Processing chocolate milk drink by low-pressure cold plasma technology

Accepted Manuscript Processing chocolate milk drink by low-pressure cold plasma technology Nathalia M. Coutinho, Marcello R. Silveira, Leonardo M. Fernandes, Jeremias Moraes, Tatiana C. Pimentel, Monica Q. Freitas, Marcia C. Silva, Renata S.L. Raices, C. Senaka Ranadheera, Fábio O. Borges, Roberto P.C. Neto, Maria Inês B. Tavares, Fabiano A.N. Fernandes, Thatyane V. Fonteles, Filomena Nazzaro, Sueli Rodrigues, A.G. Cruz PII: DOI: Reference:

S0308-8146(18)31999-X https://doi.org/10.1016/j.foodchem.2018.11.061 FOCH 23870

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

1 July 2018 22 October 2018 10 November 2018

Please cite this article as: Coutinho, N.M., Silveira, M.R., Fernandes, L.M., Moraes, J., Pimentel, T.C., Freitas, M.Q., Silva, M.C., Raices, R.S.L., Ranadheera, C.S., Borges, F.O., Neto, R.P.C., Tavares, M.I.B., Fernandes, F.A.N., Fonteles, T.V., Nazzaro, F., Rodrigues, S., Cruz, A.G., Processing chocolate milk drink by low-pressure cold plasma technology, Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem.2018.11.061

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1

Processing chocolate milk drink by low-pressure cold plasma technology

2

(Running title.: Cold plasma choc. milk drink)

3 4

Nathalia M. Coutinho1, Marcello R. Silveira1, Leonardo M. Fernandes2, Jeremias Moraes2,

5

Tatiana C. Pimentel3, Monica Q. Freitas1, Marcia C. Silva2, Renata S.L. Raices2, C.

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Senaka Ranadheera4, Fábio O. Borges5, Roberto P.C. Neto6, Maria Inês B. Tavares6,

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Fabiano A.N. Fernandes7, Thatyane V. Fonteles8, Filomena Nazzaro9, Sueli Rodrigues8,

8

A.G. Cruz2*

9

Universidade Federal Fluminense (UFF), Faculdade de Medicina Veterinária,

10

1

11

24230-340, Niterói, Brazil

12

2

13

Departamento de Alimentos, 20270-021, Rio de Janeiro, Brazil

14

3

15

4

16

School of Agriculture & Food, Melbourne, VIC 3010, Australia

17

5

18

Laboratório de Plasma e Espectroscopia Atômica, 24210-340, Niteroi, Brazil

19

6

20

Professora Eloisa Mano (IMA), 21941-598 Rio de Janeiro, Brazil

21

7

22

60440-900 Fortaleza, Ceará, Brazil

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8

24

Alimentos 60440-900 Fortaleza, Ceará, Brazil

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Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro (IFRJ), Instituto Federal do Paraná (IFPR), Paranavaí, 87703-536, Paraná, Brazil The University of Melbourne, Faculty of Veterinary & Agricultural Sciences, Universidade

Federal

Fluminense

(UFF),

Instituto

Física.

Universidade Federal do Rio de Janeiro (UFRJ), Instituto de Macromoléculas Universidade Federal do Ceará (UFC), Departamento de Engenharia Química, Universidade Federal do Ceará (UFC), Departamento de Engenharia de Istituto di Scieze dell’Alimentazione, CNR-ISA, 83100, Avellino, Italy

26 27 28 29

de

* Email:[email protected]/[email protected] (A.G.Cruz)

31

Abstract

32

This study aimed to evaluate the effect of the process time (5, 10, and 15 min)

33

and flow rate (10, 20, and 30 mL/min) of cold plasma technology on physio-

34

chemical characteristics (pH), bioactive compounds (DPPD, Total Phenolic

35

Compounds, ACE-inhibitory activity values), fatty acid composition, and volatile

36

compounds profile of chocolate milk drink. The mild (lower flow rate and

37

process time) and more severe (higher flow rate and process time) conditions

38

led to a reduction of the bioactive compounds (total phenolic compounds and

39

ACE-inhibitory activity), changes in fatty acid composition (increased saturated

40

fatty acid and decreased monounsaturated fatty acid and polyunsaturated fatty

41

acid), less favorable health indices (higher atherogenic, thrombogenic and

42

hypercholesterolemic saturated fatty acids and lower desired fatty acids), and

43

lower number of volatile compounds. In contrast, in intermediate cold plasma

44

conditions, an adequate concentration of bioactive compounds, fatty acid

45

composition, and health indices, and increased number of volatile compounds

46

(ketones, esters, and lactones) were observed. Overall, cold plasma technology

47

has proven to be an interesting alternative to chocolate milk drinks, being of

48

paramount importance the study of the cold plasma process parameters.

49

Key-words:

50

parameters; bioactive compounds; fatty acid.

51 52 53 54

chocolate

milk

drink;

cold

plasma

technology;

process

55

1. Introduction

56

Dairy products are widely consumed and recognized as important

57

components of a healthy and nutritious diet. They are considered good dietary

58

sources of calcium, protein, potassium, and phosphorus. In addition, they have

59

a high absorptive rate, availability, and relatively low cost (Pimentel et al.,

60

2017). Milk drinks are dairy products with a very promising food market, due to

61

its sustainability appeal, once it is used a by-product of the cheese industry,

62

and the health benefits associated to the bioactive peptides, antioxidants, and

63

essential amino acids from whey (Amaral et al., 2018, Panghal et al., 2018).

64

Cocoa powder has a chemical composition suitable for the flavoring of milk

65

drinks, mainly due to the polyphenols, which are associated to neuroprotective,

66

antioxidant, antimicrobial, and cardioprotective activities (Kardum & Gilbetic,

67

2018). Chocolate milk drink is the most popular product of this category

68

commercialized in Brazil (Pimentel et al., 2017).

