Composition of aroma compounds in fermented apple juice: effect of apple variety, fermentation temperature and inoculated yeast concentration Academic research paper on "Biological sciences"

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Procedía

Food Science

ELSEVIER

Procedía Food Science 1 (2011) 1709- 1716

11th International Congress on Engineering and Food (ICEF11)

Composition of aroma compounds in fermented apple juice: effect of apple variety, fermentation temperature and inoculated yeast concentration

Riekstina-Dolge Rita a *, Kruma Zanda a, Karklina Dainaa, Seglina Dalijab

a Latvia University of Agriculture, Faculty of Food Technology, Liela iela 2, LV- 3001 (rita.riekstina@,llu.Iv) b Latvia State Institute of Fruit Growing, Graudu iela 1, Dobele, LV-3701 (dalija.seglina@,lvai.lv)

Abstract

This paper reports the influence of apple variety and fermentation conditions (yeasts concentration and temperature) on the aroma compounds to fermented apple juice. Two apples variety juice were fermented using the different fermentation conditions. Apples variety 'Lietuvas Pepins' was fermented with different yeast concentration in must. The effect fermented conditions to fermented juice aroma compounds was also analyzed using headspace solid- phase microextraction and gas chromatograph- mass spectrometry (HS-SPME-GC-MS). Volatile aroma compounds were detected of apple juice, yeast and fermented juice. The main aroma compound of 'Auksis' apple juice is 2-hexenal, whereas of 'Lietuvas Pepins' acetic acid butyl ester. In fermented juices main aroma compounds were 2-hydroxyethylhydrazine, 3-methyl-1-butanol, and hexanoic acid ethyl ester, acetic acid hexyl ester.

© 20nPubhshedbyElsevierB.V.Selectionand/orpeer-review underresponsibilityofllthInternationalCongress onEngineeringandFood(ICEF11)Executive Committee.

Keywords: fermented apple juice; aroma compounds; fermentation temperature, yeast

1. Introduction

Apples are a highly favored fruit, not only because of their good storage and manufacturing properties, but also owing to their unique flavor characteristics. The most abundant volatile constituents in apples are esters (78-92% of total volatiles), alcohols (6-16% of total volatiles), aldehydes, ketones, and ethers. It is also important to mention that most of the aroma compounds in apple juice are not genuine constituents of apples, but is formed until apple juice production starts [1]. Flavor components in apple juice are numerous, and identification is considered quite complex due to the aromatic nature of apples. Eight odour-active volatiles have been identified as the most important contributors for the aroma-flavor

* Corresponding author. Tel.: +37163005647; fax: +371622829. E-mail address: rita.riekstina@llu.lv.

2211-601X © 2011 Published by Elsevier B.V. Selection and/or peer-review under responsibility of 11th International Congress on Engineering

and Food (ICEF 11) Executive Committee.

doi:10.1016/j.profoo.2011.09.252

authenticity of apple juice [2]. Apple juice is the raw material of different fermented drinks, like apple wine and cider.Aroma plays significant role in the quality of cider, and composition of volatiles depends on used technology, maturation and storage conditions [3]. Cider maturation significantly influenced the chemical composition of cider distillates. The concentrations of ethyl esters of the major organic acids of cider (lactic, acetic and succinic), as well as the contents of aromas produced by the bacteria activity (2-butanol, 2-propen-1-ol, 4-ethylguaiacol and eugenol) increased with increasing levels of cider maturation [4]. Main aroma compounds of cider are esters, higher alcohols, fatty acids, aldehydes, ketones, terpenes, lactones. And it is very important to determine influence of each compound to quality of final product. [3]. The aim of current research was to evaluate aroma composition of fermented apple juice, depending on used apple variety, fermentation temperature and yeast concentration in must.

2. Materials & Methods

2.1. Raw materials

For analysis two apple varieties 'Auksis' (Streif index 0,16, starch index 8,4) and 'Lietuvas Pepins' (Streif index 0,24, starch index 6,2 ) were used. Apples were grown in the Latvia State Institute of Fruit Growing and harvested in autumn 2010. Quality parameters of the apple juices are given in Table 1. Juice was obtained by press Voran Basket press 60K. For stabilization of juice Tannisol (Enartis, Italy) was added (concentration 10 g L-1). Tannisol capsules consist of potassium metabisulphite (9.5 g L-1), ascorbic acid (0.3 g L-1) and tannin (0.2 g L-1).

