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

LWT - Food Science and Technology 105 (2019) 224–232

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

T

Dai Chena,c, Sandrine Toussaintb, Weidong Huanga, Jicheng Zhana, Shao-Quan Liuc,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, PR 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, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Fermentation Lychee wine Diammonia phosphate Nitrogen Flavor

This study evaluated the effects of diammonia phosphate (DAP) on the non-volatile and volatile compounds of lychee wine fermented with Saccharomyces cerevisiae, when added in two different quantities (0.5 mmol/L and 1.5 mmol/L). It was found that DAP supplementation improved the utilization of ammonia and inhibited the consumption of proline and valine, which regulated the production of α-ketoglutaric, succinic and fatty acids. The addition of 0.5 mmol/L DAP improved the rate of sugar catabolism by slightly increasing yeast growth, thus inducing a higher production of glycerol than of ethanol. Additionally, more odor-active terpene derivatives (trans-β-damascenone, o-cymene, δ-guaiene) in lychee juice were retained after the fermentation added with 0.5 mmol/L DAP. However, the addition of 1.5 mmol/L DAP slowed rates of sugar metabolism and glycerol production, and significantly enhanced the production of acetic acid. Furthermore, with the exception of limonene, the higher DAP addition did not retain more terpene derivatives. These findings, therefore, suggest that a moderate addition of DAP could enhance the flavorful character of lychee wine.

1. Introduction Lychee (Litchi chinensis Sonn.) is the commercially most significant member of the Sapindaceae family, with its rose-floral and citrus-like aromas and a palatable, sweet taste. Nearly 100 major cultivars of lychee grow in tropical and sub-tropical areas (Pareek, 2015). More than 100 volatile compounds were identified in over 10 lychee cultivars since 1980, including terpene derivatives, alcohols, ketones, esters, acids, aldehydes and others (Chyau, Ko, Chang, & Mau, 2003; Feng, Huang, Crane, & Wang, 2018; Johnston, Welch, & Hunter, 1980; Wu, Pan, Qu, & Duan, 2009). Specifically, the terpene derivatives cis-rose oxide, geraniol, 3-methyl-2-buten-1-ol, linalool, α-terpineol, β-citronellol, ρ-cymene, 3-methyl-3-buten-1-ol, (E)-2-hexen-1-ol, 2-ethyl-1-hexanol, 1-octen-3-ol, ρ,α-dimethylstyrene, and 3-tert-butyl-4-hydroxyanisol were reported as the volatiles common to most lychee cultivars, contributing to the lychee odor, citrus-like and floral aroma (Wu et al., 2009). Besides terpene derivatives, 2-phenethyl alcohol is reportedly the most important alcohol compound in lychee fruits, and is likely responsible for their floral character (Chyau et al., 2003).

∗

However, most volatiles endogenous to lychee fruit (mainly terpene derivatives) have been noted to reduce dramatically to just trace levels after fermentation with Saccharomyces yeasts, resulting in the diminishment of the lychee character in the resultant wines (Chen & Liu, 2016; Wu, Zhu, Tu, Duan, & Pan, 2011). In our previous study, it was found that the utilization of non- Saccharomyces yeast (Torulaspora delbrueckii) as mono- or sequential-cultures could retain more lychee aroma-character compounds than the Saccharomyces monoculture (Chen & Liu, 2016). Besides yeast strains, nitrogen sources of musts could impact the volatile and non-volatile compounds of wine by regulating yeast metabolisms during the fermentation process. Previous studies have suggested that a single amino acid addition could markedly increase the formation of corresponding higher alcohols and esters, yet were not able to retain more of the volatiles endogenous to lychee fruits (Chen, Chia, & Liu, 2014; Chen, Vong, & Liu, 2015). Vilanova, Siebert, Varela, Pretorius, and Henschke (2012) found that a moderate addition of diammonia phosphate (DAP) (up to 350 mg N/L) significantly increased the concentrations of limonene, linalool, α-terpineol, α-ionone and β-damascenone, while a high DAP

Corresponding author. Food Science and Technology Programme, Department of Chemistry, National University of Singapore, Science Drive 3, Singapore. E-mail address: [email protected] (S.-Q. Liu).

https://doi.org/10.1016/j.lwt.2019.02.018 Received 29 November 2018; Received in revised form 22 January 2019; Accepted 6 February 2019 Available online 07 February 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.

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addition (450 mg N/L) improved the amounts of β-ionone in aromatic Albariño grape wine. However, other researchers noted little or no difference in the level of terpene derivatives after DAP addition to Shiraz and white Godello wines (Losada, Andrés, Cacho, Revilla, & López, 2011; Ugliano, Siebert, Mercurio, Capone, & Henschke, 2008). No related study has yet been carried out with respect to lychee wine fermentation. Thus, the aim of this study was to evaluate the effects of DAP additions on the fermentation and chemical composition of lychee wine fermented with Saccharomyces cerevisiae, especially with regard to the primary lychee aroma. Furthermore, it was anticipated that the result of this study could provide a suitable nitrogen condition with which to enhance lychee character in the wine.

2. Materials and methods 2.1. Fermentation of lychee juice Fig. 1. Changes in yeast population of S. cerevisiae MERIT.ferm throughout lychee wine fermentation with different treatments. Control fermentation (〇); Fermentation with 0.5 mmol/L DAP addition (▽); Fermentation with 1.5 mmol/L DAP addition (□).

Sterilized lychee juice (Litchi chinensis Sonn. var. Nuomi Ci) and a S. cerevisiae MERIT.ferm (Chr.-Han., Horsholm, Denmark) pre-culture (107 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, 1 mL 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.

