polar fat components were moved from the aqueous ethanol phase to the n-

226

hexane phase. However, this additional step influenced negatively impacted the results for SM and

227

water as a small amount of fucoxanthin moved from the aqueous ethanol phase to the n-hexane phase

228

and recovery yields were decreased below 90%. Therefore, this additional solvent partition step was

229

applied only to WM. Meanwhile, the saponification step did not influence the extraction yield in this

230

study (data not shown). Though xanthophylls such as lutein and zeaxanthin require a saponification

231

procedure for their release from esterified xanthophylls before extraction, fucoxanthin in algae does

232

not naturally exist in the esterified form.31 For this reason, the saponification step was not included in

233

the fucoxanthin analysis method of this study.

234 235

Validation of Fucoxanthin Analysis Method from Milk Solution. The quantitative analysis

236

method of fucoxanthin using HPLC was validated by various tests of linearity, limit of detection

237

(LOD), limit of quantification (LOQ), precision, accuracy, system suitability and robustness test. The

238

linearity of regression equation was determined by the analysis of fucoxanthin standard solution with

239

concentrations in the range from 0.125 to 100 µg/mL. Table 1 shows the regression equations for

240

fucoxanthin with a correlation coefficient of R2 = 1.0000, indicating good linearity. The LOD and

241

LOQ values in HPLC analysis were calculated as the minimum concentration at the signal-to-noise

242

(S/N) ratio equal to 3.3 and 10 and those of fucoxanthin were 0.027 and 0.082 µg/mL, respectively.

243

Precision data obtained with three different concentrations of fucoxanthin standard solution (10, 20

244

and 50 µg/mL) during a single day (intra-day) and over the course of 5 days (inter-day) shows

245

relative standard deviation (RSD) values under 2.2%. In addition, the accuracy test by spiking WM

246

and SM with two known concentrations of fucoxanthin (20 and 50 µg/mL) shows a minimum

247

recovery value of 96.31% when the extracted fucoxanthin values in Fig. 1B were assumed to be 100%

248

(Table 1). The system suitability result showed 0.24~0.98% RSD of peak area and retention time, 11

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 12 of 29

249

plate number 27720, 1.0 of tailing factor, and 10.24 of resolution (Table S1). This means that the

250

HPLC system and procedure is providing data with acceptable quality. The robustness data was

251

obtained by slightly changing flow rate, mobile phase and column temperature coditions.

252

Fucoxanthin recovery values (98.02~102.93%) at three concentration levels (5, 10, and 20 µg/mL)

253

were not significantly affected by these changes of the HPLC condition (Table S2). These results

254

indicate that the optimized extraction and HPLC analysis methods for fucoxanthin in milk solutions

255

used in this study are valid and can be employed to access fucoxanthin content in milk products.

256 257

Stability of Fucoxanthin in Milk Solutions and a Kinetic Study. Milk collected from a stock farm

258

is usually stored in a big tank kept at 2 ℃ for a maximum of one month before food processing.

259

Following food processing, milk products are commonly distributed and arranged on the grocery’s

260

dairy case (open shelf) maintained at 10 ℃ for one week. In this study, fucoxanthin in milk solutions

261

(WM and SM) was monitored at these two temperatures during storage periods to investigate its

262

stability in the storage process. As a negative control, fucoxanthin stability was monitored at room

263

temperature (26 ℃) for two weeks; this (storage at 26 ℃) was a relatively short period due to the

264

decay of the milk. As seen in Fig. 2, fucoxanthin was quite stable in both milk solutions (WM and

265

SM) in all tested temperature (below 20% of degradation), even though the degradation trend was

266

slightly increased with increases in temperature. However, fucoxanthin in water exhibited a clear

267

trend of instability with temperature increases. This result strongly suggests that fucoxanthin stability

268

is enhanced by some components in the two milk solutions that are absent from water. At room

269

temperature, almost 70% of added fucoxanthin in water was decomposed in two weeks (Fig. 2C).

270

Fucoxanthin in SM showed slightly more stability than WM.