Table 1. Quality parameters of apple juice

Composition 'Auksis' 'Lietuvas Pepins'

Juice yield (%) 73.39±3.5 63.27±3.5

pH 3.21±0.01 3.06± 0.01

Titratable acidity (g/L-1) 4.86±0.18 9.08±0.17

Soluble solids (g/L-1) 107.02.±0.06 101.0±0.09

Total sugars (g/L-1) 101.84 96.10

2.2. Fermentation conditions

Fermentation was performed using commercial Saccharomyces bayanis yeast EC-1118 (Lalvin, Canada) three yeast concentrations (for juice 'Lietuvas Pepins') and two fermentation temperatures were used. The EC-1118 strain is recommended for all types of wines, including sparkling, and cider. Fermentation was carried out at 16 ±1°C and 23±1°C 28 days. (Table 2). The EC-1118 strain is recommended for all types of wines, including sparkling, and ciders. The apples juice was fermented in a glass bottles (n=5). For analysis juice samples from all bottles in equal proportions were collected.

Table 2. Fermentation conditions and fermented juice composition

Apples variety Abbrev Yeasts Fermentation Titratable Soluble solids Alcohol

cells/ ml temperature, °C acidity (g/L) (g/L) (%)

'Lietuvas Pepins' LP1 5.4*107 16±1 10.06±0.06 0.88±0.13 5.3±0.09

'Lietuvas Pepins' LP2 7.3*107 16±1 9.86±0.05 1.52±0.01 5.3±0.05

'Auksis' A1 7.3* 107 16±1 6.13±0.03 0.58±0.09 5.1±0.09

'Auksis' A2 7.3* 107 23±1 n.a. n.a. n.a.

Riekstma-DolgeRita etal. / Procedia FoodSciencel (2011)1709-1716

Apples variety Abbrev Yeasts Fermentation Titratable Soluble solids Alcohol

cells/ ml temperature, °C acidity (g/L) (g/L) (%)

'Lietuvas Pepins' LP4 7.3*107 23±1 9.92±0,05 1,56±0.03 5.2±0.07

'Lietuvas Pepins' LP3 10.8*107 16±1 9.63±0.1 0.95±0.13 5.2±0.08

n.a. not analised

1.3. Datarmioatioo of volatile aroma compounds

Volatiles from apple juice and fermented drinks were extracted using solid phase microextraction (SPME). 5 g of juice were weighed in a 20 ml headspace vial and capped with a septum. SPME extraction. A divinylbenzene/carboxen/polydimethylsiloxane (DVB/Car/PDMS) fiber (Supelco Inc., Bellefonte, PA, USA) was used for headspace SPME sampling. SPME parameters were: incubation time 30 min, extraction temperature 22±2 °C, extraction duration 30 min, desorption 15 min, 250 °C. For the analysis of the SPME extracts, a Perkin Elmer Clarus 500 GC/MS and a Elite-Wax ETR (60 m x 0.25 mm i.d.; DF 0.25 ^m) was used. Working conditions were: injector 250 °C; transfer line to MSD 260 °C; oven temperature start 50 °C, hold 2 min, programmed from 50 to 100 °C at 5 °C min-1 hold 5 min, and from 100 to 210 °C at 5 °C min-1, hold 15 min; carrier gas (He) 1 ml min-1; split ratio 2:1; ionization EI+; acquisition parameters in full scan mode: scanned m/z 50-300. Compounds were identified by comparison of their mass spectra with mass spectral libraries (Nist98), and by calculation of linear retention indexes and comparison with literature data. All analyses were performed in triplicate. As a quantitative measure, the share in the total GC peak area for each compound is given.

