salto de escala1.pdf

8/18/2019 Salto de escala1.pdf

1/8

Scale-up of extremely low temperature fermentations of grape must by wheat

supported yeast cells

Panagiotis Kandylis a,*, Chryssoula Drouza b, Argyro Bekatorou a, Athanasios A. Koutinas a

a Food Biotechnology Group, Department of Chemistry, University of Patras, GR-26500 Patras, Greeceb Department of Agricultural Production and Food Science and Technology, Cyprus University of Technology, Limassol 3603, Cyprus

a r t i c l e i n f o

Article history:

Received 7 February 2010

Received in revised form 10 April 2010

Accepted 13 April 2010

Available online 18 May 2010

Keywords:

Immobilization

Wine

GC–MS

Wheat

Scale-up

a b s t r a c t

A new biocatalyst was prepared by immobilization of Saccharomyces cerevisiae AXAZ-1 yeast cells on

whole wheat grains. This biocatalyst was used for 30 repeated batch fermentations of glucose and grape

must at various temperatures. The biocatalyst retained its operational stability for a long period and it

was proved capable to produce dry wines of fine clarity even at extremely low temperatures (5 C). After

the completion of these fermentations the new biocatalyst was used in a scale-up systemof 80 L for wine

making at ambient (20C) and extremely low temperatures (2 C). The scale-up process did not affect the

fermentative ability of biocatalyst, even at low temperatures, while the produced wines had almost the

same improved aromatic profile compare to free cells as revealed by GC and GC–MS analyses. More spe-

cifically the results showed that both systems with immobilized cells (laboratory scale and 80 L bioreac-

tor) increased the formation of esters and produced wines with improved aromatic profile compared to

those with free cells. Finally an increase in the percentages of total esters and a decrease in those of

higher alcohols was observed in lower fermentation temperatures.

2010 Elsevier Ltd. All rights reserved.

1. Introduction

In recent years several immobilized cell systems have been pro-

posed for use in alcoholic fermentation, due to the several techni-

cal and economical advantages compared to free cells systems

(Kourkoutas et al., 2004). However, full industrial use is limited

to the production of sparkling wines (Fumi et al., 1988; Colagrande

et al., 1994). The supports that are used for immobilization in wine

making must be of food grade purity, low cost, abundant in nature,

easy to handle, suitable for low temperature fermentations and im-

prove wine quality. In recent years supports of food grade purity,

such as gluten pellets (Iconomopoulou et al., 2002), brewer’s spent

grains (Kopsahelis et al., 2007), sugarcane (Chandel et al., 2009),

delignified cellulosic material (Kourkoutas et al., 2002), potatoes(Kandylis and Koutinas, 2008) and corn starch gel (Kandylis

et al., 2008), have been proposed as ideal for yeast immobilization

for wine making and alcohol production. The use of alcohol resis-

tant and cryotolerant yeasts immobilized on these supports led

to low temperature fermentations producing wines with excellent

taste and aroma.

The use of immobilized cells on starchy supports in wine mak-

ing, especially at low temperatures, led to wines with improved

taste and aroma, while the reduced activation energy and the

higher reaction rate constant in the case of immobilized cells led

to the conclusion that these supports may behave as catalysts or

promoters of the enzymes involved in the process (Kandylis and

Koutinas, 2008; Kandylis et al., 2008).

Among the major cereals, wheat is the staple food in the diets of

a large segment of the world’s population. It accounts nearly

20–80% of the total food consumption in various regions of the

world. Wheat is produced worldwide in large amounts and can

be used as a support for cell immobilization reducing the cost com-

pared with gluten pellets and starch that need a costly process for

isolation. Likewise, wheat does not need the delignification cost

that is necessary in the case of brewer spent grains.

Therefore the aim of the present study was to evaluate the use

of wheat as a support for yeast immobilization suitable for ambientand low temperature fermentations and its scale-up from labora-

tory to semi-industrial scale.

2. Methods

2.1. Yeast strain and media

The alcohol resistant and cryotolerant Saccharomyces cerevisiae

AXAZ-1, isolated from Greek vineyard plantation was used in the

present study (Kopsahelis et al., 2009). It was grown on culture

medium consisting of 4 g yeast extract/L, 1 g (NH4)2SO4/L, 1g

KH2PO4/L, 5 g MgSO4 7H2O/L, and 40 g glucose monohydrate/L

0960-8524/$ - see front matter 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2010.04.031

* Corresponding author. Tel.: +30 2610 997104; fax: +30 2610 997105.

E-mail address: [email protected] (P. Kandylis).

