Accepted Manuscript

Acrylamide formation in plantain (Musa paradisiaca) chips influenced by dif-

ferent ripening stages: A correlation study with respect to reducing sugars,
amino acids and phenolic content

L. Shamla, P. Nisha

PII: S0308-8146(16)32016-7
DOI: http://dx.doi.org/10.1016/j.foodchem.2016.12.007
Reference: FOCH 20299

To appear in: Food Chemistry

Received Date: 8 August 2016

Revised Date: 1 December 2016
Accepted Date: 5 December 2016

Please cite this article as: Shamla, L., Nisha, P., Acrylamide formation in plantain (Musa paradisiaca) chips
influenced by different ripening stages: A correlation study with respect to reducing sugars, amino acids and phenolic
content, Food Chemistry (2016), doi: http://dx.doi.org/10.1016/j.foodchem.2016.12.007

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 1 Acrylamide formation in plantain (Musa paradisiaca) chips influenced by different

2 ripening stages: A correlation study with respect to reducing sugars, amino acids and

3 phenolic content

4 L Shamla and P Nisha*

5 Agro Processing and Natural Products Division,

6 CSIR-National Institute for Interdisciplinary Science and Technology,

7 Thiruvananthapuram 695019, Kerala, India.

8 *Corresponding Author

9 P. Nisha,

10 Agro Processing and Natural Products Division,

11 CSIR-National Institute for Interdisciplinary Science and Technology,

12 Thiruvananthapuram 695019, Kerala, India.

13 Tel.: +91 471 2515348

14 Fax: +91 471 2495050

15 E-mail address: pnisha@niist.res.in, bp.nisha@yahoo.com

16 Running title: Influence of ripening on acrylamide formation in plantain chips

17

1
18 Abstract

19 The effect of ripening on the formation of acrylamide in deep fried plantain chips made from

20 Nendran variety (Musa paradisiaca) was investigated. The precursors of acrylamide

21 formation, reducing sugars (glucose and fructose) and ten major amino acids, were quantified

22 during different stages of ripening using HPLC and correlated with acrylamide formation.

23 The total phenolic content and total flavonoid content were also estimated and correlated with

24 acrylamide formation. Both glucose and fructose increased during ripening and demonstrated

25 a positive correlation on formation of acrylamide (correlation coefficient of r =0.95 and 0.94

26 respectively (p<0.05), whereas asparagine, was poorly correlated (p>0.05). The decreased

27 levels of phenolic content during ripening of plantain were negatively correlated with

28 acrylamide formation in the deep fried chips prepared. Thus the selection of proper ripening

29 stage renders reduced formation of acrylamide in plantain chips to a reasonable extend.

30 Key words: Plantain, acrylamide, reducing sugars, asparagine, phenolics, deep fried chips

31

2
32 1. Introduction

33 Acrylamide is a process induced contaminant that is formed during high-temperature

34 processing (e.g., frying, baking and roasting) of carbohydrate and protein rich foods. The

35 reducing sugars and asparagine are believed to be the main precursors responsible for

36 acrylamide formation in plant-based foods (Bent, Maragh, & Dasgupta, 2012). The Maillard

37 reaction which involves the interaction between the free amino group of amino acids or

38 proteins and the carbonyl group of sugars or carbohydrates has an important role in the

39 formation of acrylamide along with time-temperature combinations. Decarboxylation and

40 deamination of the amino acid asparagine have also been suggested as a possible pathway for

41 the formation of acrylamide. Though different mechanisms have been postulated for the

42 formation of acrylamide in plant-derived foods, the concentration of asparagine and reducing

43 sugars are the major determinant factors (Friedman, 2015).

44 Banana/plantain is the fourth most important food crop in the world after rice, wheat, and

45 maize followed by potato and India is the largest producer of banana in the world (Singh,

46 Singh, Kaur, & Singh, 2016). Since plantain is a good source of carbohydrate, the formation

47 of acrylamide during high-temperature processing such as frying and baking is unavoidable.

48 A study by Daniali, Jinap, Zaidul & Hanifah (2010) on the acrylamide content of five popular

49 Malaysian fried and baked banana based snacks revealed that the acrylamide content ranged

50 from 74.0 to 7468.8 µg/ kg for banana fritter (pisang goreng), 28.9 to 243.7 µg/kg for banana

51 chips (kerepek pisang), 160.7 to 500.4 µg/kg for sweet banana chips (kerepek pisang manis),

52 not detected to 154.4 µg/kg for banana cake (kek pisang) and 31.7 to 609.1 µg/kg for banana

53 balls (cekodok pisang). Bent et al. (2012) reported that banana chips (green & ripe) and

54 banana fritters made from bananas were found to contain 100-430, 180 and 1090 µg/kg of

55 acrylamide, respectively. Effect of maturity on the formation of acrylamide in banana fritters

56 made from Musa paradisica variety, Awak and Abu has also been reported (Daniali, Jinap,

3
57 Zaidul, & Hanifah, 2013). They indicated a strong correlation between the reducing sugar

58 content and acrylamide formation as on increasing fruit maturity. But no correlation was

59 found with asparagine and acrylamide formation in banana fritters.