Statistical analysis. The differences in the volatile profiles during fermentation were analyzed using the analysis of variance (ANOVA) procedure of SPSS, Version 17.0. HSD Tukey's test was applied to compare the mean values of the volatile compounds of different fermentation conditions. p-value at 0.05 was used to determine the significant differences of content of volatiles in fermented juice samples. Mean values with standard deviations are reported.

3. Results & Discussion

3.1. Apple juices aroma compounds

An overview of the results of the aroma analysis of the apple juices is given in Tables 3 and 4. Totaly seven volatile compounds (total peak AU 2999,93 x105) were detected in 'Auksis' apple juice , (namely) - 4 esters (1 acetates, 1 hexanoates, 1 butanoate, 1 propanoate), 1 alcohols, 2 aldehydes. Apple 'Auksis' juice aroma is mainly composed of aldehydes (60,5%) and esters (35%). The main volatile compounds of 'Auksis' is 2-hexenal (50,1 %), 1 butanol, 2- methyl acetate (13,1%) and hexanal (10,5%). These aldehydes have been described as the main contributors to the green odour of apple fruit and apple juice [5].

In apple 'Lietuvas pepins' juice six volatile compounds were identified: 4 esters forming 85% of total compounds and 2 alcohols (Table 4.). In 'Lietuvas Pepins' juice comparing to 'Auksis' apple juice has higher content of aroma compounds. There were detected 6 volatile compounds of total peak AU 44 90,35*105. The main volatile compounds of 'Lietuvas Pepins' apples juice were acetic acid butylester (35,3%) acetic acid hexylester (24,2%) and 1 butanol, 2- methyl acetate (15,6%) (Table 4). The similar results were reported in Royal Gala apples where the most important volatile components were alcohols and acetates[6]. The esters and alcohols which are the products of fatty acid metabolization were the major groups in the apple juice, respectively 44% and 41% of totals volatiles [7,8] . Each group of volatile compounds gave a typical odour characteristic to the apple juice. Esters are responsible for sweet and fruity odour. 2-hexen-1-ol and 1-hexanol found in 'Lietuvas Pepins' and 'Auksis' apples juices were

also reported in apples 'Pink Lady' as one of the characteristic aroma compounds [9]. In literature butanol, was found as most abundant alcohol in the apple juice [10], but it was not identified in 'Lietuvas Pepins' and 'Auksis' juice.

Table 3. Volatile aroma compounds (AU x105± standard deviation) as measured by SPME-GC-MS in 'Auksis 'apple juice and fermented juice

Compounds Juice 'Auksis' Yeast A1 (8day) A1 (28day) A2(8day)

acetic acid n.d. 151.71±1.78 91.36 ±1.19 a 252.38 ±2.18 b 95.40 ±2.58 a

2-methyl propanoic acid n.d. 51.63±1.39 n.d. n.d. n.d.

4 methyl pentanoic acid n.d. 49.46±0.78 n.d. n.d. n.d.

octanoic acid n.d. n.d. 59.20 ±1.97 a 182.46 ±0.47 b 59.52 ±2.68 a

n-decanoic acid n.d. n.d. 31.17±0.15 n.d. 26.11±1.19

% 27.4 1.4 3.1 1.6

Esters

ethyl acetic ester n.d. n.d. 240.69 ±11.40 a 271.45 ±6.69 b 219.32±15.48 a

acetic acid 2-methylbutyl

ester n.d. n.d. 645.5±31.17 ab 683.13 ±1.58 b 613.02± 23.38a

acetic acid 2-methylbutyl

ester 394.43±10.13 n.d. n.d. n.d. n.d.

Compounds Juice 'Auksis' Yeast A1 (8day) A1 (28day) A2(8day)

acetic acid butylester 202.24±11.07 n.d. n.d. n.d. n.d.

acetic acid hexylester 216.14±8.70 n.d. 1042.30 ±7.66 c 977.16 ±17.66 b 670.22 ±8.07 a

pentafluoropropinoic acid

hexyl ester 237.90±12.66 n.d. n.d. n.d. n.d.

hexanoic acid, ethyl ester n.d. n.d. 1416.71±21.58 n.d. 689.46±15.65

hexanoic acid hexylester n.d. n.d. n.d. 906.47±14.53 n.d.

