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Natural sparkling guava wine: volatile and physicochemical characterization/Espumante natural de goiaba: caracterizacao volatil e fisico-quimica.


The guava tree (Psidium guajava L.) is a fruit tree belonging to the Mvrtaceae family that is indigenous to tropical regions of Central and South America (LIMA et al., 2010). Guava fruit has a high nutritional value and sensorial acceptance, and it is consumed both fresh and in processed forms. Fruit processing minimizes post-harvest losses by obtaining products with a longer shelf-life and high added value. An alternative to reduce such losses and improve fruit utilization consist of the production of fermented fruit beverages, which consumer surveys indicate to be promising products due to their acceptance (SANDHU & JOSHI, 1995).

Commercialization of sparkling wine lias had a considerable evolution in Brazil, as represented by the increase in consumption influenced by the warm Brazilian climate as well as the pleasant flavors and aromas of these beverages. Thus, the use of fruit species other than grapes could pose a good alternative for the production of this type of beverage. Champagne, sparkling, or natural sparkling are types of wine whose carbonic anhydrides come exclusively from a second alcoholic fermentation step, whether in bottles (champenoiselixaoiuomi method) or in large containers (Chaussepied/Charmat method), with a minimum pressure of 4 atmospheres at 20[degrees]C and an alcohol content of 10% to 13% by volume (BRASIL, 2004). During this second fermentation step, the sparkling wine develops characteristic flavor compounds (TORRESI et al., 2011). Yeast autolysis also occurs during this period, yielding the release of intracellular compounds suchas amino acids, peptides, proteins, polysaccharides, nucleic acid derivatives, and lipids. These compounds are precursors to many volatile compounds, such as vitispirane and diethyl succinate, that contribute positively to the quality of sparkling wines (BOSCH-FUSTE et al., 2007). The volatile fraction of fermented fruits is very complex, consisting of compounds with different physicochemical properties (polarity, volatility, and solubility) and a wide range of concentrations (ETIEVANT & MAARSE, 1991).

Volatile compounds that are formed in this process are characterized as the tertiary aromas of the sparkling wine. While the primary aromas arise from the fruit and are reported in younger products, secondary aromas are formed primarily during the first fermentation (UBIGLI, 2004). SOARES et al. (2007) identified the esters 3(Z)-hexenyl acetate and 3(E)-hexenyl acetate as well as the sesquiterpenes caryophyllene, [alpha]-humulene, and (3-bisabolene in ripe guava fruit. Conversely, the study by PINO & QUERIS (2011) on the characterization of guava wine described twelve active odor compounds, including (3-damascenone, ethyl octanoate, ethyl hexanoate, and ethyl butanoate.

Several analytical techniques are employed for the extraction of volatile compounds. Among them, solid-phase microextraction (SPME) has been employed for the isolation of volatile compounds from foods, including alcoholic beverages. This technique has the advantage of being faster and easier to perform than solvent extraction and distillation; it also has good reproducibility and sensitivity (PINO & QUERIS, 2010). Although, published studies exist on the production and characterization of fermented fruit products, the literature is lacking in studies about volatile compounds in natural sparkling fruit wines (such as guava) and their acceptance by consumers. Thus, the objective of the present research was the development and physicochemical and volatile characterization of a natural sparkling guava wine produced by the champenoise method.


Preparation of natural sparkling guava wine

Ripe guavas of the Paluma variety were purchased from the Polytechnic College of the Universidade Federal de Santa Maria in the municipality of Santa Maria, Rio Grande do Sul State (RS). The guavas were washed under running water and sanitized with a 200mg L1 sodium hypochlorite solution (Quimea[R], Santa Maria, RS, Brazil) for 15min. The fruit was subsequently cut in half, with the manual removal of skin and seeds. Pulp was reserved for processing.