60 Antioxidants particularly phenolic compounds have been reported to inhibit the formation of

61 acrylamide in model and various food systems. It is reported that the phenolic compounds

62 present in plantain extract could act as natural antioxidants which could help in mitigating the

63 toxicity of acrylamide consumed by the body through an antioxidant protective mechanism

64 (Jun et al., 2008). Morales, Jimenez, Garcia, Mendoza, & Beristain (2014) reported that green

65 tea, oregano & cinnamon extracts reduced the acrylamide formation in fried potatoes whereas

66 thyme and bougainvillea extracts had no effect on the formation of acrylamide. It was

67 reported that many factors such as the nature of phenolic compounds, conditions of model

68 reactions, and type of food matrices may contribute to the increase or decrease in levels of

69 acrylamide formation (Jin, Wu, & Zhang, 2013). Alternatively, these additives constitute a

70 group of phytochemicals which are highly beneficial to human health because of their

71 antioxidant properties.

72 Chips made by deep frying of raw matured plantain are a popular snack product in many

73 parts of the world. The chips prepared by deep frying the core of unripe plantain, variety-

74 Nendran (Musa paradisiaca) at a stage of maturity II, peel still completely green, is one of

75 the most favorite snack items among children as well as adults, especially in South Asian

76 countries. The plantain/banana ripening is usually divided into seven ripening stages

77 depending on its maturity (Soltani, Alimardani, and Omid, 2011) and the colours were

78 reproduced and translated to a numerical scale where stage I-entirely green; II-green with a

79 trace of yellow; III-more green than yellow; IV-more yellow than green; V-yellow with a

80 trace of green; VI-entirely yellow; VII-entirely yellow with brown speckles. Ripe plantain

81 (Nendran variety) at maturity III & IV are also used for making chips that will be slightly

4
82 sweeter in taste. A preliminary survey on the market samples carried out by the authors

83 revealed reasonable levels of acrylamide in plantain chips (unripe & ripe) which were high in

84 ripe plantain chips (Shamla & Nisha, 2014). The survey warranted a detailed investigation on

85 the influence of different ripening stages on acrylamide formation in plantain chips as there

86 were no reports on how the maturity affects the sugar & amino acid profile and the phenolic

87 content of plantain. Recent studies on the mitigation of acrylamide formation in foods

88 revealed that when polyphenols are added as antioxidants to food systems; it will influence

89 the Maillard reaction affecting formation of acrylamide in food systems (Cheng, Chen, Zhao,

90 & Zhang, 2015, Xu et al., 2014). A good documentation of the precursors such as reducing

91 sugars and asparagine in plantain along with the total phenolic & flavonoid content could be

92 helpful in understanding the formation of acrylamide in plantain chips. Therefore, the present

93 study was undertaken with an objective to investigate the changes in the composition of

94 plantain fruit during different stages of ripening and its correlation with the formation of

95 acrylamide in deep fried chips. Thus, the proper information regarding maturity will help to

96 understand the level of precursors, total phenolic & flavonoid content thereby we can reduce

97 the levels of acrylamide in the final product to a greater extend.

98 2. Materials and methods

99 2.1. Reagents and chemicals

100 Standard acrylamide (>99%), D-(+) glucose, D-(-) fructose, sodium phosphate, sodium

101 carbonate, aluminium chloride hexahydrate, potassium acetate, sodium hydroxide, gallic acid,

102 quercetin, aluminium chloride, Folin-Ciocalteau reagent, amino acid standards and o-

103 phthalaldehyde (OPA) were purchased from Sigma-Aldrich (St. Louis MO, USA). HPLC

104 water was purified on a Milli-Q system (Millipore India Pvt Ltd, Bangalore, India). Solvents

105 used were methanol, acetic acid & acetonitrile of high-performance liquid chromatography

106 (HPLC) grade. Oasis HLB (30 mg, 1 ml) solid phase extraction (SPE) cartridges were

5
107 obtained from Waters Corp. (Milliford, Massachusetts USA). Minigen syringe filters (0.22

108 µm diameter) were obtained from Genetix Biotech Asia Pvt. Ltd, New Delhi, India.

109 2.2. Sample preparation

110 A bunch of plantain with around twenty tiers, variety Nendran (Musa Paradisiaca) at stage I

111 maturity (green), was harvested from an authenticated agricultural farm in Trivandrum,

112 Kerala, India and was kept at ambient conditions. Chips were made from plantain using

113 maturity stages I - V. Two days of subsequent intervals were given for each ripening stages

114 two, three, four and five respectively, after harvesting. Plantains were peeled manually, and

115 the remaining fruit was used for making chips. The proximate composition, reducing sugars,

116 amino acids, and phenolic compounds of the peeled fruit were estimated. Fig. 1 represents

117 maturity stages I –V of plantain, plantain fruit after peeling and the chips prepared from it.

118 2.3. Preparation of plantain chips

119 As chips cannot be made from fully ripened plantain, only stages of I-V were selected for

120 preparing plantain chips in the present study. After peeling the plantain manually, the

121 remaining fruit was sliced into thin rounds with thickness of approximately 1 mm. The sliced

122 samples (500 g) were deep fried in coconut oil (3 L) using a stainless steel electrical deep fat

123 fryer (NOVA, Flomatic Industries PTE Ltd, Singapore) at 165ºC for 7 min. The temperature

124 and time of frying adopted for the study was optimized earlier based on product quality in

125 terms of texture (by rupture test of banana chips using texture analyser, TA- HDi, Stable

126 Microsystems, UK, using a ball probe) and colour (L, a and b values using ColourFlex EZ,

127 HunterLab Instruments, Virginia, USA) and sensory analysis in comparison with the

128 commercial samples. The temperature was monitored by the use of a thermometer TTX 110

129 type T temperature prob (Ebro, Germany). For each stage, chips were prepared in triplicates,

130 using the fruit from different tiers. Freshly prepared chips from each ripening stage were

131 analyzed for the acrylamide content using the procedure given under 2.9.