2.3. Statistical analysis Based on the data of triplicate fermentations for each treatment, the mean values and standard deviation were calculated and evaluated to obtain the statistical differences (at 95% confidence level) by one-way analysis of variance (ANOVA) and Scheffe's test using SPSS ® 17.0. Volatile compounds with significant differences were selected to perform principal component analysis (PCA) using software Matlab R2008a. 3. Results and discussion

2.2. Sample analysis

3.1. Yeast population

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 C18 column (150 × 3.9 mm, Waters, Dublin, Ireland) based on the method of Waters (1993). Sugars and glycerol were separated using a Zorbax carbohydrate column (150 × 4.6 mm, Agilent, Santa Clara, CA, USA) using the methods of Lee, Toh, Yu, Curran, and Liu (2013). Organic acids were quantified with a Supelcogel C-610 H column (300 × 7.8 mm, Supelco, Sigma-Aldrich, Barcelona, Spain) using the methods developed by Lee et al. (2013). Volatile compounds were analyzed using a headspace solid-phase microextraction (HSe SPME) coupled with an Agilent 7890A GC–Agilent 5975C triple-axis MS and flame ionization detector (FID). Samples were adjusted to pH 2.5 with 1 mol/L HCl and were transferred into a screw-capped headspace vial (5 mL of sample for each vial). Extraction was done using a carboxen/poly (dimethylsiloxane) fiber (Supelco, Sigma-Aldrich, Barcelona, Spain) with a SPME autosampler (CTC, Combi Pal, Switzerland) at 60 °C for 40 min under 250 rpm agitation (Chen et al., 2014). Separation was done using a 60 m × 0.25 mm capillary column (Agilent DB-FFAP, Santa Clara, CA, USA). The GC temperature was increased from 50 °C (hold for 5 min) to 230 °C (final hold for 30 min) at 5 K/min. The volatiles were identified using NIST 8.0 and Wiley 275 MS libraries, verified based on the linear retention index (LRI) and semi-quantificated based on GC-FID peak areas.

In the early stage of fermentation, the 0.5 mmol/L DAP addition significantly improved the yeast growth, rising to 5.90 × 107 CFU/mL on d 2, with approximately double the cell population in control and 1.5 mmol/L DAP added wines (2.86 × 107 CFU/mL and 3.10 × 107 CFU/mL, respectively). All yeasts reached stationary phases on d 4 with no significant difference (Fig. 1). The final yeast cell count was significantly higher in the wine added with 0.5 mmol/L DAP (1.30 × 108 CFU/mL) than that of the other wines (< 1 × 108 CFU/ mL) (Table 1). This result indicated that a moderate addition of DAP could accelerate the exponential phase of yeast growth and extend its stationary phase, while a higher DAP addition had no such effect. Similarly, some previous reports that DAP supplementation (over 3.5 mmol/L) was inconsequential to the yeast growth when the original assimilable nitrogen was over 140 mg N/L (Bell & Henschke, 2005; Lee et al., 2013; Vilanova et al., 2012). At the beginning of the winemaking process, the yeast needs to increase its population by obtaining energy from nutrients and biomass from amino acids. Both the uptake and biomass synthesis of amino acids require ATP. Compared with the amino acids, ammonia needs less ATP transported inside a yeast cell, but uses more ATP for biomass yield (Albers, Larsson, Lidén, Niklasson, & Gustafsson, 1996; Magasanik, 2003). The concentration of ammonia could be increased by adding DAP (Bell & Henschke, 2005). In this study, the moderate DAP addition improved the uptake of ammonia for biomass yield, while the higher DAP addition needed excessive ATP for its metabolism in the yeast cell, thus reducing the amount of energy available for other processes and slowing yeast growth in the early stage of fermentation. In other words, in the exponential phase of yeast growth, the consumption of nitrogen 225

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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

7

0.5 mmol/L DAP addition

1.5 mmol/L DAP addition

0.03 ± 0.00 3.52 ± 0.00a 21.97 ± 0.07 a 0.17 ± 0.02 a ∗ N.D.

9.75 ± 1.12 3.60 ± 0.02b 6.53 ± 0.30b 11.52 ± 1.35b 0.60 ± 0.05a

13.03 ± 0.65 3.59 ± 0.05ab 6.86 ± 0.19b 11.30 ± 0.56b 0.67 ± 0.01b

9.85 ± 1.37b 3.63 ± 0.02b 6.58 ± 0.15b 11.59 ± 2.16b 0.57 ± 0.03a

– – – 0.15 0.37

3.22 ± 0.14a 4.90 ± 0.19 13.42 ± 0.57

∗∗ LOQ N.D. N.D.

LOQ N.D. N.D.

0.11 ± 0.01b N.D. N.D.

0.11 0.21 0.12

LOQ N.D. 0.038 ± 0.004a N.D. 1.39 ± 0.02a N.D. 0.89 ± 0.01a N.D.

0.002 ± 0.000a N.D. LOQ 0.011 ± 0.000a 0.92 ± 0.03b 0.028 ± 0.000a 0.88 ± 0.03a 0.051 ± 0.005a

0.002 ± 0.000a N.D. LOQ 0.011 ± 0.001ab 0.91 ± 0.04b 0.027 ± 0.001a 0.87 ± 0.01a 0.056 ± 0.008a

0.003 ± 0.000a N.D. LOQ 0.010 ± 0.000b 0.92 ± 0.01b 0.030 ± 0.002a 0.83 ± 0.01b 0.055 ± 0.003a

0.002 0.008 0.012 0.004 0.010 0.018 0.016 0.018

a

Yeast Count (10 CFU/mL) pH o Brix Ethanol (mL/100 mL) Glycerol (g/100 mL) Sugars (g/100 mL) Fructose Glucose Sucrose Organic Acids (g/100 mL) Oxalic acid Citric acid Tartaric acid α-ketoglutaric acid Malic acid Pyruvic acid Succinic acid Lactic acid

LOQ

b

c

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. ∗

ammonia was easily consumed by S. cerevisiae as the preferential nitrogen source. Proline (5.71 mmol/L) and alanine (3.04 mmol/L) were the main amino acids in lychee juice. Most amino acids were depleted by all cultures, while DAP supplementation inhibited the cell uptakes of proline, valine, alanine, glycine and asparagine (Table 2). More residual glycine and asparagine were observed in the 1.5 mmol/L DAP supplemented fermentation (Table 2), while alanine, valine and proline were inhibited by both DAP treatments (Table 2). The contents of residual

and sugar should reach a good balance in energy and biomass aspects. 3.2. Ammonia and amino acids The total nitrogen concentration in the lychee juice samples was around 220 mg N/L. This increased to 236 mg N/L and 273 mg N/L after the enhancement of ammonia through the addition of 0.5 mmol/L DAP and 1.5 mmol/L DAP, respectively (Table 2). The ammonia was depleted after all fermentations (Table 2), thus indicating that the

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. (d0)

Lychee wines (d10)

Lychee juice Control

Amino acids (mmol/L) Asp Ser&Asn Glu Gly His &Gln Arg Thr Ala Pro Cys Tyr Val Met Lys Ile Leu Phe Trp

