Polyphenol Oxidase Containing Sidestreams as ... - ACS Publications

Subscriber access provided by ORTA DOGU TEKNIK UNIVERSITESI KUTUPHANESI

Article

Polyphenol oxidase containing sidestreams as emulsifiers of rumen bypass linseed oil emulsions: interfacial characterization and efficacy of protection against in vitro ruminal biohydrogenation Frederik Gadeyne, Nympha De Neve, Bruno Vlaeminck, Erik Claeys, Paul Van Der Meeren, and Veerle Fievez J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01022 • Publication Date (Web): 25 Apr 2016 Downloaded from http://pubs.acs.org on April 28, 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 38

Journal of Agricultural and Food Chemistry

1

Polyphenol oxidase containing sidestreams as emulsifiers of rumen bypass linseed

2

oil emulsions: interfacial characterization and efficacy of protection against in vitro

3

ruminal biohydrogenation

4 5

Frederik Gadeyne1, Nympha De Neve1, Bruno Vlaeminck1, Erik Claeys1, Paul Van der

6

Meeren2, Veerle Fievez1*

7 8 9

1

Laboratory for Animal Nutrition and Animal Product Quality, Faculty of Bioscience

10

Engineering, Ghent University, Proefhoevestraat 10, 9090 Melle, Belgium

11

2

12

University, Coupure Links 653, 9000 Ghent, Belgium

Particle and Interfacial Technology Group, Faculty of Bioscience Engineering, Ghent

13 14 15

* corresponding author ([email protected]; +3292649002)

16 17

1 ACS Paragon Plus Environment


18

Page 2 of 38

Abstract

19 20

The low transfer in ruminants of dietary polyunsaturated fatty acids to the milk or peripheral

21

tissues is largely due to ruminal biohydrogenation. Lipids emulsified by a polyphenol oxidase

22

(PPO) rich protein extract of red clover were shown before to be protected against this

23

breakdown after cross-linking with 4-methylcatechol. Protein extracts of thirteen other vegetal

24

resources were tested. Surprisingly, the effectiveness to protect emulsified lipids against in vitro

25

ruminal biohydrogenation largely depended on the origin of the extract and its protein

26

concentration but was not related to PPO activity. Moreover, PPO isoforms in vegetal sources,

27

effectively protecting emulsified lipids, were diverse and their presence at the emulsion interface

28

did not seem essential. Potato tuber peels were identified as an interesting biological source of

29

emulsifying proteins and PPO, particularly since protein extracts of industrial potato sidestreams

30

proved to be suitable for the current application.

31 32

Keywords: Protein sidestream - Polyphenol oxidase - Rumen Bypass - Biohydrogenation -

33

Emulsion

34


Page 3 of 38

35


Introduction

36 37

Polyphenol oxidase (PPO) is a widely distributed enzyme known for the characteristic browning

38

of damaged plant tissues as a result of polymerization of PPO-created o-quinones with

39

nucleophilic compounds1. Browning is generally unwanted in fruits and vegetables because of its

40

detrimental effect on product quality, which resulted in numerous browning prevention and

41

inhibition techniques2,3. In certain cases, however, polymerization of quinones is of primary

42

interest. The common brown color in black tea4,5 and cocoa6, for example, is the result of a PPO-

43

induced reaction. Similarly, in the field of ruminant nutrition, it has been suggested that PPO in

44

red clover is responsible for increased nitrogen use efficiency7 and the protection of lipids

45

against microbial degradation8. Recently, Gadeyne et al.9 successfully used this polymerizing

46

reaction to protect emulsified lipids against in vitro ruminal biohydrogenation (BH) and

47

oxidation during storage using a PPO-rich protein extract from red clover: due to the PPO-

48

induced conversion of the simple diphenol 4-methylcatechol (4-MC) into a quinone,

49

polymerization of the interfacial protein layer occurs, resulting in protection of the encapsulated

50

lipophilic content from degradation. Indeed, polyunsaturated fatty acids (PUFA) are to a large

51

extent subject to microbial BH in the rumen, resulting in the formation of saturated fatty acids

52

(FA) and accumulation of cis and trans isomers10. Consequently, protecting PUFA against

53

microbial degradation might enable the production of milk and meat of ruminant origin with

54

reduced amounts of saturated and trans-FA, which aligns with current public health policies11.

