Assessment of processing impacts and type of clarifier on the concentration of ochratoxin A in pekmez as a conventional grape-based product

Journal Pre-proof Assessment of processing impacts and type of clarifier on the concentration of ochratoxin A in pekmez as a conventional grape-based product Ali Heshmati, Sabah Ghadimi, Akram Ranjbar, Amin Mousavi Khaneghah PII:

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DOI:

https://doi.org/10.1016/j.lwt.2019.108882

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YFSTL 108882

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LWT - Food Science and Technology

Received Date: 4 June 2019 Revised Date:

20 September 2019

Accepted Date: 24 November 2019

Please cite this article as: Heshmati, A., Ghadimi, S., Ranjbar, A., Mousavi Khaneghah, A., Assessment of processing impacts and type of clarifier on the concentration of ochratoxin A in pekmez as a conventional grape-based product, LWT - Food Science and Technology (2019), doi: https:// doi.org/10.1016/j.lwt.2019.108882. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

1

Assessment of processing impacts and type of clarifier on the

2

concentration of ochratoxin A in pekmez as a conventional grape-based product

3

Ali Heshmati a, Sabah Ghadimia,*, Akram Ranjbara, Amin Mousavi Khaneghah b,**

4

a

Department of Nutrition and Food Safety, Nutrition Health Research Center,

5

Hamadan University of Medical Sciences, Hamadan, Iran b

6

Department of Food Science, Faculty of Food Engineering, University of

7

Campinas (UNICAMP), Rua Monteiro Lobato, 80. Caixa Postal: 6121.CEP: 13083-862.

8

Campinas. São Paulo. Brazil.

9 10 11 12

*,**

13

[email protected] (S. Ghadimi)

14

[email protected]; (A. Mousavi Khaneghah)

15

Telephone: +55(19) 3521-0091. Fax: +55(19) 3521-2153

Corresponding authors.

16 17 18 19 1

20

Abstract

21

Pekmez can be considered as one of the most favored traditional grape's

22

products, however, it can be contaminated with ochratoxin A (OTA). In this

23

regard, for the first time, the effects of unit operations including washing, juicing

24

(crushing and pressing), clarification with the aid of various clarifier (bentonite,

25

white soil, and gelation) and boiling on the concentration of OTA in artificially

26

spiked

27

operations utilized in pekmez production led to OTA reduction while the range -

28

mean of OTA reduction in washing, juicing (crushing and pressing), clarification

29

and boiling stages were recorded as

30

6.13±3.16%,

31

respectively. Among the clarifiers used, the highest (23.76±7.90%) and lowest

32

(11.48±9.37%) mean reduction of OTA was attributed to gelatin and bentonite,

33

respectively. Therefore,

34

OTA. Generally speaking, the OTA overall reduction range and mean during

35

the

36

respectively, which depended on the initial concentration of OTA and unit

37

operations, the type and concentration of clarifier. The optimum condition of

38

OTA reduction was obtained in samples containing initial OTA content of 5 µg

39

kg-1 which were clarified with 3% of gelatin.

40

Keywords:

41

Ochratoxin A clarifiers

pekmez

samples

were

investigated.

findings,

all

unit

and

1.54-37.43%-10.44±3.85%,

the utilization of gelatin could remove higher levels of

production

Pekmez

to

7.36-16.53%-12.19±4.60%, 2.51-8.32%-

4.24-16.55%-16.38±10.25%,

pekmez

According

process

ochratoxin

A;

was

Grape

2

25.84-54.67

pekmez;

and

Mycotoxin

37.52±6.54%,

in

pekmez;

42

1.

Introduction

43

In recent years, there is a high tendency to use natural sweeteners instead of

44

refined sugar in different food products (Ajibola, Chamunorwa, & Erlwanger,

45

2012; Grembecka, 2015). In this regard, pekmez, as one of the traditional

46

grape products in some countries, particularly in Iran and Turkey, due to its

47

attractive properties, such as a high level of fructose, can be considered as a

48

natural sweetener (Akbulut, Çoklar, & Özen, 2008; Karababa & Develi Isikli,

49

2005). Additionally, the consumption of pekmez could offer notable amounts of

50

minerals

51

(Heshmati Ghadimi, Ranjbar, & Khaneghah, 2019). It is a molasses-like syrup

52

which is produced after juicing, clarification and condensing of the fruit mainly

53

grape, plum, mulberry, sugarcane, sugarbeet, fig, and apricot (Heshmati et al.,

54

2019; Karababa & Develi Isikli, 2005; Tosun, Yıldız, Obuz, & Seçkin, 2014).