6
132 2.4. Chemical compositional analysis

133 The standard procedures of AOAC (2005) were used for the determination of moisture, ash,

134 crude fat and protein contents of all stages of raw plantain. Triplicate samples of all the five

135 stages of plantain were oven-dried at 100°C transferred to a desiccator, and allowed to cool at

136 room temperature for moisture content. The sample weights were recorded before and after

137 heat treatment in a muffle furnace (550°C for 12 h) for the ash content determination. Micro-

138 Kjeldahl method was used for the protein estimation with nitrogen to protein conversion

139 factor of 6.25 and fat content was determined using Soxhlet extraction. Total carbohydrate

140 was calculated using the difference method.

141 2.5. Determination of reducing sugars by HPLC

142 The reducing sugars, glucose and fructose of plantain fruit (fresh samples after peeling as

143 mentioned under 2.2), were measured using Shimadzu HPLC (Kyoto, Japan) technique using

144 a reversed phase Supelcosil LC-NH2 column (25cm × 4.6mm, 5µm) equipped with a

145 Refractive Index detector. The standards of glucose and fructose in the concentration range 5-

146 15 mg/mL were used for quantification. The sample extraction was carried out according to

147 Vivanti, Firotti, and Friedman (2006) with slight modifications. For the sugar analysis, the

148 sample preparation was as follows. 1 g of plantain fruit (after peeling) paste was mixed with

149 10 ml acetonitrile/water (8.5:1.5 v/v) and stirred for 5-15 min. The suspension was

150 centrifuged at 1700 g for 10 min, and the supernatant was passed through 0.22 µm syringe

151 filter. The mode of elution used was isocratic with the mobile phase consisting of (85:15 v/v)

152 acetonitrile/water at a flow rate of 1 mL/min.

153 2.6. Determination of amino acids by HPLC

154 Ten amino acids namely asparagine, glutamine, serine, glycine, threonine, valine, isoleucine,

155 leucine, phenylalanine and lysine were detected and quantified by the HPLC method (Georgi,

156 Rittmann, & Wendisch, 2005). The amino acid analysis was performed using Shimadzu

7
157 HPLC system (Kyoto, Japan) containing a binary pump delivery system (LC-20AD), robotic

158 autosampler (SIL-10AP), column thermostat (CTO-20A) and a fluorescence detector (RF-10

159 AxL). Briefly, the standard amino acids and the samples were automatically derivatized with

160 OPA by programming the robotic autosampler. The derivatized amino acids were detected by

161 the fluorescence detector by an excitation emission wavelength λ = 340/450 nm using Zorbax

162 Eclipse-AAA column, 5 µm, 150 × 4.6 mm (Agilent), at 40°C. Mobile phase A was 40 mM

163 NaH2PO4, adjusted to pH 7.8 with NaOH, while mobile phase B was

164 acetonitrile/methanol/water (45/45/10 v/v/v). The separation was obtained at a flow rate of 2

165 mL/min with a gradient program that allowed 0-1.9 min at 0% B, followed by an 18.1 min

166 step that raised eluent B to 57%, 18.6 to 22.3 min at 100 % B and then equilibration at 0% B

167 was performed in a total analysis time of 30 min.

168 2.7. Total Phenolic (TPC) & Flavonoid Content (TFC)

169 Nendran fruit after peeling, at different ripening stages, was freeze dried (VirTis, Genesis,

170 U.S.A), powdered and extracted with methanol (1:10 w/v) at ambient temperature until the

171 solvent become colorless. The extracts were filtered through Whatman No.1 filter paper and

172 were evaporated to dryness in a rotary evaporator (Buchi, Switzerland) under reduced

173 pressure at 35°C. Finally, the extracts were washed with 10 ml of methanol and stored at 4°C

174 for further analysis.

175 Folin-Ciocalteau reagent was used for the total phenolic content measurement as described by

176 Singleton & Rossi (1965). The results were expressed as mg GAE/g dry weight of extract by

177 measuring the absorbance at 760 nm using a multimode reader (Synergy, Biotek, USA). TFC

178 was measured based on the aluminum chloride colorimetric method described by Chang,

179 Yang, Wen, & Chem (2002) with quercetin as standard. The absorbance was measured at 415

180 nm using multimode reader (Synergy, Biotek, USA) and the results were calculated as

181 milligram quercetin equivalents (mg QE/g dry weight of extract).

8
182 2.8. Quantification of polyphenols by HPLC

183 The freeze dried samples as mentioned under 2.7 was extracted using methanol (1:10 w/v) at

184 ambient temperature until the solvent become colorless. These methanolic extracts and

185 eleven reference compounds (gallic acid, catechol, chlorogenic acid, syringic acid, p-

186 coumaric acid, ferulic acid, ellagic acid, cinnamic acid, quercetin, kaempferol & apigenin in

187 1mg/ml concentration) were prepared in methanol and were filtered through 0.45µm PTFE

188 syringe filter. Filtrate (20 µL) was injected into an HPLC system (Shimadzu, Japan)

189 containing two LC 20AD preparative liquid chromatography pump units, a reverse-phase

190 Phenomenex, Luna® C18 column (250×4.6mm i.d.; 5 mm), a column oven (CTO-20 AC

191 VP), a system controller (SCL-20A VP), a Rheodyne injector (USA) with a loop of 20 µL

192 volume and a diode array detector (DAD; SPD-M20A VP) was used for the analysis of

193 polyphenols.