55

However, the seasonal availability and the low-fat content of red clover limits the potential

56

industrial applicability of the current technology. Nevertheless, PPO or tyrosinase activity has

57

been reported in almost any organism, both in plants, fungi, bacteria and animals12. Although

58

only PPO extracted from red clover has been used to create protection against ruminal BH,

59

extrapolation to other resources could be hypothesized. In this perspective, industrial sidestreams

60

of plant resources are widely available, some of which are now used in animal feeds or 3 ACS Paragon Plus Environment


Page 4 of 38

61

discarded, although they still might contain valuable molecules and characteristics. Therefore,

62

the aim of this paper is to assess the possibility to protect PUFA against ruminal BH using 4-MC

63

as diphenolic cross-linking mediator after emulsification using protein extracts from different

64

plant sources or possible sidestreams as emulsifiers. In addition, protein extracts and emulsions

65

were further characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis

66

(SDS-PAGE), PPO zymography, conventional PPO activity assays and protein load tests to get

67

further insight in factors affecting the protection efficiency (PE) in the rumen.

68 69

Materials and methods

70 71

Materials

72

Different vegetal sources were used for a screening test depending on their availability, formerly

73

reported PPO activity in literature13-23 or potential as upgradable industrial sidestream24-26: apple

74

peels (Malus domestica Mill. cv. Jonagold), artichoke flower leaves (Cynara scolymus L.),

75

broccoli stems (Brassica oleracea L. convar. botrytis var. cymosa), carrot peels (Daucus carota

76

subsp sativus (Hoffm.) Schübl & G. Martens), cauliflower florets, stems and leaves (Brassica

77

oleracea L. convar. botrytis var. botrytis), eggplant pulp (Solanum melongena L.), pineapple

78

peels (Ananas comosus (L.) Merr.), potato tuber peels (Solanum tuberosum L.), red clover stems

79

& leaves (Trifolium pratens L. cv. Lemmon), spinach leaves (Spinacia olereacea L.) and tomato

80

stems & leaves (Solanum lycopersicum L.). Plant sources were either bought in a local grocery

81

store or kindly provided by the Institute for Agricultural and Fisheries Research (ILVO,

82

Belgium). Immediately upon arrival in the laboratory, plant material was cut into fine pieces,

83

snap frozen in liquid nitrogen and stored at -80 °C until further use.

84

In addition to these sources, sidestreams of the production process of two potato processing

85

companies were collected including solid (cutter scraps and slivers) as well as liquid sidestreams


Page 5 of 38


86

after steam peeling, but were taken before any blanching step was performed. Samples were

87

stored at -80 °C until further use.

88

Crude linseed oil (40, 194, 169 and 586 mg per g total FA of C18:0, C18:1n-9, C18:2n-6 and

89

C18:3n-3, respectively) was a gift from Dumoulin (Kortrijk, Belgium). 4-MC was purchased

90

from Sigma-Aldrich (Bornem, Belgium). All other chemicals were of analytical grade and were

91

purchased from Sigma-Aldrich (Bornem, Belgium), Merck (Darmstadt, Germany), Carl-Roth

92

(Karlsruhe, Germany) or VWR (Heverlee, Belgium), unless stated otherwise. Any centrifugal

93

step was performed using a refrigerated Beckman J2-HS centrifuge (Beckman Coulter, Brea,

94

California, USA).

95 96

Preparation of protected PUFA emulsions and experimental setup

97

Emulsions were prepared as described by Gadeyne et al.9 In brief, a three-step process was

98

performed: first, proteins were isolated from frozen plant material by extraction in 0.1 M sodium

99

phosphate buffer (SPB) with ascorbic acid (pH=7), protein precipitation with acetone (4:1

100

volumetric acetone to SPB ratio) and recovery of soluble protein in 0.01 M SPB (pH=7); second,

101

this protein extract was used to emulsify oil with a high speed Ultra-Turrax (T25 Basic, Ika

102

Werke, Staufen, Germany) for pre-emulsification and passed 5 times at 25 MPa through a

103

microfluidizer (M110S, Microfluidics Corporation, Newton, Massachusetts, USA); third,

104

emulsions were treated with 4-MC and shaken for 24 h to induce protein-phenol complexing. All

105

emulsions were prepared using 20 mg linseed oil per ml of extract and stored at 4 C.

