Changes in Volatile Compounds and Some Physicochemical ...

Murat Yilmaztekin and Kubra Sislioglu

The changes in volatile compounds and some physicochemical properties of European Cranberrybush (Viburnum opulus L.) were investigated during traditional fermentation. Using the principal component analysis (PCA), relations between volatile compounds and fermentation were associated with dynamics of these compounds. In total, 58 volatile compounds were identified, 3-methylbutanoic acid (25.4% to 66.4% of identified volatile compounds) being the major constituent in raw, 2-, 3-, and 4-mo fermented European Cranberrybush fruits, while 2-octanone was dominant in 1-mo fermented sample with a 30% of the total identified volatiles. The amount of total volatile compounds was increased in the 1st month of fermentation and then decreased gradually in the following months. Acids were the dominant volatile compounds in raw and 3- to 4-mo fermented European Cranberrybush. Ketones and alcohols had the highest percentage in total volatile compounds in the 2nd and 3rd months of fermentation, respectively. Abstract:

Keywords: European Cranberrybush (Viburnum opulus L.), fermentation, volatile compounds, gas chromatography-mass spectrometry

The fermented fruit juice of European cranberrybush is a traditional drink consumed by people living in middle Anatolian region. It has an astringent taste and the length of the traditional fermentation is important to mask the astringency and obtain a more pleasant taste. This study has reported the changes in volatile compounds of fruits during the fermentation. The results demonstrated that the length of fermentation affects the volatile composition of fruits significantly. The identification of the principal compounds produced during the fermentation can be of help in searching for indicators of off-flavor and in deciding when to stop the fermentation process to avoid production of off-flavor compounds.

Practical Application:

Introduction

2013). European Cranberrybush fruits contain high amount of ˇ polyphenolics (Cesonien˙ e and others 2008), ascorbic acid (Rop and others 2010), and L-malic acid (C ¸ am and Hisil 2007). It has been reported that berry juice is rich in chlorogenic acid which constitutes 54% of the total phenolic content (Velioglu and others 2006). This amount is much higher compared to some widely consumed juices and nectars (C ¸ am and Hisil 2007). The berries of European Cranberrybush were also reported as a source of flavonoids, including (+)-catechin and (−)-epicatechin, quercetin glycosides (Velioglu and others 2006) and proanthocyanidins (Zayachkivska and others 2006). In addition, they contain ˇ carotenoids (Gavrilin and others 2007; Cesonien˙ e and others 2008) and high concentration of phenolics which brings in strong radical scavenging capacity (Sagdic and others 2006; C ¸ am and ˇ Hisil 2007; Cesonien˙ e and others 2008). Antimicrobial activity of dried fruits (Sagdic and others 2006) and seed oil (Yilmaz and others 2008) of gilaburu was also reported. In the light of these findings, it may be postulated that V. opulus berries can be regarded as a promising fruit containing bioactive components which might be utilized for food, nutraceutical, and medicinal purposes. Sensory quality is among the most important factor in food applications, and from this point of view the majority of widely MS 20141393 Submitted 8/13/2014, Accepted 2/6/2015. Authors are with Inonu Univ., Faculty of Engineering, Dept. of Food Engineering, 44280, Malatya, Turkey. consumed berries’ and fruits’ distinct aroma profiles determine the consumer preferences. European Cranberrybush berries Direct inquiries to author Yilmaztekin (E-mail: [email protected]). also possess some typical smell notes, which are rather disliked European Cranberrybush (Viburnum opulus L.) belongs to the plant family Caprifoliaceae. V. opulus var. opulus which is widespread ˇ in eastern, northeastern, western, and central Europe (Cesonien˙ e and others 2012), central Anatolia and Black Sea region of Turkey (Altan and Maskan 2004). European Cranberrybush which is widely known as gilaburu in Turkey, is a red colored fruit having special astringent taste, mostly grown around the Kayseri city in Turkey (Dinç and others 2012; Cam and others 2007). Many species of this fruit become popular as garden or landscape plants because of their showy flowers, berries, and fragrance, while some of its species have edible fruits which can be eaten either raw or jammed (Yilmaz and others 2013). The fruit contains a high amount of vitamin C and phenolic compounds and has a strong special flavor due to presence of valeric acid (Dinç and others 2012). The juice of the gilaburu fruit is a traditional drink for people living in Kayseri and it is thought to have a preventive effect on kidney disorders as well as menstrual and stomach cramps (Soylak and others 2002). It is believed that gilaburu cures hypertension, asthma, digestion problems, and common cold (Ulger and others

R C 2015 Institute of Food Technologists

doi: 10.1111/1750-3841.12836 Further reproduction without permission is prohibited

Vol. 80, Nr. 4, 2015 r Journal of Food Science C687

C: Food Chemistry

Changes in Volatile Compounds and Some Physicochemical Properties of European Cranberrybush (Viburnum opulus L.) During Ripening Through Traditional Fermentation

Volatiles of European cranberrybush . . .