The must was obtained from pulp homogenized in a blender (Britania[R], Sao Paulo, Sao Paulo state (SP), Brazil). Pectinolytic enzymes (LAFAZYM enzyme system, Laffort[R], Bordeaux, France; 0.02g [L.sup.-1]) and sulfur dioxide (Veronese, Caxias do Sul, RS, Brazil; lOmg L1) were added to the must. Sucrose (Uniao, Sao Paulo, SP, Brazil; 243g [L.sup.-1]) was added until the must reached 16.2[degrees]Brix. Fermentation was conducted in 5L containers and initiated with the addition of commercial yeast (Saccharomvces cerevisiae, Laffort[R], Bordeaux, France; 0.2g [L.sup.-1]). At the end of the alcoholic fermentation, the fermented base product was transferred and bottled for the production of the sparkling wine, following the method described by ZOECKLEIN (2002). The fermented base was extracted from the bottles, followed by the addition of sucrose (Uniao, Sao Paulo, SP, Brazil; 24g [L.sup.-1]) and yeast (Saccharomvces cerevisiae. Laffort Bordeaux, France). A bentonite- and silicon dioxide-based coadjuvant (Pentagel[R], Diepoldsau, Switzerland; 0.1 Og [L.sup.-1]) and an ammonium sulfate- and thiamine-based fermentation activator (Thiazote, Laffort[R], Bordeaux, France; 0.10g [L.sup.-1]) were subsequently added. The product was bottled again and capped with a polyethylene plug. The remuage was performed for a period of three months, followed by degorgement and sealing with a cork stopper and wire protection. Sparkling wine was stored for two months. The entire process was carried out at 20-25[degrees]C on the premises of a commercial winery (Velho Amancio, Santa Maria, RS, Brazil). Cleaning was carried out at 5[degrees]C. All experiments were performed in triplicate.

Physicochemical analysis

Natural sparkling guava wine was analyzed for some of its physicochemical parameters. The pH was determined by direct reading of the sample using a pH potentiometer (Digimed[R], DM-22, Campo Grande, SR Brazil). Total acidity was determined using titration by neutralization up to a pH of 8.2 and is expressed as mEq [L.sup.-1] of tartaric acid. Meanwhile, the volatile acidity was determined by steam distillation followed by titration and is expressed in mEq [L.sup.-1] of acetic acid. Alcoholic strength was determined by Gibertini[R] distillation (Gibertini, Italy) according to AMEREME & OUGH (1980). Reducing sugar content was determined according to Lane and Eynon's method (ZOECKLEIN, 2002). All analyses were performed in triplicate.

Determination of volatile compounds

Analysis of volatile compounds was carried out using the headspace solid-phase microextraction technique. DVB/Car/PDMS fibers (Supelco[R], Sigma-Aldrich, Saint Louis, Missouri, USA; 50/30(im, 2cm long) were used in the procedure. Two hundred milliliters of sample was degassed in an ultrasound bath (Unique[R], USC-800, Indaiatuba, SP, Brazil) at 2[degrees]C for 40min. Sample aliquots of lOmL containing 3g of sodium chloride (Merck[R], Darmstadt, Germany) were packed in 20mL vials and immediately sealed with a screw cap containing a polytetrafluoroethylene (PTFE) septum. Volatile compound extraction was carried out at 35[degrees]C with 50min of fiber exposure to the sample headspace. The vial containing the sample was maintained for 5min under the same conditions prior to the extraction. Sample was stirred throughout the entire analysis period. Extractions were performed in triplicate.

