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Accepted Manuscript

Effects of diammonia phosphate addition on the chemical constituents in lychee wine fermented with Saccharomyces cerevisiae

Dai Chen, Sandrine Toussaint, Weidong Huang, Jicheng Zhan, Shao-Quan Liu

PII:

S0023-6438(19)30108-2

Reference:

YFSTL 7831

To appear in:

LWT - Food Science and Technology

Received Date: 29 November 2018

Revised Date:

Accepted Date: 6 February 2019

22 January 2019

Revised Date: Accepted Date: 6 February 2019 22 January 2019 Please cite this article as: Chen,

Please cite this article as: Chen, D., Toussaint, S., Huang, W., Zhan, J., Liu, S.-Q., Effects of diammonia phosphate addition on the chemical constituents in lychee wine fermented with Saccharomyces cerevisiae, LWT - Food Science and Technology (2019), doi: https://doi.org/10.1016/j.lwt.2019.02.018.

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Effects of diammonia phosphate addition on the chemical constituents in lychee

wine fermented with Saccharomyces cerevisiae

Dai Chen a,c , Sandrine Toussaint b , Weidong Huang a , Jicheng Zhan a , Shao-Quan Liu c,d*

a Beijing Key Laboratory of Viticulture and Enology, College of Food Science and

Nutritional Engineering, China Agricultural University (CAU), Tsinghua East Road

17, Haidian District, Beijing, 100083, P. R. China;

b Medtronic France, 27 quai Alphonse Le Gallo, CS 30001, 92513

Boulogne-Billancourt cedex, France;

c Food Science and Technology Program, Department of Chemistry, National

University of Singapore (NUS), 3 Science Drive 3, Singapore 117543, Republic of

Singapore

d National University of Singapore (Suzhou) Research Institute, 377 Lin Quan Street,

Suzhou Industrial Park, Jiangsu 215123, P. R. China

Running title: Impact of DAP additions on lychee wines

*Corresponding author. Postal address: Food Science and Technology Programme,

Department of Chemistry, National University of Singapore, Science Drive 3,

Singapore. Tel.: +65 6516 2687; fax: +65 6775 7895.

E-mail address: chmlsq@nus.edu.sg (Shao-Quan Liu)

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Abstract

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This study evaluated the effects of diammonia phosphate (DAP) on the

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non-volatile and volatile compounds of lychee wine fermented with Saccharomyces

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cerevisiae, when added in two different quantities (0.5 mmol/L and 1.5 mmol/L). It

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was found that DAP supplementation improved the utilization of ammonia and

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inhibited the consumption of proline and valine, which regulated the production of

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α-ketoglutaric, succinic and fatty acids. The addition of 0.5 mmol/L DAP improved

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the rate of sugar catabolism by slightly increasing yeast growth, thus inducing a

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higher production of glycerol than of ethanol. Additionally, more odor-active terpene

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derivatives (trans-β-damascenone, o-cymene, δ-guaiene) in lychee juice were retained

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after the fermentation added with 0.5 mmol/L DAP. However, the addition of 1.5

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mmol/L DAP slowed rates of sugar metabolism and glycerol production, and

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significantly enhanced the production of acetic acid. Furthermore, with the exception

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of limonene, the higher DAP addition did not retain more terpene derivatives. These

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findings, therefore, suggest that a moderate addition of DAP could enhance the

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flavorful character of lychee wine.

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Keywords

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Fermentation; lychee wine; diammonia phosphate; nitrogen; flavor.

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42 1. Introduction

43 Lychee (Litchi chinensis Sonn.) is the commercially most significant member of

44 the Sapindaceae family, with its rose-floral and citrus-like aromas and a palatable,

45 sweet taste. Nearly 100 major cultivars of lychee grow in tropical and sub-tropical

46 areas (Pareek, 2015). More than 100 volatile compounds were identified in over 10

47 lychee cultivars since 1980, including terpene derivatives, alcohols, ketones, esters,

48 acids, aldehydes and others (Chyau, Ko, Chang, & Mau, 2003; Feng, Huang, Crane,

49 & Wang, 2018; Johnston, Welch, & Hunter, 1980; Wu, Pan, Qu, & Duan, 2009).

50 Specifically, the terpene derivatives cis-rose oxide, geraniol,

51 3-methyl-2-buten-1-ol, linalool, α-terpineol, β-citronellol, ρ-cymene,

52 3-methyl-3-buten-1-ol, (E)-2-hexen-1-ol, 2-ethyl-1-hexanol, 1-octen-3-ol,

53 ρ,α-dimethylstyrene, and 3-tert-butyl-4-hydroxyanisol were reported as the volatiles

54 common to most lychee cultivars, contributing to the lychee odor, citrus-like and

55 floral aroma (Wu et al., 2009). Besides terpene derivatives, 2-phenethyl alcohol is

56 reportedly the most important alcohol compound in lychee fruits, and is likely

57 responsible for their floral character (Chyau et al., 2003)

58 However, most volatiles endogenous to lychee fruit (mainly terpene derivatives)

59 have been noted to reduce dramatically to just trace levels after fermentation with

60 Saccharomyces yeasts, resulting in the diminishment of the lychee character in the

61 resultant wines (Chen & Liu, 2016; Wu, Zhu, Tu, Duan, & Pan, 2011). In our previous

62 study, it was found that the utilization of non- Saccharomyces yeast (Torulaspora

63 delbrueckii) as mono- or sequential-cultures could retain more lychee aroma-character

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compounds than the Saccharomyces monoculture (Chen & Liu, 2016).

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Besides yeast strains, nitrogen sources of musts could impact the volatile and

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non-volatile compounds of wine by regulating yeast metabolisms during the

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fermentation process. Previous studies have suggested that a single amino acid

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addition could markedly increase the formation of corresponding higher alcohols and

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esters, yet were not able to retain more of the volatiles endogenous to lychee fruits

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(Chen, Chia, & Liu, 2014; Chen, Vong, & Liu, 2015).