0.57 0.34 0.34 0.10 0.37 0.28 0.06 3.04 5.71 ND 0.05 0.35 ND 0.07 0.06 ND 0.10 ND

NH3 (mmol/L)

*

± ± ± ± ± ± ± ± ±

0.12a 0.04 0.03 0.01a 0.02 0.03 0.01 0.32a 0.33a

± 0.01 ± 0.02a ± 0.01 ± 0.01 ± 0.02

The total nitrogen concentrations (mg N/L) Total **

0.05 ND ND 0.05 ND ND ND ND 1.52 ND ND 0.12 ND ND ND ND ND ND

0.5 mmol/L DAP addition

± 0.01b

0.04 ND ND 0.04 ND ND ND 0.08 3.62 ND ND 0.29 ND ND ND ND ND ND

± 0.01b

± 0.08b

± 0.00b

± 0.01b

± 0.01b

± 0.01b ± 0.15c

± 0.01c

1.5 mmol/L DAP addition

0.20 ND ND 0.16 ND ND ND 0.08 5.56 ND ND 0.23 ND ND ND ND ND ND

± 0.02c

± 0.01c

± 0.01b ± 0.79a

± 0.01c

ND

ND

ND

24.31 ± 1.31a

57.08 ± 2.61b

87.22 ± 11.80c

a,b,c

Statistical analysis at 95% confidence level with same letters indicating no significant difference; ND, not detected. NH3 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). ∗

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that of sucrose, which was fastest in the fermentation added with 0.5 mmol/L DAP and slowest in the fermentation added with 1.5 mmol/ L DAP (Fig. 2b and c). This indicated that DAP supplementation could impact sugar metabolism by affecting the yeast population. On the other hand, neither oBrix reduction nor ethanol production was significantly altered in any of the fermentations (Table 1, Fig. 2d). This implied that the endogenous nitrogen concentration of lychee juice was able to complete the fermentation process and that DAP supplementation could not affect the fermentation rate, despite the lack of preferred nitrogen sources in the control juice. Similar results were reported by Lee et al. (2013) in durian wine fermentation. DAP supplementation significantly affected the production rate of glycerol (Fig. 2e) via glyceropyruvic fermentation (Takagi et al., 2005). In the control and 0.5 mmol/L DAP added fermentations, glycerol was quickly produced to its peak content on d 2, and reduced to around 0.6 g/100 mL on d 4, remaining relatively stable until d 10 (Fig. 2e). The final concentration of glycerol (0.67 g/100 mL) in the wine added with 0.5 mmol/L DAP was significantly higher than that in control wine (0.60 g/100mL), increasing the level of sweetness in the wine (Ugliano & Henschke, 2009). The addition of 1.5 mmol/L DAP delayed the production of glycerol, reaching its highest concentration on d 4, and its final concentration (0.57 g/100 mL) was lowest in all wines (Fig. 2e). The trend of glycerol production was consistent with the rates of sugar consumptions, implying that the changes of sugar catabolism were mainly due to glyceropyruvic fermentation, rather than alcoholic fermentation. However, a previous study reported that glycerol production was positively correlated with DAP supplementation (Ugliano et al., 2008; Ugliano, Travis, Francis, & Henschke, 2010). Furthermore, glycerol production might be affected by yeast strains (Vilanova et al., 2007). Additionally, glycerol could act as a compatible solute to counter osmotic pressure and thus was regulated by other osmoprotectants, such as proline (Cronwright, Rohwer, & Prior, 2002). The highest concentration of proline in the lychee wine with 1.5 mmol/L DAP addition could reduce the need for glycerol production (Table 1, Table 2).

alanine were similar in both DAP added fermentations, while no residual alanine was detected in the control fermentation (Table 2). DAP additions markedly reduced the uptake of valine (Table 2), suggesting that it was a less preferred nitrogen source, possibly. Because valine contains only general amino acid permease (Gap1p) for transportation (Albers et al., 1996). The consumption rate of proline was found to be negatively correlated with the DAP supplementation, reducing from 73.4% (control fermentation) to 36.6% and 2.7% in the 0.5 mmol/L and 1.5 mmol/L DAP added fermentations, respectively (Table 2). This indicated that the DAP addition had a more significant impact on the metabolism of proline than it did on other amino acids. Actually, proline is excluded from assimilable nitrogen sources, since yeast is unable to catabolize proline under anaerobic conditions (Deed, Van Vuuren, & Gardner, 2011). Proline was much consumed in the control wine, suggesting that the lychee juice was deficient in preferred nitrogen sources, and under which condition the activities of Gap1p and proline permease increased (Bell & Henschke, 2005). Proline could be metabolized as an intermediate for the production of α-ketoglutaric (α-KG) (Arias-Gil, Garde-Cerdan, & Ancin-Azpilicueta, 2007), thus regulating the concentration of α-KG. Proline could also serve as an osmoprotectant in yeast cells, balancing the hyperosmotic conditions between the yeast cells and the wine matrix (Takagi, Takaoka, Kawaguchi, & Kubo, 2005). 3.3. Sugar catabolism Sucrose and glucose were completely metabolized, while a trace quantity of fructose was present at the end of all fermentations. Sucrose was dramatically hydrolyzed in the first 4 d of fermentations (Fig. 2a) by yeast extracellular invertase (Ostergaard, Olsson, & Nielsen, 2000). Thus, the rate of sucrose hydrolysis was positively correlated with the rate of yeast growth (Figs. 1 and 2a). The concentrations of glucose and fructose during the fermentations were a net balance between utilization by S. cerevisiae and the release of sucrose. The consumption trends of glucose and fructose were similar to

Fig. 2. Changes in sucrose (a), glucose (b), fructose (c), ethanol (d) and glycerol (e) during lychee wine fermentation with different treatments. Control fermentation (〇); Fermentation with 0.5 mmol/L DAP addition (▽); Fermentation with 1.5 mmol/L DAP addition (□). 227

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related to more NADPH being produced via the pentose phosphate pathway, in which the key enzyme phosphogluconate dehydrogenase (GND) was up-regulated under nitrogen deprivation (He et al., 2018). Further research could elucidate this possibility.