55

such

The

as

calcium,

contamination

of

magnesium,

food

iron,

products

potassium,

with

copper,

mycotoxins

and

in

zinc

various

56

conditions along the production chain always is the point of concern (Heshmati

57

& Mozaffari Nejad, 2015; Mashak, Sohi, Heshmati, & Nejad, 2016). Moreover,

58

the prevalence of some fungal genera in food products can be addressed as a

59

warning

60

Elmi, & Mousavi Khaneghah, 2017; Khaneghah, Fakhri, Raeisi, Armoon, &

61

Sant'Ana, 2018; Majeed, Khaneghah, Kadmic, & Khan, 2017; Oteiza et al.,

62

2017). In this context, the mycotoxins as a serious concern for human health

63

can be defined as secondary metabolites secreted by some species belonging

64

mainly

signal

to

the

of

mycotoxin

Aspergillus,

contamination

Fusarium,

(Amirahmadi,

Penicillium, 3

and

Shoeibi,

Alternaria

Rastegar,

genera

65

(Amirahmadi et al., 2017; Mahmood Fashandi, Abbasi, & Mousavi Khaneghah,

66

2018; Majeed et al., 2017; Mousavi Khaneghah, Eş, Raeisi, & Fakhri, 2018;

67

Nabizadeh et al., 2018). They can result in reduced product quality as well as

68

further fails to meet the required criteria for the human health and animals

69

consumptions

70

Nejad, & Sant’Ana, 2017; Khaneghah, Fakhri, Raeisi, et al., 2018; Khaneghah,

71

Fakhri,

72

Sant’Ana, 2018; Rastegar et al., 2017)

&

(Campagnollo

Sant'Ana,

2018;

et

al.,

2016;

Khaneghah,

Heshmati,

Martins,

Zohrevand,

von

Hertwig,

Khaneghah,

Bertoldo,

&

73

Some fungus such as Alternaria spp, Eurotium spp, Penicillium spp, Botrytis

74

cinerea, Aspergillus spp, Cladosporium spp, and Rhizopus spp, can grow on

75

the grape (Aydogdu & Gucer, 2009; Oteiza et al., 2017), while Aspergillus spp

76

and Penicillium spp could produce mycotoxins such as ochratoxin A (OTA)

77

aflatoxins (AFs). In this context, the grape and derived products such as raisin,

78

currant, and pekmez may be contaminated with some mycotoxins including

79

OTA and AFs (Akdeniz, Ozden, & Alpertunga, 2013; Arici, Gümüs, & Kara,

80

2004; Heshmati et al., 2019; Heshmati & Mozaffari Nejad, 2015; Khiabani &

81

Mohamadi Sani, 2015; Kollia, Kanapitsas, & Markaki, 2014; Oteiza et al., 2017;

82

Tosun et al., 2014; Varga & Kozakiewicz, 2006).

83

The consumption of contaminated foods by OTA as one of the wide

84

detected mycotoxins in food chains can result in some adverse effects on

85

health

86

immunosuppressive

87

2015; Quintela, Villarán, López de Armentia, & Elejalde, 2013; Wang et al.,

such

as and

nephropathies, hepatotoxic

effects

4

carcinogenesis, (Heshmati

&

teratogenic,

Mozaffari

Nejad,

88

2018).

89

Cancer, OTA was classified as Group 2B (possibly carcinogenic to human)

90

(Majeed et al., 2017; Ostry, Malir, Toman, & Grosse, 2017). The occurrence of

91

OTA is one of the major concerns in pekmez, mainly can be correlated with

92

mediocre quality grapes used as raw materials or contamination by the fungus

93

during

94

samples was investigated by Arici et al. (2004). According to Tosun et al.

95

(2014),

96

contaminated with OTA. Moreover, based on the findings of Akdeniz et al.

97

(2013), 23 (92%) out of 25 investigated pekmez samples were contaminated

98

by OTA. Also, considering to report of Mohamadi Sani (2013), the pekmez

99

samples collected from Khorasan province, Iran was contaminated by OTA

100 101

However,

according

processing.

organic,

The

to

the

incidence

home-made,

and

International

of

OTA

Agency

was

commercial

for

reported

pekmez

Research

among

samples

on

pekmez

can

be

(Mohamadi Sani, 2013). Based on previously conducted investigations, the processing of food

102

products

could

reduce

103

(Heshmati, 2015; Heshmati et al., 2019; Khaneghah, Martins, et al., 2018). In

104

this regard, the investigation of the fate of mycotoxins during the production of

105

different traditional products such as pekemz attracted notable attention. The

106

findings of Heshmati et al. (2019) demonstrated that the concentration of

107

Aflatoxin

B1

108

(AFG2),

was

109

according to Arici et al. (2004), the OTA content of pekmez was increased

110

during manufacturing, although the role of each unit operation used in pekmez

(AFB1),

the

levels

Aflatoxin

significantly

of

contaminations,

B2 (AFB2),

reduced

during

5

Aflatoxin the

especially

mycotoxins

G1 (AFG1), Aflatoxin

pekmez

processing.