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Vilanova, Siebert, Varela, Pretorius, & Henschke (2012) found that a moderate

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addition of diammonia phosphate (DAP) (up to 350 mg N/L) significantly increased

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the concentrations of limonene, linalool, α-terpineol, α-ionone and β-damascenone,

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while a high DAP addition (450 mg N/L) improved the amounts of β-ionone in

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aromatic Albariño grape wine. However, other researchers noted little or no difference

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in the level of terpene derivatives after DAP addition to Shiraz and white Godello

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wines (Losada, Andrés, Cacho, Revilla, & López, 2011; Ugliano, Siebert, Mercurio,

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Capone, & Henschke, 2008). No related study has yet been carried out with respect to

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lychee wine fermentation.

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Thus, the aim of this study was to evaluate the effects of DAP additions on the

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fermentation and chemical composition of lychee wine fermented with

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Saccharomyces cerevisiae, especially with regard to the primary lychee aroma.

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Furthermore, it was anticipated that the result of this study could provide a suitable

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nitrogen condition with which to enhance lychee character in the wine.

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2. Materials and methods

2.1. Fermentation of lychee juice

Sterilized lychee juice (Litchi chinensis Sonn. var. Nuomi Ci) and a S. cerevisiae

MERIT.ferm (Chr.-Han., Horsholm, Denmark) pre-culture (10

7

CFU/mL) were

prepared using the same method as mentioned by Chen et al. (2014).

Three groups of lychee wine fermentations (250 mL prepared lychee juice) were

carried out in sterile Erlenmeyer flasks, each supplemented with different amounts of

DAP (control, 0.5 mmol/L DAP and 1.5 mmol/L DAP) by adding 0 mL, 1mL and 3

mL of 1.65 g/100 mL sterilized DAP (Sigma-Aldrich, Oakville, ON, Canada) solution,

respectively. All flasks were fitted with non-absorbent cotton wool and covered with

aluminum foil to establish semi-aerobic conditions in the early stages of fermentation.

S. cerevisiae MERIT.ferm pre-culture (2.5 mL) was inoculated into each flask for the

10 d fermentation at 20°C.

2.2. Sample analysis

Yeast growth was monitored using the spread plating method on a potato

dextrose agar (Oxoid, Hampshire, England). °Brix and pH were measured using a

refractometer (ATAGO, Tokyo, Japan) and a pH meter (Metrohm, Switzerland),

respectively.

The Shimadzu HPLC system was used to analyze non-volatile compounds

(including sugars, glycerol, organic acids and amino acids). Amino acids were

analyzed with a Waters AccQ-Tag Nova-Pak C

18 column (150 × 3.9 mm, Waters,

Dublin, Ireland) based on the method of Waters (1993). Sugars and glycerol were

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108 separated using a Zorbax carbohydrate column (150 × 4.6 mm, Agilent, Santa Clara,

109 CA, USA) using the methods of Lee et al. (2013). Organic acids were quantified with

110 a Supelcogel C-610 H column (300 × 7.8 mm, Supelco, Sigma-Aldrich, Barcelona,

111 Spain) using the methods developed by Lee et al. (2013).

112 Volatile compounds were analyzed using a headspace solid-phase microextraction

113 (HS- SPME) coupled with an Agilent 7890A GC–Agilent 5975C triple-axis MS and

114 flame ionization detector (FID). Samples were adjusted to pH 2.5 with 1 mol/L HCl

115 and were transferred into a screw-capped headspace vial (5 mL of sample for each

116 vial). Extraction was done using a carboxen/poly (dimethylsiloxane) fiber (Supelco,

117 Sigma-Aldrich, Barcelona, Spain) with a SPME autosampler (CTC, Combi Pal,

118 Switzerland) at 60 °C for 40 min under 250 rpm agitation (Chen et al., 2014).

119 Separation was done using a 60 m × 0.25 mm capillary column (Agilent DB-FFAP,

120 Santa Clara, CA, USA). The GC temperature was increased from 50

o C (hold for 5

121 min) to 230

o C (final hold for 30 min) at 5 K/ min. The volatiles were identified using

122 NIST 8.0 and Wiley 275 MS libraries, verified based on the linear retention index

123 (LRI) and semi-quantificated based on GC-FID peak areas.

124 2.3. Statistical analysis

125 Based on the data of triplicate fermentations for each treatment, the mean values

126 and standard deviation were calculated and evaluated to obtain the statistical

127 differences (at 95% confidence level) by one-way analysis of variance (ANOVA) and

128 Scheffe's test using SPSS

® 17.0. Volatile compounds with significant differences

129 were selected to perform principal component analysis (PCA) using software Matlab

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130 R2008a.

131 3. Results and discussion

132 3.1. Yeast population

133 In the early stage of fermentation, the 0.5 mmol/L DAP addition significantly

134 improved the yeast growth, rising to 5.90 ×10

7 CFU/mL on d 2, with approximately

135 double the cell population in control and 1.5 mmol/L DAP added wines (2.86 ×10

7

136 CFU/mL and 3.10 ×10

7 CFU/mL, respectively). All yeasts reached stationary phases

137 on d 4 with no significant difference (Fig. 1). The final yeast cell count was

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significantly higher in the wine added with 0.5 mmol/L DAP (1.30 ×10

8

CFU/mL)

139 than that of the other wines (<1×10

8 CFU/mL) (Table 1). This result indicated that a

140 moderate addition of DAP could accelerate the exponential phase of yeast growth and

141 extend its stationary phase, while a higher DAP addition had no such effect. Similarly,

142 some previous reports that DAP supplementation (over 3.5 mmol/L) was

143 inconsequential to the yeast growth when the original assimilable nitrogen was over

144 140 mg N/L (Bell & Henschke, 2005; Lee et al., 2013; Vilanova et al., 2012).