271

In order to understand fucoxanthin degradation kinetics, the fucoxanthin samples from the 12

ACS Paragon Plus Environment

Page 13 of 29

Journal of Agricultural and Food Chemistry

272

storage stability test were analyzed through three equations of kinetic order. Among the zero to

273

second order equations, the zero order equation showed the most accurate result in terms of the R2

274

values and the degradation rate constant (k) as presented in Table 2. However, degradation of

275

astaxanthin and ß-carotene has been demonstrated to follow first order kinetics in milk solutions and

276

systems stabilized with milk proteins.6,13 The k values in Table 2 reveal two clear increasing

277

tendencies based on the food matrix and the temperature. In addition, there is a significant gap in the

278

k values between milk systems and water. These results follow the trends of the fucoxanthin

279

degradation graphs in Fig. 2.

280

The solution of astaxanthin in WM degraded more than that of SM because fat acids present in

281

WM make astaxanthin more difficult to diffuse in the solution and form micelles with milk

282

proteins.13, 32 Conversely, astaxanthin can interact more easily with milk proteins in SM, and can

283

achieve stability more efficiently by forming micelles. Therefore, fat content is major factor for the

284

stability difference between WM and SM. Another point to be considered is protein content in the

285

milk solutions. As SM is produced by removing fat from WM, protein content in SM (4.29%, w/w)

286

is relatively increased from that of WM (3.04%, w/w). Milk proteins such as casein and whey protein

287

isolates are good encapsulation materials which stabilize materials by the free radical scavenging

288

function of sulfhydryl and nonsulfhydryl amino acids.33 Thus, SM containing greater amounts of

289

these proteins showed more stability than WM and the significant difference in k values between

290

milk solutions and water may be due to the lack of proteins in the latter. Temperature is a critical

291

factor in fucoxanthin degradation with several studies reporting fucoxanthin degradation with

292

temperature increases.5, 9 Even though the temperature used in those experiments was generally room

293

temperature, the trend of increasing fucoxanthin degradation with increasing temperature is an

294

undoubted fact. This degradation effect was also demonstrated below room temperature as shown in

295

Table 2. Regardless of the food matrices, degradation rate constant of fucoxanthin always showed 13

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 14 of 29

296

increased tendency with temperature increases. Several isomers (13-cis, 13’-cis and 9-cis

297

fucoxanthin) are reported (but not analyzed) as the degradation product of fucoxanthin in solution by

298

thermal treatment.9

299 300

Effect of Pasteurization Method on Fucoxanthin Stability. Pasteurization is an essential step in

301

milk production and distribution that protects humans from infection of pathogenic bacteria.34 Until

302

now, various pasteurization methods were developed and applied in the milk industry. The heat

303

treatment method used for pasteurization can be categorized into three typical types based on the

304

temperatures and treatment times used: Low Temperature Long Time (LTLT), 65 ℃/30 min; Hot

305

Temperature Short Time (HTST), 75 ℃/15 sec; Ultra-High Temperature (UHT), 135 ℃/2 sec. Table

306

3 shows fucoxanthin recovery values over 91% after its treatment by these pasteurization steps,

307

indicating that most fucoxanthin was retained during all tested pasteurization processes. In this

308

experiment, the three factors of temperature, time, and food matrix are variables impacting the

309

fucoxanthin recovery value. However, we did not determine any factors influencing consistently to

310

fucoxanthin recovery value among these three variables in Table 3. Yet, this result is meaningful

311

because it indicates that fucoxanthin is stable during the industrial process of pasteurization.

312 313

Effect of Drying Method on Fucoxanthin Stability. Three kinds of drying processes were also

314

applied to WM and SM solutions and investigated for fucoxanthin recovery. The drying process in

315

the milk industry is used to produce dried milk powder utilized in various foods. Among the tested

316

methods, the freeze-drying method demonstrated the highest recovery value and the hot air-drying

317

method showed the lowest recovery value (Fig. 3). Among WM and SM, the retained fucoxanthin

318

amounts were always high in SM. This result was similar with that of the stability test discussed

319

above. Thus, it could be assumed that the contents of fat and protein in both food systems influenced 14