octanoic acid , ethyl ester n.d. n.d. 1023.25±58.23 a 2327.84±10.91 b 990.23±50.09 a

decanoic acid ethyl ester n.d. n.d. 460.84±9.69 a 864,81±8.91 b 287.11±11.32 a

undecanoic acid ethyl ester n.d. n.d. 81.56±4.44 n.d. 31.88±2.15

9-Decenoic acid, ethyl

ester n.d. n.d. 50.82±0.76 n.d. 20.09±0.80

acetic acid 2-phenylethyl

ester n.d. n.d. 14.88±0.45 n.d. 20.64±0.72

% 35.2 n.d. 31.2 42.8 32.9

Alcohols

1-propanol, 2-methyl n.d. 41.12±0.56 n.d. n.d. n.d.

1-hexanol, 2-ethyl n.d. 50.70±1.28 n.d. n.d. n.d.

1-hexanol n.d. n.d. 246.79±5.39 273.20±0.71 226.93±159.96

2-hexen-1-ol 133.29±8.04 n.d. n.d. n.d. n.d.

2- hydroxyethylhydrazine/4-

penten-2-ol n.d. 138.75±2.32 n.d. n.d. n.d.

2-hydroxyethylhydrazine n.d. n.d. 5590.48±39.31 5167.67±86.63 4972.39±204.62

1-butanol, 3-methyl n.d. 381.51±11.3 1772.79±57.55 1874.57±6.99 2259.75±52.27

8-heptadecanol n.d. n.d. n.d. 60.12±1.36

phenylethyl alcohol n.d. 58.73±0.72 126.37±3.91 257.64±0.68 287.95±14.20

Riekstiaa-DelgeRíta etal. / Procedía FoodSciencel (2011)1709-1716

Compounds Juice 'Auksis' Yeast A1 (8day) A1 (28day) A2(8day)

% 4.4 57.6 62.2 54.1 67.5

Aldehydes

hexanal 313.59±12.25 n.d. n.d. n.d. n.d.

2-hexenal 1502.34±73.67 n.d. n.d. n.d. n.d.

% 62.5 n.d. n.d. n.d. n.d.

a - values are expressed as means of the peak area unites of each compound (n=3), b - n.d.: not detected

3.2. Fermented juice aroma compounds

Three sources of aroma compounds in fermented juices - apple juice, yeast, and yeast metabolites -were analysed. Some compounds - aldehydes hexanal and 2-hexenal and alcohols 2-hexen-1-ol - found in apple juice were not possible to identified in fermented apple juices. A total of 16 volatile compounds were detected in 'Auksis' apples fermented juice at the early stage of fermentation 13 at the 28 day of fermentation. In the majority of juices alcohols (67, 5%), esters (31%) and acids (1,6%) were detected after 8th day of fermentation and alcohols (54,1%), esters (42,8%) and acids (3,2%) after 28th day of fermentation. The main groups of compounds that forms the fermentation bouquet are the acids, alcohols and esters and, to a lesser extent, aldehydes and ketones [11]. Acetic acid, phenylethyl alcohol and 3-methyl-1-butanol, were characteristic compounds of yeasts. Among higher alcohols determined, all the strains produced 3-«methyl-1-butanol that is the major higher alcohol in wine [12,13]. 3-methyl-1-butanol and 2-methyl-1-butanol also plays the major role in the apple juices quality. Both substances are not genuine constituents of the apple fruit, but are inevitably formed during apple juice production, probably through transamination and decarboxylation of the amino acids leucine and isoleucine, respectively [14]. n-decanoic acid, hexanoic acid ethylester, decanoic acid ethylester, 9-Decenoic acid, ethyl ester were detected after 8th day of fermentation of 'Auksis', but were not detected after 28th day of fermentation. Whereas opposite tendency were observed for hexanoic acid hexylester and 8-heptadecanol. They were detected after 28th day of fermentation, but were not detected at early stage of fermentation. Characteristic apple aroma compound acetic acid butylester was detected in fermented juice only at the initial stage of fermentation. Hydroxyethylhydrazine was found as the main volatile aroma compound in all fermented aplle juices. Acetic acid butylester, acetic acid hexylester, decanoic acid ethylester, octanoic acid, decanoic acid found in analysed fermented juices, are also characteristic aroma compounds in ciders made from from 'Fuji' apples [15]. It is possible to identify higher concentrations of apple juice aroma compounds in fermented juices with lower concentration of inoculated yeast. Acetic acid, 1-butanol, 3-methyl phenylethyl alcohol found in fermented juices, are typical yeast aroma compounds. Others aroma are developing during fermentation process. The highest content of aroma compounds was detected in LP4 (with lower concentration of yeast, followed by LP3 fermented with lower concentration of yeast compared to LP4 at higher temperature. Content of acetic acid increased during fermentation process, and the highest content was detected in juice with the lowest concentration of inoculated yeast.