145 At the beginning of the winemaking process, the yeast needs to increase its

146 population by obtaining energy from nutrients and biomass from amino acids. Both

147 the uptake and biomass synthesis of amino acids require ATP. Compared with the

148 amino acids, ammonia needs less ATP transported inside a yeast cell, but uses more

149 ATP for biomass yield (Albers, Larsson, Lidén, Niklasson, & Gustafsson, 1996;

150 Magasanik, 2003).

151 The concentration of ammonia could be increased by adding DAP (Bell &

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152 Henschke, 2005). In this study, the moderate DAP addition improved the uptake of

153 ammonia for biomass yield, while the higher DAP addition needed excessive ATP for

154 its metabolism in the yeast cell, thus reducing the amount of energy available for other

155 processes and slowing yeast growth in the early stage of fermentation. In other words,

156 in the exponential phase of yeast growth, the consumption of nitrogen and sugar

157 should reach a good balance in energy and biomass aspects.

158 3.2. Ammonia and amino acids

159 The total nitrogen concentration in the lychee juice samples was around 220 mg

160 N/L. This increased to 236 mg N/L and 273 mg N/L after the enhancement of

161 ammonia through the addition of 0.5 mmol/L DAP and 1.5 mmol/L DAP, respectively

162 (Table 2). The ammonia was depleted after all fermentations (Table 2), thus indicating

163 that the ammonia was easily consumed by S. cerevisiae as the preferential nitrogen

164 source.

165 Proline (5.71 mmol/L) and alanine (3.04 mmol/L) were the main amino acids in

166 lychee juice. Most amino acids were depleted by all cultures, while DAP

167 supplementation inhibited the cell uptakes of proline, valine, alanine, glycine and

168 asparagine (Table 2). More residual glycine and asparagine were observed in the 1.5

169 mmol/L DAP supplemented fermentation (Table 2), while alanine, valine and proline

170 were inhibited by both DAP treatments (Table 2). The contents of residual alanine

171 were similar in both DAP added fermentations, while no residual alanine was detected

172 in the control fermentation (Table 2). DAP additions markedly reduced the uptake of

173 valine (Table 2), suggesting that it was a less preferred nitrogen source, possibly.

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174 Because valine contains only general amino acid permease (Gap1p) for transportation

175 (Albers et al., 1996).

176 The consumption rate of proline was found to be negatively correlated with the

177 DAP supplementation, reducing from 73.4% (control fermentation) to 36.6% and 2.7%

178 in the 0.5 mmol/L and 1.5 mmol/L DAP added fermentations, respectively (Table 2).

179 This indicated that the DAP addition had a more significant impact on the metabolism

180 of proline than it did on other amino acids. Actually, proline is excluded from

181 assimilable nitrogen sources, since yeast is unable to catabolize proline under

182 anaerobic conditions (Deed, Van Vuuren, & Gardner, 2011). Proline was much

183 consumed in the control wine, suggesting that the lychee juice was deficient in

184 preferred nitrogen sources, and under which condition the activities of Gap1p and

185 proline permease increased (Bell & Henschke, 2005).

186 Proline could be metabolized as an intermediate for the production of

187 α-ketoglutaric (α-KG) (Arias-Gil, Garde-Cerdan, & Ancin-Azpilicueta, 2007), thus

188 regulating the concentration of α-KG. Proline could also serve as an osmoprotectant

189 in yeast cells, balancing the hyperosmotic conditions between the yeast cells and the

190 wine matrix (Takagi, Takaoka, Kawaguchi, & Kubo, 2005).

191 3.3. Sugar catabolism

192 Sucrose and glucose were completely metabolized, while a trace quantity of

193 fructose was present at the end of all fermentations. Sucrose was dramatically

194 hydrolyzed in the first 4 d of fermentations (Fig. 2a) by yeast extracellular invertase

195 (Ostergaard, Olsson, & Nielsen, 2000). Thus, the rate of sucrose hydrolysis was

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196 positively correlated with the rate of yeast growth (Fig. 1 and Fig. 2a).

197 The concentrations of glucose and fructose during the fermentations were a net

198 balance between utilization by S. cerevisiae and the release of sucrose. The

199 consumption trends of glucose and fructose were similar to that of sucrose, which was

200 fastest in the fermentation added with 0.5 mmol/L DAP and slowest in the

201 fermentation added with 1.5 mmol/L DAP (Fig. 2b and Fig. 2c). This indicated that

202 DAP supplementation could impact sugar metabolism by affecting the yeast

203 population.

204 On the other hand, neither

o Brix reduction nor ethanol production was

205 significantly altered in any of the fermentations (Table 1, Fig. 2d). This implied that

206 the endogenous nitrogen concentration of lychee juice was able to complete the

207 fermentation process and that DAP supplementation could not affect the fermentation

208 rate, despite the lack of preferred nitrogen sources in the control juice. Similar results

209 were reported by Lee et al. (2013) in durian wine fermentation.

210 DAP supplementation significantly affected the production rate of glycerol (Fig.

211 2e) via glyceropyruvic fermentation (Takagi et al., 2005). In the control and 0.5

212 mmol/L DAP added fermentations, glycerol was quickly produced to its peak content

213 on d 2, and reduced to around 0.6 g/100 mL on d 4, remaining relatively stable until d

214 10 (Fig. 2e). The final concentration of glycerol (0.67 g/100 mL) in the wine added

215 with 0.5 mmol/L DAP was significantly higher than that in control wine (0.60% w/v),

216 increasing the level of sweetness in the wine (Ugliano & Henschke, 2009). The

217 addition of 1.5 mmol/L DAP delayed the production of glycerol, reaching its highest

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218 concentration on d 4, and its final concentration (0.57 g/100 mL) was lowest in all

219 wines (Fig. 2e).