194 The HPLC analysis was performed according to Arun et al (2015) with some modifications.

195 Two solvent systems were used as mobile phases for the analysis in which solvent A was a

196 mixture of methanol, acetic acid & water in the ratio 10:2:88 and methanol-acetic acid-water

197 in the ratio 90:2:8 was used as solvent B with the gradient program 0–15 min 15% B, 16–20

198 min 50% B, 21–35 min 70% B, 36–50 min 100% and finally the column was regenerated in

199 10 min. 20 µl was used for the HPLC analysis and the flow rate was set at 1ml/min. The

200 phenolic acids were detected at a wavelength of 280 nm. Comparing the retention time of

201 peaks for the standards, the sample peaks were identified for the individual phenolic acids.

202 The compounds were confirmed by spiking with corresponding authentic phenolic standards.

203 Shimadzu CLASS-VP version of 6.14 SP1software is used for the data acquisition and

204 analysis.

205 2.9. Analysis of acrylamide by HPLC

9
206 The analysis of acrylamide was performed as reported by Shamla et al., (2014). Finely

207 ground fresh chips samples (4.0 g) were defatted four times using hexane (15 ml) followed by

208 vigorous shaking (5 min) and was filtered. The residue was dried under vacuum and was

209 analysed for acrylamide. Acrylamide was extracted with acetone using ultrasonication (Elma,

210 Germany) at 40 °C for about 30 min. It was filtered and the filtrate was evaporated to dryness

211 and the residue was dissolved and made up to 2 ml with water. The cleanup of the extracts

212 were done by passing through Oasis HLB catridge (Waters Corporation, Milford,

213 Massachusetts USA) and finally through 0.45 µm syringe filter before HPLC analysis. A

214 known amount of standard acrylamide was used as the internal standard.

215 The HPLC used for the analysis of acrylamide was a Shimadzu HPLC system (Japan)

216 consisting of two LC-8A chromatography pump units, column oven (CTO-10AC VP),

217 system controller (SCL-10A VP), rheodyne injector (USA) with a loop of 20 µL volume,

218 diode array detector (DAD; SPD-M10A VP). The HPLC column used was Atlantis ® dC 18

219 (4.6X 250 mm i.d.; 5µm). An isocratic elution pattern was adopted for the separation of the

220 analyte, and 100% Milli-Q water was used as the mobile phase. The column temperature was

221 set at 25°C; the flow rate was maintained at 0.5 ml/min while the detection was performed at

222 206 nm. The injection volume was 20 µL and the retention time for AA was 6.8 min.

223 3. Statistical analysis

224 Statistical analysis of all data was performed using SPSS version 12.0 (SPSS, Inc., Chicago,

225 IL). Duncan’s post-hoc comparison of means was carried out to determine significant

226 differences for proximate compositional analysis along with precursor compositions which

227 include reducing sugars, amino acids, total phenolic & flavonoid content determinations for

228 the respective ripening stages of Nendran. The term significant is used to indicate differences

229 for which p < 0.05. The correlations between reducing sugars, amino acids, total phenolic &

230 flavonoid content in all the five stages of Nendran variety were studied using Pearson's

10
231 correlation coefficient. Three independent analysis were performed for each stage wise

232 analysis of acrylamide. Values are expressed as mean± standard deviation of minimum three

233 experiments.

234 4. Results and discussion

235 4.1. Proximate Compositional Analysis of plantain at different stages of ripening

236 Table 1 summarizes the proximate composition of plantain at five different ripening stages.

237 The statistical significance of each value was noted by Duncan's test. The moisture content

238 was increased during the process of ripening, from stage one (55.20 g/100 g) to stage five

239 (63.95 g/100 g). Stover & Simmonds (1987) reported that during fruit ripening the sugar

240 concentration increases in the pulp than in the peel thus promoting a difference in osmotic

241 pressure. As a result of transpiration to the atmosphere and osmosis to the pulp, the peel loses

242 water causing an increase in the fresh weight of the pulp as the fruit ripens. A significant

243 increase in protein, ash, and fat content were observed during different ripening stages of

244 plantain. Baiyeri, Aba, Otitoju, & Mbah (2011) reported higher ash content in ripe plantain

245 which was due to the release of mineral elements as a result of the tissue breakdown during

246 ripening. The carbohydrate content decreases while the sugar content increases from stage I

247 to V of plantain. This is because as the plantain ripens, the starch content decreases due to the

248 conversion of starch to sugar.

249 4.2. Reducing sugars

250 Table 2 summarizes the concentrations of fructose and glucose in the Nendran variety of

251 plantain at five different stages of ripening. The HPLC profiling of reducing sugars (glucose

252 and fructose) in all the five ripening stages of plantain is given in supplementary data

253 (Supplementary Fig.1). Results from our study indicated that both glucose and fructose

254 content increased during ripening, as expected. The glucose and fructose content increased

255 from 4.83 to 15.15% and 4.76 to 15.70%, respectively, from stages I-V. As a result of fruit

11
256 ripening associated with plantains, the cell wall gets ruptured, and the fruit becomes very

257 soft, resulting in an increase in starch breakdown and a simultaneous increase in the

258 concentration of soluble sugars. The liberation of these soluble sugars as on ripening may be

259 attributed to the activities of various enzymes such as starch phosphorylase and amylase

260 (Mohan, Rajesh, Zuhra, and Vijitha, 2014).