Determination of volatile compounds was performed using a Shimadzu QP-2010Plus gas chromatograph coupled to a mass spectrometer (GC/MS). The SPME fiber containing the isolate was thermally desorbed in the GC/MS injector at a temperature of 250[degrees]C for 2min in the splitless mode. The separation of volatile compounds occurred on a capillary column consisting of fused silica coated with a ZB-5MS non-polar stationary phase (30m x 0.25mm x 0.25(im; Phenomenex, USA). Temperature program of the column started with 2 minutes at 35[degrees]C, followed by a 4[degrees]C/min temperature ramp until reaching 80[degrees]C and then one other temperature ramp (30[degrees]C min1) until 200[degrees]C was reached, where it remained in isothermal conditions for 5min. Helium (purity grade 5.0; White Martins, Osasco, SP, Brazil) was used as a carrier gas with a constant flow rate of 1.2mL [min.sup.-1]. The GC/MS interface and the ionization source were maintained at 250[degrees]C. Quadrupole mass analyzer was operated in the sweep mode (35-350/;/ [z.sup.-1]). Volatile compound quantification was performed through internal standardization by adding the internal standard 3-octanol (Sigma Aldrich, 10[micro]L of an ethanol solution at 82.2mg [L.sup.-1]) to the sample according to BERNARDI et al. (2014). Volatile compounds were identifiedby comparing analyte mass spectra with those in the NIST 05 spectral library and the experimental retention index (RI) of the analyte with the theoretical RI reported in the literature (NIST, El-Sayed, 2014). Experimental RIs were calculated from the retention times of a homologous series of alkanes (C8-C22) obtained under the same chromatographic conditions of the sample. Ethyl acetate, ethyl hexanoate, ethyl octanoate, 2-methyl1-butanol, 3-methyl-1-butanol, 1-hexanol, 1-octanol, phenylethyl alcohol, and hexanoic acid were positively identified by comparing the spectra and chromatographic peak RIs of samples and analytical standards. Odor thresholds of the volatile compounds identified in natural sparkling guava wine were estimated by comparison to values reported from the literature, shown in table 1, according to PINO & QUERIS (2011).


Physicochemical composition of natural sparkling guava wine

Few studies in the literature have employed fruits other than grapes in the production of natural sparkling wines (JIANQIANG et al., 2008; CARVALHO, 2009). Thus, the present results were discussed based on standards used for sparkling wines according to Law 10970 of 2004 that dictates the standards and quality for wine and the derivatives of grapes and wine (BRASIL, 2004).

The natural sparkling guava wine had a pH of 3.6 [+ or -] 0.2. Avalue below 4.0 makes this product less susceptible to acetic bacteria action, which could deteriorate the product (LOPES & SILVA, 2006). Results of the total and volatile acidity were 114.1 [+ or -] 1.7 and 13.1 [+ or -] 0.6mEq [L.sup.-1] of tartaric acid and acetic acid, respectively. Alcohol content was reported to be 12.0 [+ or -] 0.30% by volume at 20[degrees]C. Results allowed classification of the natural sparkling guava wine as being within the limits established by the Brazilian legislation of 50-130 and <20mEq [L.sup.-1] for the total acidity and volatile acidity, respectively, and as having an alcoholic graduation between 10 and 13%. The sugar content was calculated as 4.2 [+ or -] 0.6g [L.sup.-1], which places this sparkling wine in the "extra brut" category (sparkling wines up to 6g sugar [L.sup.-1] are categorized as "extra brut") (BRASIL, 2004).

Volatile compounds of the naturai sparkling gua\>a wine

Eighty-nine volatile compounds were detected, of which 42 were tentatively identified and 9 were positively identified by comparison with standards. Among the identified compounds, one can find 26 esters, 10 alcohols, 9 terpenes, 3 ketones, and 3 acids (Table 1). The remaining 37 unidentified volatile compounds comprised 9.42% of the total chromatogram area on average.