220 The trend of glycerol production was consistent with the rates of sugar

221 consumptions, implying that the changes of sugar catabolism were mainly due to

222 glyceropyruvic fermentation, rather than alcoholic fermentation. However, a previous

223 study reported that glycerol production was positively correlated with DAP

224 supplementation (Ugliano, 2008; Ugliano, Travis, Francis, & Henschke, 2010).

225 Furthermore, glycerol production might be affected by yeast strains (Vilanova et al.,

226 2007). Additionally, glycerol could act as a compatible solute to counter osmotic

227 pressure and thus was regulated by other osmoprotectants, such as proline

228 (Cronwright, Rohwer, & Prior, 2002). The highest concentration of proline in the

229 lychee wine with 1.5 mmol/L DAP addition could reduce the need for glycerol

230 production (Table 1, Table 2).

231 3.4. pH and organic acids

232 The pH value was not significantly different in wines (Table 1). Malic and

233 succinic acids represented over 98% of the total organic acids in the lychee juice

234 (Table 1). After fermentation, succinic and tartaric acids had slightly decreased, while

235 approximately 40% of malic acid had been reduced. Trace concentrations of α-KG,

236 pyruvic and lactic acids were produced during fermentations, in concurrence with the

237 results of a previous lychee wine study (Chen et al., 2014).

238 Most organic acids showed no significant difference after DAP supplementation,

239 however, the succinic acid and α-KG were significantly reduced after 1.5 mmol/L

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240 DAP addition (Table 1). As mentioned above, α-KG can be synthetized via proline

241 metabolism (Takagi, et al., 2005) and combined with ammonium to produce amino

242 acids for cell growth (Magasanik, 2003). In the 1.5 mmol/L DAP added fermentation,

243 the lowest amount of α-KG was produced from proline metabolism, while the most

244 was consumed during ammonium metabolism, and thus, the final content of α-KG

245 was lowest. These entered into the TCA cycle, resulting in the lowest value of

246 succinic acid in the 1.5 mmol/L DAP added wine (Table 1). Succinic acid reduction

247 positively affects wine sensory evaluation by reducing the unusual salty and bitter

248 taste (Ugliano & Henschke, 2009). Similarly, some previous studies have shown that

249 DAP addition reduced the concentration of α-KG and succinic acids (Torrea et al.,

250 2011).

251 3.5. Volatiles

252 A range of volatiles (50 volatiles and 72 volatiles, excluding ethanol) were

253 respectively detected in lychee juice and wines. In the lychee juice, aldehydes and

254 terpene derivatives were two most abundant volatiles (relative peak area, RPA 25.53%

255 and 24.34%, respectively), followed by alcohols (RPA 19.75%), esters (RPA 15.36%)

256 and ketones (RPA 6.65%) (Table 3). After fermentations, esters and alcohols were

257 significantly produced (RPA over 70% and 20%, respectively) in the lychee wines,

258 with fewer amounts of acids and terpene derivatives (RPA 2% - 3%) and trace levels

259 of aldehydes and ketones (RPA<1%) (Table 3). A total of 33 volatile compounds had

260 significantly different contents after DAP additions, including 12 terpene derivatives,

261 6 esters, 5 ketones, 4 acids, 2 alcohols, 2 aldehydes, and 2 other volatiles (Table 3).

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262 3.5.1. Volatile acids

263 Acetic acid was the only volatile acid detected in the lychee juice, while the

264 other three acids (octanoic, decanoic and 9-decenoic acids) were produced after

265 fermentation (Table 3). The content of 9-decenoic acid was negatively correlated with

266 DAP additions (Table 3). Conversely, the RPA of acetic acid was positively correlated

267 with the DAP values (Table 3). During alcoholic fermentation, acetic acid is produced

268 from acetaldehyde, releasing NADH to restore the redox balance (Rodicio & Heinisch,

269 2009). Amino acid metabolisms could also generate NADH. The generated NADH

270 could be used during glutamate synthesis by ammonia (Magasanik, 2003). In this

271 study, the DAP supplementation might result in more NADH being consumed by

272 ammonia and less NADH being produced by amino acids. The cell therefore needed

273 to generate more NADH via other reactions, including the oxidative formation of

274 acetic acid from acetaldehyde (Bely, Rinaldi, & Dubourdieu, 2003). Similar results

275 were reported by Torrea et al. (2011).

276 The levels of fatty acids (C

8 and C

10 ) were both the lowest in the lychee wine

277 with 0.5 mmol/L DAP addition and highest in the 1.5 mmol/L DAP added wine (Table

278 3). Fatty acids are generated from acetyl-CoA by consuming NADPH, and

279 acetyl-CoA is activated from acetic acid (Cherry et al., 2012). Thus, the high

280 concentration of acetic acid could result in the high production of fatty acids in the 1.5

281 mmol/L DAP added. Compared with the level in the 0.5 mmol/L DAP added wine, the

282 higher production of fatty acids in the control wine might be related to more NADPH

283 being produced via the pentose phosphate pathway, in which the key enzyme

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284 phosphogluconate dehydrogenase (GND) was up-regulated under nitrogen

285 deprivation (He et al., 2018). Further research could elucidate this possibility.

286 3.5.2. Alcohols (excluding ethanol)

287 Most of the alcohols were reduced to trace levels after fermentation. High levels

288 of isoamyl alcohol, 2-phenylethyl alcohol and butanol were produced with no

289 significant difference (Table 3). The DAP supplementation reduced the amounts of

290 1-hexanol and o-butylphenol after fermentations (Table 3). Similar results were

291 reported by Vilanova et al. (2012), who noted that a nitrogen addition (total nitrogen

292 350 mg N/L) could significantly reduce the concentration of 1-hexanol.