3.4. pH and organic acids The pH value was not significantly different in wines (Table 1). Malic and succinic acids represented over 98% of the total organic acids in the lychee juice (Table 1). After fermentation, succinic and tartaric acids had slightly decreased, while approximately 40% of malic acid had been reduced. Trace concentrations of α-KG, pyruvic and lactic acids were produced during fermentations, in concurrence with the results of a previous lychee wine study (Chen et al., 2014). Most organic acids showed no significant difference after DAP supplementation, however, the succinic acid and α-KG were significantly reduced after 1.5 mmol/L DAP addition (Table 1). As mentioned above, α-KG can be synthetized via proline metabolism (Takagi et al., 2005) and combined with ammonium to produce amino acids for cell growth (Magasanik, 2003). In the 1.5 mmol/L DAP added fermentation, the lowest amount of α-KG was produced from proline metabolism, while the most was consumed during ammonium metabolism, and thus, the final content of α-KG was lowest. These entered into the TCA cycle, resulting in the lowest value of succinic acid in the 1.5 mmol/L DAP added wine (Table 1). Succinic acid reduction positively affects wine sensory evaluation by reducing the unusual salty and bitter taste (Ugliano & Henschke, 2009). Similarly, some previous studies have shown that DAP addition reduced the concentration of α-KG and succinic acids (Torrea et al., 2011).

3.5.2. Alcohols (excluding ethanol) Most of the alcohols were reduced to trace levels after fermentation. High levels of isoamyl alcohol, 2-phenylethyl alcohol and butanol were produced with no significant difference (Table 3). The DAP supplementation reduced the amounts of 1-hexanol and o-butylphenol after fermentations (Table 3). Similar results were reported by Vilanova et al. (2012), who noted that a nitrogen addition (total nitrogen ≤ 350 mg N/ L) could significantly reduce the concentration of 1-hexanol. 3.5.3. Esters Six esters were affected by the DAP additions, including methyl dodecanoate, isoamyl dodecanoate, ethyl hexanoate, ethyl pentadecanoate. ethyl 9-decenoate and ethyl 9-hexadecenoate (Table 3). Ethyl hexanoate was increased from 1.62% (RPA) to over 1.80% (RPA) in the lychee wine after the DAP additions (Table 3), potentially enhancing the fruity flavor and strawberry aroma. Several previous studies have presented similar results (Ugliano et al., 2008, 2010; Vilanova et al., 2012). In this work, trace levels of methyl dodecanoate and isoamyl dodecanoate were only produced in the wine added with 0.5 mmol/L DAP, while ethyl pentadecanoate was only undetectable in this treatment (Table 3). These long-chain fatty acid esters may not affect the sensory quality of the resultant wine, with high odor detection thresholds. The addition of 1.5 mmol/L DAP could significantly lower the synthesis of ethyl 9-decenoate and ethyl 9-hexadecenoate, which might be related to the lowest production of the precursor 9decenoic acid in this treatment (Table 3) (Jackson, 2008, pp. 270–331; Ugliano & Henschke, 2009).

3.5. Volatiles A range of volatiles (50 volatiles and 72 volatiles, excluding ethanol) were respectively detected in lychee juice and wines. In the lychee juice, aldehydes and terpene derivatives were two most abundant volatiles (relative peak area, RPA 25.53% and 24.34%, respectively), followed by alcohols (RPA 19.75%), esters (RPA 15.36%) and ketones (RPA 6.65%) (Table 3). After fermentations, esters and alcohols were significantly produced (RPA over 70% and 20%, respectively) in the lychee wines, with fewer amounts of acids and terpene derivatives (RPA 2%–3%) and trace levels of aldehydes and ketones (RPA < 1%) (Table 3). A total of 33 volatile compounds had significantly different contents after DAP additions, including 12 terpene derivatives, 6 esters, 5 ketones, 4 acids, 2 alcohols, 2 aldehydes, and 2 other volatiles (Table 3).

3.5.4. Terpene derivatives A total of 17 terpene derivatives were found in the lychee juice (Table 3). Generally, nearly half of terpene derivatives were affected by DAP additions and the RPAs of the terpene derivatives were highest in the wines supplemented by 0.5 mmol/L DAP (2.99%), followed by the 1.5 mmol/L DAP added wine (2.40%) and then the control wine (2.19%) (Table 3). Most of the volatiles were significantly reduced after fermentations, with the exception of trans-β-damascenone. A significant amount of trans-β-damascenone was produced in the wine added with 0.5 mmol/L DAP, but it was undetectable in the other wines (Table 3). Trans-β-damascenone is a powerful odorant in wine, with a low odor threshold (0.05 μg/L), thus the significant increase of this compound could considerably enhance apple and rose flavors (Guth, 1997). Similar results were obtained by Vilanova et al. (2012), although Ugliano et al. (2008) observed that this compound was not associated with YAN supplementation. Another 5 volatiles endogenous to lychee juice were also affected by the DAP additions. As with the trans-β-damascenone, o-cymene and δguaiene were only found in the wine added with 0.5 mmol/L DAP. Two times of limonene remained in the wine supplemented by 1.5 mmol/L DAP, while trans-β-ocimene (sweet herbal) was depleted in this treatment (Table 3). Limonene is an important lychee flavor character compound in lychee fruit, and can enhance the citrus flavor of resultant wine (Feng et al., 2018; Wu et al., 2009). Both DAP treatments in this study retained significantly higher amounts of 3-carene (sweet citrus flavor) than the control, as well as slightly higher levels of cis-rose oxide (Table 3). The latter volatile compound has been suggested to significantly enhance the lychee odor in wine owing to its low odor threshold (0.2 μg/L) (Guth, 1997). There were nine terpene derivatives produced after fermentations in this work, among which p, α-dimethyl styrene, o-allyltoluene, α-ocimene, p-cymene and perillen were affected by the different DAP

3.5.1. Volatile acids Acetic acid was the only volatile acid detected in the lychee juice, while the other three acids (octanoic, decanoic and 9-decenoic acids) were produced after fermentation (Table 3). The content of 9-decenoic acid was negatively correlated with DAP additions (Table 3). Conversely, the RPA of acetic acid was positively correlated with the DAP values (Table 3). During alcoholic fermentation, acetic acid is produced from acetaldehyde, releasing NADH to restore the redox balance (Rodicio & Heinisch, 2009). Amino acid metabolisms could also generate NADH. The generated NADH could be used during glutamate synthesis by ammonia (Magasanik, 2003). In this study, the DAP supplementation might result in more NADH being consumed by ammonia and less NADH being produced by amino acids. The cell therefore needed to generate more NADH via other reactions, including the oxidative formation of acetic acid from acetaldehyde (Bely, Rinaldi, & Dubourdieu, 2003). Similar results were reported by Torrea et al. (2011). The levels of fatty acids (C8 and C10) were both the lowest in the lychee wine with 0.5 mmol/L DAP addition and highest in the 1.5 mmol/L DAP added wine (Table 3). Fatty acids are generated from acetyl-CoA by consuming NADPH, and acetyl-CoA is activated from acetic acid (Cherry et al., 2012). Thus, the high concentration of acetic acid could result in the high production of fatty acids in the 1.5 mmol/L DAP added. Compared with the level in the 0.5 mmol/L DAP added wine, the higher production of fatty acids in the control wine might be 228