69

The processing of dairy products includes the heat treatment of milk,

70

aiming the destruction of all pathogenic microorganisms and reduction of the

71

spoilage microorganisms (Cappato et al., 2017, Amaral et al., 2017). The high

72

temperature causes thermal degradation and autoxidation of fats, leading to

73

the formation of secondary compounds, such as aldehydes, ketones, and

74

carboxylic

75

(Sarangapani, Keogh, Dunne, Bourke & Cullen, 2017; Gavahian, Chu,

76

Khaneghah, Barba & Misra, 2018). Furthermore, the oxidation results in

77

changes in the protein structure and fatty acid composition, reduction in the

78

nutrient value, and degradation of the sensory quality (Coutinho et al., 2018).

acids,

which

can

have

negative

effects

in

human

health

79

Currently, there is a significant concern of efforts to improve the food

80

science and technology focused on the efficiency, sustainability, and

81

development of alternatives to traditional thermal processes (Cappato et al.,

82

2018). The non-thermal processes aim to improve the level of food safety

83

standards, to increase the shelf life while maintaining important food quality

84

attributes, such as nutritional, physical, and sensory characteristics, preserving

85

unstable bioactive compounds and modulating enzyme activity, avoiding the

86

undesirable effects generated by heat treatments (Coutinho et al., 2018).

87

Plasma is often referred to as the fourth state of matter in compliance

88

with the increasing order of energy from solid, to liquid, to gas, ultimately to

89

the ionized state-plasma (Misra et al., 2018). Atmospheric pressure cold plasma

90

(ACP) is a relatively new emerging non-thermal technology, which is produced

91

by the ionization of gas with electrical discharges at room temperature and

92

atmospheric pressure. The ionized gas consists of free electrons, ions, and

93

neutral particles, as well as reactive species (such as superoxide, hydroxyl

94

radicals, nitric oxide, ozone, and others) in constant interaction, with enough

95

electrical energy to break the covalent bonds and induce numerous chemical

96

reactions (Liao et al., 2017; Coutinho et al., 2018).

97

The compounds produced by cold plasma have been widely reported as

98

effective in microbial inactivation. The microbial inactivation may occur by

99

chemical interaction of radicals, reactive species, or charged particles with the

100

cell membranes, by damage to membranes and internal cellular components by

101

the UV radiation, or broken the DNA strands by the UV light (Pinela & Ferreira,

102

2015). However, cold plasma technology has been shown to modify or interact

103

with food components such as proteins, lipids, water, carbohydrates, and

104

phenolic compounds (Bahrami et al., 2016; Sarangapani et al., 2017,

105

Rodríguez, Gomes, Rodrigues & Fernandes, 2017). Although higher gas flow

106

rates and longer processing times can increase the number of collisions and the

107

possibilities of the reactive species acting on the microorganisms, resulting in

108

increased decontamination efficiency (Liao et al., 2017), their influence on the

109

quality parameters of the products has not been extensively studied. Previous

110

studies have evaluated the impact of cold plasma processing on the quality

111

parameters of cheese (Yong et al., 2015), milk (Korachi et al., 2015) and milk

112

fat (Saragapani et al., 2017). Furthermore, the effect of cold plasma on whey

113

proteins is also reported (Segat, Misra, Cullen & Innocente, 2015; Tammineedi,

114

Choudhary, Perez-Alvarado & Watson, 2013). To the best of our knowledge,

115

there are no studies about the application of cold plasma in milk drinks.

116

In this context, this study aimed to evaluate the effect of the cold plasma

117

process parameters (processing time and gas flow rate) on the: (1) physio-

118

chemical characteristics (pH), (2) bioactive compounds (DPPD, Total Phenolic

119

Compounds, ACE-inhibitory activity values), (3) fatty acid composition, and (4)

120

volatile compounds profile of chocolate milk drink, when compared to a

121

pasteurized product (control, 72-75°C/15 s).

122

2.

Materials and methods

123

2.1

Chocolate milk drink processing

124

The chocolate milk drinks were made according to the methodology

125

proposed by Castro et al. (2013), with some adaptations. Pasteurized milk (3%

126

fat, Betânia, Fortaleza, Brazil) and reconstituted whey powder (70/30% (v/v);

127

Alibra, São Paulo, Brazil) were mixed, and cocoa powder 1.5% (w/v) (Nestlé,

128

Rio de Janeiro, Brazil), colorless gelatin powder 0.5% (w/v) (Royal, São Paulo,

129

Brazil), and organic crystal sugar 10% (w/v) (Native, São Paulo, Brazil) were

130

added. The samples were homogenized until complete dissolution of the

131

ingredients. Then, the control chocolate milk drink formulation was pasteurized

132

at 63-65 ºC for 30 min in a digital water bath (Solab, São Paulo, Brazil) and

133

immediately cooled (4 °C). The other chocolate milk drink formulations (T1-T9;

134

Table 1) were submitted to the cold plasma treatment.

135

For cold plasma processing, the Plasma Etch PE-50 Venus (Plasma Etch

136

Inc, USA) apparatus was used, consisting of an aluminum chamber with a

137

horizontal electrode. The equipment operated with a 400 W and 50 kHz power

138

supply (continuous variable with an automatic matching network) connected to

139

the mains; vacuum was generated by a two-stage pump with a capacity of 5

140

m3/min, and the gas flow was controlled by computerized valves. The system

141

was fully automated and controlled by the Plasma Etch, Inc. computer program

142

provided by the equipment manufacturer. For each treatment, 120 mL of

143

beverage was divided into three 50 mL sealed falcon tubes, which were

144

introduced into the process chamber and subjected to plasma treatment. All

145

experiment was performed at room temperature (21-25 ºC) and using nitrogen

146

as a gas. The samples were treated with cold plasma at gas flow rates of 10,

147

20, and 30 mL/min and processing times of 5, 10, and 15 min totalizing 9

148

treatments (Table 1). The process parameters were chosen considering the

149

results of the preliminary tests and based on previous studies with fruit juices

150

(Rodríguez et al., 2017; Alves Filho et al., 2018).

151

2.2

pH

152

The pH of the chocolate milk drinks was measured using a pH meter

153

(AKSO, AK103, São Leopoldo/RS, Brazil) calibrated with buffer solutions of pH 4

154

and pH 7.

155

2.4

156

The bioactive compounds were extracted as described by Cappato et al

157

(2018). Briefly, 1 g milk drink was mixed with 30 mL ethanol/water (50:50, v/v)

158

and shaken at 200 rpm at room temperature for 1 h. After that, the extract was

159

filtered under vacuum, the volume was completed to 50 mL, and stored under

160

refrigeration until analysis.