C: Food Chemistry

(Mettler Toledo SevenMulti, Columbus, Ohio, U.S.A.). Titratable acidity was determined according to AOAC (1980) by titrating the sample (20-mL fruit juice plus 80 g water) with 0.1 N NaOH to an endpoint of pH 8.2 at 20 °C. Titratable acidity was expressed as lactic acid equivalent in percentage. The sample temperature was 20 °C for each analysis. The amount of total phenolics in the fruit juice was determined as gallic acid equivalent by using the Folin–Ciocalteu reagent method (Slinkard and Singleton, 1977). The reagent was prepared by diluting a stock solution of Folin–Ciocalteu reagent (SigmaAldrich Chemie GmbH) with distilled water (1:10, v/v). Samples (1 mL, 2 replicates) were placed into test cuvettes, and 5 mL of Folin–Ciocalteu phenol reagent and 4 mL of Na2 CO3 (7.5%) were added. The absorbance of the samples was measured at 765 nm using a Shimadzu model UV-1700 UV/VIS spectrophotometer (Shimadzu Corporation, Kyoto, Japan) after incubating at 20 °C for 1 h. The results were expressed as milligrams of gallic acid equivalent (GAE) per 100 g of fruit juice. The total nitrogen content was measured using the Kjeldahl method according to AOAC (1980). An exact amount of sample was weighed and mineralized in a glass tube after the addition of concentrated sulfuric acid, a small amount of selenium as catalyzer, and copper (II) oxide. At first, the glass tube was heated to approximately 200 °C and, after the addition of a solution containing 40% hydrogen peroxide, to approximately 400 °C until a clear light blue solution was obtained. After cooling, a few milliliters of water were added and then the tube was placed into the Kjeldahl apparatus (Simsek Laborteknik, Turkey) for the determination of the nitrogen content. Sodium hydroxide (0.01 N) was added in excess, and the total nitrogen present was distilled as ammonia. Materials and Methods The excess of NaOH was then back-titrated with 0.01 N HCl in Fruit samples the presence of a mixed indicator (bromocresol green and methyl The bunches with mature fruits of European Cranberrybush red). The results were expressed as grams of total nitrogen per (Viburnum opulus L.) were harvested in October 2013 from the kilogram of fruit juice. All analyses were conducted in triplicate vicinities of Kayseri province, Turkey. Bunches with berries were for each sample. picked from the bottom, middle and top parts of the plants selecting 4 different bushes. Liquid–liquid extraction of volatile compounds Liquid–liquid extraction of volatile compounds was performed Chemicals using the method of Yilmaztekin (2014). Briefly, 100 g of clear All chemicals and reagents used in this work were pur- fruit juice and 50 mL of freshly distilled dichloromethane were chased from Merck (Darmstadt, Germany) and Sigma Chemicals transferred in a 500-mL Erlenmeyer flask and immediately cooled (Sigma-Aldrich, Steinheim, Germany). to 4 °C in an ice bath. Five microliters of a mixture of 4-nonanol (2.46 mg/mL in ethanol), γ -valerolactone (3.98 mg/mL in Fermentation procedure ethanol), and cyclohexyl butanoate (2.78 mg/mL in ethanol) Bunches from different bushes of the European Cranberrybush was added as internal standards before extraction. The mixture fruits were mixed, washed 3 times with tap water and placed in was agitated at 750 rpm during 30 min under a gaseous nitrogen plastic drums. The plastic drums containing fruits were filled with saturated atmosphere. Later, the mixture was centrifuged at tap water to overflowing, closed tightly and allowed to ferment 10000 × g at 4 °C during 15 min. The aqueous phase was in a dark place at room temperature (25 °C). Raw and fermented removed and the organic phase was dehydrated with Na2 SO4, fruits were frozen and stored at −20 °C before further use. Fruits filtrated through glass fiber, and the filtrate was collected in a were defrosted at 24 °C for 1 h before the production of fruit 260 mL flask. The filtrated sample was distilled in a Vigreux juice. The stalks were removed and the berries were pressed by column followed by a Dufton column at 40 °C, to concentrate hand with a muslin cloth. The obtained juice was centrifuged in a the sample approximately to 0.5 mL. The organic extract was Hettich model 320 R centrifuge (Hettich, Tuttlingen, Germany) stored at −20 °C in a 1.5 mL vial until the analysis by GC-FID at 9000 rpm for 10 min. The supernatant of the clear juice and GC-MS. The extraction was performed in triplicate. was poured into glass bottles and kept at 4 °C before analysis. Fermentation trials were conducted in triplicate. GC-FID and GC-MS analysis All analyses were performed on a Shimadzu QP 2010 Plus Physicochemical analysis (Shimadzu, Kyoto, Japan) GC equipped with an AOC-20i/20s Soluble solids, reported as Brix value, were determined with a autosampler, a flame ionization detector and a MS-QP 2010 sedigital refractometer (Mettler Toledo, Columbus, Ohio, U.S.A.). ries mass selective detector. The GC was fitted with a TRB-WAX The pH of fruit juices was recorded using a digital pH-meter (Teknokroma, Barcelona, Spain) fused silica capillary column by the consumers. In the Kayseri region of Turkey, European Cranberrybush fruits are allowed to stand in plastic drums containing tap water at a dark place and room temperature for about 4 mo. This traditional fermentation and sometimes addition of small amounts of sugar to the fruit juice minimize the effect of astringency caused by mostly phenolic compounds. However, fermentation conditions such as temperature and time affects the taste of the fruit juice and there is not a standard procedure for the fermentation of European Cranberrybush fruits except the trial-and-error method. Scientific information on the composition of volatile compounds of European Cranberrybush volatile compounds is limited to the study carried out by Kraujalyte and others (2012). There are 41 identified compounds, 3-methyl-butanoic acid being the major constituent in studied European Cranberrybush cultivars. Moreover, to the best of our knowledge, there were no attempts to identify the changes in the aroma compounds of European Cranberrybush fruits during traditional fermentation. In a previous study, a total of 21 bacteria belonging to 6 different lactic acid bacteria species were isolated from traditionally fermented European Cranberrybush (Yapar 2008), and this gives some hints about the possible production of some volatile compounds during fermentation. The aim of this study was to examine some physicochemical properties and volatile compounds of European Cranberrybush fruits during the traditional fermentation. For this purpose the European Cranberrybush fruits grown in the Kayseri region of Turkey were studied under controlled traditional fermentation conditions.