261 4.3. Amino acids

262 The amino acid concentrations of major amino acids found in all the five ripening stages of

263 plantain are listed in Table 2. Asparagine is the major amino acid which are reported to

264 influence the formation of acrylamide. The asparagine content ranged from 1.80, 2.98, 1.37,

265 1.42 & 1.89 mg/g respectively for the ripening stages of I to V. The amino acid glutamine

266 increased up to stage III and then decreased. The amino acids serine, isoleucine, leucine &

267 phenylalanine exhibited an increase up to stage four and then decreased. The glycine content

268 did not show any significant differences in the concentration during ripening. Threonine

269 increased from stage one to three and then decreased. There is a slight increase found for the

270 amino acid valine from stage one to two and then decreased. Ketiku (1973) reported that

271 during fruit ripening, the concentration of lysine, methionine, histidine, proline,

272 phenylalanine was increased. Khawas et al. (2014) reported that there is a decline in the

273 essential amino acid content as on increasing fruit maturity of culinary banana (Musa ABB).

274 However, in the present study, the change in amino acids during ripening did not follow a

275 pattern, as reported earlier.

276 4.4. TPC & TFC contents in five different ripening stages of Nendran

277 Many factors such as variety, cultivation, species, area, ripeness, harvesting time, climatic

278 conditions, storage time and environment directly contribute to the phenolic content of fruits.

279 Among these, the environmental factors which include agronomic (biological culture,

280 greenhouses or fields, hydroponic culture, fruit yield per tree, etc.) or climatic (sun exposure,

12
281 soil type, rainfall) have a significant role in the total phenolic content. However, the

282 important factor that affects the concentration of polyphenols is the fruit maturity (Iqbal &

283 Bhanger, 2006). In the present study, it was noted that the total phenolic content decreased

284 significantly with increase in the ripeness of the plantain (Fig.2). The total phenolic content

285 showed a decline from 275.34 to 31.92 mg GAE from stage I to V of plantain with a

286 statistical significance of p ≤ 0.05. The increase of polyphenol oxidase activity together with

287 the increase in the polymerization of leucoanthocyanidins and the hydrolysis of astringent

288 arabinose ester of hexahydrodiphenic acid contributes to the loss of astringency may be

289 attributed to the decrease in the total phenolic content upon fruit ripening (Parr & Bolwell,

290 2000). The TFC exhibited a similar decline upon fruit ripening. The flavonoid content in the

291 unripe stage is significantly higher (Fig.2) when compared to the ripened stage of plantain

292 (7.505 to 0.573 mg/g in stage I to V, respectively). This may be because of the fact that

293 during ripening, different phenolic compounds condenses forming complex phenolic

294 compounds such as tannins and lignin, etc, which cannot be determined by the present

295 analytical method employed (Ben-ahmed, Ben-rouina, Sensoy, & Boukhriss, 2009).

296 4.5. HPLC quantification of polyphenols in different stages of Nendran ripening

297 The individual phenolic compounds in each stage were quantified by HPLC (Supplementary

298 data Fig. 2). Gallic acid & chlorogenic acid was present in higher amounts in Nendran variety

299 of plantain. As can be seen, the concentration of the phenolic compounds decreased

300 significantly (p≤ 0.05) upon fruit ripening (Table.2), which is in accordance with the increase

301 in acrylamide content.

302 From the above studies, it was found that there was a change in the concentration of the

303 precursors of acrylamide formation viz., reducing sugars, amino acid and asparagine, during

304 the ripening of Nendran. Phenolic compounds are found to decrease during the ripening

305 process. In order to understand how the chemical changes during the ripening influence the

13
306 formation of acrylamide when exposed to high-temperature processing like deep frying, deep

307 fried chips were prepared from plantain stages I - V, as a model. The occurrence of

308 acrylamide in the chips was estimated and correlated with the precursors as well as the

309 phenolic content, to understand the effect of ripening on the formation of acrylamide.

310 4.6. Estimation of Acrylamide

311 Deep fried chips were prepared from plantain at different stages of ripening and the formation

312 of acrylamide during the deep frying process was estimated using HPLC. It was interesting to

313 note that the acrylamide content in the chips were increased with ripening. The bar graph

314 showing acrylamide concentrations in plantain chips from stage I to stage V of ripening is

315 given in fig.3 and the values are 49.8 ± 2.4, 185.6 ± 3.5, 240.2 ± 3.6, 315.6 ± 24.1 and 2062.0

316 ± 26.9 µg/kg respectively. The acrylamide concentrations were significantly different (p ≤

317 0.05) for each ripening stages of Nendran variety of plantain. There are very few reports on

318 the occurrence of acrylamide in banana/plantain products. Acrylamide levels in banana

319 fritters made from over ripe banana are reported to range between 268-3585 µg/kg (Daniali et

320 al., 2010). Another study by Bassama, Brat, Bohuon, & Hocine, (2011) reported the

321 acrylamide levels up to 900 µg/kg in plantain-based products. The acrylamide content in

322 commercial samples of chips made from green and ripe banana, collected from Caribbean

323 market, was reported to be 100 – 430 and 180 µg/kg (Bent at al., 2012). Mulla, Annapure,

324 Bharadwaj, & Singhal, (2016) reported a higher level of acrylamide content in chips made

325 from ripe banana, which is correlated to its higher reducing sugar content. They also reported

326 that the formation of acrylamide in plantain is in the same magnitude as that of potato, rye

327 and wheat products. The results from the present study are in accordance with our previous

328 study on market samples where the occurrence of acrylamide in deep fried chips prepared

329 from raw and ripe plantain was found to range from 14.73-1690.48 and 24.81-1959.80 µg/kg

330 respectively (Shamla et al., 2014).