106

Three experiments were performed to assess the potential of different sources as emulsifier to

107

protect against ruminal degradation. Emulsions in experiment 1 were created using protein

108

extracts from the thirteen selected vegetal sources mentioned in the Materials sections.

109

Emulsions (before addition of diphenol) were prepared in triplicate, starting from three different

110

protein extracts. Afterwards, 4-MC was added to obtain a final concentration of 0, 10, 20 or 40

111

mM 4-MC. Experiment 2 attempted to perform a first screening using industrial sidestreams of 5 ACS Paragon Plus Environment


Page 6 of 38

112

potato processing facilities. Samples were taken from two companies at similar places in the

113

production process and kept as replicates throughout the experiment. Extraction procedures of

114

solid and liquid sidestreams aimed at obtaining protein concentrations of at least 1 mg per ml

115

solution. Therefore, extraction of solid sidestreams was as described before9. Volumes of liquid

116

byproducts needed for extraction in 0.1 M SPB with ascorbic acid were determined based on

117

preliminary analysis of the protein content. Final 4-MC concentrations of emulsions were 0 or 20

118

mM 4-MC. Finally, in experiment 3 four vegetal sources were selected from the first experiment

119

for further characterization of proteins of the continuous phase and present at the interface: carrot

120

peels, cauliflower florets, eggplant pulp and potato tuber peels. Protein solutions were obtained

121

as described before, but were diluted with 0.01 M SPB to obtain similar protein concentrations

122

across treatments. Emulsions were prepared in triplicate starting from the same protein solution

123

and contained a final concentration of 0 or 40 mM 4-MC.

124 125

Protein extract characterization

126

Spectrophotometric analysis

127

Protein solutions were characterized by measuring the PPO activity and the concentration of total

128

protein and protein-bound phenol (PBP). The adaptations described by Winters and Minchin27

129

were used to perform a modified Lowry assay, in order to determine total protein and PBP

130

concentration simultaneously. Protein content and PBP were expressed as mg of protein per ml

131

extract and mg tyrosine-equivalents per mg protein, respectively, assuming 1 g of bovine serum

132

albumin, used for the standard solution, contains 0.043 g tyrosine-equivalents. Further, PPO

133

activity was determined by measuring the absorbance at 400 nm each 5 seconds for 1 min. For

134

this, 0.75 ml 0.04 M 4-MC (containing 5 mM HCl to prevent autoxidation) was added to a

135

solution of 2.2 ml 0.01 M SPB (pH=7) and 50 µl of protein extract. The increase in absorbance

136

of the initial linear part of the curve was retained as a measure of activity. PPO activity was

137

finally expressed in nkatal by recalculating the increase in absorbance per time unit to amounts 6 ACS Paragon Plus Environment

Page 7 of 38


138

of formed quinones using a standard series of 4-methylbenzoquinone, obtained by treating 3 ml

139

of 4-MC with 3 ml 0.045 M sodium periodate and measuring the absorbance at 400 nm after 10

140

min. Specific PPO activity was expressed as µkatal per mg protein. All spectrophotometric

141

analyses were done in triplicate (analytical replicates).

142

Gel electrophoretic analysis

143

Next to spectrophotometric analyses of protein and PPO activity, gel electrophoresis was

144

performed to visualize different protein patterns in the extracts using a vertical slab gel system

145

(SE600, Hoefer Scientific Instruments, Holliston, Massachusetts, USA). Gels were prepared as

146

described by Greaser et al.28 Briefly, separating gels contained 10 or 12 % of total acrylamide

147

and N,N′-methylene-bis-acrylamide. In experiment 1, wells were loaded with the protein

148

solution, to which SDS (2 %, w/v), approximately 15 % sucrose (w/v) and 0.3 mg/ml

149

bromophenol blue were added. Twenty µl of this mixture was loaded per well, while volumes

150

loaded in experiment 3 varied from 10 to 50 µl per well, depending on the protein concentration