293 3.5.3. Esters

294 Six esters were affected by the DAP additions, including methyl dodecanoate,

295 isoamyl dodecanoate, ethyl hexanoate, ethyl pentadecanoate. ethyl 9-decenoate and

296 ethyl 9-hexadecenoate (Table 3).

297 Ethyl hexanoate was increased from 1.62% (RPA) to over 1.80% (RPA) in the

298 lychee wine after the DAP additions (Table 3), potentially enhancing the fruity flavor

299 and strawberry aroma. Several previous studies have presented similar results

300 (Ugliano et al., 2008; Ugliano et al., 2010; Vilanova et al., 2012). In this work, trace

301 levels of methyl dodecanoate and isoamyl dodecanoate were only produced in the

302 wine added with 0.5 mmol/L DAP, while ethyl pentadecanoate was only undetectable

303 in this treatment (Table 3). These long-chain fatty acid esters may not affect the

304 sensory quality of the resultant wine, with high odor detection thresholds. The

305 addition of 1.5 mmol/L DAP could significantly lower the synthesis of ethyl

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306 9-decenoate and ethyl 9-hexadecenoate, which might be related to the lowest

307 production of the precursor 9-decenoic acid in this treatment (Table 3) (Jackson, 2008;

308 Ugliano & Henschke, 2009).

309 3.5.4. Terpene derivatives

310 A total of 17 terpene derivatives were found in the lychee juice (Table 3).

311 Generally, nearly half of terpene derivatives were affected by DAP additions and the

312 RPAs of the terpene derivatives were highest in the wines supplemented by 0.5

313 mmol/L DAP (2.99 %), followed by the 1.5 mmol/L DAP added wine (2.40 %) and

314 then the control wine (2.19%) (Table 3).

315 Most of the volatiles were significantly reduced after fermentations, with the

316 exception of trans-β-damascenone. A significant amount of trans-β-damascenone was

317 produced in the wine added with 0.5 mmol/L DAP, but it was undetectable in the

318 other wines (Table 3). Trans-β-damascenone is a powerful odorant in wine, with a

319 low odor threshold (0.05 ug/L), thus the significant increase of this compound could

320 considerably enhance apple and rose flavors (Guth, 1997). Similar results were

321 obtained by Vilanova et al. (2012), although Ugliano et al. (2008) observed that this

322 compound was not associated with YAN supplementation.

323 Another 5 volatiles endogenous to lychee juice were also affected by the DAP

324 additions. As with the trans-β-damascenone, o-cymene and δ-guaiene were only

325 found in the wine added with 0.5 mmol/L DAP. Two times of limonene remained in

326 the wine supplemented by 1.5 mmol/L DAP, while trans-β-ocimene (sweet herbal)

327 was depleted in this treatment (Table 3). Limonene is an important lychee flavor

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328 character compound in lychee fruit, and can enhance the citrus flavor of resultant

329 wine (Feng et al., 2018; Wu et al., 2009). Both DAP treatments in this study retained

330 significantly higher amounts of 3-carene (sweet citrus flavor) than the control, as well

331 as slightly higher levels of cis-rose oxide (Table 3). The latter volatile compound has

332 been suggested to significantly enhance the lychee odor in wine owing to its low odor

333 threshold (0.2 ug/L) (Guth, 1997).

334 There were nine terpene derivatives produced after fermentations in this work,

335 among which p, α-dimethyl styrene, o-allyltoluene, α-ocimene, p-cymene and perillen

336 were affected by the different DAP treatments. Both DAP additions produced seven

337 times more of p, α-dimethyl styrene and o-allyltoluene than in the control wine (Table

338 3). p, α-Dimethyl styrene was reported as the common aromatic compound in lychee

339 fruit (Wu et al., 2009). However, α-ocimene (floral and wet cloth note), p-cymene

340 (floral, grassy) were not found in either of the DAP added wines and perillen (woody

341 note) was undetectable in the 1.5 mmol/L DAP added wine (Table 3).

342 3.5.5. Aldehydes, ketones and others

343 Nearly all aldehydes were depleted after fermentations, with only trace levels of

344 p-tolualdehyde and benzaldehyde remaining in the control wine and DAP treatment

345 wines, respectively (Table 3). Benzaldehyde has been detected in some lychee

346 cultivars (Fei Zi Xiao and sweetheart), possessing an almond-like, fragrant and sweet

347 odor (Feng et al., 2018; Wu et al., 2009).

348 There were seven ketones detected in the lychee juice, of which

349 1,4-dimethyl-3-cyclohexenyl methyl ketone (contributing fruity flavor) was

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350 significantly higher in the lychee wines with DAP additions (Table 3). Four ketones

351 were produced during the fermentations, all of which were affected after the DAP

352 additions. 2-Nonanone, with a green fruity and cheesy flavor, was only produced after

353 the DAP additions (Table 3). The addition of 1.5 mmol/L DAP increased the

354 concentration of 2-undecanone (fruity and fatty odor) and cyclodecene, while 1.5

355 mmol/L DAP could not produce 2-methyl tetrahydrothiophen-3-one (Table 3).

356 Of the other volatiles, pentadecane was only undetectable in the lychee wine

357 with 1.5 mmol/L DAP addition, while 1,4-dimethyl-7-(1-methylethyl)azulene was

358 only retained in the 0.5 mmol/L DAP added wine (Table 3).

359 3.4. PCA analysis

360 The 33 volatiles, all significantly changed in the different treatments, were

361 selected to analyze the correlation between DAP additions and volatile compounds

362 (Fig. 2). The first principal component (PC1) explained 58.70% of the total variance

363 and separated the lychee juice from the lychee wines. 1-Hexanol, benzaldehyde,

364 p-tolualdehyde, 1,4-dimethyl-3-cyclohexenyl methyl ketone, limonene,

365 trans-β-ocimene, α-ocimene, 3-carene, δ-guaiene, trans-β-damascenone, α-calacorene

366 and 1,4-dimethyl-7-(1-methylethyl)azulene were all found in the same part of lychee

367 juice, indicating that these volatiles contributed to its main character being different

368 from the lychee wines (Fig. 2).