C688 Journal of Food Science r Vol. 80, Nr. 4, 2015

Volatiles of European cranberrybush . . . Table 1–Some physicochemical properties raw and fermented European Cranberrybush (Viburnum opulus L.) fruit juice. Samplesa

pH Brix Titratable acidity (% lactic acid) Total phenolics (mg/100 g gallic acid) Total dry matter (%) Total nitrogen (gr/kg) Ash (%)

I 2.77 10.35 2.42 413 8.51 2.09 0.32

± ± ± ± ± ± ±

II 0.0a 0.1b 0.0c 3.5a 0.0c 0.1b 0.0a

2.81 10.06 2.04 457 8.36 1.42 0.27

± ± ± ± ± ± ±

III 0.0ab 0.1b 0.1b 4.2b 0.0c 0.2ab 0.0a

2.82 8.84 1.78 453 7.37 1.33 0.24

± ± ± ± ± ± ±

IV 0.0ab 0.2a 0.0a 3.5b 0.0b 0.3ab 0.0a

2.85 8.66 1.78 464 7.30 1.04 0.25

± ± ± ± ± ± ±

V 0.0b 0.3a 0.0a 1.4b 0.0b 0.2a 0.0a

2.84 8.45 1.75 483 7.00 0.69 0.23

± ± ± ± ± ± ±

0.0b 0.1a 0.1a 4.2c 0.1a 0.2a 0.0a

Note: In the same row, means with different italic letters significantly differed in ANOVA test (P < 0.05, n = 3). a I: raw fruit, II: 1-mo fermented fruit, III: 2-mo fermented fruit, IV: 3-mo fermented fruit, V: 4-mo fermented fruit.

(60 m × 0.25 mm i.d. and 0.25-μm film thickness). Helium was used as the carrier gas at a flow rate of 1 mL/min. The column was maintained at 40 °C for 5 min after injection, then programmed at 3 °C/min to 240 °C, which was maintained for 15 min. The total run time including oven cooling was 86 min. The same oven temperature program was used for both the flame ionization and the mass selective detector analysis. Injector, transfer line and ionsource temperatures were 250 °C. All mass spectra were acquired in electron-impact (EI) mode; the ionization voltage was 70 eV; the mass range was 35 to 450 m/z; scanning rate 1 scan/s. A mixture of n-alkanes (C8 -C40 ) was injected under the above temperature program to calculate the linear retention indexes of each compound. Concentrations of volatile compounds were expressed as internal standards equivalents (μg/kg) and average of 3 replicates. The peaks were identified by comparison of the obtained mass spectra of the relevant chromatographic peaks with spectra of the NIST (Natl. Inst. of Standards and Technology, Gaithersburg, Md., U.S.A.) and Wiley libraries. In addition, the compounds were tentatively identified by comparing the experimental retention indexes with the theoretical ones, which were obtained from the Flavornet and the Pherobase databases. Peak enrichment on coinjection with authentic reference compounds was also carried out. The comparison of the MS fragmentation pattern with those of pure compounds and mass spectrum database search was performed using the MS spectral databases.

contents of raw European Cranberrybush were lower, while Brix, titratable acidity and total nitrogen contents were higher than the values reported by Yapar (2008) and Akbulut and others (2008). There were significant differences between the raw and fermented fruit juices with respect to the values of all physicochemical properties. All the samples analyzed were found to be acidic in character. Their pH values ranged between 2.77 and 2.85. The pH values of samples were almost remained constant during the fermentation. In contrast to our results, Yapar (2008) found that the pH decreased during the fermentation. In lactic acid fermentations a decrease in pH is expected due to production of lactic acid in the medium (Adams and Nicolaides 1997). For an optimal course of lactic acid fermentation, the content of proteins that neutralize emergent acids must be minimal (Kopec 2000). As mentioned before, in our study, total nitrogen values were higher than that of Yapar (2008) and Akbulut and others (2008), and the insufficient decrease of pH could be related to high values of nitrogen content of fruits. The Brix values of raw and fermented fruit of European Cranberrybush were significantly different, especially after 2 mo of fermentation (P < 0.05). At the end of the fermentation the Brix value was determined as 8.45. The amount of total phenolics significantly increased, while the amount of total nitrogen decreased during fermentation (P < 0.05). Similar results were also reported by Yapar (2008).