Bioactive compounds

161

The antioxidant capacity was determined by the 1,1-diphenyl-2-

162

picrylhydrazyl (DPPH) method, which is based on the quantification of free

163

radical-scavenging activity, as described by Brand-Williams, Cuvelier, and Berset

164

(1995). Readings were measured at 517 nm every minute, until the absorbance

165

reduction and stabilization. The total antioxidant activity was calculated using

166

Eq. 1.

167 168

% DPPH = (initial absorbance

control

- absorbance

initial absorbance

extract)

X 100 (Eq. 1)

control

169 170

The total phenolic content (TPC) was determined according to Georgé,

171

Brat, Alter, and Amiot (2005) with modifications. Each extract of the chocolate

172

milk drinks (1 mL) was mixed with 1 mL of Folin–Ciocalteu reagent (diluted in

173

water 1:10). After 3 min of reaction, 1.5 mL of 10% (w/w) sodium carbonate

174

solution was added. The mixture was stirred and kept at room temperature for

175

2 h in the dark and the absorbance was measured at 725 nm. Results were

176

expressed as gallic acid equivalents (GAE)/g of chocolate milk drink, using gallic

177

acid as a reference standard.

178

The ACE inhibitory activity was determined using the methodology

179

proposed by Amaral et al. (2018), and the results were expressed as % ACE

180

inhibition, calculated using the Eq. 2: ACE inhibition (%) = [1− (C − D) / (A − B)] × 100

181

(2)

182

where A is the absorbance with ACE and without sample; B is the

183

absorbance without ACE and sample; C is the absorbance with ACE and sample,

184

and D is the absorbance without ACE and with the sample.

185

2.5

Fatty acid composition

186

The total lipids in chocolate milk drinks were cold-extracted and the fatty

187

acid methyl esters (FAMEs) were transesterified according to previous studies

188

(Cappato et al., 2018, Martins et al., 2018). The identification and quantification

189

of fatty acids were determined through a gas chromatograph GC-MS (Agilent

190

Technologies 7890A/5975C-GC/MS, Santa Clara, USA) with CTC PAL sampler

191

(Agilent Technologies) operating in the split-injection mode, using a software

192

for data acquisition and system control (Agilent MassHunter Quantitative

193

Analysis). A DB-FFAP column (polyethylene glycol modified with nitro

194

terephthalic acid, of 15 m long, 0.10 mm internal diameter, 0.10 µm film

195

thickness, Agilent Technologies) was used for FAMEs separation, using helium

196

as a carrier gas at a flow rate of 0.5 mL/min. Injector (split of 1:100) was set in

197

240 °C, and mass spectrometry detector (MSD) was acquired in full scan

198

analysis at an m/z range of 40-400. The oven temperature was initially set at

199

70 °C for 1 min and then programmed to increase to 115 °C at a rate of 45

200

°C/min. Further, the temperature increased at a rate of 40 °C/min until 175 °C

201

and then increased at a rate of 30 °C/min until 240 °C held for 4.23 min. FAMEs

202

were identified by the comparison of the retention time of the reference

203

standard containing 37 fatty acid methyl esters (Sigma-Aldrich, St. Louis, MO,

204

USA, 18919-1AMP), and the mass spectra were compared with the NIST

205

spectra library 11.

206

The atherogenic and thrombogenic indices (AI and TI, Batista et al.,

207

2017, Sperry et al., 2018), the desired fatty acids (DFA, Barlowska et al., 2018),

208

and the hypercholesterolemic saturated fatty acids (HSFA, Barlowska et al.,

209

2018) were calculated according to Eq. (3), (4), (5), and (6), respectively. AI = (C12:0 + 4 × C14:0 + C16:0)/[Σ MUFA + Σ PUFA(n-6) (n-3)] (Eq.

210 211

3) TI = (C14:0 + C16:0 + C18:0)/[0.5 × Σ MUFA + 0.5 × Σ PUFA(n-6) + 3

212 213

× Σ PUFA(n-3) + (n-3)/(n-6)] (Eq. 4)

214

DFA = MUFA + PUFA + C18:0 (Eq. 5)

215

HSFA = C12:0 + C14:0 + C16:0 (Eq. 6)

216

2.6

Volatile compounds

217

The volatile compounds were analyzed using the methodology by

218

Condurso, Verzera, Romeo, Ziino & Conte (2008). All extractions were

219

performed by solid phase microextraction using an SPME fiber of 50/30 μm

220

Divinylbenzene/Carboxen/Polydimethylsiloxane

221

Bellefonte, PA, USA). The identification of the volatile compounds was done by

222

CG-EM (Agilent Technologies, 7890A-5975C) with a CTC PAL sampler 120

(DVB/CAR/PDMS)

(Supelco,

223

(Agilent Technologies). The analysis conditions were: fiber injection, with no

224

splitless flow division of the mobile phase, injector temperature of 240 °C

225

(mobile phase flow rate of 2 mL/min); oven temperature initially set at 45 °C

226

for 5 min, with a temperature ramp programmed to increase to 80 °C at a rate

227

of 10 °C/min, followed by a new ramp at 5 ºC/min until 240ºC, and holding for

228

25 min. A CP-Wax 52 CB column (60 m long, 0.25 mm internal diameter, 0.25

229

µm film thickness, Agilent Technologies) and a mass spectrometry detector

230

(MSD) in the range of 40-500 m/z was used. The compounds were identified

231

according to the linear retention index (LRI) of each compound and calculated

232

according to Van den Dool and Kratz equation, comparing with the LRI of

233

alkane standards of 8-40 carbons (Sigma, 40147-U).

234

2.7

Statistical analysis

235

The process was repeated three times, and the analyses were performed

236

in triplicate. The results were presented as means ± standard deviation and

237

analyzed by ANOVA followed by the Tukey's test (p-value ≤ 0.05) using the

238

software XLSTAT 2018.5 (Adinsoft, Paris, France).

2393 3.Results and discussion

3.4 240

3.1 pH values

241

The chocolate milk drinks presented pH values in the range of 6.33-6.88

242

(Table 2), corroborating previous studies with milk drinks (Janiaski, Pimentel,

243

Cruz & Prudencio, 2016), and the pasteurized beverage presented the lowest

244

pH value (6.33) (p ≤ 0.05). During the heat treatment, lactose undergoes

245

reactions resulting in compounds such as formic acid, pyruvic acid, acetic acid,

246

among others. Formic acid is primarily responsible for the increased acidity of

247

milk subjected to high temperatures (Dursun, Güler & Sekerli, 2017).