G2

However,

111

production was not investigated in detail. Therefore, the assessment of the

112

influence of unit operation on the OTA level among products such as pekmez

113

is a matter of concern. Thus, for the first time, the present study was aimed to

114

investigate

115

concentration. Besides, the influence of various types of clarifier on OTA

116

removal was investigated.

the

effects

of

each

unit

of

pekmez

processing

on

the

OTA

117 118

2.

Materials and methods

119

2.1. Materials

120

The OTA standard (10 µg mL-1) was bought from Sigma-Aldrich (St. Louis, MO,

121

US).

122

bicarbonate, and acetone, phosphate buffer saline (PBS), all in the analytical

123

grade were obtained from Merck (Darmstadt, Germany).

The

chemical

reagents

such

as

methanol,

acetonitrile,

sodium

124

Grape samples (variety of Asgari) were collected from a vineyard located

125

in Malayer city (Iran) (October 2017). The collected samples were washed with

126

tap water in order to remove dust and other physical contaminations. OTA level

127

in the grape samples was measured while the OTA levels in these samples

128

were lower than the limit of detection (LOD; 0.10 µg kg-1). White soil was

129

acquired from the mountains located in Malayer city of Iran. Immunoaffinity

130

columns (IAC), Bentonite (purity of 98%) and gelatin (purity of 99%), was

131

purchased from Libios (Pontcharra- sure-Turdine, France), Sina tolid (Tehran,

6

132

Iran) and Arya Inc. (Karaj, Iran), respectively. Ultra-pure water was prepared by

133

a Milli-Q system (Millipore, Milford, MA, USA).

134

2.2.

Methods

135

2.2.1.

Spiking OTA into grape and pekmez production

136

As

finding

difficult,

mentioned

139

Based on the maximum acceptable limits (10 µg kg-1) proposed by Institute of

140

Standards and Industrial Research of Iran for grape and its products such as

141

pekmez (ISIRI, 2010), in the current study, samples with 50, 100, and 150%,

142

i.e., 5, 10, and 15 µg kg-1 of OTA were used. Therefore, in order to obtain OTA-

143

spiked grape samples, a stock solution of OTA in methanol (1000 µg L-1) was

144

prepared. Then, 500, 1000 and 1500 µL from stock solution was added into

145

100 g of grape samples to obtain sample containing 5, 10 and 15 µg kg-1 of

146

spiked OTA. Due to this fact that the volume of spiked solutions was low, they

147

were diluted with methanol up to a volume of 10 mL and then sprayed on grape

148

samples. Afterward, the grape samples were poured into a mechanical mixer

149

(Model 40979, Gastroback GmbH, Hollenstedt, Germany) and agitated slowly

150

for one h to help homogeneous distribution of OTA (Camenzuli et al., 2018).

151

Samples were placed in room temperature for 24 h to infiltrate OTA into them.

152

For ensuring of OTA infiltration into a grape, the skin of grape was removed

153

and then the OTA concentration in the remained part was measured according

154

to

was 7

grape

the

employed. One of the crucial factors for this study was the spike levels of OTA.

concentration

OTA-spiked

with

138

OTA

was

samples

concentrations

2.2.2.

OTA

contaminated

137

section

of

naturally

approximately

samples

equal

to

were

expected

155

spiked levels, i.e. 5, 10 and 15 µ kg-1. Hence, it was observed that OTA was

156

penetrated grape samples during storage at room temperature. Subsequently,

157

the grape samples were soaked in water for 5 min and rinsed under tap water

158

for 20 s in order to follow the conventional pekmez procedure. In the next

159

stage, samples were crushed entirely and pressurized by the aid of a fruit juicer

160

(Pars Khazar, Tiger 700 Watts, Tehran, Iran) to produce juice (initial Brix of the

161

juice was 21.5 ºBx). The grape skin and seed were separated by passing

162

crushed samples through Whatman filter paper (No. 2). The obtained juice

163

containing suspended particles was called "must” needs to be clarified. During

164

clarification,

165

However, in the conventional method of pekmez production, only white soil is

166

used, in this study, in addition to white soil, two types of clarifier agents, i.e.,

167

bentonite or

168

incorporated into the must. Afterward, the samples were stirred for one h with

169

the aid of a mechanical stirrer (Mtops Co., Seoul, Korea) at room temperature

170

and were kept for 24 h at room temperature. Then samples were filtered by

171

Whatman filter paper (No. 2) to remove the used clarifier agents (The Brix of

172

the juice after the clarification was recorded as 22.3 °Bx). The obtained solution

173

poured

174

subjected gas stove flame (Singer Model of SG-ECO 5051, Tehran, Iran) to be

175

concentrated (72 °Bx). Each treatment (each unit operation) was performed in

176

triplicate,

177

concentrations, were reported.

solid

into

compounds

gelatin

a

and

in

stainless

two

were

levels

steel

average,

as

separated

of

1.5

container

well

and

and

as

the

8

from

the

3%

boiled

w/w,

at

standard

liquid

were

100-110

deviation

medium.

separately

°C

of

while

OTA

178

To determine the reduction percentage of OTA from the first unit operation, i.e.