151

of the samples, in order to load equal amounts of protein. Separation was carried out at constant

152

current. After the bromophenol front reached the bottom of the gel, proteins were fixed in a 20 %

153

methanol / 10 % acetic acid solution and visualized by staining with Coomassie brilliant blue

154

R250 according to Claeys et al.29, followed by scanning with a Bio-Rad computing densitometer

155

(model CDS-100; Bio-Rad, Temse, Belgium). Further, similar gels were used to visualize PPO

156

activity, based on the method described by Rescigno et al.30 For this, 10 % gels were prepared

157

similarly as for the protein visualization. The presence of SDS is of particular interest as it is

158

known to activate PPO isoforms from its latent state, in contrast to many enzymes which are

159

inactivated by SDS.31 After proteins were separated, gels were gently washed with demineralized

160

water and soaked in 0.1 M SPB (pH=7) for 3 min. Next, the gels were transferred to a solution

161

containing 80 mM 4-MC (dissolved in 0.1 M SPB, pH=7). After 5 min, gels were washed in the

162

original SPB solution and immediately transferred to a 16 mM 4-amino-N,N-diethylaniline

163

sulfate solution, which reacts with 4-methylbenzoquinone, resulting in the visualization of PPO 7 ACS Paragon Plus Environment


Page 8 of 38

164

isoforms as purple blue bands. Zymograms were scanned at least 10 minutes after addition of 4-

165

amino-N,N-diethylaniline sulfate.

166 167

Emulsion characterization

168

The particle size distribution of emulsions was assessed immediately after preparation with a

169

Mastersizer S (Malvern Instruments, Malvern, UK) as described before9. Droplet sizes were

170

characterized in terms of volume-weighted mean diameter (D43), surface-weighted mean

171

diameter (D32), median volume-weighted distribution value D[v,0.5] or 90 % percentile of the

172

volume-weighted distribution D[v,0.9]. Specific surface areas (in m²/g oil) were calculated as SSA

173

= 6 / [D32 x ρ], assuming a linseed oil density ρ of 930 kg/m³.

174

Further, the protein load of emulsions from experiment 3 was determined. To separate proteins

175

present in the continuous phase from proteins at the interface after emulsification, 25 ml of

176

emulsion was centrifuged for at least 30 min (30000 g; 4° C). The protein fraction of the

177

continuous phase was obtained by removing 5 ml of the subnatant with a fine needle and was

178

filtered through a 0.20 µm cellulose syringe filter (Chromafil, Macherey-Nagel, Düren,

179

Germany). These unadsorbed proteins were further characterized using the modified Lowry and

180

gel electrophoretic procedures (vide supra). As demonstrated before32, the aqueous phases

181

obtained after filtration only contained negligible volumes of oil (data not shown). Accordingly,

182

adsorbed protein in the continuous phase can be assumed negligible as well. Protein loads were

183

expressed as mg protein per m² interfacial area or relatively to the initial protein concentration in

184

the aqueous extract (g per 100 g protein). Next, to recover the interfacial proteins for gel

185

electrophoresis, an extra 15 ml was removed from the centrifuged sample and the remaining

186

fraction was washed twice by redispersing in fresh 0.01 M SPB (pH=7), thorough vortexing and

187

centrifuging for at least 30 min (30000 g; 4° C). Then, 1 % SDS solution was added to the

188

remaining fraction, vortexed and gently shaken for 1 h to allow interfacial proteins to be replaced

189

by SDS, before centrifuging for at least 30 min (30000 g; 4° C). Finally, 5 ml of the subnatant 8 ACS Paragon Plus Environment

Page 9 of 38


190

was removed and passed through a 0.20 µm cellulose syringe filter to obtain the adsorbed protein

191

fraction for gel electrophoretic analysis.