248

The cold plasma process parameters had a significant impact on the pH of

249

the chocolate milk drinks (p ≤ 0.05), once higher treatment time and flow rate

250

led to lower pH values (p ≤ 0.05). The effect of cold plasma treatment in the

251

pH values is mainly due to the interaction of plasma reactive species with the

252

moisture of the food products. In liquid products, such as milk drinks, plasma

253

species reacts with water, forming acidic compounds (Yong et al., 2015).

254

Higher flow rates and longer processing times result in the formation of a

255

higher quantity of acidogenic molecules, which decreases pH of the medium

256

(Yong et al., 2015).

257

The parameter pH is a quality attribute in most of the processed food

258

products, thus, any drastic change can lead to an undesirable impact on flavor,

259

texture, and shelf life of the product (Pankaj, Wan & Keener, 2018). Milk drinks

260

are characterized as refreshing, light, genuine thirst quencher, healthful, and

261

less acidic than fruit juice (Panghal et al., 2018). Therefore, the increased pH of

262

the cold plasma-treated chocolate milk drinks may be interesting from the

263

consumer point of view, as a product with low acidity and high intensity of

264

chocolate flavor is expected.

3.5 265

3.2 Bioactive compounds

266

Table 2 shows the mean values of DPPH, TPC, and ACE inhibitory activity

267

of the chocolate milk drinks, with values of 8.76 to 9.24 μg Eq. TE/g, 4.26 to

268

22.53 mg GAE/100 mL, and 7.11 to 13.75%, respectively. Differences were

269

observed between the pasteurization and cold plasma treatments and among

270

the cold plasma processed products (p ≤ 0.05). Therefore, the study of the cold

271

plasma

272

maintenance of the bioactive compounds of milk, whey, or cocoa powder,

273

besides the comparison with a product subjected to the traditional processing

274

method (pasteurization).

process

parameters

is

of

paramount

importance

aiming

the

275

The TPC of the chocolate milk drinks is associated with the components of

276

the beverage formulations, including milk, whey, and cocoa powder. In fact,

277

polyphenols are found in considerable amounts in ruminant milk, originating

278

mainly from the secondary metabolism of the plants ingested by the animals.

279

Furthermore, polyphenols, tannins, and flavonoids can also be found in cocoa

280

(Monteiro et al., 2018).

281

The chocolate milk drinks submitted to cold plasma processing (T1-T9)

282

presented lower TPC when compared to the pasteurized product (p ≤ 0.05).

283

The phenolic compounds are generally considered heat-stable, and the

284

occasional loses in the different thermal processes are probably due to

285

lixiviation (Gomez-Gomez, Borges, Minatel, Luvizon & Lima, 2018). Higher

286

temperature may disintegrate the phenolic-cell wall matrix bond, enhancing the

287

phenolic extraction and resulting in higher TPC in the heat-treated products

288

(Gomez-Gomez et al., 2018). On the other hand, the energetic electrons

289

produced by plasma discharge can dissociate the oxygen molecules originating

290

single oxygen atoms, which combines with an oxygen molecule (O2) to form

291

ozone gas. Phenolic compounds are very susceptible to ozone attack, as ozone

292

acts on the aromatic compounds resulting in the formation of hydroxylated and

293

quinone compounds (Almeida et al., 2015).

294

The cold plasma process parameters had a significant impact on the TPC

295

values, with higher TPC in the products submitted to higher flow rates and

296

longer processing times (p ≤ 0.05). The increased number of reactive species

297

of the products subjected to more drastic conditions promoted the rupture of

298

the cell membranes and the release of the phenolic compounds, increasing their

299

concentration in the medium (Almeida et al., 2015), which may compensate the

300

impact of ozone on these compounds.

301

Phenolic compounds are a rich group of secondary metabolites with

302

renowned pharmacological and biological effects, which are responsible for

303

color, flavor, and aroma of numerous fruits, flowers, vegetables, and even

304

spices. They also play very important health effects, such as antioxidant,

305

antitumor, antitussive, analgesic, anti-inflammatory and hepatoprotective

306

activities, among others (Gomez-Gomez et al., 2018).

307

Considering the antioxidant activity of the beverages, only the treatments

308

subjected to the cold plasma, T5 (20 mL/min, 10 min) and T9 (30 mL/min, 15

309

min), presented lower antioxidant activity when compared to the pasteurized

310

product (p ≤ 0.05), thus high flow rates and longer treatment times led to a

311

decrease in the antioxidant activity (p ≤ 0.05). These results are due to a

312

higher quantity of plasma reactive species is obtained, which can react with the

313

amino acid present in the product, leading to protein denaturation, resulting in

314

the breakdown of the peptides with antioxidant activity. Although the cold

315

plasma treatment originated chocolate milk drinks with lower TPC, the

316

antioxidant activity was maintained similar to the pasteurized product (except

317

for T5 and T9).

318

The processing of whey proteins, which contain high levels of specific

319

dipeptides as glutamylcysteine, promotes the synthesis of glutathione, which is

320

an important antioxidant (Park & Nam, 2015). β-lactoglobulin is another

321

example of the peptide from milk protein with higher antioxidant activity when

322

compared to that of butylated hydroxyanisole, a common synthetic antioxidant

323

ingredient used in the food industry to prevent product deterioration (Nielsen,

324

Beverly, Qu & Dallas, 2017). In addition to milk proteins, the cocoa bean and its

325

products including cocoa powder, cocoa liquor, and dark chocolate are

326

considered a rich source of phenolic compounds and flavonoids and exhibit

327

great antioxidant capacity (Moreira et al., 2018).

328

The influence of the cold plasma technology on the ACE inhibitory

329

activity was dependent on the cold plasma process parameters. In the mild

330

conditions (10-20 mL/min, 5-15 min), the cold plasma treated samples (T1-T6)

331

presented lower ACE inhibitory activity when compared to the pasteurized

332

product (p ≤ 0.05). More severe conditions (longer time and higher gas flow

333

rate) resulted in a higher inhibitory potential, thus the highest ACE inhibitory

334

activities (13.29-13.75%) were found in the products subjected to the highest

335

gas flow rate (30 mL/min, T7-T9). Possibly, the most drastic conditions resulted

336

in a partial whey protein denaturation, resulting in the exposure of bioactive

337

peptides and higher ACE inhibition (Amaral et al., 2018).