Table 4. Volatile aroma compounds (AU x105± standard deviation) as measured by SPME-GC-MS in 'Lietuvcs Pepins ' apple juice and fermented

Compound_Juice LP_LP1(8day)_LP1(28day)_LP2(8day)_LP2(28day)_LP3(8day)

acetic acid n.d 96.82 ±4.91b 197.35±6.80c 81.40±3.48 a 229.43±0.67d 343.17±0.87f

octanoic acid n.d 1.39 54.04± 0.99d 57.22± 1.89d 126.13±3.80g 117.40± 0.54f

n-decanoic acid n.d 18.80±0.71 n.d 33.78±0.25 n.d 49.82±1.03

% n.d 2.2 2.3 2.2 3.2 3.7

Esters

ethyl acetate 1-butanol, 3-methyl acetate 1 butanol, 2- methyl acetate n.d. n.d. 701.46±61.02 118.3±2.37a 371.94±22.64e n.d 281.89±11.12c 281.69 ±2.35c 290.52±1.23 124.67±3.58a 374.18±7.72e n.d 242.30±0.73b 218.18±7.93b 234.67±4.51 570.45 ±4.19e 602.92 ±4.78f n.d

acetic acid butylester 1586.78±55.75 499.86±15.95 n.d 494.59±26.95 n.d 752.17±4.60 n.d

acetic acid hexylester 1086.51±94.12 645.04±26.79 375.40±13.69 666.30±18.36 237.51±4.27 1009.66±19.78

hexanoic acid, ethyl ester n.d. 426.47±25.90 n.d 414.66±11.37 n.d 565.60±3.85 n.d

octanoic acid , ethyl ester n.d. 313.47± 8.93c 872.,37±11.99e 325.78±10.20c 832.66±4.15e 720.48± 8.27d

decanoic acid ethyl ester undecanoic acid ethyl ester n.d. n.d. 106.12 ±1.76e 21.48±0.40 119.73 ±2.45f n.d 93.30 ±0.08d 28.01±1.40 103.81±2.36e n.d 189.30± 2.61g 115.27±1.20 n.d

hexanoic acid hexyl ester n.d. n.d 1126.83±36.57 n.d 963.20±12.19 n.d

% 82.5 35.9 32.7 32.2 25.7 32.5

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Compound_Juice LP_LP1(8day)_LP1(28day)_LP2(8day)_LP2(28day)_LP3(8day)

Alcohols

1-hexanol 508.18±29.14 538.89±9.36e 407.18 ±1.72d 569.59±21.86f 358.07±7.34b 884.04 ±4.04g

2-hydroxyethylhydrazine n.d. 2556.42±101.81a 4766.85±101.81c 3736.02±134.51b 5515.97±119.27d 5715.64±111..30

1-butanol, 3-methyl n.d. 1124.13±6.69b 1573.49 ±33.52e 1247.08 ±7.14c 1517.97 ±1.72d 2055.51± 2.76g

8-heptadecanol n.d. n.d. 261.75±7.88 n.d. 199.31±1.33 n.d.