192 193

Assessment of protection against ruminal biohydrogenation

194

Batch in vitro incubations were performed to assess protection of PUFA in emulsions against

195

ruminal BH as described before9. In brief, incubation flasks contained 1 ml emulsion, 250 mg

196

hay and 24 ml buffer/rumen fluid solution in a 4:1 ratio, which has been shown before10 to be

197

appropriate to estimate BH of C18:2n-6 or C18:3n-3. Rumen contents were collected before the

198

morning feeding from three rumen fistulated sheep, which were fed grass hay ad libitum and a

199

grain based concentrate (200 g/day), combined and filtered before incubation. Fistulation was

200

approved by the ethical commission (file number 114, 2009) of the Institute for Agricultural and

201

Fisheries Research (ILVO, Belgium). Flasks were flushed with CO2 and incubated under

202

intermittent shaking at 39 °C for 24 h in a batch culture incubator (Edmund Bühler GmbH,

203

Hechingen, Germany). All emulsions were incubated in duplicate (analytical replicates). To

204

monitor the quality of the incubations, gas composition33, pH (Hanna Instruments, Temse,

205

Belgium) and volatile FA34 was assessed. Gas composition was converted to absolute gas

206

production rates based on the pressure accumulation in the flask (Infield 7C handheld read-out

207

device equipped with a T1 Stitch-Tensiometer; UMS GmbH, München, Germany). Incubation

208

characteristics are not reported as no major differences were observed between treatments,

209

indicating changes in the extent of BH were not due to changes in microbial activity.

210

To calculate BH, 5 ml of incubation fluid was taken before and after incubation, freeze-dried and

211

analyzed for long chain FA after direct transesterification using gas chromatograph, according to

212

Gadeyne et al.9 Peaks were identified based on their retention times, compared to external

213

standards (GLC463, Nu-Check Prep Inc., Elysian, Minnesota, USA). Quantification of FA

214

methyl esters was based on the area of the internal standard (C21:0 for experiment 1 and C13:0

215

for experiments 2 and 3) and on the conversion of peak areas to the weight of FA by a theoretical 9 ACS Paragon Plus Environment


Page 10 of 38

216

response factor for each FA35,36. Finally, BH (%), which is the disappearance of C18:3n-3 after

217

incubation, was calculated as [(proportion of C18:3n-3 in total C18 FA)0h – (proportion of

218

C18:3n-3 in total C18 FA)24h] × 100 / (proportion of C18:3n-3 in total C18 FA)0h. PE of

219

C18:3n-3 (%) was calculated as [(BH of C18:3n-3)non-protected (0 mM) – (BH of C18:3n-3)protected (10,

220

20 or 40 mM)]

221

C18 FA BH, was calculated as [(proportion of C18:0 in total C18 FA)24h – (proportion of C18:0

222

in total C18 FA)0h] × 100 / [(proportion of C18:3n-3 and C18:2n-6 in total C18 FA)0h –

223

(proportion of C18:3n-3 and C18:2n-6 in total C18 FA)24h].

× 100 / (BH of C18:3n-3)non-protected (0 mM). The formation of C18:0, the end product of

224 225

Statistics

226

Results were analyzed with the MIXED procedure of SAS (SAS Enterprise Guide 7, SAS

227

Institute Inc., Cary, North Carolina, USA). Analytical replicates were averaged prior to statistical

228

analysis. Linear regression analyses and two-sample T-tests were performed using the REG and

229

TTEST procedure of SAS, respectively.

230

For experiment 1, the following model was used: Yij = µ + Pi + Dj + Pi × Dj + εij, with Yij the

231

variable of interest, µ the overall mean, Pi the fixed effect of protein source (i = apple peel,

232

artichoke flower leaf, broccoli stem, etc.), Dj the fixed effect of diphenol concentration (j = 0, 10,

233

20 or 40 mM 4-MC) and εij the residual error. Hierarchical cluster analysis was performed on

234

C18:3n-3 BH using 10, 20 and 40 mM 4-MC to distinguish between protein source using the

235

CLUSTER and TREE procedures of SAS with average linkage.

236

In experiment 2 and 3, the same model as for experiment 1 was used, with Pi the fixed effect of

237

protein source (i = solid or liquid sidestreams or i = carrot peel, cauliflower floret, eggplant pulp

238

or potato tuber peel for experiment 2 and 3, respectively).

239

Principal component analysis was performed to determine components which account for most

240

of the variation in PE. The PRINCOMP procedure of SAS was applied using data from the 40


Page 11 of 38


241

mM 4-MC emulsions of experiment 1 and 3, whereby PE of C18:3n-3, protein concentration,

242

PPO activity and D32 were included as variables.