369 Different lychee wines were separated by the second principal component (PC2)

370 explaining 25.11% of the total variance. The lychee wine with 0.5 mmol/L DAP

371 addition was in the negative part of PC2, as were acetic acid, 2-nonanone, 2-methyl

16

ACCEPTED MANUSCRIPT

372

tetrahydrothiophen-3-one, 2-undecanone, methyl dodecanoate, isoamyl dodecanoate,

373

ethyl hexanoate, perillen, o-allyltoluene, p, α-dimethyl styrene and pentadecane (Fig

374

2). These volatiles can contribute sweet, fruity, fatty and cheesy flavors to lychee wine.

375

The other wines and volatiles (mainly fatty acids and ethyl esters) were all found in

376

the positive part of PC2. The lychee wine supplemented with 1.5 mmol/L DAP was

377

placed between the control wine and the 0.5 mmol/L DAP added wine and near to the

378

x-axis, suggesting that the effects of the DAP additions on volatile compounds were

379

not consistent with their concentrations.

380

381

4. Conclusion

382

Both quantities of DAP additions affected the nitrogen metabolism of the lychee

383

wine by enhancing the utilization of ammonia and reducing the consumptions of less

384

preferred amino acids (mainly proline and valine). This led to the higher production of

385

acetic acid and reductions of α-KG and succinic acid. As the main product of

386

alcoholic fermentation, ethanol showed no significant differences after the DAP

387

additions. The moderate DAP addition (0.5 mmol/L) significantly improved the rate

388

of yeast growth, thus increasing the rates of sugar consumption and glycerol

389

production in the early stage of fermentation, while higher DAP addition (1.5 mmol/L)

390

slowed these rates. In the primary volatiles of lychee fruits, the moderate DAP

391

addition was best able to retain trans-β-damascenone, o-cymene, and δ-guaiene, while

392

the high DAP addition retained more limonene. Both DAP supplementations

393

contributed to higher amounts of 3-carene, and benzaldehyde. Therefore, these results

17

ACCEPTED MANUSCRIPT

394

indicated that a moderate DAP addition could be a convenient way to retain the

395

lychee odor-active compounds in lychee wine fermented with S. cerevisiae.

396

397

Acknowledgments

398

This work was supported by an ARF grant from the Ministry of Education of

399

Singapore (WBS No. R-143-000-507-112) and China Postdoctoral Science

400

Foundation Grant (Grant No. 2017M610129). The first author would like to thank the

401

International Postdoctoral Exchange Fellowship Program (CAU) for financial support.

402

403

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22

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Table 1 Oenological parameters of lychee juice (d0) and lychee wines (d10) fermented with S. cerevisiae MERIT.ferm, with and without DAP addition.

Lychee juice

(d0)

Lychee wines (d10)

Control

0.5 mmol/L

DAP addition

1.5 mmol/L

DAP addition

LOQ

Yeast Count

(10 7 CFU/mL)

0.03 ± 0.00 a

pH 3.52

± 0.00 a

o Brix

21.97 ± 0.07 a

9.75 ± 1.12

b

3.60 ± 0.02 b

6.53 ± 0.30 b

13.03 ± 0.65 c

3.59 ± 0.05 ab

6.86 ± 0.19 b

9.85 ± 1.37 b

3.63 ± 0.02

6.58 ± 0.15

b

b

Ethanol

(mL/100 mL)

Glycerol

(g/100 mL)

Sugars (g/100 mL)

0.17 ± 0.02 a

* N.D.

11.52 ± 1.35 b

0.60 ± 0.05 a

11.30 ± 0.56 b

0.67 ± 0.01 b

11.59 ± 2.16 b

0.57 ± 0.03 a

Fructose

3.22 ± 0.14 a

** LOQ

LOQ

0.11 ± 0.01

Glucose

4.90 ± 0.19

N.D.

N.D.

N.D.

Sucrose

13.42 ± 0.57

N.D.

N.D.

N.D.

Organic Acids (g/100 mL)

b

-

-

-

0.15

0.37

0.11

0.21

0.12

Oxalic acid

Citric acid

Tartaric acid

α-ketoglutaric

acid

Malic acid

LOQ

N.D.

0.038 ± 0.004 a

N.D.

1.39 ± 0.02 a

0.002 ± 0.000 a

N.D.

LOQ

0.011 ± 0.000 a

0.92 ± 0.03 b

0.002 ± 0.000 a

N.D.

LOQ

0.011 ± 0.001 ab

0.91 ± 0.04 b

0.003 ± 0.000 a

N.D.

LOQ

0.010 ± 0.000 b

0.92 ± 0.01 b

0.002

0.008

0.012

0.004

0.010

Pyruvic acid

Succinic acid

N.D.

0.89 ± 0.01 a

0.028 ± 0.000 a

0.88 ± 0.03 a

0.027 ± 0.001 a

0.87 ± 0.01 a

0.030 ± 0.002 a

0.83 ± 0.01 b

0.018

0.016

Lactic acid

N.D.

0.051 ± 0.005 a

0.056 ± 0.008 a

0.055 ± 0.003 a

0.018

a,b,c Statistical analysis ANOVA (n = 3) at 95% confidence level with same letters indicating no significant difference * N.D., not detected; ** LOQ, limit of quantification

ACCEPTED MANUSCRIPT

Table 2 Amino acid and ammonia contents of lychee juice (d0) and lychee wines (d10) fermented with S. cerevisiae MERIT.ferm, with and without DAP addition.