Esters were the predominant volatile compounds in natural sparkling guava wine sample, totaling a concentration of 1934.5(ig [L.sup.-1]. Compounds with the highest concentrations included ethyl octanoate (383.0[micro]g [L.sup.-1]), isobutyl acetate (335.0[micro]g [L.sup.-1]), ethyl 3(E)-hexenoate (176.0[micro]g [L.sup.-1]), ethyl decanoate (116.0[micro]g [L.sup.-1]), hexyl acetate (106.3[micro]g [L.sup.-1]), and 2-phenylethyl acetate (100.l[micro]g [L.sup.-1]). Ethyl octanoate, in this concentration range, may have contributed fruit/floral scent notes to the aroma of the beverage, as its concentration is 75 times higher than the perception threshold (TORRESI et al., 2011). This analyte can come from the matrix itself, as reported by CHEN et al. (2006), who reported a concentration of 159[micro]g [L.sup.-1] in guava. PINO & QUERIS (2011) reported the same compound at a concentration of 235.4[micro]g [L.sup.-1] in fermented guava and observed a relationship with the primary fermentation. Furthermore, other identified esters are also reported in the guava matrix, such as methyl hexanoate (2.78[micro]g [L.sup.-1]), ethyl hexanoate (396[micro]g [L.sup.-1]), ethyl decanoate (116[micro]g [L.sup.-1]) and 2-phenylethyl acetate (100[micro]g [L.sup.-1]) (SCHREIER & ID STEIN, 1985; CHEN et al., 2006; SOARES et al., 2007). Ethyl hexanoate and (E)-ethyl cinnamate compounds present in wine samples are described as fruity, strawberry, sweet, and floral flavor compounds (AZNAR et al., 2001).

Higher alcohols presented the highest total concentration in natural sparkling guava wine (4038.80[micro]g [L.sup.-1]), of which 3-methyl-1-butanol was the most abundant and may contribute to the characteristic odors of whiskey, malt, wine, banana, and sweet (SOUZA et al., 2010). This compound has already been reported in natural sparkling blackberry wine by JIANQIANG et al. (2008). Of the alcohols identified, 3-(Z)-hexenol (269.0[micro]g [L.sup.-1]), 1-hexanol (227.0[micro]g [L.sup.-1]), 1-octanol (5.1[micro]g [L.sup.-1]) and phenylethyl alcohol have been described in guava fruit "(SCHREIER & IDSTEIN, 1985) and guava wine (PINO & QUERIS, 2011). In the present research, phenethyl alcohol was the analyte with the second highest concentration; it had been reported as the largest odoriferous component in Finnish sherry and berry wines (NYKANEM, 1986). Higher alcohols (2- and 3-methyl-1-butanol, 1-hexanol) are compounds formed during the alcoholic fermentation process (PINO & QUERIS, 2011) and are; therefore, characteristic of wines and fermented beverages (SOUZA, 2010). These compounds are also abundant in Brazilian "brut" sparkling wines (RIZZON et al., 2000).

The terpenes present in natural sparkling guava wine amounted to 99.5[micro]g [L.sup.-1], with 9 different substances identified. PINO & QUERIS (2011) reported that this class represented only 0.2% of the volatile fraction of fermented guava, with six terpenes identified, all of which are also present in the fruit matrix. Of these six compounds, only two were identified in natural sparkling guava wine: [alpha]-terpineol and [beta]-damascenone. The former (as well as citronellol) can be formed during yeast autolysis during the second fermentation step (TORRESI et al., 2011). The compound (E)-6,10-dimethyl-5,9undecadien-2-one, also known as geranyl acetone, is classified as a norisoprenoid derived from the degradation of long chain terpenes ([beta]-carotene and lycopene), conferring a floral aroma to ripe fruit (LEWINSOHN et al., 2005; CENTENO & RUST, 2009). C13-norisoprenoids, such as vitispirane (21.50[micro]g [L.sup.-1]), [beta]-ionone (10.40[micro]g/L) and trans-[beta] -damascenone (1.10[micro]g [L.sup.-1]), were identified in the sparkling wine. Formation of these compounds may be related to the degradation of pigments in the guava matrix (MERC AD ANTE, 1999). These compounds are known for their contribution to the aroma of fruits and their derivatives. Vitispirane can be used to differentiate between young and aged sparkling wines (TORRESI et al., 2011) (2011). [beta]-Damascenone; although, reported in low concentrations, has a low odor threshold value (0.1[micro]g [L.sup.-1]) and can thus significantly influence the aroma of the product.