243

Differences were assigned at the 0.05 significance level and differences among least square

244

means evaluated using Tukey’s multiple comparison test.

245 246

Results

247 248

Experiment 1: screening of different plant sources

249

In the first experiment, a screening test was performed to assess whether PPO containing protein

250

extracts from different sources could be used to protect PUFA against ruminal BH. Therefore,

251

thirteen possible vegetal sources were selected, proteins extracted and used as emulsifiers.

252

Protein concentrations of the extracts largely differed (Table 1), ranging from 0.204 to 2.73 mg

253

of protein per ml extract for apple peel and potato tuber peel, respectively. Negligible

254

concentrations of PBP were observed in the extracts (Table 1), except for artichoke flower leaf

255

(10.5 mg tyrosine equivalents per mg protein), which was also reflected in a dark discoloration

256

during the extraction procedure prior to acetone precipitation. The protein extracts from the latter

257

also contained the highest specific PPO activity (5.23 µkatal per mg protein). Substantial

258

amounts of quinones were formed in most extracts after addition of 4-MC, ranging from 1.53

259

nkatal for pineapple peel to 116 nkatal for artichoke flower leaf (Table 1). Very low activities

260

were found for broccoli, carrot and cauliflower.

261

Gel electrophoretic analysis was performed to separate proteins according to their molecular

262

weights. Large variation in molecular weight and intensity of protein bands within and between

263

vegetal extracts was observed (Figure 1A). Also, reactivity towards 4-MC of these proteins,

264

attributed to the activity of PPO (Figure 1B), varied to a large extent among the protein sources.

265

Most sources showed one or more bands indicating PPO activity, except for carrot peel.

266

Remarkably, protein sources were quite diverse in PPO isoforms as indicated by the lack of 11 ACS Paragon Plus Environment


Page 12 of 38

267

uniformity in PPO bands, showing proteins with varying molecular weight being responsible for

268

PPO activity. No relation between the spectrophotometric PPO activity (Table 1) and intensity of

269

bands after electrophoretic analysis was observed: for example, no (detectable) or very low PPO

270

activity was measured spectrophotometrically for protein extracts of cauliflower, broccoli,

271

spinach and pineapple, whereas intense bands were detected on gels. Furthermore, intensities of

272

bands with high PPO activity (Figure 1B) were not related to intensities of protein as such

273

(Figure 1A), meaning for some sources PPO represented only a minor proportion of the proteins.

274

Emulsion droplet sizes and specific surface areas (Table 1) illustrate the protein concentrations

275

of the extracts were sufficient to obtain stable emulsions with a D32 ranging from 0.67 to 3.91

276

µm for cauliflower floret and carrot peel, respectively, associated with the highest (9.69 m²/g oil)

277

and lowest (1.67 m²/g oil) specific surface area. Artichoke, broccoli, cauliflower and spinach

278

protein extracts resulted in emulsions with the smallest droplet sizes, with 90 % of the droplet

279

population showing a diameter below 10 µm. Regression analysis revealed no linear link

280

between D32 of the emulsions and the protein concentration of the different extracts (p=0.927).

281

In order to create protection, 4-MC was added to the emulsions to obtain final concentrations of

282

0, 10, 20 or 40 mM. Results for BH of C18:3n-3 are depicted in Figure 2A, while the associated

283

PE for 40 mM 4-MC emulsions are presented in Table 1. Numeric averages and standard

284

deviations for BH of C18:3n-3 and C18:2n-6 as well as the PE of C18:3n-3 and the formation of

285

C18:0 for all treatments are reported in Supplement 1. On average across all protein sources,

286

96.3 % of C18:3n-3 in emulsions without 4-MC was hydrogenated after 24 h in vitro incubation.

287

Upon addition of 4-MC, a decrease in BH of C18:3n-3 was observed with the extent of the

288

decrease depending on the source of protein used as emulsifier. C18:3n-3 BH of emulsions

289

containing 10 mM 4-MC, prepared using potato tuber peel, was significantly lower compared

290

with other protein sources (p

Polyphenol Oxidase Containing Sidestreams as ... - ACS Publications

Recommend Documents