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Table 3 Volatile compounds (excluding ethanol, GC-FID peak area × 106) 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) Peak area

Acids Acetic acid Octanoic acid 9-Decenoic acid Decanoic acid Alcohols Butanol Isoamyl alcohol 3-Isopentenyl alcohol Prenyl alcohol 1-Hexanol 1-Octen-3-ol Dihydromyrcenol Benzenemethanol 2-Phenylethyl alcohol Phenol o-Butylphenol 2,4-Di-tert-butylphenol Ester Methyl octanoate Methyl decanoate Methyl dodecanoate Compounds identified in this study

1457 1847 2335 2272

1.10 ± 0.23a ∗∗ N.D. N.D. N.D.

1154 1218 1254 1330 1360 1452 1467 1902 1945 2018 2180 2311

N.D. N.D. 2.48 1.13 2.18 2.20 1.83 0.49 1.46 0.25 0.36 1.05

1382 1591 1802

N.D. N.D. N.D.

LRI

Lychee juice (d0)

± ± ± ± ± ± ± ± ± ±

0.14 0.28 0.16a 0.25 0.11 0.02 0.21a 0.04 0.04a 0.15a

RPA 1.62 1.62 – – – 19.75 – – 3.64 1.67 3.21 3.24 2.70 0.71 2.15 0.36 0.53 1.54 15.36 – – –

Peak area

2.70 3.14 0.64 2.59

± ± ± ±

RPA

0.19b 0.25ab 0.14a 0.22a

12.70 ± 1.04a 29.57 ± 0.60a N.D. N.D. 0.26 ± 0.01b N.D. N.D. N.D. 16.20 ± 2.20b N.D. 0.97 ± 0.03b 0.75 ± 0.12b 0.13 ± 0.02a 0.49 ± 0.04a N.D.

3.12 0.93 1.08 0.22 0.89 20.85 4.38 10.20 – – 0.09 – – 5.59 – 0.33 0.26 73.01 0.04 0.17 –

Control (d10)

Add 0.5 mmol/L DAP (d10)

Add 1.5 mmol/L DAP (d10)

Peak area

Peak area

2.83 2.86 0.26 2.00

± ± ± ±

RPA

0.58b 0.44a 0.03b 0.01b

14.05 ± 1.25a 27.65 ± 1.53a N.D. N.D. 0.21 ± 0.05b N.D. N.D. N.D. 16.68 ± 2.95b N.D. N.D. 0.65 ± 0.07b 0.13 ± 0.01a 0.45 ± 0.07a 0.14 ± 0.03

2.75 0.98 0.99 0.09 0.69 20.45 4.85 9.55 – – 0.07 – – – 5.76 – – 0.22 72.18 0.04 0.16 0.05

3.74 4.00 0.20 2.82

± ± ± ±

RPA

0.57c 0.29b 0.04b 0.23a

12.76 ± 0.03a 31.95 ± 6.63a N.D. N.D. N.D. N.D. N.D. N.D. 18.67 ± 1.34b N.D. 0.46 ± 0.07a 0.72 ± 0.05b 0.15 ± 0.01a 0.44 ± 0.06a N.D.

3.56 1.24 1.32 0.07 0.93 21.38 4.23 10.58 – – – – – – 6.18 – 0.15 0.24 71.18 0.05 0.15 –

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

14.91 – – – – – 0.45 – – – – – – – – – – – – – – –

9.48 ± 1.17a 4.70 ± 0.27a 0.22 ± 0.01a 25.28 ± 4.08a 1.40 ± 0.11a 0.09 ± 0.01a 74.78 ± 18.06b 34.58 ± 0.04a 0.35 ± 0.08a 21.62 ± 4.36a 0.78 ± 0.10a 0.25 ± 0.04a 4.88 ± 0.20a 6.20 ± 0.76a 1.00 ± 0.21a 2.74 ± 0.09a 0.25 ± 0.03a 0.81 ± 0.05a 1.24 ± 0.22a 2.37 ± 0.20a 3.65 ± 0.83a 5.31 ± 0.72a

3.27 1.62 0.08 8.72 0.48 0.03 25.79 11.92 0.12 7.46 0.27 0.09 1.68 2.14 0.35 0.95 0.08 0.28 0.43 0.82 1.26 1.83

9.20 ± 0.77a 5.65 ± 0.31b 0.28 ± 0.02a 29.51 ± 0.66a 1.32 ± 0.22a 0.10 ± 0.01a 76.06 ± 14.52b 30.18 ± 0.51a 0.34 ± 0.05a 20.03 ± 2.86a 0.82 ± 0.17a N.D. 4.76 ± 0.71a 4.92 ± 0.74ab 0.96 ± 0.13a 2.71 ± 0.37a 0.28 ± 0.03a 0.84 ± 0.11a 1.35 ± 0.04a 2.22 ± 0.19a 3.47 ± 0.24a 4.98 ± 0.74a

3.18 1.95 0.10 10.19 0.46 0.03 26.27 10.43 0.12 6.92 0.28 – 1.64 1.70 0.33 0.94 0.10 0.29 0.47 0.77 1.20 1.72

10.35 ± 0.46a 5.48 ± 0.62b 0.25 ± 0.05a 28.31 ± 4.42a 1.47 ± 0.30a N.D. 88.46 ± 13.19b 22.66 ± 4.85b 0.37 ± 0.05a 21.77 ± 0.16a 0.67 ± 0.07a 0.28 ± 0.06a 4.17 ± 0.69a 3.97 ± 0.20b 0.97 ± 0.14a 2.56 ± 0.46a 0.24 ± 0.01a 0.75 ± 0.09a 1.09 ± 0.17a 2.41 ± 0.26a 3.93 ± 0.62a 5.18 ± 0.25a

3.43 1.81 0.08 9.37 0.49 – 29.29 7.50 0.12 7.21 0.22 0.09 1.38 1.31 0.32 0.85 0.08 0.25 0.36 0.80 1.30 1.72

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

– 1222 1324 1430 1530 1630 1638 1693 1741 1844 2049 2130 2255 2283 2465 2481 1513 1724 1547 1756 1116 1660

10.14 ± 1.38a N.D. N.D. N.D. N.D. N.D. 0.31 ± 0.06a N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.