Only three compounds in the ketone class were identified: 6-methylhept-5-en-2-one, 2-pentanone, and acetophenone; PINO & QUERIS (2011) identified all three in the volatile fraction of fermented guava. The 6-Methylhept-5-en-2-one (4.90[micro]g [L.sup.-1]) and acetophenone (2.80[micro]g [L.sup.-1]) were also reported in guava (SCHREIER & IDSTEIN, 1985).

Three acids were quantified: hexanoic acid (1.0[micro]g [L.sup.-1]), octanoic acid (44.8[micro]g [L.sup.-1]), and decanoic acid (34.5[micro]g [L.sup.-1]). Hexanoic acid (also known as capric acid) is responsible for an unpleasant, pungent, and cheese-like odor; decanoic acid is also described as unpleasant with rancid notes (MOREIRA et al., 2012). According to PINO & QUERIS (2011), these acids have already been identified in the guava matrix, and in the present study, were above the threshold value of sensory perception (Table 1).

No aldehyde compounds were reported in the natural sparkling guava wine samples. SOARES et al. (2007) reported the abundance of aldehyde compounds in unripe guava as well as their decrease associated with an increase in esters throughout maturation process. A reduction of the aldehyde content was indicated during the aging of sparkling wine (TORRESI et al., 2011).


Natural sparkling wine that was produced presented a complex composition of fruity and floral aromas, determined by its volatile composition in which many compounds that are likely responsible for the beverage's aroma and flavor were identified. Among the compounds of higher sensory relevance, we found ethyl octanoate, ethyl hexanoate, phenethyl acetate, [beta]-(E)-damascenone, (E)-ethylcinnamate, 2-methylbutyl acetate, 3-methyl-1-butanol, ethyl 3-(E)-hexenoate, and methyl 5-hexenoate. Furthermore, the use of the method traditionally applied to grapes, the champenoise method, enabled the production of a natural sparkling guava wine with physicochemical characteristics equivalent to those of sparkling wines made from grapes.

Received 11.05.15 Approved 06.07.17 Returned by the author 07.24.17 CR-2015-1509.R2


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Silvana Maria Michelin Bertagnolli (1) * Gabrieli Bernardi (2) Jossie Zamperetti Donadel (1) Aline de Oliveira Fogaca (3) Roger Wagner (4) Neidi Garcia Penna (4)

(1) Programa de Pos-graduacao em Ciencia e Tecnologia dos Alimentos (PPGCTA), Centro de Ciencias Rurais (CCR), Universidade Federal de Santa Maria (UFSM), 97050-790, Santa Maria, RS, Brasil. E-mail: 'Corresponding author.

(2) Departamento de Engenharia e Tecnologia Ambiental, Universidade Federal de Santa Maria (UFSM), Campus Frederico Westphalen, Frederico Westphalen, RS, Brasil.

(3) Curso de Farmacia, Centro Universitario Franciscano (Unifra), Santa Maria, RS, Brasil.

(4) Departamento de Tecnologia e Ciencia dos Alimentos (DTCA), Centro de Ciencias Rurais (CCR), Universidade Federal de Santa Maria (UFSM). Santa Maria. RS. Brasil.
Table 1--Volatile compounds identified in natural sparkling guava

Class       No.               Compounds               ID

             1            Ethyl isobutvrate            b
             2             Isobutyl acetate            b
             3              Ethyl lactate              b
             4            Isopentyl acetate            b
             5          2-Methylbutyl acetate          b
             6             Methyl hexanoate            b
             7             Ethyl hexanoate             a
             8           3(E)-ethyl hexenoate          a
             9              Hexyl acetate              b
            10            5-Methyl hexenoate           b
            11           2(E)-ethyl hexenoate          b
            12             2-Ethyl fiiroate            b
            13             Methyl octanoate            b
Esters      14        3-Ethyl-3-hydroxyhexanoate       b
            15              Ethyl benzoate             b
            16             Ethyl octanoate             a
            17            Phenethyl acetate            b
            18           Isopentyl hexanoate           b
            19          2-Phenylethyl acetate          b
            20             Ethyl Decanoate             b
            21     Ethyl-3-methylbutyl butanedioate    c
            22            Diethyl succinate            b
            23           Phenylethyl lactate           c
            24           Isopentyl octanoate           b
            25           (E)-ethyl cinnamate           b
            26         Isopropyl tetradecanoate        b
            27            [alpha]-Terpineol            b