Compound Juice LP LP1(8day) LP1(28day) LP2(8day) LP2(28day) LP3(8day)

phenylethyl alcohol n.d. 93.70 ±1.46a 315.41± 14.23f 127.82 ±4.68b 226.10 ±1.54e 196.10 ±1.30d

% 19.5 61.92 67.22 67.82 71.22 63.72

a - values are expressed as means of the peak area unites of each compound (n=3), b - n.d.: not detected

Acknowledgements

The research has been done within the National Research Programme "Sustainable use of local resources (earth, food, and transport) - new products and technologies (Nat Res)" (2010.-2013.) Project no. 3. Sustainable use of local agricultural resources for development of high nutritive value food products (Food)".

References

[1] Dixon, J., Hewett, E. W. 2001. Temperature of hypoxic treatment alters volatilecomposition of juice from 'Fuji' and 'Royal Gala' apples. Postharvest Biology andTechnology, 22, 71-83.

[2] Cunningham, D. G., Acree, T. E., Barnard, J., Butts, R. M., Braell, P.A. 1986. Charm analysis of apple volatiles. Food Chemistry, 19, 137-147.

[3] Mangas, J.J.,Gonzalez.,M.P., Rodriguz, R.&Blaco, D. 1996. Solidphase extraction and determination of trace aroma and flavour components in cider by GC-MS. ChaamaSaanpin. 42.101-105.

[4] Rodriguez Madrera R., Picinelli Lobo A., Mangas. Alonso, J.J. 2009. Effect of cider maturation on the chemical and sensory characteristics of fresh cider spirits. Food Research International, January 2010, Pages 70-78.

[5] Zheng, C. H., Kim, T. H., Kim, K. H., Leem, Y. H., & Lee, H. J. 2004. Characterization of potent aroma compounds in Chrysanthemum coronarium L. (Garland) using aroma extract dilution analysis. Flavour and Fragrance Journal, 19, 401-405.

[6] Young, H., Gilbert, J. M., Murray, S.H., Bal R. D. 1995. Causal effects of Aroma Compounds On Royal Gala Apple Flavours. Journal of the Science of Food Agriculture, 1996, 71, 29-336.

[7] Echeverri'a, G., Graell, J., Lo' pez, M. L., & Lara, I. 2004. Volatile production, quality and aroma-related enzyme activities during maturation of 'Fuji' apples. Postharvest Biology and Technology, 31, 217-227.

[8] Song, J., & Bangerth, F. 2003. Fatty acids as precursors for aroma volatile biosynthesis in pre-climacteric and climacteric apple fruit. Postharvest Biology and Technology, 30, 113-121.

[9] Elss S, Preston C., Appel M., Heckel F., Schreier P. 2006. Influence of technological processing on apple aroma analysed by high resolution gas chromatography-mass spectrometry andon-line gas chromatography-combustion/pyrolysis-isotoperatio mass spectrometry. Food Chemistry 1998 269-276 .

[10] Karlsen, A. M., Aaby, K., Sivertsen, H., Baardseth, P., & Ellekjar, M. R. 1999. Instrumental and sensory analysis of fresh Norwegian and imported apples. Food Quality and Preference, 10, 305-314.

[11]. Lambrechts, M.G. & I.S. Pretorius. 2000. Yeast and its importance to wine aroma. South African Journal of Enology and Viticulture 21:97-129.

[12] Romano, P., Capece, A., Serafino, V., Romaniello, R., Poeta, C., 2008. Biodiversity of wild strains of Saccharomyces cerevisiae as tool to complement and optimize wine quality. World J. Microbiol. Biotechnol. 24, 1797-1802.

[13] Garde-Cerda' n, T., Ancz'n-Azpilicueta, C., 2007. Effect of SO2 on the formation and evolution of volatile compounds in wines. Food Control 18, 1501-1506.

[14]. Hey, M., Kürbel, P., Hopf, I., & Dietrich, H. (2007). Untersuchung sortenreiner Apfelsaftaromen. Flüssiges Obst, 02, 6267.

[15] Peng B , Yue T, Yuan Y 2009 Analysis of key aroma components in cider from Shaanxi (China) duji apple. International Journal of Food Science & Technology 44,610-615

Presented at ICEF11 (May 22-26, 2011 - Athens, Greece) as paper FPE339.