14
331 The results from the study indicated that the acrylamide content in the product was increased

332 with increase in ripening. As discussed earlier, the increase in the level of acrylamide in the

333 chips with the increase in the ripening of plantain may be correlated to the change in the

334 precursors. Further to confirm this, statistical correlation were carried out to find out the

335 correlation between the levels of reducing sugars, amino acids, and phenolic compounds.

336 5. Correlating acrylamide formation with reducing sugars, amino acids, total phenolic

337 & flavonoid content in different ripening stages of plantain

338 The correlation between the acrylamide content in the final products with reducing sugars,

339 amino acids and the five major phenolics in the five ripening stages of plantain (Nendran)

340 were examined using the Pearson correlation (r), and the results are shown in Table 3. The

341 correlation between reducing sugar contents (glucose and fructose) and acrylamide levels in

342 the Nendran variety yielded high positive r values of 0.95 and 0.94 respectively for glucose

343 and fructose. Positive correlation was reported earlier between the formation of acrylamide

344 and reducing sugars in banana fritters made from Abu & Awak varieties of banana (Daniali et

345 al. 2013). Reducing sugars are also correlated with acrylamide formation in chips (Mulla et

346 al., 2016)

347 The results from the present study indicated that the increase in acrylamide content correlates

348 positively with the reducing sugar content as on increasing fruit ripening. The reducing

349 sugars fructose and glucose are important for acrylamide formation via Maillard reaction

350 pathway, and glucose was found to generate more acrylamide than fructose (Robert et al.,

351 2004). Correlation between the precursors and acrylamide formation has been reported for

352 potato and potato products. The highly reactive aldehyde group in glucose is reported to be

353 responsible for the greater acrylamide formation compared to ketohexose group in fructose.

354 In contrary to this, Rydberg, Eriksson, Tareke, Karlson, Ehrenberg, & Tornqvist (2005)

355 reported that fructose plays a major role in the formation of acrylamide in potato system.

15
356 Pollien, Lindinger, Yeretzian, & Blank (2003) reported in a study that, fructose generate

357 more acrylamide than glucose when heated with asparagine at 150ºC, in a model system. It is

358 reported that glucose and fructose concentrations in the tubers significantly correlated with

359 acrylamide formation in the products (Wicklund, Ostlie, Lothe, Knutsen, Brathen & Kita,

360 2006). Becalski et al., (2004) reported that the acrylamide formation in French fries can be

361 effectively controlled by the use of potatoes with low levels of sugar.

362 It was found that the asparagine which is reported to be one of the major precursors for

363 acrylamide formation demonstrated a weaker correlation with acrylamide formation. All the

364 other amino acids except, threonine and leucine, exhibited a negative correlation with the

365 formation of acrylamide. However, the correlation coefficient ‘r' for threonine and leucine

366 were, much below the acceptable range. State of maturity and storage conditions influences

367 sugar levels which directly affects the formation of acrylamide in fried potato products

368 (Abong, Okoth, Imungi, & Kabira, 2009). Friedman & Levin (2008) reported that the

369 concentration of asparagine does not have a significant effect on acrylamide formation in

370 potatoes. Yoshida et al. (2005) reported that among reducing sugars and asparagine, reducing

371 sugars in potato tubers play the limiting factors for acrylamide formation in potato chips. As

372 reported in the case of potatoes by various researchers, it was observed from the present study

373 that the changes of precursors during ripening of plantain influence the formation of

374 acrylamide during the deep frying process for making chips and thus the formation of

375 acrylamide can be controlled by selecting proper stage of maturity.

376 Previous studies suggest that phenolic compounds can influence the acrylamide formation

377 (Zhu, Cai, Ke, & Corke, 2009; Morales et al., 2014). Considering the influence of

378 polyphenols on acrylamide formation, the TPC & TFC contents were evaluated for all the

379 ripening stages of Nendran. It was found that the acrylamide formation correlated negatively

380 with the TPC and TFC with r values of -0.585 & -0.575 respectively. Five major phenolic

16
381 compounds in plantain viz gallic acid, chlorogenic acid, syringic acid, p-coumaric acid &

382 quercetin quantified at stages I -V were also correlated with acrylamide content in the chips

383 prepared. It was found that all the five compounds exhibited a negative correlation with the

384 acrylamide content in chips indicating that the decrease in the phenolic content during

385 ripening may be one of the reasons for promoting the formation of acrylamide in chips during

386 frying.

387 Earlier reports suggest that plant polyphenols comprise a large group of compounds having

388 different structures having a greater influence on the formation of acrylamide on heating

389 (Zhu, et al., 2009). Polyphenols act as free radical scavengers thereby inhibiting the chain

390 reaction by the combination of the hydrogen atom of phenolic –OH with free radicals.