179

grape washing, to the last unit operation, i.e. boiling and concentration, applied

180

for pekmez production the Equation 1 was used while the reduction percentage

181

of OTA during each unit operation was calculated with Equation 2:

182



Reduction % =















!



× 100 Equation 1



183 Reduction % =

OTA concentration before each unit operation − OTA concentration after each unit operation OTA concentration before each unit operation × 100 Equation 2

184 185

2.2.2. OTA determination

186

OTA

measurement

187

investigations

188

Heshmati et al., 2017). For the extraction of OTA, 25 g of grape, must or

189

pekmez samples (prepared in the previous section) was weighted then 40 mL

190

of ultra-pure water, 60 mL of acetone and 0.3 g of sodium bicarbonate powder

191

were added and mixed for 5 min with the aid of a magnetic stirrer (Mtops Co.,

192

Seoul,

193

(Whatman No. 1), and 10 mL of filtrate was collected. Forty mL of phosphate-

194

buffered saline (PBS) was added, and afterward, 40 mL was collected and then

195

passed through the Immunoaffinity column (Puri-Fast OTA HCS

196

sensitivity) The OTA was eluted from the column with the aid of 1.5 mL of

with

Korea).

The

was some

carried out

according

modifications

mixture

was

then

to our

(Heshmati

filtered

9

&

by

previously conducted

Mozaffari

the

aid

Nejad,

of

filter

IAC,

2015;

paper

high

197

methanol and acetic acid mixture (1:1 v/v) and poured in the vial and were kept

198

at -18 °C until the analysis (maximum one month).

199

The OTA analysis was done by an HPLC instrument (Waters, 2695,

200

Milford, MA, USA) equipped with an analytical column RP C18 (250 mm 4.6

201

mm, i.d., 5 mm) (Waters, ODS1, Milford, MA, USA) and a fluorescence

202

detector (Waters, 2475, Milford, MA, USA). The used mobile phase consisted

203

of acetonitrile, ultra-pure water, methanol, and acetic acid (a volumetric ratio of

204

39: 30: 30: 1 V/V/V/V). The speed of the mobile phase was adjusted as 1

205

mL/min while the injection volume was set as 50 µL. The measurement was

206

carried out at excitation and emission wavelengths of 365 nm and 333 nm,

207

respectively (Heshmati & Mozaffari Nejad, 2015).

208 209

2.2.3.

Analytical method validation

210

The validation, accuracy, precision, the linearity of the method used in the

211

current

212

recovery. OTA was spiked into the grape, must and pekmez samples at three

213

levels of 5, 10 and 15 µg kg-1. The OTA extraction and measurement was done

214

according

215

percentage was calculated:

216

Recovery % =

study

were

to

the

4

investigated.

method

5

6 7 5

Accuracy

mentioned in



was

section

× 100 (Equation 3)

10

determined

2.3.

by

Then,

the

the

aid

of

recovery

217

The precision was assessed through spiking of OTA in mentioned levels

218

and their measurement in three consecutive days. The ratio of 3 and 10 times

219

the signal area to noise area was considered for LOD and LOQ, respectively.

220

In

order

to

investigate

the

impact

of

each

unit

operation

on

the

221

concentration of OTA and also to eliminate negative effects of diluting and

222

concentrating, the OTA content in the grape, must and pekmez samples were

223

presented as dry weight (DW).

224 225

2.3.

Statistical analysis

226

For statistical analysis of the data, SPSS Version 16.00 (SPSS Inc., Chicago,

227

IL, USA) was used. The comparison among mean was made by One-way

228

analysis of variance (One-way ANOVA) and turkey’s test. The differences were

229

considered as significant while P < 0.05. All treatments were done in triplicate.

230 231

3.

Results and Discussion

232

3.1.

The validation of the OTA analysis

233

The LOD for OTA in three matrices investigated (grape, must and pekmez)

234

were measured as 0.10, 0.13 and 0.18 µg kg-1 while the corresponded values

235

for LOQ were recorded as 0.32, 0.44 and 0.60 µg kg-1, respectively (Table 1).

236

The correlation coefficient (R2) obtained from the calibration curves was higher

237

than 0.99, indicating the excellent accuracy of the established method. The

11

238

results of the recovery of OTA in three levels of 5, 10, and 15 µg kg-1 were

239

presented in Table 2.

240

3.2.