ACS Paragon Plus Environment

Page 15 of 29

Journal of Agricultural and Food Chemistry

320

fucoxanthin stability in the drying process. In both food systems, fucoxanthin recovery depended on

321

temperature and treatment times during the drying processes. In terms of treatment times, hot air-

322

drying and freeze-drying methods required quite a long time (48 hr and 72 hr, respectively) and

323

spray-drying required a relatively shorter time (below 10 second). In terms of temperature, there

324

were significant differences between each method (45 ℃ for hot air-drying, 108~180 ℃ for spray-

325

drying, -120 ℃ for freeze-drying method). Even though we could not determine either variable as

326

the more statistically significant factor, the mixed effect of these two factors in fucoxanthin recovery

327

was significant in both food systems.

328

In conclusion, fucoxanthin, a highly valuable marine carotenoid, was used to fortify two kinds of

329

milk solutions and its analytical method was developed and validated through LOD, LOQ, precision,

330

accuracy, system suitability and robustness tests. In addition, fucoxanthin stability was investigated

331

during storage, pasteurization, and drying processes in order to check the feasibility of fucoxanthin

332

application in milk products. As a result, the content of fats and milk proteins and temperature were

333

suggested as critical factors for fucoxanthin stability in these processes and it was proved that

334

fucoxanthin can be a suitable functional material for milk fortification. Thus, biological functions of

335

fucoxanthin such as anti-obesity activity can be expected from milk products fortified with

336

fucoxanthin. At present, fucoxanthin is not as popular as lutein and astaxanthin due to its short

337

supply in the global market. Nevertheless, this study suggests the plausibility of fucoxanthin

338

application to the food industry.

339 340

ACKNOWLEDGEMENT

341

This research was supported by a grant from Marine Biotechnology Program (2MP0360) funded by

342

Ministry of Oceans and Fisheries, Korea and an intramural grant (2Z04690) from KIST Gangneung 15

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

343

Page 16 of 29

Institute of Natural Products

344 345

346

Supporting Information. System suitability and robustness data of the developed fucoxanthin

347

analysis method by HPLC-DAD. This material is available free of charge via the Internet at

348

http://pubs.acs.org

349 350

■ REFERENCE

351

(1) Peng, J.; Yuan, J. P.; Wu, C. F.; Wang, J. H. Fucoxanthin, a marine carotenoid present in brown

352

seaweeds and diatoms: metabolism and bioactivities relevant to human health. Mar Drugs. 2011, 9,

353

1806-1828.

354

(2) D´Orazio, N.; Gemello, E.; Gammone, M. A.; Girolamo, M.; Ficoneri, C.; Riccioni, G.

355

Fucoxanthin: A treasure from the sea. Mar. Drugs 2012, 10, 604-616.

356

(3) Miyashita, M. The carotenoid fucoxanthin from brown seaweed affects obesity. Lipid Tech. 2009,

357

21, 186-190.

358

(4) Kanazawa, K; Ozaki, Y.; Hashimoto T.; Das, S. K.; Matsushita, S.; Hirano, M.; Okada, T.;

359

Komoto, A.; Mori, N.; Nakatsuka, M. Commercial-scale preparation of biofunctional fucoxanthin

360

from waste parts of brown sea algae Laminalia japonica. Food Sci. Technol. Res. 2008, 14, 573-582.

361

(5) Kim, S. M.; Jung, Y. J.; Kwon, O. N.; Cha, K. H.; Um, B. H.; Chung, D. H.; Pan, C. H. A

362

potential commercial source of fucoxanthin extracted from the microalga Phaeodactylum

363

tricornutum. Appl. Biochem. Biotechno. 2012, 166, 1843-1855. 16

ACS Paragon Plus Environment

Page 17 of 29

Journal of Agricultural and Food Chemistry

364

(6) Achir, N.; Randrianatoandro, V. A.; Bohuon, P.; Laffargue, A.; Avallone, S. Kinetic study of β-

365

carotene and lutein degradation in oils during heat treatment. Eur. J. Lipid Sci. Technol. 2010, 112,

366

349-361.