338

Bioactive peptides (BAPs) derived from milk proteins have been

339

considered functional ingredients with health-promoting effects. They are

340

derived from both casein and whey proteins and may modulate different

341

biological and physiology properties with health benefits, including antioxidant,

342

ACE inhibition, antimicrobial, antihypertensive, antithrombotic, opioid agonist

343

and antagonist activities, mineral binding, and immunomodulatory properties

344

(Park & Nam, 2015; Nielsen, Beverly, Qu & Dallas, 2017). ACE-inhibitory

345

peptides from milk and dairy products have attracted interest for their possible

346

use as a natural alternative to drugs, for reducing blood pressure through the

347

binding and ACE inhibition, because some milk peptides are absorbed into the

348

bloodstream as α- and k-casein fragments (Nielsen, Beverly, Qu & Dallas,

349

2017). Antioxidant and ACE inhibitory activities have also been reported for the

350

peptides and hydrolysates from cocoa (Sarmadi, Ismail & Hamid, 2011).

351

The present results indicate that milder processing conditions (lower gas

352

flow rates and processing times) resulted in chocolate milk drinks with lower

353

TPC and ACE inhibitory activity while maintaining the antioxidant activity similar

354

to the pasteurized product. In the case of more severe processing conditions

355

(higher gas flow rates and processing times), higher TPC and ACE inhibitory

356

activity were observed, with a decrease in antioxidant activity (only for T9). The

357

chocolate milk drinks subjected to intermediate cold plasma conditions

358

exhibited bioactive compounds in the middle range and preservation of the

359

antioxidant activity. The results demonstrated that the selection of the suitable

360

cold plasma process parameters allows obtaining chocolate milk drinks with

361

improved nutritional quality when compared to the pasteurized product, mainly

362

concerning the ACE inhibitory activity.

3.6 363

3.3 Fatty acid composition

364

The fatty acid composition of the chocolate milk drinks is presented in

365

Table 3. A total of 13 fatty acids were detected, including the saturated chain

366

fatty acids (SFAs, C4:0, and C18:0), monounsaturated fatty acids (MUFAs,

367

C14:1, C16:1, and C18:1) and polyunsaturated fatty acids (PUFAs, C18:2, and

368

C18:3). The major fatty acids identified in the samples were oleic (19.17-60.16

369

g/100g), palmitic (12.99-32.15 g/100g), stearic (6.72-16.46 g/100g), and

370

myristic (4.82-12.12 g/100g) acids. Monteiro et al. (2018) reported that the

371

saturated fatty acids comprise 70% of milk composition and the long chain fatty

372

acids palmitic (C16:0), myristic (C14:0), stearic (C18:0), oleic (C18:1n-9), and

373

linoleic (C18:2n-6) are characteristic of milk fat. The authors also reported that

374

the cocoa butter contains stearic (C18:0), oleic (C18:1n-9), and palmitic

375

(C16:0) acids. MUFAs are protective against Metabolic Syndrome and

376

Cardiovascular Disease Risk Factors (Sperry et al., 2018).

377

There was an impact of the cold plasma treatment on the fatty acid profile

378

of the chocolate milk drinks (p ≤ 0.05), with changes observed for all fatty

379

acids evaluated. In mild (T1, 10 mL/min, 5 min) and severe conditions (30

380

mL/min, T7, T8 and T9), there was an increase in SFA (butanoic, hexanoic,

381

octanoic, decanoic, dodecanoic, myristic, palmitic, and stearic acids) and a

382

decrease in both MUFA (myristoleic, palmitoleic and oleic acids) and PUFA

383

(linoleic and linolenic acids) (p ≤ 0.05) when compared to the pasteurized

384

product. The changes in fatty acids may be due to the oxygen radicals

385

produced during plasma treatment, including ozone, that react with the

386

unsaturated fatty acids and break down the double bonds, leading to an

387

increase in SFA (Gavahian et al., 2018). The more unsaturated the fatty acids,

388

the more susceptible to the action of the plasma reactive species, because the

389

energy needed for abstraction of a hydrogen atom is significantly lower in

390

double bonds than CH bonds linked elsewhere (272 kJ/mol vs. 422 kJ/mol)

391

(Gavahian et al., 2018).

392

The chocolate milk drinks subjected to intermediate cold plasma treatment

393

conditions (T2-T6) presented an improved fatty acid profile when compared to

394

the pasteurized product, with a reduction in stearic acid and an increase in

395

myristoleic acid (T4), linoleic (T5), and PUFA (T3) levels (p ≤ 0.05). In addition,

396

mild and drastic conditions had higher AI, TI, and HSFA, and lower DFA (p ≤

397

0.05), while the products submitted to intermediate conditions had similar

398

indices (AI, TI, HSFA, and DFA) when compared to the pasteurized product (p

399

> 0.05). A lipid fraction with high quality must contain low AI, TI, and HSFA

400

levels, which can inhibit the aggregation of platelets, preventing the appearance

401

of coronary diseases, with health benefits in humans (Sperry et al., 2018).

402

Therefore,

403

processing conditions (T2-T6) have proven to be more suitable for the

404

manufacture of chocolate milk drinks.

405

considering

the

health-associated

effects,

the

intermediate

3.4 Volatile compounds

406

More than 30 volatile organic compounds (VOC’s) were identified in the

407

control and the plasma-treated chocolate milk drink (Table 4), including 9

408

carboxylic acids, 8 ketones, 7 alcohols, 1 aldehyde, 3 esters, 1 furan, and 2

409

lactones. Overall, qualitative changes were observed between the products

410

processed by cold plasma and pasteurization, and the differences were related

411

to the cold plasma process parameters. The chocolate milk drinks processed at

412

mild (T1) and severe (T7-T9) cold plasma conditions presented 15-18 VOC,

413

while the pasteurized product presented 19 compounds. However, the products

414

submitted to intermediate conditions (T2, T3, T4, and T6) presented a higher

415

number of VOC (22-26), suggesting that these conditions provided protection to

416

the volatile compounds.