Compounds identified in this study

LRI

Lychee juice (d0)

Isoamyl decanoate Isoamyl dodecanoate Carvyl acetate Hexyl acetate Heptyl acetate Phenylethyl acetate Aldehydes Hexanal trans-2-Hexenal Benzaldehyde o-Tolualdehyde p-Tolualdehyde Terpene derivatives Limonene trans-β-Ocimene α-Ocimene m-Cymene

Control (d10)

Control (d10)

Peak area

RPA

1864 2068 1232 1263 1366 1825

N.D. N.D. N.D. N.D. N.D. N.D.

1082 1224 1540 1669 1670

5.25 7.05 0.32 2.03 2.70

1183 1241 1545 1256

1.07 ± 0.27a 1.44 ± 0.08a N.D. N.D.

– – – – – – 25.53 7.72 10.37 0.47 2.99 3.98 24.34 1.57 2.11 – –

± ± ± ± ±

0.82 1.18 0.05a 0.48a 0.36a

Peak area 5.17 N.D. 0.29 0.32 0.18 3.14

± 0.34 ± ± ± ±

RPA a

0.02a 0.07a 0.03a 0.37a

N.D. N.D. N.D. N.D. 0.34 ± 0.01b 0.25 0.10 0.31 0.69

± ± ± ±

0.02b 0.01b 0.03 0.12a

1.78 – 0.10 0.11 0.06 1.08 0.12 – – – – 0.12 2.19 0.09 0.04 0.11 0.24

Add 0.5 mmol/L DAP (d10)

Add 1.5 mmol/L DAP (d10)

Peak area

Peak area

4.04 0.30 0.34 0.31 0.21 3.02

± ± ± ± ± ±

RPA a

0.52 0.01 0.01a 0.03a 0.01a 0.55a

N.D. N.D. 0.07 ± 0.01b N.D. N.D. 0.25 ± 0.02b 0.11 ± 0.01b N.D. 0.78 ± 0.07a

1.40 0.10 0.12 0.11 0.07 1.04 0.02 – – 0.02 – – 2.99 0.09 0.04 – 0.27

5.04 N.D. 0.33 0.30 0.17 3.19

RPA a

± 0.56 ± ± ± ±

0.05a 0.02a 0.04a 0.28a

N.D. N.D. 0.08 ± 0.02b N.D. N.D. 0.49 ± 0.02c N.D. N.D. 0.76 ± 0.08a

1.67 – 0.11 0.10 0.06 1.06 0.03 – – 0.03 – – 2.40 0.16 – – 0.25

(continued on next page) 229

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Table 3 (continued) ∗

Compounds identified in this study

LRI

Lychee juice (d0) Peak area ± 0.07a

o-Cymene p-Cymene Terpinolene cis-Rose oxide trans-Rose oxide

1263 1271 1272 1346 1365

2.01 N.D. 0.34 2.82 1.21

Compounds identified in this study

LRI

Lychee juice (d0)

± 0.03 ± 0.19a ± 0.10

RPA

Peak area

RPA

Peak area

RPA

2.96 – 0.51 4.15 1.78

N.D. 0.54 ± 0.01 N.D. 0.49 ± 0.03b N.D.

– 0.19 – 0.17 –

0.83 ± 0.01b N.D. N.D. 0.64 ± 0.04b N.D.

0.29 – – 0.22 –

N.D. N.D. N.D. 0.64 ± 0.01b N.D.

– – – 0.21 –

Control (d10) Peak area

– 1.18 – 0.28 – 2.63 0.87 1.73 1.80 0.35 – – 0.20 0.32 1.24 0.58 0.36 6.65 3.81 0.92 0.48 0.56

0.07 0.60 0.22 0.24 0.88 N.D. N.D. 0.23 N.D. N.D. 0.22 1.04 N.D. N.D. N.D. 0.32 N.D.

N.D. 0.80 N.D. N.D. N.D. 1.79 0.59 1.18 1.22 0.23 N.D. N.D. 0.08 0.22 0.84 0.39 0.25

1204 1294 1339 1343

2.59 0.62 0.33 0.38

Compounds identified in this study

LRI

Lychee juice (d0)

± 0.01a

± ± ± ± ±

0.24 0.11 0.09a 0.13a 0.06a

± ± ± ± ±

0.01a 0.07 0.16 0.01a 0.05

± ± ± ±

0.31 0.01 0.06 0.07

Add 1.5 mmol/L DAP (d10)

Peak area

RPA

1411 1416 1441 1445 1469 1472 1503 1605 1545 1718 1748 1777 1830 1840 1867 1927 2267

Add 0.5 mmol/L DAP (d10)

RPA

Peak area Perillen 1,3-Di-tert-butylbenzene o-Allyltoluene p, α-Dimethyl styrene 1,3,8-p-Menthatriene Neroloxide α-Terpinene γ-Terpinene 3-Carene δ-Guaiene Farnesene ar-Curcumene trans-β-Damascenone Calamenene β-Pinene α-Calacorene Junipene Ketones 4-Methyl-2-heptanone Acetoin 6-Methyl-5-hepten-2-one 4,6-Dimethyl-2-heptanone

2-Nonanone 1,4-Dimethyl-3-cyclohexenyl methyl ketone 2-Methyl tetrahydrothiophen-3-one 2-Undecanone Butyrolactone Cyclodecene 6-Methoxy-1-acetonaphthone Others trans-Pinane Toluene Pentadecane 4,7-Dimethylbenzofuran p-Acetyltoluene 2,6-Dimethylnaphthalene 1,3-Dimethylnaphthalene Cadalin 1,4-dimethyl-7-(1-methylethyl)azulene

Control (d10)

± ± ± ± ±

0.01a 0.08b 0.01a 0.04a 0.15a

± 0.06b

± 0.05a ± 0.16a

± 0.01bc

N.D. N.D. N.D. N.D.