391 Besides the antioxidant capacity of polyphenols, some other mechanistic pathway may be

392 influencing the formation of acrylamide along with structure, concentration & reaction

393 conditions which have not been given a correct explanation so far. In a study by Constantinou

394 & Koutsidi (2016), it is reported that, in Maillard reaction systems, phenolic antioxidants may

395 react with sugar fragments and/or reactive carbonyl compounds, forming adducts through

396 electrophilic aromatic substitution reactions and inhibits acrylamide formation. The number

397 and position of phenol hydroxyls of flavonoid compounds play an important role in

398 controlling the formation of acrylamide. Furthermore, corresponding parameters, including

399 the number, positions and spatial steric effects of functional hydroxyls, and the related mode-

400 of-actions remain largely uncertain (Zhang, Mengmeng, Wanga, Cheng, 2016). In general,

401 the more phenolic hydroxyls are present in the chemical structures of flavanols and

402 derivatives, the greater the change in the antioxidant attributes of the Maillard reaction

403 products, no matter whether the formation of acrylamide is reduced or promoted. The

404 flavanols and derivatives may contribute their active phenolic hydroxyl groups to the

17
405 Maillard reaction, thus affecting the final antioxidant activity of the MRP (Huang, Wang,

406 Chen, Zhang, 2016).

407 The study, therefore, documents the chemical changes during ripening of plantain and also

408 highlight how these changes in reducing sugars, amino acids, polyphenol & flavonoid content

409 during ripening affect the formation of acrylamide during the preparation of deep-fried chips.

410 The changes in the concentration of phenolic compounds during ripening and its effect on

411 acrylamide formation have not been reported earlier, as per the knowledge. Thus our study

412 recommends stages I and II, which possess less reducing sugars and higher phenolic content,

413 for making chips with lower acrylamide content.

414 6. Conclusions

415 Formation of acrylamide during deep frying of plantain showed a positive correlation with

416 respect to reducing sugars, whereas a poor correlation were observed with amino acids. Five

417 major phenolic compounds namely gallic acid, chlorogenic acid, syringic acid, p-coumaric

418 acid & quercetin were quantified in all the stages of plantain ripening and found to decrease

419 during ripening. All these five compounds also exhibited a negative correlation with

420 acrylamide formation in chips indicating that higher phenolic content in raw plantain along

421 with reduced levels of reducing sugars in the initial ripening stages minimized the formation

422 of acrylamide in deep fried plantain chips. Higher the phenolic content, more the antioxidant

423 activity which would act as free radical scavengers thereby inhibits the chain reaction

424 minimizing acrylamide formation. Thus by selecting the proper ripening stage of raw

425 material with lesser sugar content and higher phenolic content, the formation of acrylamide in

426 the final product can be reduced to a reasonable level. The study also warrants the importance

427 of similar studies that could help to formulate strategies and guidelines for reducing

428 acrylamide in a variety of high temperature processed food products that can bring down the

429 exposure of this toxicant to the population.

18
430 Acknowledgements

431 L. Shamla thanks the Council of Scientific and Industrial Research (CSIR, New Delhi, India)

432 for the Senior Research Fellowship. The project was financed under the fast track young

433 scientist award scheme of Department of Science & Technology, Ministry of Science and

434 Technology, Government of India.

435 Conflict of Interest

436 The authors declare that there are no conflicts of interest.

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550 Table Legends

23
551 1. Table 1. Proximate composition (% of nutrient) in the pulp of raw plantains

552 (Nendran) from stages I-V

553 2. Table 2. Reducing sugars (mg/100gm), free aminoacids (mg/g) and polyphenol

554 (mg/g) contents (fresh wt.) of Nendran fruit at five different maturity stages (n=3))

555 3. Table 3. Pearson’s correlation between acrylamide formation and precursor

556 concentrations in Nendran variety of all the five stages

557 Figure Legends

558 1. Fig.1 Five different ripening stages of Nendran (I-V)

559 A-raw plantain with skin; B- plantain fruit after peeling; and C- fried chips

560 2. Fig.2 TPC& TFC contents of Nendran pulp extract as on increasing ripening

561 (* indicates the values are significantly different)

562 3. Fig. 3 Bar graph showing acrylamide concentrations for five different ripening

563 stages(I-V) of plantain (Nendran) [* represents the values are significantly

564 different]

565

24
566

567

568 Fig.1 Different ripening stages of Nendran (I-V) used in the study
569 A-raw plantain with skin; B- plantain fruit after peeling; and C- fried chips
570

571

572

573

574

575

576

577

578

579

580

581

582

583

584

585

25
586

587

588 Fig. 2 TPC& TFC contents of Nendran pulp extract as on increasing ripening
589 (* indicates the values are significantly different)
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617

26
618
619
2500

Acrylamide Conc. (µg/kg)

2000

1500

1000

500
* *
*
0 *
I II III IV V
620 Ripening stages of Nendran
621
622
623 Fig.3 Bar graph showing acrylamide concentrations for five different ripening stages(I-V) of
624 plantain (Nendran) [* represents the values are significantly different]
625