417

The application of cold plasma at low flow rates and lower processing

418

times (T1) resulted in the absence of some carboxylic acids (decanoic acid,

419

hexadecenoic acid, and tetradecanoic acid) and presence of some alcohols (2-

420

ethyl-1-hexanol and 1-pentanol) when compared to the pasteurized product.

421

The application of more drastic conditions (T7-T9) resulted in the non-

422

identification of some carboxylic acids (decanoic acid, isobutyric acid, octanoic

423

acid, and tetradecanoic acid), furans (3-furan methanol) and lactones (4-

424

hydroxydihydrofuran-2-(3H)-one and 2-hydroxy-gamma-butyrolactone) and the

425

identification of some alcohols (2-methylbutan-1-ol, 2-ethyl-1-hexanol and DL-

426

2,3-butanediol). Alcohols may be formed by the decomposition of fatty acids

427

hydroperoxides or the reduction of aldehydes (Liu et al., 2015), corroborating

428

the changes in the fatty acid composition observed in the present study (Table

429

3). Amaral et al. (2018) reported that severe non-thermal treatments can cause

430

loss of volatile compounds and alteration of the sensory and functional

431

properties of the products. In these conditions, other compounds can also be

432

formed.

433

Lactone has been explicitly identified as the primary odorant of milk

434

products (Liu et al., 2015) and responsible for fruity, nutty, and dairy aromas

435

(Mahajan, Goddik & Qian, 2004). The lactone 4-hydroxydihydrofuran-2-(3H)-

436

one represents the caramel-like odor of cocoa (Afoakwa, Paterson, Fowler &

437

Ryan, 2008). Furthermore, the carboxylic acids can contribute to the harsh,

438

nutty and cocoa-like odor of dark chocolate, cocoa liquor, and cocoa powder,

439

and products with low concentrations or absence of carboxylic acids lose the

440

characteristic chocolate flavor (Liu et al., 2015). Therefore, the mild and more

441

drastic cold plasma conditions resulted in products with the absence of volatile

442

compounds that are important to the odor and flavor characteristics of the

443

chocolate milk drinks.

444

The

intermediate

processing

conditions

(T2-T6)

resulted

in

the

445

maintenance of some compounds that were not observed in the pasteurized

446

product, such as ketones (2-hydroxycyclopent-2-en-1-one, 2-methyldihydro-

447

3(2H)-thiophenone, dihydroxyacetone, pyranone), alcohols (2-ethyl-1-hexanol

448

and DL-2,3-butanediol), and esters (hexanoic acid, ethyl ester; octanoic acid,

449

ethyl ester, and ethyl tiglate). In addition, products without the presence of 2-

450

nonanone (T3 and T6), 4-hydroxydihydrofuran-2-(3H)-one (T3), and 2-hydroxy-

451

gamma-butyrolactone (T5) were also observed.

452

Alcohols are responsible for producing the desirable flavor notes of

453

interest, and the compound 2,3 butanediol, which was detected only in the cold

454

plasma-treated products, is associated with a sweet citrusy odor (Caprioli et al.,

455

2016) and cocoa butter (Moreira et al., 2018). Esters are correlated to fruit

456

notes (Moreira et al., 2017). The presence of 2-pentanone is associated to

457

sweet, fruity, banana, and fermented aroma in cocoa beans (Hinneh et al.,

458

2018), while dihydro-2-methyl-3(2h)-thiophenone is associated with fruity and

459

berry aromas (Li, Yu, Curran & Liu, 2011). Therefore, the use of intermediate

460

flow rates provided a protection of flavor compounds, which is important for the

461

maintenance of the aroma and flavor characteristics of the chocolate milk

462

drinks. The absence of 2-nonanone in some cold plasma-treated formulations is

463

also interesting, as this compound is responsible for the “ketone” and “stale”

464

flavor of heat-treated milk (Dursun et al., 2016). However, it is also associated

465

with the sweetness of cocoa (Liu et al., 2015).

466

All formulations presented the compounds 3-methyl butanoic acid, acetic

467

acid, butanoic acid, hexanoic acid, 2-heptanone, 1-methoxy-2-propane, 3-

468

methylbutan-1-ol

469

hydroxymethylfurfural. These compounds are associated with the malty-

470

chocolate odor (methylbutan-1-ol, Caprioli et al., 2016), sweet taste of cocoa

471

(3-methyl butanoic acid, Afoakwa et al., 2008), cheesy (3-methyl butanoic acid,

472

Liu et al., 2015), sour taste (acetic acid, Afoakwa et al., 2008), buttery flavor

473

(butanoic acid, Afoakwa et al., 2008), sweet and pungent odor (hexanoic acid,

474

Afoakwa et al., 2008), sweet and citrusy odor (2-heptanol, Moreira et al.,

475

2017), caramel (5-hydroxymethylfurfural, Vásquez-Araújo et al., 2008), rancid

476

and sour odor (hexanoic acid, butanoic acid, Amaral et al., 2018) and green

477

odor (2-pentanol, Moreira et al., 2017) associated to milk, cocoa powder, or

478

whey used in the present formulations. The compound 2-heptanone is found in

479

milk and dairy products, including cheese, butter, and yogurts (Amaral et al.,

480

2018). The most important carboxylic acids in cocoa powder are acetic acid, 2-

481

methylpropanoic acid, 3-methyl butanoic acid, and hexanoic acid (Liu et al.,

482

2015). Acetic acid has been widely identified as an important flavor enhancer of

483

cocoa and chocolate, by imparting sour, buttery, and sweaty notes, while the

484

lack of this compound may break the desirable balance of the chocolate flavor

485

(Liu et al., 2015). Acetic acid is also found in sweet whey powder and is

(except

T5),

2-pentanol,

2-heptanol,

and

5-

486

responsible for its unique aroma (Mahajan, Goddik & Qian, 2004). The

487

compounds 2-butanone, dimethyl sulfide, ethanol, and 2-propanone, known to

488

cause off-flavor in milk and dairy products (Korachi et al., 2015), were not

489

identified in all chocolate milk drinks.

490

The results indicate that the use of mild and severe processing conditions

491

resulted in products with the absence of some important volatile compounds.

492

However, when intermediate cold plasma conditions were applied, a higher

493

level of ketones, esters, and lactones was observed, demonstrating that the

494

compounds important to chocolate and milk aroma and flavor were maintained

495

when compared to the pasteurized product.