Lychee wines (d10)

Lychee juice

 

(d0)

Control

0.5 mmol/L DAP addition

1.5 mmol/L DAP addition

Amino acids (mmol/L)

 

Asp

0.57 ± 0.12 a

0.05 ± 0.01

b

0.04

± 0.01

b

0.20

± 0.02

c

Ser&Asn

0.34 ± 0.04

ND

ND

ND

Glu

0.34 ± 0.03

ND

ND

ND

Gly

0.10 ± 0.01 a

0.05 ± 0.01

b

0.04

± 0.01

b

0.16

± 0.01

c

His &Gln

0.37 ± 0.02

ND

ND

ND

Arg

0.28 ± 0.03

ND

ND

ND

Thr

0.06 ± 0.01

ND

ND

ND

Ala

3.04 ± 0.32 a

ND

0.08 ± 0.01

b

0.08

± 0.01

b

Pro

5.71 ± 0.33 a

1.52 ± 0.08

b

3.62

± 0.15

c

5.56

± 0.79

a

Cys

ND

ND

ND

ND

Tyr

0.05 ± 0.01

ND

ND

ND

Val

0.35 ± 0.02 a

0.12 ± 0.00

b

0.29

± 0.01

c

0.23

± 0.01

c

Met

ND

ND

ND

ND

Lys

0.07 ± 0.01

ND

ND

ND

Ile

0.06 ± 0.01

ND

ND

ND

Leu

ND

ND

ND

ND

Phe

0.10 ± 0.02

ND

ND

ND

Trp

ND

ND

ND

ND

NH

3

*

ND

ND

ND

(mmol/L)

The total nitrogen concentrations (mg N/L)

**

Total

24.31 ± 1.31 a

57. 08 ± 2.61 b

87.22 ± 11.80 c

a,b,c Statistical analysis at 95% confidence level with same letters indicating no significant difference;

ND, not detected.

* NH 3 concentrations of lychee juice were 2.69 ± 0.27 mmol/L (control), 3.80 ± 0.66 mmol/L (0.5

mmol/L DAP addition) and 6.45 ± 0.62 mmol/L (1.5 mmol/L DAP).

**The total nitrogen concentrations of lychee juice were 220.55 ± 15.74 mg N/L (control), 236.07 ±

21.26 mg N/L (0.5 mmol/L DAP addition), and 273.26 ± 20.69 mg N/L (1.5 mmol/L DAP addition).

ACCEPTED MANUSCRIPT

Table 3. Volatile compounds (excluding ethanol, GC-FID peak area ×10 6 ) and their relative peak areas (% RPA) in lychee juice (d0) and lychee

wines (d10) fermented with S. cerevisiae MERIT.ferm, with and without DAP addition.

* Compounds identified in this study

LRI

Lychee juice (d0)

Control (d10)

 

Add 0.5 mmol/L DAP (d10)

Add 1.5 mmol/L DAP (d10)

Peak area

RPA

Peak area

RPA

Peak area

RPA

Peak area

RPA

Acids

1.62

3.12

2.75

3.56

Acetic acid

1457

1.10 ± 0.23 a

1.62

2.70 ± 0.19

b

0.93

2.83 ± 0.58

b

0.98

3.74 ± 0.57 c

1.24

Octanoic acid

1847

** N.D.

-

3.14 ± 0.25

ab

1.08

2.86 ± 0.44

a

0.99

4.00 ± 0.29 b

1.32

 

a

b

9-Decenoic acid

Decanoic acid

2335

2272

N.D.

N.D.

-

-

0.64 ± 0.14

2.59 ± 0.22

a

0.22

0.89

0.26 ± 0.03

2.00 ± 0.01

b

0.09

0.69

0.20 ± 0.04 b

2.82 ± 0.23 a

0.07

0.93

Alcohols

19.75

20.85

20.45

21.38

Butanol Isoamyl alcohol 3-Isopentenyl alcohol Prenyl alcohol

1154

N.D.

-

12.70 ± 1.04

a

4.38

14.05 ± 1.25

a 12.76 ± 0.03 a

4.85

4.23

1218

N.D.

-

29.57 ± 0.60

a

10.20

27.65 ± 1.53

a 31.95 ± 6.63 a N.D. N.D. N.D. N.D. N.D. N.D. 18.67 ± 1.34 b N.D. 0.46 ± 0.07 a 0.72 ± 0.05 b

9.55

-

-

0.07

-

-

-

5.76

-

-

0.22

10.58

1254

2.48 ± 0.14

3.64

N.D.

-

N.D.

-

1330

1.13 ± 0.28

1.67

N.D.

-

N.D.

-

1-Hexanol

1-Octen-3-ol

1360

1452

2.18 ± 0.16

2.20 ± 0.25

a

3.21

3.24

0.26 ± 0.01 b

N.D.

0.09

-

0.21 ± 0.05 b

N.D.

-

-

Dihydromyrcenol Benzenemethanol 2-Phenylethyl alcohol Phenol o-Butylphenol

2,4-Di-tert-butylphenol

1467

1.83 ± 0.11

2.70

N.D.

N.D.

-

1902

0.49 ± 0.02

0.71

N.D.

-

N.D.

-

1945

2018

1.46 ± 0.21

0.25 ± 0.04

a

2.15

0.36

16.20 ± 2.20 b

N.D.

5.59

-

16.68 ± 2.95 b

N.D.

6.18

-

 

a

 

2180

2311

0.36 ± 0.04

1.05 ± 0.15

a

0.53

1.54

0.97 ± 0.03 b

0.75 ± 0.12 b

0.33

0.26

N.D.

0.65 ± 0.07 b

0.15

0.24

Ester

15.36

73.01

72.18

71.18

Methyl octanoate Methyl decanoate Methyl dodecanoate

1382

N.D.