367

(7) Savia-Trujillo, L.; Sun, Q.; Um, B. H.; Park, Y.; McClements, D. J. In vitro and in vivo study of

368

fucoxanthin bioavailability from nanoemulsion-based delivery systems: Impact of lipid carrier type.

369

J. Funct. Foods 2015, 17, 293-304.

370

(8) Quan, Jie.; Kim, S. M.; Pan, C. H.; Chung, D. H. Characterization of fucoxanthin-loaded

371

microspheres composed of cetyl palmitate-based solid lipid core and fish gelatin-gum Arabic

372

coacervate shell. Food Res. Int. 2012, 50, 31-37.

373

(9) Zhao, D.; Kim, S. M.; Pan, C. H.; Chung, D. H. Effects of heating aerial exposure and

374

illumination on stability of fucoxanthin in canola oil. Food Chem. 2014, 145, 505-513.

375

(10) Ravi, H.; baskaran, V. Biodegradable chitosan-glycolipid hybrid nanogels: A novel approach to

376

encapsulate fucoxanthin for improved stability and bioavailability. Food Hydrocolloid. 2014, 43,

377

717-725.

378

(11) Augustin, M. A.; Sanguansri, L.; Oliver, C. M. Functional properties of milk constituents:

379

Application for microencapsulation of oils in spray-dried emulsions – A minireview. Dairy Sci.

380

Technol. 2010, 90, 137-146 .

381

(12) Mensi, A.; Borel, P.; Goncalves, A.; Nowicki, M.; Gleize, B.; Roi, S.; chobert, J. M.; Haertlé, T.;

382

Reboul, E. β-Lactoglobulin as a vector for β-carotene food fortification. J. Agric. Food Chem. 2014,

383

62, 5916-5924.

384

(13) Mezquita, P. C.; Barragán Huerta, B. E.; Palma Ramírez, J. C.; Ortíz Hinojosa, C. P. Milks 17

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 18 of 29

385

pigmentation with astaxanthin and determination of colour stability during short period cold storage.

386

J. Food Sci. Technol. 2015, 52, 1634-1641.

387 388

389 390

(14) Schweiqert, F.; Hurtienne, A.; Bathe, K. Improved extraction procedure for carotenoids from human milk. Int. J. Vitam. Nutr. Res. 2000, 70, 79-83. (15) Rebecca, Y.; McCormick, M.; Yachetti, S.; Burgher, A. M.; Kong, K.; Walsh, J. A method for the measurement of lutein in infant formula. Food Nutri. Sci. 2011, 2, 145-149.

391

(16) Arora, S. Fortification of milk and milk products for value addition. In Chemical analysis of

392

value added dairy products and their quality assurance; Sharma, R., Mann, B.; Division of dairy

393

chemistry national dairy research institute: Karnal, India, 2011; 29-35.

394

(17) Amorim-carrilho, K. T.; Cepeda, A.; Fente, C.; Regal, P. Review of methods for analysis of

395

carotenoids. Trend. Anal. Chem. 2014, 56, 49-73.

396

(18) Abidov, M.; Ramazanov, Z.; Seifulla, R.; Grachev, S. The effects of xanthigenTM in the weight

397

management of obese premenopausal women with non-alcoholic fatty liver disease and normal liver

398

fat. Diabetes Obes. Metab. 2010, 12, 72-81.

399

(19) Kim, S. M.; Kang, S. W.; Jeon, J. S.; Jung, Y. J.; Kim, W. R.; Kim, C. Y. Determination of major

400

phlorotannins in Eisenia bicyclis using hydrophilic interaction chromatography: Seasonal variation

401

and extraction characteristics. Food Chem. 2013, 138, 2399-2406.

402

(20) Shabir, G. A. Validation of high-performance liquid chromatography methods for

403

pharmaceutical analysis: Understanding the differences and similarities between validation

404

requirements of the US Food and Drug Administration, the US Pharmacopeia and the International

405

Conference on Harmonization. J. Chromatogr. A. 2003, 987, 57-66. 18

ACS Paragon Plus Environment

Page 19 of 29

Journal of Agricultural and Food Chemistry

406

(21) Kim, Y. G.; Seo, H. S.; Won, C. H. Analytical method validation of oxiracetam using HPLC.