Impact of unit operation on OTA concentration

241

The mean OTA concentration and its reduction percentage during pekmez

242

processing were shown in Table 3. Depending on the initial OTA concentration

243

of grape, the washing stage and juicing stage (crushing and pressing) could

244

reduce the level of this mycotoxin in the range of 7.36-16.53% and 2.51-8.32%,

245

with mean of 12.19±4.60% and 6.13±3.16%, respectively (Table 3).

246

There is little information regarding the changes in OTA during the

247

washing step. In this context, considering to findings of Arici et al. (2004), no

248

data was presented regarding the effects of washing, juicing (crushing and

249

pressing) on OTA while they reported only the changes in OTA amount after

250

pressing. The changes in mycotoxin during washing and, juicing (crushing and

251

pressing) in other fruits such as apple were reported previously. In a previously

252

conducted study, the average reduction of aflatoxins (AFs) in pekmez such as

253

AFB1 and AFB2, AFG1 and AFG2 during washing step was 19.3, 22.2, 23.7 and

254

34.3%, respectively. Moreover, the juicing (crushing and pressing) processing,

255

caused a reduction about 8.2, 14.8, 12.4, and 22.7%, in AFB1 and AFB2, AFG1

256

and AFG2, respectively (Heshmati et al., 2019). According to Sant’Ana et al.

257

(2008),

258

contamination. It seemed that the efficiency of washing, juicing (crushing and

259

pressing) in OTA removal was lower than AFs and patulin. However, the

the

washing

of

apple

could

eliminate

12

80%

of

patulin

(PAT)

260

observed variation in findings among different investigations can be correlated

261

with the differences in solubility in water of investigated mycotoxins. In this

262

regard, the order of mycotoxin solubility reduction in water can be presented as

263

following: PAT: 163,000 mg L-1 > AFG1:424 mg L-1 > AFB2: 392 mg L-1 > AFB1:

264

233 mg L-1 > OTA: 25.6 mg L-1 (Karlovsky et al., 2016). It believed that some

265

parts of OTA were bonded the grape skin and seed while they were eliminated

266

at the stages of juicing (crushing and pressing) with removing of skin and seed.

267

In

268

concentration of OTA in must was defined as lower than the spiked levels.

269

However, according to Grazioli et al. (2006), during crushing and maceration,

270

OTA was transferred from the grape into must, and furthermore, the levels of

271

OTA in must were increased.

the

current

investigation,

due

to

the

reduction

mentioned

above,

the

272

The OTA reduction level during the clarification is strongly depended on

273

the initial OTA content of grape, the type, and concentration of clarifier agents.

274

The OTA reduction during this stage by bentonite, white soil, and gelatin

275

ranged

276

considering initial OTA concentrations and clarifier levels, the mean of OTA

277

reduction

278

13.92±10.28%,

279

clarifiers, the highest (23.76±7.90%) and lowest (11.48±9.37%) mean reduction

280

of OTA was attributed to gelatin and bentonite, respectively. OTA was mostly

281

adsorbed onto clarifier agents by using of the negative charge on the carboxyl

282

group of their phenylalanine moiety (Castellari, Versari, Fabiani, Parpinello, &

1.95-26.21%

by

1.54-28.72%

bentonite, and

white

23.76±7.90%,

and

14.99-37.43%

soil,

and

gelatin

respectively.

13

(Table

was

Therefore,

3).

Without

11.48±9.37%, among

the

283

Galassi, 2001). Due to the difference in charge values of clarifiers, their

284

potencies for OTA removal was varied. Another reason for discrepancies in the

285

efficiency of clarifier agents for removal of OTA can be associated with the

286

differences in the active surface area of the clarifier (Lasram et al., 2008).

287

In previous studies, the impact of different clarifiers and fining agents on

288

OTA removal in wine were investigated (Grazioli, Fumi, & Silva, 2006; Leong,

289

Hocking, Varelis, Giannikopoulos, & Scott, 2006; Sun et al., 2017; Var, Kabak,

290

& Erginkaya, 2008). In this context, based on the findings of Grazioli et al.

291

(2006), the reduction in OTA as result of

292

strongly depended on the type and the extent of adjuvant (a compound in the

293

form of a powdered riddling agent which is added to sparkling of wine to

294

remove sediment). Due to the absorption of OTA by solid particles during the

295

step of racking (solid-liquid separation stage, which often is referred as filtering

296

or fining without any type clarifier); 4 and 9% of OTA were eliminated in white

297

and red wine production, respectively (Leong et al., 2006a). The differences

298

between the results of the current study and other research regarding clarifiers

299

effects in OTA removal could be related to the type and concentration of the

300

used clarifier, initial OTA concentration and clarification time and food matrix,

301

as well as the molecular weight of used clarifier such as gelatin (Quintela et al.