Statistical analysis Data obtained from physicochemical, sensory and volatile compound analyses were expressed as the means ± standard error. Statistical differences between the samples were evaluated by ANOVA (one way). Duncan’s multiple comparison tests were used as post hoc tests, P < 0.05 was considered to be significant. Concentrations of aroma compounds with data standardization were also subjected to principal component analysis (PCA). Compounds in each process were studied separately. All statistical analyses were performed using IBM SPSS Statistics 21 Software (SPSS Inc., Chicago, Ill., U.S.A.).

Volatile compounds of European Cranberrybush during ripening The amounts and changes in volatile compounds of raw and fermented European Cranberrybush fruits during traditional fermentation were given in Table 2. Fifty-eight volatile compounds were identified in fruit juices which belong to the following chemical classes: acids (10), alcohols (27), aldehydes (3), esters (3), ketones (12), and lactones (3). The amount of total volatile compounds was significantly different in raw and fermented European Cranberrybush fruits (P < 0.05). Total ion chromatogram (TIC) of 4-mo fermented European Cranberrybush was given in Figure 1. The most abundant compounds were acids, followed by alcohols, ketones, esters, aldehydes, and lactones in raw European Cranberrybush. In the 1st month of the fermentation, the concentration of total volatile compounds was at the highest level among the raw and fermented fruits and then decreased gradually until the end of the fermentation. The abundant volatile compounds were ketones and alcohols in the 1st and 2nd month of fermentation, respectively. Then, the acids have dominated the last 2 mo of fermentation. Changes in total amounts of different classes of volatile compounds during fermentation were given in Figure 2 and 3. The number and the concentration of volatile compounds increased with the

Results and Discussion Physicochemical analyses The physicochemical properties of raw and fermented European Cranberrybush fruits are given in Table 1. The pH, Brix, titratable acidity, total phenolics, total dry matter, total nitrogen, and ash contents were determined. The pH, Brix, titratable acidity, total phenolics, total dry matter, total nitrogen, and ash contents of raw European Cranberrybush fruit juice were established as 2.77%, 10.35%, 2.42%, 413 mg/100 g, 8.51%, 2.09 gr/kg and 0.32%, respectively. The pH, total phenolics, total dry matter and ash


C: Food Chemistry

Physicochemical properties

Volatiles of European cranberrybush . . . Table 2–Volatile compounds identified in raw and fermented European Cranberrybush (Viburnum opulus L.) fruit juice (µg/kg). Samplesa Compound

LRIb

I

II

III

IV

V

Acetic acidc 2-Methylpropionic acidd Butanoic acidc 3-Methylbutanoic acidc Pentanoic acidc 3-Methylpentanoic acidd Hexanoic acidc Heptanoic acidc Octanoic acidc Nonanoic acidc Subtotal

1450 1567 1628 1668 1738 1793 1846 1954 2063 2170

190 ± 6.9a nda nda 1354 ± 55a nda 74.8 ± 6.7a 13.5 ± 0.7a nda 5.0 ± 0.3a 11.4 ± 1.7a 1649a

157 ± 1.4a nda nda 1592 ± 32c nda 90.9 ± 7.9ab 16.9 ± 1.0a nda 9.3 ± 0.7b 76.8 ± 2.5d 1943b

190 ± 5.6a 5.4 ± 0.5d 2.5 ± 0.3b 1604 ± 16c 10.9 ± 0.7b 96.9 ± 2.5ab 16.9 ± 1.0a 6.9 ± 1.3b 12.8 ± 0.3c 71.7 ± 4.1d 2018b

307 ± 11b 4.1 ± 0.3c 3.3 ± 0.2c 1408 ± 11ab 12.1 ± 0.9c 106 ± 6.3b 25.6 ± 1.3b 8.3 ± 0.9b 11.2 ± 0.6b 38.9 ± 1.9c 1923b

346 ± 13c 2.8 ± 0.3b 4.1 ± 0.1d 1549 ± 59bc 9.9 ± 0.5b 97.4 ± 4.1ab 28.9 ± 2.0b 8.9 ± 0.9b 8.9 ± 1.0b 22.7 ± 2.7b 2078b

3-Hexanold 2-Methyl-1-propanolc 1-Butanolc 3-Methyl-1-butanolc 2-Hexanolc 1-Pentanolc 6-Methyl-1-heptanolc 4-Methyl-2-pentanolc 5-Methyl-2-hexanold 1-Hexanolc Z-3-Hexenold E-2-Hexenold 2-Octanolc Z-6-Undecen-2-old 2-Ethyl-1-hexanolc 2-Nonanolc 1-Octanolc 2-Decanold 7-Methyl-4-octanold 1-Nonanolc Z-3-Nonenold 4-Methyl-1-heptanold Citronellolc Benzyl alcoholc Phenylethyl alcoholc 5-Ethyl-2-heptanold 1-Tetradecanold Subtotal