407

Anal. Sci. Technol. 2010, 23, 587-594.

408

(22) Rimawi, F. A. Development and validation of a simple reversed-phase HPLC-UV method for

409

determination of oleuropein in olive leaves. J. Food. Drug. Anal. 2014, 22, 285-289.

410

(23) Angelo, T.; Pires, F. Q.; Gelfuso, G. M.; Silva, J. K. R.; Gratieri, T.; Cunha-filho, M. S. S.

411

Development and validation of a selective HPLC-UV method for thymol determination in skin

412

permeation experiments. J. Chromatogr. B. 2016, 1022, 81-86.

413

(24) Khachik, F.; Spangler, C. J.; Smith, J. C. Identification, quantification and relative

414

concentrations of carotenoids and their metabolites in human milk and serum. Anal. Chem. 1997, 69,

415

1873-1881.

416

(25) König, K.; Goethel, S. F.; Rusu, V. M.; Vogeser, M. Deproteination of serum samples for LC-

417

MS/MS analyses by applying magnetic micro-particles. Clin. Biochem. 2013, 46, 652-655.

418

(26) Kim, S. M.; Kang, S. W.; Kwon O. N.; Chung, D. W.; Pan, C. H. Fucoxanthin as a major

419

carotenoid in Isochrysis aff. galbana: Characterization of extraction for commercial application. J.

420

Korean Soc. Appl. Biol. Chem. 2012, 55, 477-483.

421

(27) Shang, Y. F.; Kim, S. M.; Lee, W. J.; Um, B. H. Pressurized liquid method for fucoxanthin

422

extraction from Eisenia bicyclis (Kjellman) Setchell. J. Biosci. Bioeng. 2011, 111, 237-241.

423

(28) Howe, J. A.; Tanumihardjo, S. A. Evaluation of analytical methods for carotenoid extraction

424

from biofortified maize (Zea mays sp.). J. Agric. Food Chem. 2006, 54, 7992-7997.

425

(29) Barba, A. I. O.; Hurtado, M. C.; Mata, M. C. S.; Ruiz, V. F.; de Tejada, M. L. S. Application of a

426

UV-vis detection-HPLC method for a rapid determination of lycopene and β-carotene in vegetables. 19

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 20 of 29

427

Food Chem. 2006, 95, 328-336.

428

(30) Fratianni, A.; Criscio, T. D.; Mignogna, R.; Panfili, G. Carotenoids, tocols and retinols evolution

429

during egg pasta – making process. Food Chem. 2012, 131, 590-595.

430

(31) Crupi, P.; Toci, A. T.; Mangini, S.; Wrubi, F.; Rodolfi, L.; Tredici, M. R.; Coletta, A.; Antonacci,

431

D. Determination of fucoxanthin isomers in microalgae(Isochrysis sp.) by high-performance liquid

432

chromatography coupled with positive electrospray ionization. Rapid Commun. Mass Spectrom.

433

2013, 27, 1027-1035.

434

(32) Pérez-Gálvez, A.; Mínguez-Mosquera, M. I. Esterification of xanthophylls and its effect on

435

chemical behavior and bioavailability of carotenoids in the human. Nutr. Res. 2005, 25, 631–640.

436

(33) Ribeiro H. S.; Ax, K.; Schubert, H. Stability of lycopene emulsions in food systems. J. Food Sci.

437

2003, 68, 2730–2734.

438

(34) LeJeune, J. T.; Rajala-Schultz, P. J. Unpasteurized milk: A continued public health threat. Clin.

439

Infect. Dis. 2009, 48, 93-100.

440 441

442

20

ACS Paragon Plus Environment

Page 21 of 29

443

Journal of Agricultural and Food Chemistry

Figure captions

444 445

Figure 1. The HPLC chromatogram (A) and comparison of fucoxanthin recovery values before and

446

after n-hexane-partition (B) from fucoxanthin-fortified solutions. Initial fucoxanthin concentration of

447

8 µg/mL was assumed as 100%.

*P