496

This is the first study on the use of cold plasma technology in chocolate

497

milk drinks. Future studies are required to evaluate the effect of cold plasma on

498

the sensory characteristics (Silva et al., 2018) and consumer acceptance

499

(Belsito et al., 2017) of the products.

500 501

4. Conclusion

502

The cold plasma technology is a non-thermal alternative for the

503

processing of chocolate milk drinks, and the processing parameters are of

504

paramount importance. In mild (lower flow rate and process time) and more

505

severe (higher flow rate and process time) conditions, reductions of the

506

bioactive compounds (total phenolic compounds and ACE-inhibitory activity),

507

changes in fatty acid composition (increased saturated fatty acid and decreased

508

monounsaturated fatty acid and polyunsaturated fatty acid), less favorable

509

health indices (higher atherogenic and thrombogenic indices, and high

510

saturated fatty acids and lower desired fatty acids levels), and lower volatile

511

compounds levels were observed. In contrast, under intermediate cold plasma

512

conditions, a suitable concentration of bioactive compounds, fatty acid

513

composition, health indices, and increased number of volatile compounds

514

(ketones, esters, and lactones) was observed. Therefore, it is advisable to use a

515

flow rate of 20 mL/min and a process time of 5 min to process chocolate milk

516

drinks.

517 518

Acknowledgments

519

This research was financially supported from the Conselho Nacional de

520

Desenvolvimento Científico e Tecnológico (CNPq) and the Coordenação de

521

Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

522

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Barrachina, A. A. (2008). Investigation of aromatic compounds in toasted

733

almonds used for the manufacture of turrón. European Food Research and

734

Technology, 227, 243-254.

735 736

Yong, H. I., Kim, H. J., Park, S., Kim, K., Choe, W., Yoo, S. J., & Jo, C. (2015).

737

Pathogen inactivation and quality changes in sliced cheddar cheese treated

738

using flexible thin-layer dielectric barrier discharge plasma. Food Research

739

International, 69, 57-63.

740 741 742

Table 1. Chocolate milk drink samples processed by cold plasma.

743 744 745 746 747 748 749 750 751 752 753 754 755 756 757

Treatments

Time (min)

Gas flow (mL/min)

T1 T2 T3 T4 T5 T6 T7 T8 T9

5 10 15 5 10 15 5 10 15

10 10 10 20 20 20 30 30 30

758 759 760 761 762

763 764 765 766 767 768 769 770 771 772 773

Table 2. pH and bioactive compounds values in chocolate milk drink after processed by cold plasma.

Treatments

pH

DPPH

Phenolics

ACE

Pasteurized

6.33 ± 0.01e

9.16 ± 0.01ab

22.53 ± 0.71a

12.65 ± 0.28b

T1

6.86 ± 0.01ab

9.14 ± 0.01ab

5.07 ± 0.19de

7.11 ± 0.17e

T2

6.85 ± 0.01abc

9.24 ± 0.02a

4.26 ± 0.07e

9.54 ± 0.47d

T3

6.88 ± 0.01a

9.18 ± 0.01ab

4.39 ± 0.05e

9.88 ± 0.01cd

T4

6.88 ± 0.01a

9.2 ± 0.01ab

5.88 ± 0.05d

10.72 ± 0.09c

T5

6.86 ± 0.01ab

9.01 ± 0.01c

4.29 ± 0.02e

10.56 ± 0.15c

T6

6.82 ± 0.01cd

9.1 ± 0.01bc

19.25 ± 0.14c

10.45 ± 0.16cd

T7

6.82 ± 0.01bc

9.1 ± 0.01bc

20.81 ± 0.07b

13.29 ± 0.39ab

T8

6.83 ± 0.01bc

9.11 ± 0.01abc

20.14 ± 0.07bc

13.75 ± 0.04a

T9

6.78 ± 0.01d

8.76 ± 0.1d

19.58 ± 0.41c

13.67 ± 0.31a

* Values expressed as mean ± standard deviation. DPPH values is expressed in μg Eq. TE/g. Phenolics are expressed by g Eq. GAE/g. ACE is expressed in %. a-e Means followed by the same letters in the columns are statistically different by the Tukey test (P < 0.05). See Table 1 for formulations.

Table 3. Fatty acids profile (g/100g fat) of chocolate milk drink submitted to cold plasma technology.

Fatty Acids Butanoic (C4:0)

Pasteurized

T1

1.66 ±

0.09a

4.14 ±

0.11a

2.49 ± 1.93 ±

0.13c

0.85 ±

0.06c

2.03 ±

0.02c

1.95 ±

0.31cd

1.6 ±

0.16cd

0.7 ±

0.04cd

1.81 ±

0.15cd

T4 1.87 ±

0.47d

1.51 ±

0.32d

0.66 ±

0.10d

1.77 ±

0.17d

T5

T

1.59 ±

0.09cd

0.7 ±

0.01cd

Decanic (C10:0)

1.80±

0.05cd

Dodecanoic (C12:0)

1.19 ± 0.01c

2.78 ± 0.05a

1.3 ± 0.05c

1.24 ± 0.05c

1.18 ± 0.15c

1.17 ± 0.07c

1.2 ±

Myristic (C14:0)

5.27 ± 0.12cd

12.12 ± 0.25a

5.59 ± 0.45c

5.14 ± 0.28cd

4.99 ± 0.48cd

4.82 ± 0.13d

5.16 ±

Palmitic (C16:0)

14.59 ± 0.3c

32.15 ± 0.11a

13.75 ± 0.67cd

13.3 ± 0.23d

13.61 ± 1.12cd

12.99 ± 0.50d

13.4 ±

Stearic (18:0)

8.34 ± 0.2c

16.46 ± 0.08a

6.72 ± 0.07d

7.18 ± 0.26d

7.29 ± 0.75d

6.76 ± 0.15d

6.98 ±

Octanoic (C8:0)

3.94 ±

0.07a

0.23c

T3

2.04 ±

Hexanoic (C6:0)