1013 1087 1140 1203 1216 1246 1247 1306 1315 1349 1381 1402 1417 1458 1487 1516 1556 1618 1650 1659 1684 1762 1766 1880 1916 1970 2023

42.8 ± 3.7a nda nda 65.2 ± 6.5a nda 5.0 ± 0.7a 4.2 ± 0.3a nda nda 85.0 ± 1.9b 36.9 ± 1.3b 13.8 ± 1.2b nda nda 23.7 ± 2.4c nda nda nda nda nda nda nda nda nda nda nda 39.2 ± 1.1c 316a

48.2 ± 3.9a 58.0 ± 0.6d 5.8 ± 0.4b 209 ± 12b 23.5 ± 2.8c 11.0 ± 0.6b 4.2 ± 0.4a nda 79.6 ± 1.6d 132 ± 5.1c 24.5 ± 1.4a 4.4 ± 0.4a 1376 ± 64d nda 19.0 ± 0.9bc 42.2 ± 2.5d nda nda 264 ± 7.3d 30.1 ± 3.2b nda nda nda nda 42.2 ± 0.9b nda 31.9 ± 0.4b 2406d

50.4 ± 5.9a 47.8 ± 2.8c nda 283 ± 10d 22.1 ± 2.1c 12.2 ± 0.5b 3.4 ± 0.5a 10.1 ± 0.6b 80.1 ± 0.8d 86.6 ± 4.0b 23.0 ± 2.2a 3.3 ± 0.3a 1394 ± 30d 84.2 ± 2.2c 17.5 ± 1.7abc 56.2 ± 2.1e nda 15.5 ± 1.5c 165 ± 13c 39.6 ± 1.6b nda nda 6.1 ± 0.7c 12.8 ± 1.3b 96.2 ± 3.6c nda 15.2 ± 0.1a 2524d

38.8 ± 2.0a 30.1 ± 2.1b nda 270 ± 7.5cd 7.5 ± 0.7b 13.8 ± 1.1b 3.9 ± 0.4a 10.1 ± 0.2b 35.0 ± 2.1c 55.4 ± 2.4a 20.0 ± 1.2a 2.8 ± 0.5a 709 ± 15c 28.1 ± 2.1b 13.2 ± 1.5ab 27.1 ± 2.1c 10.4 ± 1.4b 8.0 ± 1.3b 40.6 ± 2.2b 68.5 ± 2.5c 12.8 ± 0.6b nda 4.2 ± 0.5bc 25.4 ± 3.2c 128 ± 5.9d 4.9 ± 0.7b 14.4 ± 1.8a 1583c

43.5 ± 2.5a 33.6 ± 2.3b nda 240 ± 0.7bc 2.9 ± 0.5ab 15.8 ± 2.4b 4.0 ± 0.7a 11.2 ± 0.5b 23.8 ± 2.4b 53.6 ± 2.7a 21.7 ± 0.7a 3.0 ± 0.2a 473 ± 9.7b nda 12.0 ± 0.7a 14.1 ± 0.3b 11.0 ± 0.4b nda 4.6 ± 0.7a 75.2 ± 4.2c 17.2 ± 2.0c 1.7 ± 0.5b 3.6 ± 0.7b 24.2 ± 1.8c 107 ± 5.8c 6.0 ± 1.2b 11.4 ± 1.6a 1213b

2-Ethyl-hexanald Octanalc Nonanalc Subtotal

1188 1290 1396

3.5 ± 0.3b 1.4 ± 0.2b 10.1 ± 0.8a 11.5a

nda nda 9.0 ± 0.1a 9.0a

nda nda 11.8 ± 1.1ab 11.8ab

nda nda 15.7 ± 1.8bc 15.7bc

nda nda 16.1 ± 0.3c 16.1c

Ethyl 3-methylbutanoatec Methyl 2-methyllactated Methyl 2-hydroxydecanoated Subtotal

1072 1240 2002

nda 1.3 ± 0.9a 23.6 ± 2.6ab 25.0a

nda 2.0 ± 0.1a 44.9 ± 0.5c 46.9c

nda 2.1 ± 0.2a 18.6 ± 1.1a 20.7a

12.2 ± 1.8b 2.1 ± 0.3a 21.6 ± 1.0ab 36.0b

12.5 ± 1.7b 2.6 ± 0.6a 27.2 ± 3.2b 42.2bc

2-Hexanonec 1-(3-Ethylcyclobutyl)-ethanoned 2-Heptanonec 3-Hydroxy-2-butanonec 2-Octanonec 7-Octen-2-oned 6-Methyl-5-heptene-2-onee 4-Hydroxy-4-methyl-2-pentanoned 2-Nonanonec 2-Decanonec 5-Ethyl-2-heptanonec 6-Dodecanoned Subtotal