626

27
627

Stages I II III IV V

Moisture 55.20±0.30a 57.40±0.03b 55.87±0.04c 60.76±0.02d 63.95±0.04e

Total ash 3.47±0.03a 4.86±0.02b 5.22±0.00c 5.85±0.02d 6.18±0.08e

Crude fat 0.24±0.05a 0.45±0.04b 0.68±0.01c 0.86±0.01d 1.20±0.03e

Crude protein 2.73±0.02a 2.89±0.05b 2.90±0.04b 2.95±0.01b 3.08±0.02c

Carbohydrate 55.23±0.09a 43.98±0.07b 35.58±0.03c 32.48±0.02d 28.95±0.05e

628 Table 1
629 Proximate composition (% of nutrient) in the pulp of raw plantains (Nendran) from stages I-
630 V
631 Values are means ± SE (n = 3). Means values in the same row followed by a different superscript
632 letter are significantly (p ≤ 0.05) different

633

634

635

636

637

638

639

640

641

642

643

644

645

646

647

648

28
649

650 Table 2

651 Reducing sugars (mg/100gm), free aminoacids (mg/g) and polyphenol (mg/g) contents (fresh
652 wt.) of Nendran fruit at five different maturity stages (n=3)

653

Precursors Stage I Stage II Stage III Stage IV Stage V

Reducing
Sugars
(g/100gm)
Glucose 4.83 ± 0.28a 6.24 ± 0.10 b 8.42 ± 0.08 c 9.41 ± 0.06 d 15.15 ± 0.15
Fructose 4.76 ± 0.05a 6.28 ± 0.10b 8.65 ± 0.09c 9.75 ± 0.17 d 15.70 ± 0.07 e
Aminoacids
(mg/g)
Asparagine 1.80 ± 0.05a 2.96 ± 0.02 b 1.37 ± 0.04 c 1.45 ± 0.09c 1.89 ± 0.04d
Glutamine 0.15 ± 0.07 a 0.50 ± 0.03 b 0.59 ± 0.02c 0.43 ± 0.04d 0.06 ± 0.05 a
Serine 1.34 ±0.09a 1.37 ± 0.02 a 1.78 ± 0.03b 1.78 ± 0.03b 1.23 ± 0.03 c
Glycine 2.00 ± 0.08 a 2.01 ± 0.02 a 2.00 ± 0.02 a 2.00 ± 0.03 a 2.00 ± 0.03 a
Threonine 1.64 ± 0.05a 2.09 ± 0.02 b 2.91 ± 0.01 c 2.70 ± 0.02d 2.59 ± 0.02 e
Valine 1.27 ± 0.01a 1.36 ± 0.03 b 1.29 ± 0.01 c 1.31 ± 0.04 c 1.28 ± 0.01 a
Isoleucine 2.38 ± 0.01a 2.69 ± 0.01 b 2.95 ± 0.03 c 3.04 ± 0.02d 2.57 ± 0.08 e
Leucine 1.25 ± 0.02a 1.30 ± 0.02 b 1.36 ± 0.05b 1.47 ± 0.02b 1.40 ± 0.09 b
Phenylalanine 1.10 ± 0.02a 0.93 ± 002b 1.46 ± 0.07 c 1.62 ± 0.03d 0.76 ± 0.01 e
Lysine 0.60 ± 0.02a 0.70 ± 0.01 b 0.52 ± 0.09 c 0.55 ± 0.01 c 0.42 ± 0.04 d
Polyphenols
(mg/g)
Gallic acid 11.26±0.509 a 4.08±0.170 b 1.23±0.448 c 0.21±0.056d 0.13±0.063 e
Chlorogenic 12.20±0.777 a 10.82±0.462 b 3.38±0.103 c 1.33±0.285d 0.80±0.063e
acid
Syringic acid 3.61±0.213a 1.19±0.190b 0.37±0.171c 0.036±0.008 d -
p-Coumaric 3.01±0.859a 0.41±0.063b 0.12±0.037c 0.038±0.009 d 0.028±0.010e
acid 9.38±0.438a 7.45±0.478b 1.85±0.581c 0.21±0.119d 0.086±0.026e
Quercetin
654 Values are mean ± SE (n = 3). Mean values in the same row followed by a different superscript letter
655 are significantly different (p ≤ 0.05)

656

29
657

658

659

660 Table 3

661 Pearson correlation between acrylamide formation and precursor concentrations in Nendran
662 variety of all the five stages

Precursors Pearson P-value R2

correlation (r)
Reducing Sugars
Glucose 0.947 0.015 0.78
Fructose 0.936 0.019 0.87

Aminoacids
Asparagine -0.034 0.957 0.00
Glutamine -0.560 0.326 0.31
Serine -0.492 0.392 0.24
Glycine -0.030 0.960 0.00
Threonine 0.320 0.599 0.10
Valine -0.257 0.675 0.06
Isoleucine -0.210 0.735 0.04
Leucine 0.342 0.573 0.11
Phenylalanine -0.572 0.313 0.33
Lysine -0.728 0.163 0.53

Polyphenols
Gallic acid -0.490 0.402 0.24
Chlorogenic acid -0.594 0.290 0.35
Syringic acid -0.974 0.005 0.24
p-Coumaric acid -0.400 0.504 0.16
Quercetin -0.500 0.316 0.33

663

664

665

666

667

30
668

669

670 Highlights

671
Plantain of five different ripening stages were selected for preparing
672 banana chips
673
Acrylamide formation was correlated with reducing sugars, aminoacids &
674 phenolics
675
Reducing sugars increased on ripening and correlated positively with
676 acrylamide formation
677
TPC & TFC decreased on ripening, correlated negatively with acrylamide
678 formation
679
Acrylamide in plantain chips can be minimized by proper selection of
680 maturity stage
681

682

683

684
685

31