302

2013).

clarification during winemaking,

303

Gelatin has a positive charge, which could play an active role in OTA

304

removal (Castellari et al., 2001). The efficiency of gelatin for OTA removal

305

depended on the presence of other proteins in the treated sample. Leong, 14

306

Hocking,

Varelis,

307

gelatin resulted in a notable decline in levels of OTA among Shiraz wine

308

samples (did not contain detectable proteins) while compared with Semillon

309

wine

310

proteins in the Shiraz wine, OTA was probably to exist in a freely-soluble form.

311

Therefore, it easily can be bound to the gelatin, while grape-derived proteins

312

that existed in the Semillon wine could compete with gelatin for the binding of

313

OTA. The reduction of OTA by gelatin in the current study was lower while

314

compared with previous investigations (Castellari et al., 2001; Lasram et al.,

315

2008; Sun et al., 2017). In a previous study by Lasram et al. (2008), the

316

clarification of red wine with gelatin (0.1 mL L-1) resulted in OTA reduction

317

around 58%. Also, according to Castellari et al. (2001), the reduction in OTA in

318

the wine with initial OTA concentration of 3.78 and 1.50 ng mL-1 that clarified

319

by gelatin (1 g L-1) was 37% and 17%, respectively. Sun et al. (2017) reported

320

that

321

concentration of 5 and 20 ng mL-1 resulted in the reduction of 80% and 60% of

322

this

323

(positively charged), bentonite (negatively charged) and egg white (positively

324

charged), the treatment with 0.20 mg/mL of egg white for 48 h was chosen as

325

the best OTA removal method (Sun et al., 2017). Besides, Leong et al. (2006a)

326

found between two different doses of gelatin (0.05 mg mL-1 and 0.15 mg mL-1),

327

only high dose of this clarifier, i.e., 0.15 mg/mL-1 showed removing properties,

328

while it could remove 37% of OTA. The OTA reduction amount by gelatin in our

samples

the

(containing

addition

mycotoxin,

Giannikopoulos,

of

&

Scott (2006)

grape-derived

gelatin

respectively.

(0.2

While

mg

proteins).

mL-1)

among

15

found

that

Because

into

three

wine

application

of

with

clarifiers,

the

lack

initial

i.e.,

of

of

OTA

gelatin

329

study was similar to research done by Anli et al. (2011) which gelatin (0.1 mg

330

mL-1) could remove 14.3% of OTA in wines with an initial concentration of 0.51

331

µg L-1.

332

Bentonite, as an absorbent natural smectite clay, is a layered aluminium

333

silicate with a negative charge and slightly positive polar terminal regions or

334

edges (Jahed, Khodaparast, & Khaneghah, 2014; Jalali, Jahed, Khodaparast,

335

Limbo, & Khaneghah, 2014, Kulkarni & Shaw, 2016). Each smectite particle of

336

bentonite is composed of thousands of submicroscopic platelets. The faces of

337

these platelets carry a negative charge, while edges have a slightly positive

338

charge. The net negative charge of the platelet is greatly balanced by sodium

339

ions. These charge-balancing ions are associated with platelet faces and are

340

termed “exchangeable” because they are readily substituted by other cations

341

(Kulkarni & Shaw, 2016). OTA can be bonded to the proteins due to ionic

342

interactions.

343

consequently are precipitated (Castellari et al., 2001; Jahed, Khodaparast, &

344

Khaneghah,

345

Leong et al, 2006a). The low efficiency of bentonite compared with gelatin and

346

white soil in the removal of OTA can be associated with the competition

347

between grape proteins and OTA for binding to bentonite. In this context,

348

according

349

concentrations of 0.5 and 2.5 g L-1 into Semillon wine decreased OTA around

350

37% and 67%, respectively (Leong et al., 2006a). In another investigation,

351

bentonite (0.5 g L-1) reduced 20.3% of OTA in white wine (Anli, Vural, &

Furthermore,

2014;

to

Jalali,

Leong

et

the

proteins

Jahed,

al.

can

be

Khodaparast,

(2006b),

adsorbed

Limbo,

incorporation

16

&

of

to

bentonite

Khaneghah,

bentonite

and

2014;

in

the

352

Bayram, 2011). According to Sun et al. (2017) bentonite (0.12, 0.16 and 2 mg

353

mL-1) reduced 10% of the OTA (5, 10 and 20 ng mL-1) in wine while their

354

findings were similar to the current investigation. Also, an OTA reduction of

355

about 8% in wine samples as a result of the incorporation of bentonite (1 g L-1)

356

was

357

combination of gelatin (to bind OTA) and bentonite (to bind and precipitate the

358

gelatin) for the elimination of OAT could be more influential than combinations

359

of gelatin and other clarifiers such as silica gel or gallotannin (Latasteet al.

360

2004).

reported

by

Castellari

et

al.

(2001).