1082 1090 1183 1283 1287 1333 1339 1361 1390 1496 1822 2109

nda nda nda 8.0 ± 0.6b nda nda 10.0 ± 0.7b 11.1 ± 0.5a nda nda nda nda 29.1a

66.8 168 217 27.4 2245 126 6.1 62.2 95.7 21.4 10.2

± 3.3d ± 3.9d ± 6.7d ± 2.1c ± 79d ± 5.4d ± 0.2a ± 2.1c ± 3.8d ± 1.2d ± 0.7c nda 3045e

32.6 ± 2.9c 73.6 ± 4.5c 110 ± 6.5c 41.7 ± 1.6d 1255 ± 49c 57.0 ± 3.7c 4.8 ± 0.8a 23.9 ± 3.6b 44.0 ± 2.5c 9.8 ± 0.6c 5.9 ± 0.9b 44.1 ± 2.0c 1702d

16.2 ± 2.2b 26.1 ± 2.5b 38.1 ± 2.1b nda 614 ± 19b 18.3 ± 1.6b 4.0 ± 0.8a 13.6 ± 1.3a 20.4 ± 1.4b 5.0 ± 0.9b nda 13.1 ± 0.2b 769c

9.1 ± 0.4 b nda 21.2 ± 1.4b nda 448 ± 2.9b 9.8 ± 0.6ab 5.1 ± 0.6a 15.1 ± 1.5a 11.5 ± 2.1b 1.6 ± 0.5a nda nda 522b

Acids

C: Food Chemistry Alcohols

Aldehydes

Esters

Ketones

(Continued)


Volatiles of European cranberrybush . . . Table 2–Continued. Samplesa Compound

LRIb

I

II

III

IV

V

Delta-valerolactonec Gamma-butyrolactonec Gamma-nonalactoned Subtotal Total

1621 1635 2042

nda 6.6 ± 0.6a nda 6.6a 2040a

nda 6.0 ± 0.2a 27.7 ± 2.6c 33.8c 7484d

nda 12.4 ± 0.8b 23.1 ± 0.8c 35.5c 6312c

5.9 ± 1.0b 12.3 ± 0.5b 11.4 ± 1.7b 29.6bc 4356b

5.0 ± 0.6b 15.2 ± 0.7c 3.8 ± 0.6a 24b 3895b

Note: In the same row, means with different italic letters significantly differed in ANOVA test (P < 0.05, n = 3). a I: raw fruit, II: 1-mo fermented fruit, III: 2-mo fermented fruit, IV: 3-mo fermented fruit, V: 4-mo fermented fruit. b Linear retention index. LRI of the compounds were confirmed as available from The Flavornet database (http://www.flavornet.org/flavornet.html) and The Pherobase database (http://www.pherobase.com/database/kovats). c Identified by GC-MS spectra, LRI, and using reference compounds. d Tentatively identified by GC-MS spectra and LRI. nd: not determined.

initiation of fermentation, which indicated that most of the volatile compounds formed during fermentation. Acids were the dominant volatile compounds in raw European Cranberrybush fruits. The amount of total acid increased in the 1st month of fermentation, and remained at the same level throughout the fermentation (P > 0.05). The concentration of 3-methylbutanoic acid was at the highest level among the other acids in all samples. Kraujalyte and others (2012) found similar results reporting, 3-methylbutanoic acid, very important odor active compound, to be a dominant volatile compound in Shukshinskaya, Kiyevskaya Sadovaya, and Krasnaya Grozd cultivars of Viburnum opulus. The odor of this compound was described as possessing strong “dirty socks” and “old cheese” notes (Kraujalyte and others 2012). It is reported that 3-methylbutanoic may form as a side metabolism product in the biosynthesis of branched amino acid isoleucine (Schwab and others 2008; Kraujalyte and others 2012). The second most abundant compound was acetic acid in all samples. Its concentration increased especially in 3rd and 4th months of fermentation. The presence of acetic can be attributed to lactic acid fermentation, as acetic acid is one of the characteristic products of lactic acid fermentation (Adams and Nicolaides 1997). Although acetic acid in fermented products has antimicrobial effects, high acetic acid concentrations are not typically desirable from sensory and quality point of view (Lee and others 1999).

Alcohols were the second dominant compounds in all samples except the 2nd month of fermentation, while their total amount was at the highest level among the other classes of volatiles in the 2nd month of fermentation, and began to decrease afterwards (P < 0.05). The dominant compound was 2-octanol among the other alcohols in fermented European Cranberrybush fruits, followed by 3-methyl-1-butanol. Interestingly, ethyl alcohol was not detected in fermented fruits. The absence of ethyl alcohol could be related to production procedure, as it may remove with water phase after fermentation. The other formed alcohols were 2-methyl-1-propanol, 5-methyl-2-hexanol, 2-nonanol, 7-methyl-4-octanol, 1-nonanol, benzyl alcohol, and phenylethyl alcohol during fermentation. These compounds are related to herbaceous notes and can be recognized by their strong and pungent smell and taste (Garc´ıa-Carpintero and others 2012). Especially, 3-methyl-1-butanol may contribute to the complexity of aroma, although it can produce unpleasant notes at high levels. Additionally, C6 alcohols, such as 1-hexanol, 3-hexanol, Z-3-hexenol, and E-2-hexenol were determined in raw European Cranberrybush fruits which are derived from the membrane lipids via the lipoxygenase pathway. The amount of C6 alcohols were at the highest level in raw European Cranberrybush fruits, while their concentrations decreased throughout the fermentation. C6 alcohols are related to vegetal and herbaceous aromas in