            28               Citronellol               b
            29               Vitispirane               b
            30                 B-ionone                b
            31          [beta]-(E)-damascenone         b
Terpenes    32             [beta]-Farnesene            b
            33      [alpha]-(Z)-bisabolene epoxide     b
            34             Geranyl acetone             b
            35             [beta]-Nerolidol            b
            36               2-Pentanone               b

            37         6-Methyl-hept-5-en-2-one        b
Ketones     38               Acetophenone              b
            39           2-Methyl-1-butanol            a

            40           3-Methyl-1-butanol            a
            41              3-(Z)-hexenol              b
            42                1-Hexanol                a
            43            2-Ethyl-hexan-l-ol           b
Alcohols    44                1-Octanol                a
            45              Phenylethanol              a
            46              3-(Z)-nonenol              b
            47                1-Nonanol                b
            48               2-Undecanol               b
            49              Hexanoic acid              a

            50              Octanoic acid              b
Acids       51              Decanoic acid              b

Class      [RI.sub.Exp]   ([[mu].sub.g]/L)     SD         Odor

               705             63.40          5.10         --
               744             14.60          9.02        66.0
               814             18.20          4.26       1400.0
               881             335.00         8.30         --
               883             60.20          2.37        30.0
               927              2.80          0.27         --
               1003            396.00         5.26        30.0
               1009            176.00         4.17        14.0
               1016            106.30         3.00         --
               1018             7.30          1.54         2.0
               1045             3.90          0.11         --
               1053            11.20          0.26         --
               1140             4.40          0.18         --
Esters         1143             1.51          0.75         --
               1174            51.10          0.59         --
               1198            383.00         6.08         5.0
               1242            21.60          0.31        0.50
               1249             4.50          0.29         --
               1254            100.10         2.30        250.0
               1384            116.00         8.95        200.0
               1427            21.40          0.55         --
               1430             5.80          0.11        70.0
               1434             2.90          0.81         --
               1442             4.20          5.40         --
               1460            21.60          1.37        1.10
               1781             1.50          0.79         --
               1190            19.70          0.31        330.0

               1224             2.70          0.12         --
               1272            21.50          0.03         --
               1308            10.40          0.18         --
               1379             1.10          0.03        0.10
Terpenes       1453             0.90          0.13         --
               1470             5.30          0.19         --
               1449            24.50          0.27         --
               1555            13.40          0.08         --
               742              1.90          0.16         --

               988              4.90          1.38         --
Ketones        1062             2.80          0.32        65.0
               744             33.60          6.43        280.0

               741            2434.00         63.67       280.0
               861             269.00         10.82       385.0
               874             227.00         12.88       600.0
               1031             1.80          0.07        270.0
Alcohols       1073             5.10          0.20         --
               1132           1059.00         33.29      1100.0
               1163             1.10          0.20        200.0
               1178             6.70          0.28         --
               1258             1.50          0.33         --
               991              1.00          0.48       3000.0

               1194            44.80          6.64
Acids          1376            34.50          3.57       10000.0

(a) Reliability of identification: a) positively identified through
the use of standards; b) tentatively identified through agreement of
the mass spectrum and RIexp with the literature; c) tentatively
identified through agreement of the mass spectrum with the
literature. (b) Published odor threshold data (PINO & QUERIS. 2011).
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Title Annotation:produccion de alimentos; texto en ingles
Author:Bertagnolli, Silvana Maria Michelin; Bernardi, Gabrieli; Donadel, Jossie Zamperetti; de Oliveira Fog
Publication:Ciencia Rural
Date:Sep 1, 2017
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