Some

studies

demonstrated

a

361

One of the major compounds of white soil as another clarifier agent

362

used in the current study is silicon dioxide (silica gel). Castellari et al. (2001)

363

found that silica gel has the ability to absorb OTA. These authors declared that

364

the range of OTA reduction by silica gel (type A, narrow pore diameter and

365

surface area of 175-450 m2 g-1) with a positive charge, silica gel (type B,

366

medium pore size and surface area of 175-450 m2 g-1) with a negative charge

367

and silica gel (type C, large pore size surface area of 175-450 m2 g-1) with

368

negative charge were 18-34%, 7-11%, and 1-3%, respectively. In conclusion,

369

the authors declared that the positively charged silica gel offered a good affinity

370

for OTA removal, whereas the negatively charged silica gel had less efficacy

371

(Castellari et al., 2001).

372

During the boiling and concentration of pekmez, the reduction range

373

OTA was noted as 6.66-14.94% (Table 3), with mean of 10.44±3.85%, despite

374

this fact that the OTA is a relatively heat-stable mycotoxin during thermal 17

375

processing (Oteiza et al., 2017). Little information regarding the OTA stability

376

during pekmez boiling was reported, although the changes in AFs during

377

pekmez boiling and PAT during concentration, boiling and pasteurization of

378

apple

379

Rosenthal, & de Massaguer, 2008). In a previous study, the reduction range of

380

AFB1, AFB2, AFG1 and AFG2 during boiling and concentration of pekmez was

381

6.2-25.6%,

382

34.6%, 15.1% and 42.9%, respectively (Heshmati et al., 2019). The reduction

383

of

384

reported (Sant’Ana et al., 2008). These variations can be correlated with the

385

differences

386

stability, and wet weight or dry weight used for reporting of results. It has been

387

reported that the presence of proteins and carbohydrate could increase the

388

degrading of OTA and aflatoxins during heat treatment (Raters and Matissek,

389

2008).

390

juice

was

previously

4.6-100.0%,

9.4%-14.06%

for

in

Generally was

6.7-25.0%

patulin

heating

the

and

OTA

range

and

during

time

speaking, in

documented

of

(Heshmati

et

15.6-100.0%,

concentration

temperature

reduction

2019;

with

of

juice

pekmez

also

was

structure,

production

process

392

37.52±6.54%. The findings regarding OTA reduction during pekmez production

393

was in contrast with findings of Arici and Kara (2014) where OTA level in

394

pekmez increased 5–6 times higher while compared with grape juice (Arici et

395

al., 2004).

18

with

14.8%,

391

396

3)

Sant’Ana,

of

mycotoxin

the

(Table

mean

apple

used,

during

25.84-54.67%

al.,

a

mean

of

397

4. Conclusions

398

In this study, the impact of each unit operation of pekmez production on OTA

399

was investigated. Besides, the influence of various clarifiers on OTA changes

400

was assessed. All stages and unit operations applied in pekmez production

401

could result in the reduction in OTA levels. Generally speaking, the OTA

402

reduction range during the pekmez production was 25.84-54.67%, with a mean

403

of

404

11.48-23.76%,

405

assigned for gelatin). The utilization of gelatin instead of white soil, which is

406

traditionally used in pekmez production, could remove higher levels of OTA.

407

However, the price of gelatin is substantially higher while compared with white

408

soil, its utilization might result in further increases in the quality and safety of

409

pekmez.

410

Acknowledgements

411

This study (plan No. 9605103036) has been funded by Vice-Chancellor of

412

Research and Technology of Hamadan University of Medical Sciences and

413

Health Services.

37.52±6.54%.

The

while

mean it

OTA

depended

reduction on

clarifier

414 415

Conflict of interest

416

No conflict of interest

417 19

during agent

the

clarification

(highest

ranged

removal

was

418

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27

Table1. Linearity range, limit of detection (LOD) and limit of quantification (LOQ) for OTA in grape, must and pekmez Matrix

Range of linearity (µg kg-1)

Grape Must Pekmez

0.5-20 0.5-20 0.8-25

Equation of calibration curve Y=80125X+25689 Y=81568X+28125 Y=81424X+33022

R2

LOD (µg kg-1)

LOQ (µg kg-1)

0.996 0.992 0.994

0.10 0.13 0.18

0.32 0.44 0.60

Table 2. The mean recovery percentage for various levels of spiked OTA in grape, must and pekmez Matrix

Spiked level (µg kg-1)

Grape

RSD

5 10 15

Recovery percentage 104.13 91.28 87.54

Must

5 10 15

91.53 90.84 89.25

8.57 8.36 3.92

Pekmez

5 10 15

77.46 78.37 81.54

2.88 3.59 5.74

5.52 4.14 4.72

Table 3. The change in OTA concentration and its reduction percentage during pekmez processing