Add 0.5 mmol/L DAP (d10)

Add 1.5 mmol/L DAP (d10)

RPA

Peak area

RPA

Peak area

RPA

0.03 0.21 0.08 0.08 0.30 – – 0.08 – – 0.08 0.36 – – – 0.11 – 0.41 – – – –

0.11 0.55 2.00 1.96 0.85 N.D. N.D. 0.23 0.17 0.10 0.23 0.97 0.20 N.D. N.D. 0.27 N.D.

0.04 0.19 0.69 0.68 0.29 – – 0.08 0.06 0.03 0.08 0.34 0.07 – – 0.09 – 0.57 – – – –

N.D. 0.50 1.64 1.90 0.80 N.D. N.D. 0.39 0.15 N.D. 0.26 0.89 N.D. N.D. N.D. 0.35 N.D.

– 0.17 0.54 0.63 0.26 – – 0.13 0.05 – 0.09 0.29 – – – 0.12 – 0.58 – – – –

Control (d10)

± ± ± ± ±

0.03a 0.05b 0.24b 0.37b 0.09a

± ± ± ± ± ±

0.02b 0.04b 0.02b 0.03a 0.05a 0.02b

± 0.04b

N.D. N.D. N.D. N.D.

0.21 0.32 N.D. 0.45 N.D. 0.55 0.22

1387 1526 1537 1598 1653 1735 2073

N.D. 0.26 ± 0.06a N.D. N.D. 0.17 ± 0.05 N.D. 0.16 ± 0.03a

N.D. 0.08 0.18 0.40 N.D. 0.39 0.15

1.58 1.25 N.D. 0.40 0.22 0.25 0.28 N.D. 0.42

– 0.03 0.06 0.14 – 0.13 0.05 0.32 – – 0.04 – – 0.08 – 0.12 –

0.25 0.27 0.22 0.41 N.D. 0.35 0.14

1033 1042 1502 1724 1798 2031 2031 2242 2243

– 0.39 – – 0.26 – 0.23 6.77 2.33 1.84 – 0.59 0.33 0.37 0.41 – 0.62

0.13 0.02 0.05a 0.04

± 0.03a

N.D. N.D. N.D. N.D.

0.09 0.09 0.08 0.14 – 0.12 0.05 1.06 – – 0.04 – – 0.07 – 0.15 0.12

Peak area

± ± ± ±

± 0.01c

Peak area

RPA

N.D. N.D. 0.11 ± 0.02a N.D. N.D. 0.24 ± 0.06a N.D. 0.33 ± 0.04a N.D.

± 0.06a ± 0.22a

RPA

Peak area

± 0.40 ± 0.05

± 0.11b ± 0.03b

Add 1.5 mmol/L DAP (d10)

RPA

± 0.03a ± 0.01a

0.06b 0.23b 0.31b 0.15a

Add 0.5 mmol/L DAP (d10)

Peak area

± 0.01b ± 0.01a ± 0.02a

± ± ± ±

N.D. N.D. 0.12 N.D. N.D. 0.20 N.D. 0.43 0.36

± ± ± ±

0.04a 0.07a 0.02a 0.02ab

± 0.02a ± 0.04a

a

± 0.02

± 0.04a ± 0.09a ± 0.07a

± 0.03a ± 0.03a ± 0.01ab ± 0.01b ± 0.06a

N.D. N.D. N.D. N.D. N.D. 0.31 ± 0.03a N.D. 0.44 ± 0.01a N.D.

RPA 0.07 0.11 – 0.15 – 0.18 0.07 0.88 – – – – – 0.10 – 0.15 –

a,b,c,

Statistical analysis at 95% confidence level with same letters indicating no significant difference. Exclude ethanol. ∗∗ N.D.: not detected. ∗

treatments. Both DAP additions produced seven times more of p, αdimethyl styrene and o-allyltoluene than in the control wine (Table 3). p, α-Dimethyl styrene was reported as the common aromatic compound in lychee fruit (Wu et al., 2009). However, α-ocimene (floral and wet cloth note), p-cymene (floral, grassy) were not found in either of the DAP added wines and perillen (woody note) was undetectable in the 1.5 mmol/L DAP added wine (Table 3).

Benzaldehyde has been detected in some lychee cultivars (Fei Zi Xiao and sweetheart), possessing an almond-like, fragrant and sweet odor (Feng et al., 2018; Wu et al., 2009). There were seven ketones detected in the lychee juice, of which 1,4dimethyl-3-cyclohexenyl methyl ketone (contributing fruity flavor) was significantly higher in the lychee wines with DAP additions (Table 3). Four ketones were produced during the fermentations, all of which were affected after the DAP additions. 2-Nonanone, with a green fruity and cheesy flavor, was only produced after the DAP additions (Table 3). The addition of 1.5 mmol/L DAP increased the concentration of 2-undecanone (fruity and fatty odor) and cyclodecene, while 1.5 mmol/L DAP could not produce 2-methyl tetrahydrothiophen-3-one (Table 3).

3.5.5. Aldehydes, ketones and others Nearly all aldehydes were depleted after fermentations, with only trace levels of p-tolualdehyde and benzaldehyde remaining in the control wine and DAP treatment wines, respectively (Table 3). 230

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compounds were not consistent with their concentrations. 4. Conclusion Both quantities of DAP additions affected the nitrogen metabolism of the lychee wine by enhancing the utilization of ammonia and reducing the consumptions of less preferred amino acids (mainly proline and valine). This led to the higher production of acetic acid and reductions of α-KG and succinic acid. As the main product of alcoholic fermentation, ethanol showed no significant differences after the DAP additions. The moderate DAP addition (0.5 mmol/L) significantly improved the rate of yeast growth, thus increasing the rates of sugar consumption and glycerol production in the early stage of fermentation, while higher DAP addition (1.5 mmol/L) slowed these rates. In the primary volatiles of lychee fruits, the moderate DAP addition was best able to retain trans-β-damascenone, o-cymene, and δ-guaiene, while the high DAP addition retained more limonene. Both DAP supplementations contributed to higher amounts of 3-carene, and benzaldehyde. Therefore, these results indicated that a moderate DAP addition could be a convenient way to retain the lychee odor-active compounds in lychee wine fermented with S. cerevisiae. Acknowledgments