-

0.13 ± 0.02 a

0.04

0.13 ± 0.01 a

0.04

0.16

0.05

0.15 ± 0.01 a 0.44 ± 0.06 a N.D.

0.05

1591

N.D.

-

0.49 ± 0.04 a

0.17

0.45 ± 0.07 a 0.14 ± 0.03

0.15

1802

N.D.

-

N.D.

-

-

ACCEPTED MANUSCRIPT

Table 3. (Cont’d)

Compounds identified in this study

Lychee juice (d0)

Control (d10)

Add 0.5 mmol/L DAP (d10)

Add 1.5 mmol/L DAP (d10)

LRI

 

Peak area

RPA

Peak area

RPA

Peak area

RPA

Peak area

RPA

Ethyl acetate Ethyl hexanoate Ethyl heptanoate Ethyl octanoate Ethyl pelargonate Ethyl furoate Ethyl decanoate Ethyl 9-decenoate Ethyl undecanoate Ethyl dodecanoate Ethyl myristate Ethyl pentadecanoate Ethyl hexadecanoate Ethyl 9-hexadecenoate Ethyl stearate Ethyl oleate Propyl octanoate Propyl decanoate Isobutyl octanoate Isobutyl decanoate Isoamyl acetate Isoamyl octanoate

-

10.14 ± 1.38 a

14.91

9.48 ± 1.17

a

3.27

9.20 ± 0.77

a

3.18

10.35 ± 0.46 a

3.43

1222

N.D.

-

4.70 ± 0.27

a

1.62

5.65 ± 0.31

b

1.95

5.48 ± 0.62 b

1.81

1324

1430

N.D.

N.D.

 

a

0.08

8.72

0.28 ± 0.02

29.51 ± 0.66

a

0.10

10.19

0.25 ± 0.05 a

28.31 ± 4.42 a

0.08

9.37

-

-

0.22 ± 0.01

25.28 ± 4.08

a

 

a

1530

N.D.

-

1.40 ± 0.11

a

0.48

1.32 ± 0.22

a

0.46

1.47 ± 0.30 a

0.49

1630

N.D.

-

0.09 ± 0.01

a

0.03

0.10 ± 0.01

a

0.03

N.D.

-

1638

0.31 ± 0.06 a

0.45

74.78 ± 18.06

b

25.79

76.06 ± 14.52

b

26.27

88.46 ± 13.19 b

29.29

1693

N.D.

-

34.58 ± 0.04

a

11.92

30.18 ± 0.51

a

10.43

22.66 ± 4.85 b

7.50

1741

N.D.

-

0.35 ± 0.08

a

0.12

0.34 ± 0.05

a

0.12

0.37 ± 0.05 a

0.12

1844

2049

N.D.

N.D.

21.62 ± 4.36

0.78 ± 0.10

a

a

 

7.46

0.27

20.03 ± 2.86

0.82 ± 0.17

a

6.92

0.28

21.77 ± 0.16 a

0.67 ± 0.07 a 0.28 ± 0.06 a 4.17 ± 0.69 a 3.97 ± 0.20 b 0.97 ± 0.14 a 2.56 ± 0.46 a 0.24 ± 0.01 a 0.75 ± 0.09 a 1.09 ± 0.17 a 2.41 ± 0.26 a 3.93 ± 0.62 a 5.18 ± 0.25 a

7.21

0.22

-

-

a

2130

N.D.

-

0.25 ± 0.04 a

0.09

N.D.

-

0.09

2255

N.D.

-

4.88 ± 0.20 a

1.68

4.76 ± 0.71 a

1.64

1.38

2283

N.D.

-

6.20 ± 0.76 a

2.14

4.92 ± 0.74 ab

1.70

1.31

2465

N.D.

-

1.00 ± 0.21 a

0.35

0.96 ± 0.13 a

0.33

0.32

2481

N.D.

-

2.74 ± 0.09 a

0.95

2.71 ± 0.37 a

0.94

0.85

1513

N.D.

-

0.25 ± 0.03 a

0.08

0.28 ± 0.03 a

0.10

0.08

1724

N.D.

-

0.81 ± 0.05 a

0.28

0.84 ± 0.11 a

0.29

0.25

1547

N.D.

-

1.24 ± 0.22 a

0.43

1.35 ± 0.04 a

0.47

0.36

1756

N.D.

-

2.37 ± 0.20 a

0.82

2.22 ± 0.19 a

0.77

0.80

1116

N.D.

-

3.65 ± 0.83 a

1.26

3.47 ± 0.24 a

1.20

1.30

1660

N.D.

-

5.31 ± 0.72 a

1.83

4.98 ± 0.74 a

1.72

1.72

ACCEPTED MANUSCRIPT

Table 3. (Cont’d)

Compounds identified in this study

Lychee juice (d0)

Control (d10)

Add 0.5 mmol/L DAP (d10)

Add 1.5 mmol/L DAP (d10)

 

LRI

Peak area

RPA

Peak area

RPA

Peak area

RPA

Peak area

RPA

Isoamyl decanoate Isoamyl dodecanoate Carvyl acetate Hexyl acetate Heptyl acetate Phenylethyl acetate

1864

N.D.

-

5.17 ± 0.34

a

1.78

4.04 ± 0.52

a

1.40

5.04 ± 0.56 a N.D.

1.67

2068

N.D.

-

N.D.

-

0.30 ± 0.01

0.10

-

1232

N.D.

-

0.29 ± 0.02

a

0.10

0.34 ± 0.01

a

0.12

0.33 ± 0.05 a

0.11

1263

N.D.

-

0.32 ± 0.07

a

0.11

0.31 ± 0.03

a

0.11

0.30 ± 0.02 a

0.10

 

a

a

1366

1825

N.D.

N.D.

-

-

0.18 ± 0.03

3.14 ± 0.37

a

0.06

1.08