(x1,000,000) 1.75 TIC 1.50 1.25 1.00 0.75 0.50 0.25 0.00 20.0

25.0

30.0

35.0

40.0

45.0

50.0

55.0

60.0

65.0

Figure 1–Total ion chromatogram (TIC) of 4-mo fermented European Cranberrybush. Vol. 80, Nr. 4, 2015 r Journal of Food Science C691

C: Food Chemistry

Lactones


a

b

b

Concentration (μg/kg)

2000

b

1500 1000 500 0 II

III

IV


d

2500

c

1500 1000

b

a

500 0

I

II

III

IV

V

15

bc

c

ab

a a

10 5 0 I

II

III

a

IV

V

Figure 2–Evolution of total acids (A), total alcohols (B), and total aldehydes (C) in the raw and fermented European Cranberrybush (Viburnum opulus L.) fruits. I: raw fruit, II: 1-mo fermented fruit, III: 2-mo fermented fruit, IV: 3-mo fermented fruit, V: 4-mo fermented fruit. Labeled error bars indicate the standard error for each treatment (n = 3). Different letters indicate significant differences among samples (P < 0.05).


bc

a

20 10 0 II

III

IV

V

B

total ketones 3500 3000 2500 2000 1500 1000 500 0

e d c a I

II

III

IV

b V

C

total lactones

C

total aldehydes 20

30

I

d

2000

b

40

B

total alcohols 3000

c

50

V


I

A

total esters



2500

b

2-octanone, 7-octen-2-one, 6-methyl-5-heptene-2-one, 4hydroxy-4-methyl-2-pentanone, 2-nonanone, 2-decanone, 5-ethyl-2-heptanone, and 6-dodecanone) and 3 lactones (deltavalerolactone, gamma-butyrolactone, and gamma-nonalactone) were identified in raw and fermented European Cranberrybush fruits. Nonanal was the most abundant straight-chain aliphatic aldehyde detected in raw fruits. 2-ethyl-hexanal and octanal were not detected in fermented fruits, while the amount of nonanal was increased throughout the fermentation. The amount of total ketones was at the highest level among the all classes of compounds in the 1st month of fermentation, but hereafter the concentration of the ketones started to decrease throughout the fermentation (P < 0.05). The most dominant ketone was 2-octanone, formed by the beginning of fermentation. Contrary to the result given by Kraujalyte and others (2012) we could not determine 2-octanone in raw fruits of European Cranberrybush. 3-hydroxy-2-butanone,

A

total acids


C: Food Chemistry

fermented beverages (Etiévant 1991) and usually have a negative effect on quality when their concentration is above their odor threshold values (Garc´ıa-Carpintero and others 2012). In total, 3 different ester compounds (ethyl 3-methylbutanoate, methyl 2-methyllactate, and methyl 2-hydroxydecanoate) were identified in raw and fermented European Cranberrybush fruits. There were significant differences between the amount of esters in raw and fermented fruits, except methyl 2-methyllactate (P < 0.05). Ethyl 3-methylbutanoate appeared after 3 mo of fermentation. The amount of total esters was at the highest level in the 1st month of fermentation. Esters are responsible for the fruity aroma in fermented beverages, their origin being mainly fermentative (Ugliano and Henschke 2009). Among the carbonyl compounds 3 aldehydes (2-ethylhexanal, octanal and nonanal), 12 ketones (2-hexanone, 1-(3ethylcyclobutyl)-ethanone, 2-heptanone, 3-hydroxy-2-butanone,

40 35 30 25 20 15 10 5 0

c

c

bc b

a I

II

III

IV

V

Figure 3–Evolution of total esters (A), total ketones (B), and total lactones (C) in the raw and fermented European Cranberrybush (Viburnum opulus L.) fruits. I: raw fruit, II: 1-mo fermented fruit, III: 2-mo fermented fruit, IV: 3-month fermented fruit, V: 4-mo fermented fruit. Labeled error bars indicate the standard error for each treatment (n = 3). Different letters indicate significant differences among samples (P < 0.05).

6-methyl-5-heptene-2-one, and 4-hydroxy-4-methyl-2pentanone were not detected in raw fruits among the other ketones. Three lactones were identified and quantified in raw and fermented European Cranberrybush fruits. The concentration of the most dominant compound gamma-nonalactone was at the highest level at the 1st month of fermentation and then decreased through the last 2 mo.