The type of applied unit operation

-1

a

W W/P W/P/B1.5 W/P/B3 W/P/S1.5 W/P/S3 W/P/G1.5

-1

Initial concentration (µg kg ) of OTA in grape 5 4.17±0.14 (16.53%*) c * 3.83±0.19 (23.47% ; ** 8.32% ) c 3.75±0.23 (25%; 1.95%) ef 2.82±0.13 (43.53%; 26.21%) c 3.76±0.19 (24.73%; 1.54%) ef 2.79±0.18 (44.2%; 28.72%) b

e

2.91±0.23 (41.8%; 23.65%) efg

W/P/G3

2.72±0.11

W/P/B1.5/C W/P/B3/C W/P/S1.5/C W/P/S3/C W/P/G1.5/C W/P/G3/C

3.43±0.11 (31.47%; 8.46%) gh 2.64±0.02 (47.13%; 6.23%) 3.19±0.05e (36.13%; 14.98%) h 2.53±0.12 (49.4%; 9.18%) i 2.49±0.02 (50.13%; 13.94%) i 2.27±0.05 (54.67%; 16.55%)

d

(45.65, 37.43%)

a

10 8.73±0.1 (12.67%) bc 8.51±0.11 (14.87%; 2.51%) b

11.71±0.43 (21.93%; 8.82%) de 11.51±0.49 (23.29%; 10.4%)

d

11.81±0.15 (21.27%; 8.02%) def 11.17±0.32 (25.51%; 13.65%) ef 10.59±0.45 (29.42%; 17.52%) def 11.08±0.32 (26.11%; 14.99%) ef 10.98±0.41 (26.78%; 6.19%) f f 10.5±0.45 (30%; 8.52%) def 11.12±0.15 (25.84%; 5.82%) f 10.7±0.47 (28.64%; 4.24%) g 9.23±0.7 (38.47%; 12.53%) g 9.32±0.71 (37.87%; 15.74%)

7.81±0.17 (21.87%; 8.23%) gf 6.73±0.13 (32.7%; 23.33%) 6.6±0.3

6.53±0.04

a

15 13.9±0.35 (7.36%) c 12.84±0.23 (14.4%; 7.58%) b

c

8.27±0.62 (17.33%; 2.94%) ef 6.94±0.06 (30.63%; 18.52%)

fgh

Average (µg kg )

(34.03%; 22.48 %) ghi

e

(34.73%; 26.52%)

7.17±0.08 (28.33%; 13%) i 6.21±0.05 (37.9%; 10.48%) ef 6.93±0.08 (30.73%; 11.31%) hi 6.29±0.06 (37.13%; 6.57%) j 5.81±0.06 (41.9%; 11.82%) j 5.71±0.12 (42.9%; 12.51%)

de

d

a

10 8.93±4.87 (12.19%) b

c

8.39±4.51 (17.58%; 6.13%) d 7.91±3.99 (21.42%; 4.57%) ef 7.09±4.35 (32.49%; 18.38%) d 7.79±4.03 (22.62%; 5.93%) 6.9±4.19

efg

(34.14%; 21.9%)

gh

6.7±3.84 (35.09%; 21.21%) fgh 6.78±4.19 (35.48%; 26.31%) e 7.19±3.78 (28.86%; 9.22%) h 6.45±3.94 (38.34%; 8.41%) f 7.08±3.97 (30.9%; 10.7%) h 6.51±4.09 (38.39%; 6.66%) i 5.84±3.37 (43.5%; 12.76%) i 5.77±3.53 (45.14%; 14.94%)

W: Washing stage; P: Juicing (crushing and pressing) stage; B1.5: Bleaching stage by bentonite (1.5 g/100 g); B3: Bleaching stage by bentonite (3 g/100 g); S1.5: Bleaching stage by white soil (1.5 g/100 g); S3: Bleaching stage by white soil (3 g/100 g); .5: G1.5: Bleaching stage by gelatin (1.5 g/100 g); G3: Bleaching stage by gelatin (3 g/100 g); C:Concentration stage. Different letters (a-j) indicated significant differences among value OTA concentrations within each column (P<0.05). *The first value within parenthesis indicated OTA reduction percentage in comparison with the initial concentration of this mycotoxin (5, 10 and 15 -1 µg kg ) in grape. **The second value within parenthesis indicated OTA reduction percentage in comparison with previous stage.

Grape Spiking of ochratoxin A (5, 10 and 15 µg kg-1) Storage at room temperature for 24 h

Washing

Juicing (Crushing and pressing)

Filtration

Clarification Addition of clarifier (bentonite, gelatin, white soil) Filtration

Boiling and concentration

Pekmez Fig 1. Schematic of pekmez production procedure

Highlights •

Ochratoxin A (OTA) in different pekmez stages of production was investigated.

•

The concentration of OTA was decreased during processing of pekmez.

•

OTA reduction during pekmez production was ranged between 25.84-54.67%.

•

Type and concentration of clarifying agents Influence the removal of OTA

•

Gelatin had the highest impact on OTA removal (23.76%).

Conflict of interest The authors declare that they have no conflict of interest.