Fig. 3. Bi-plot of principal component analysis of selected volatile compounds in lychee juice and wines. Lychee juice (★); control lychee wine (●); lychee wine with 0.5 mmol/L DAP addition (▲); lychee wine with 1.5 mmol/L DAP addition (▼). (1) Acetic acid; (2) Octanoic acid; (3) 9-Decenoic acid; (4) Decanoic acid; (5) 1-Hexanol; (6) o-Butylphenol; (7) Benzaldehyde; (8) pTolualdehyde; (9) 2-Nonanone; (10) 1,4-Dimethyl-3-cyclohexenyl methyl ketone; (11) 2-Methyl tetrahydrothiophen-3-one; (12) 2-Undecanone; (13) Cyclodecene; (14) Methyl dodecanoate; (15) Ethyl hexanoate; (16) Ethyl 9decenoate; (17) Ethyl pentadecanoate; (18) Ethyl 9-hexadecenoate; (19) Isoamyl dodecanoate; (20) Limonene; (21) trans-β-Ocimene; (22) α-Ocimene; (23) o-Cymene; (24) p-Cymene; (25) Perillen; (26) o-Allyltoluene; (27) p, αDimethyl styrene; (28) 3-Carene; (29) δ-Guaiene; (30) trans-β-Damascenone; (31) α-Calacorene; (32) Pentadecane; (33)1,4-Dimethyl-7-(1-methylethyl)azulene.

This work was supported by an academic research fund (ARF) grant from the Ministry of Education of Singapore (Singapore, WBS No. R143-000-507-112) and China Postdoctoral Science Foundation Grant (China, Grant No. 2017M610129). The first author would like to thank the International Postdoctoral Exchange Fellowship Program (CAU) for financial support. References Albers, E., Larsson, C., Lidén, G., Niklasson, C., & Gustafsson, L. (1996). Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation. Applied and Environmental Microbiology, 62, 3187–3195. Arias-Gil, M., Garde-Cerdan, T., & Ancin-Azpilicueta, C. (2007). Influence of addition of ammonium and different amino acid concentrations on nitrogen metabolism in spontaneous must fermentation. Food Chemistry, 103, 1312–1318. Bell, S. J., & Henschke, P. A. (2005). Implications of nitrogen nutrition for grapes, fermentation and wine. Australian Journal of Grape and Wine Research, 11, 242–295. Bely, M., Rinaldi, A., & Dubourdieu, D. (2003). Influence of assimilable nitrogen on volatile acidity production by Saccharomyces cerevisiae during high sugar fermentation. Journal of Bioscience and Bioengineering, 96, 507–512. Chen, D., Chia, J. Y., & Liu, S. Q. (2014). Impact of addition of aromatic amino acids on non-volatile and volatile compounds in lychee wine fermented with Saccharomyces cerevisiae MERIT.ferm. International Journal Of Food Microbiology, 170, 12–20. Chen, D., & Liu, S. Q. (2016). Impact of simultaneous and sequential fermentation with Torulaspora delbrueckii and Saccharomyces cerevisiae on non-volatiles and volatiles of lychee wines. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 65, 53–61. Chen, D., Vong, W. C., & Liu, S. Q. (2015). Effects of branched-chain amino acid addition on chemical constituents in lychee wine fermented with Saccharomyces cerevisiae. International Journal of Food Science and Technology, 50, 2519–2528. Cherry, J. M., Hong, E. L., Amundsen, C., Balakrishnan, R., Binkley, G., Chan, E. T., et al. (2012). Saccharomyces genome database: The genomics resource of budding yeast. Nucleic Acids Research, 40, D700–D705. Chyau, C.-C., Ko, P.-T., Chang, C.-H., & Mau, J.-L. (2003). Free and glycosidically bound aroma compounds in lychee (Litchi chinensis Sonn.). Food Chemistry, 80, 387–392. Cronwright, G. R., Rohwer, J. M., & Prior, B. A. (2002). Metabolic control analysis of glycerol synthesis in Saccharomyces cerevisiae. Applied and Environmental Microbiology, 68, 4448–4456. Deed, N. K., Van Vuuren, H. J. J., & Gardner, R. C. (2011). Effects of nitrogen catabolite repression and di-ammonium phosphate addition during wine fermentation by a commercial strain of S. cerevisiae. Applied Microbiology and Biotechnology, 89, 1537–1549. Feng, S., Huang, M., Crane, J. H., & Wang, Y. (2018). Characterization of key aromaactive compounds in lychee (Litchi chinensis Sonn.). Journal of Food and Drug Analysis, 26, 497–503. Guth, H. (1997). Quantitation and sensory studies of character impact odorants of different white wine varieties. Journal of Agricultural and Food Chemistry, 45, 3027–3032. He, Q., Yang, Y., Yang, S., Donohe, B. S., Wychen, S. V., Zhang, M., et al. (2018). Oleaginicity of the yeast strain Saccharomyces cerevisiae D5A. Biotechnology for Biofuels, 11, 258.

Of the other volatiles, pentadecane was only undetectable in the lychee wine with 1.5 mmol/L DAP addition, while 1,4-dimethyl-7-(1methylethyl)azulene was only retained in the 0.5 mmol/L DAP added wine (Table 3). 3.6. PCA analysis The 33 volatiles, all significantly changed in the different treatments, were selected to analyze the correlation between DAP additions and volatile compounds (Fig. 3). The first principal component (PC1) explained 58.70% of the total variance and separated the lychee juice from the lychee wines. 1-Hexanol, benzaldehyde, p-tolualdehyde, 1,4dimethyl-3-cyclohexenyl methyl ketone, limonene, trans-β-ocimene, αocimene, 3-carene, δ-guaiene, trans-β-damascenone, α-calacorene and 1,4-dimethyl-7-(1-methylethyl)azulene were all found in the same part of lychee juice, indicating that these volatiles contributed to its main character being different from the lychee wines (Fig. 3). Different lychee wines were separated by the second principal component (PC2) explaining 25.11% of the total variance. The lychee wine with 0.5 mmol/L DAP addition was in the negative part of PC2, as were acetic acid, 2-nonanone, 2-methyl tetrahydrothiophen-3-one, 2undecanone, methyl dodecanoate, isoamyl dodecanoate, ethyl hexanoate, perillen, o-allyltoluene, p, α-dimethyl styrene and pentadecane (Fig. 3). These volatiles can contribute sweet, fruity, fatty and cheesy flavors to lychee wine. The other wines and volatiles (mainly fatty acids and ethyl esters) were all found in the positive part of PC2. The lychee wine supplemented with 1.5 mmol/L DAP was placed between the control wine and the 0.5 mmol/L DAP added wine and near to the xaxis, suggesting that the effects of the DAP additions on volatile 231

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