Principal component analysis (PCA) Principal component analysis (PCA) was performed to determine the most important aroma compounds on raw and fermented European Cranberrybush fruits. The two 1st principal components (PCs) are sufficient to explain the maximum variation in all samples. In our case, the PC1 and PC2 explained 45.31% and 38.88% variances, respectively. Loadings and scores plots are shown in Figure 4A and B. On the one hand, PC1 on the

positive axis was highly influenced by some volatile compounds such as 3-methylbutanoic acid (c41), 3-methyl-1-butanol (c9), 2-octanol (c27), phenylethyl alcohol (c50), 2-octanone (c15), and gamma-nonalactone (c55). The concentration of these compounds increased during the 1st and 2nd month of fermentation (II and III). Compounds with the highest concentrations were 3-methylbutanoic acid (c41), 3-methyl-1-butanol (c9), 2-octanol (c27), and 2-octanone c(15). On the other hand, PC1 negative axis grouped compounds with high concentration in raw (I) fruits (Figure 4B) such as 2-ethyl-hexanal (c8), Z-3-hexenol (c23), E-2-hexenol (c26), 2-ethyl-1-hexanol (c30). In addition, the concentration of these compounds decreased during the fermentation process. Regarding PC2, most of the aroma compounds located in the positive axis of the plane: explaining that the compounds formed during the 2nd, 3rd, and 4th month of fermentation. The concentration of these compounds decreased in the last 2 mo

Figure 4–Principal component analysis the first 2 principal components: (A) loading plot for aroma compounds (compound1: 3-hexanol, c2:ethyl 3-methylbutanoate, c3: 2-hexanone, c4: 2-methyl-1-propanol, c5: 1-(3-ethylcyclobutyl)-ethanone, c6: 1-butanol, c7: 2-heptanone, c8: 2-ethyl-hexanal, c9: 3-methyl-1-butanol, c10: 2-hexanol, c11: methyl 2-methyllactate, c12: 1-pentanol, c13: 1-octanol, c14: 3-hydroxy-2-butanone, c15: 2-octanone, c16: octanal, c17: 4-methyl-2-pentanol, c18: 5-methyl-2-hexanol, c19: 7-octen-2-one, c20: 6-methyl-5-heptene-2-one, c21: 1-hexanol, c22: 4-hydroxy-4-methyl-2-pentanone, c23: Z-3-hexenol, c24: 2-nonanone, c25: nonanal, c26: E-2-hexenol, c27: 2-octanol, c28: acetic acid, c29: (Z)-6-undecen-2-ol, c30: 2-ethyl-1-hexanol, c31:2-decanone, c32: 2-nonanol, c33: 1-octanol, c34: 2-methylpropionic acid, c35: 2-decanol, c36: delta-valerolactone, c37: butanoic acid, c38: gamma-butyrolactone, c39: 7-methyl-4-octanol, c40: 1-nonanol, c41: 3-methylbutanoic acid, c42: Z-3-nonenol, c43: pentanoic acid, c44: 4-methyl-1-heptanol, c45: citronellol, c46: 3-methylpentanoic acid, c47: 5-ethyl-2-heptanone, c48: hexanoic acid, c49: benzyl alcohol, c50: phenylethyl alcohol, c51: heptanoic acid, c52: 5-ethyl-2-heptanol, c53: methyl 2-hydroxydecanoate, c54: 1-tetradecanol, c55: gamma-nonalactone, c56: octanoic acid, c57: 6-dodecanone, c58: nonanoic acid). (B) Score plot from raw and fermented European Cranberrybush (Viburnum opulus L.) fruits. I: raw fruit, II: 1-mo fermented fruit, III: 2-mo fermented fruit, IV: 3-mo fermented fruit, V: 4-mo fermented fruit.


C: Food Chemistry



C: Food Chemistry

of fermentation (IV and V) (Figure 4B). However, we observed that the aroma compounds were grouped on the negative axis, that is, acetic acid (c28), hexanoic acid (c48), phenylethyl alcohol (c50), nonanal (c25), 5-ethyl-2-heptanol (c52), and gamma-butyrolactone (c38). These compounds were the most important at the 1st month of fermentation (II) (Figure 4B). The score distribution for the two 1st PCs (Figure 4B) showed 4 separate groups, at 4 mo of fermentation. The 1st and 2nd months of fermentation (II and III) were located on the positive side of PC1, while raw, 3- and 4-mo fermented fruits (I, IV, and V) were found in the negative side. The last 3 mo of fermentation (III, IV, and V) were clustered on the positive axis of PC2, but the raw and 1-mo fermented fruits (I and II) were found in the negative axis of PC2.

Conclusion The volatile compounds of European Cranberrybush (Viburnum opulus L.) fruits during traditional fermentation were studied for the first time. The amount and variety of volatile compounds have changed throughout the fermentation. The acids formed the dominant group of volatile compounds in all samples, with 3-methylbutanoic acid being the major constituent. Ketones and alcohols had the highest share in total volatile compounds in the 2nd and 3rd months of fermentation. The total amount of volatile compounds was at the highest level in the 1st month of fermentation and then began to decrease gradually. The PCA discriminated the stage of fermentation as 3 groups (1st month, 2nd month and 3rd–4th month). The identification of the principal compounds produced during the fermentation can be of help in searching for indicators of off-flavor and as a fermentation index. This information can also be used in deciding when to stop the fermentation process to avoid production of compounds with off-flavor.

Conflicts of Interest The authors declare no conflict of interest.

Author Contributions M. Yilmaztekin and K. Sislioglu designed the study and interpreted the results. M. Yilmaztekin drafted the manuscript.

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