4.84 ±

0.18a

T2

0.05cd

2.07 ±

0.06cd

2.26 ±

1.61 ±

0.07cd

1.73 ±

0.68 ±

0.05d

0.74 ±

1.73 ±

0.07d

1.8 ±

∑SFA

35.53 ± 0.73c

78.1 ± 0.67a

34.67 ± 1.69cd

32.91 ± 0.97cd

32.87 ± 3.54cd

31.83 ± 1.11d

33.27 ±

Myristoleic (14:1n-9)

1.26 ± 0.08bc

0.45 ± 0.04e

1.41 ± 0.1ab

1.38 ± 0.04abc

1.44 ± 0.12a

1.29 ± 0.02abc

1.23 ±

1.14 ±

0.01c

3.25 ±

0.27a

19.17 ±

0.61c

57.33 ±

1.83a

20.77 ±

0.66c

61.99 ±

1.46a

1.05 ±

0.01d

0.08 ±

0.01e

1.13 ±

0.01d

3.81 ±

0.16a

5.42 ±

0.2a

Palmitoleic (C16:1n-9) Oleic (C18:1n-9) ∑MUFA Linoleic (C18:2n-6) Linolenic (C18:3n-3) ∑PUFA

3.08 ±

0.1a

56.95 ±

0.87a

61.29 ±

0.69a

2.93 ±

0.01b

0.25 ±

0.01abc

3.04 ±

0.25ab

0.30 ±

0.02ab

3.34 ±

0.23ab

0.57 ±

0.05c

0.78 ±

0.05c

3.27 ±

0.35a

58.83 ±

1.24a

63.48 ±

0.93a

3.3 ±

0.03ab

0.32 ±

0.06a

3.62 ±

0.03a

0.52 ±

0.03c

0.74 ±

0.02c

3.24 ±

0.11a

59.04 ±

3.29a

63.73 ±

3.53a

3.2 ±

0.01ab

0.2 ±

0.01cd

3.40 ±

0.02ab

0.52 ±

0.07c

0.76 ±

0.11c

3.14 ±

0.23a

3.28 ±

60.16 ±

1.06a

58.81 ±

64.59 ±

0.81a

63.32

3.33 ±

0.22a

3.17 ±

0.25 ±

0.08abc

0.24 ±

3.58 ±

0.30ab

3.41 ±

0.49 ±

0.02c

0.53 ±

0.71 ±

0.04c

0.75 ±

3.18 ±

0.04b

0.57 ±

0.02c

TI

0.86 ±

0.03c

DFA

72.81± 0.52a

38.36± 0.74c

72.05± 1.61a

74.28± 1.22a

74.42± 2.80a

74.93± 0.96a

73.71±

HSFA

21.05± 0.44c

47.05± 0.30a

20.64± 1.18c

19.68± 0.56c

19.78± 1.75c

18.98± 0.70c

19.76±

AI

774 775 776 777 778 779 780

*Values are expressed as mean ± standard deviation. Analysis performed in triplicate. a-e Means with different lowercase superscripts in the same row indicate presence of statistical difference (P < 0.05) among treatments control (pasteurization) and cold plasma by Tukey Test. SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acid. AI = (C12:0 + 4 C14:0 + C16:0)/[∑MUFA + ∑PUFA(n-6) and (n-3)]; TI = (C14:0 + C16:0 + C18:0)/[0.5 x ∑MUFA + 0.5 x ∑PUFA(n6) + 3 x ∑PUFA(n- 3) + (n-3)/(n-6)]; DFA = MUFA + PUFA + C18:0; HSFA = C12:0 + C14:0 + C16:0. See Table 1 for formulations.

Table 781 4. Volatile compounds of chocolate milk drink after pasteurization and cold plasma technology. 782 783

unds

LRI*

Pasteurized

T1

T2

T3

T4

T5

T6

T7

T8

19 X

18 X

25 X

22 X

26 X

19 X

26 X

17

15

1656

X

X

1434

X

X

X

X

X

X

X

X

X

1615

X

X

X

X

X

X

X

X

X

2250

X

−

X

X

X

X

X

−

−

1829

X

X

X

X

X

X

X

X

X

2877

X

−

X

X

X

X

X

X

X

1556

X

X

X

X

X

X

X

−

X

2040

X

X

X

X

X

X

X

X

−

X

−

−

X

X

−

X

9

6

8

9

9

8

9

− 6

− 6

ntified

acid

d

2667

d

ylic acids 1161

X

X

X

X

X

X

X

X

X

nt-2-en-1-one (2H)-

1756

−

−

X

−

−

−

X

−

−

1519

−

−

X

X

X

X

X

−

−

anone

1286

X

X

X

X

X

X

X

X

X

2066

−

−

X

−

X

−

X

−

−

1976

−

−

−

X

X

−

X

X

−

2246

−

−

X

−

X

−

X

1376

X

X

X

−

X

X

−

− −

− X

3

3

7

4

7

4

7

3

3

ide

s

ol

1202

X

X

X

X

X

−

X

X

X

ol

1203

−

−

−

−

−

−

−

−

−

1475

−

X

X

−

−

−

−

−

X

1241

−

X

−

−

−

−

−

−

−

1110

X

X

X

X

X

X

X

X

X

1306

X

X

X

X

X

X

X

X

X

1525

−

−

X

X

X

X

X

3

5

5

4

4

3

4

X 4

X 5

X

X

X

X

X

X

X

X

X

1

1

1

1

1

1

1

1

1

s X

urfural

des

hyl ester

X

−

−

X

X

X

X

X

yl ester

−

−

X

−

−

−

X

−

−

X

−

−

X

−

−

−

−

X

X

0

0

1

2

2

2

− 2

− 0

− 0

l

X

s

furan-2(3H)-one

X

a-butyrolactone

X

es

784 785 786 787 788

X

X

X

X

X

−

X

X

1

1

1

1

1

0

1

1

X

X

X

−

X

X

X

X

−

X

X

X

X

X

−

X

X

−

2

2

2

1

2

1

2

2

0

*LRI – Linear Retention Index. VOC’s - volatile organic compounds . (−) Means not detected. X=presence of the compounds in the formulations.. See Table 1 for formulations.

 Chocolate milk drink manufactured using cold plasma technology; 32

789

 All the operational conditions improved pH values;

790

 Milder and more severe operational conditions decreased total phenolic

791 792 793

content, ACE values and volatile compounds;  Milder and more severe operational conditions proportionated less favorable health fatty acid indexes;

794

33