Bioresource Technology 101 (2010) 7484–7491

Contents lists available at ScienceDirect

Bioresource Technology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o r t e c h

http://dx.doi.org/10.1016/j.biortech.2010.04.031mailto:[email protected]://www.sciencedirect.com/science/journal/09608524http://www.elsevier.com/locate/biortechhttp://www.elsevier.com/locate/biortechhttp://www.sciencedirect.com/science/journal/09608524mailto:[email protected]://dx.doi.org/10.1016/j.biortech.2010.04.031


2/8

at 30 C and centrifuged at 5000 rpm for 10 min. All media were

sterilized at 130 C and 1.5 atm for 15 min. Must of roditis grape

variety, was provided from the local wine industry ‘‘Achaia Clauss”,

with 11.3–12.1 initial oBe density (199–211 g/L), total acidity of 6–

7 g tartaric acid/L and sulphur dioxide content of 60 mg/L.

2.2. Preparation of support and cell immobilization

Whole wheat grains were boiled for 15 min and sterilized at

130 C and 1.5 atm for 15 min. Cell immobilization was carried

out at 30 C by mixing 100 g (dry weight, 140 g wet weight) of

the support and 16 g wet weight AXAZ-1 cells in a 1-L glass cylin-

der containing 800 mL of 12% (w/v) glucose culture medium. The

system was allowed to ferment until the density of the fermented

liquid reached 0–0.5 oBe. The fermented liquid was decanted and

the support was washed two times with 12% (w/v) glucose culture

medium.

2.3. Enumeration of immobilized cells and determination of cell

viability

Representative 10 g portions of duplicate wheat grains samples

taken after immobilization were blended with 90 mL of sterilized

ringer solution (1/4 strength) and subjected to 10 serial dilutions.

The enumeration of immobilized yeast cells made on Malt agar

and on an agar with the following (w/v) content: glucose 4%, yeast

extract 0.4%, (NH4)2SO4 0.1%, KH2PO4 0.1% and MgSO4 7H2O 0.5%

after incubation at 30 C for at least 72 h (Kandylis and Koutinas,

2008). The number of immobilized cells on wheat grains was

approximately constant during fermentations (9.6 107 cells g

wet wheat, corresponding to 6.35 103 g dry weight cells/g wet

wheat) with small reduction with the drop of fermentation

temperature.

2.4. Fermentations

The immobilized biocatalyst was used for 30 repeated batch fer-mentations without any agitation. The fermentations were carried

out initially at 30 C using 400 mL glucose medium (11.5 oBe) then

400 mL must was used and the temperature was successively de-

creased to 25, 20, 15, 10 and 5 C. When the fermentation was

completed the liquid was collected for analyses and the support

was washed twice with 200 mL of glucose medium or must and

then used for the next fermentation batch. For comparison must

fermentations using free cells were also carried out.

2.5. Pilot-plant operation

2.5.1. 80-L bioreactor

The main bioreactor was a vertical cylinder made from stainless

steel, 0.90 m tall with a 0.34 m internal diameter and 80 L capacity.A second tank was used for the grape must. It was a vertical cylin-

der made from stainless steel, 0.80 m tall with a 0.34 m internal

diameter and 70 L capacity. Grape must was transferred from that

tank to the main bioreactor through a volumetric pump. For cell

growth air was supplied through a bacteriostatic filter using a

compressor.

2.5.2. Cell growth and cell immobilization

The growth of S. cerevisiae cells was carried out inside the 80 L

bioreactor. More specifically, 40 L of grape must (2 oBe) enriched

with 58 g (NH4)2SO4 and 19g KH2PO4 and 80 g of S. cerevisiae

F{FZ-1 cells were added in the 80 L bioreactor. Air, supplied by

a compressor, passed through a bacteriostatic filter and was spread

into the fermentation medium. The whole left for 24 h to fermentand then 10 kg of boiled wheat grains and 10 L grape must (12 oBe)

was added. The whole left to ferment for 8 h, without air supply.

Then the fermentation medium decanted and the biocatalyst

washed twice with 10 L of grape must (11.5 oBe) for the removal

of free cells.

2.5.3. Fermentations

The immobilized biocatalyst was used for eight repeated batch

fermentations without any agitation at several temperatures rang-ing from 20 to 2 C and using 50 L grape must (11.5 oBe). When the

fermentation was completed the liquid was collected for analyses.

The support was washed twice with grape must for the removal of

any free cell and then was used for the next fermentation batch.

2.6. Analyses

2.6.1. Determination of residual sugar, ethanol and acidities

Fermentation kinetics were performed by measuring the Be

density at various time intervals. Residual sugar, ethanol, total

and volatile acidity were determined as described previously

(Kandylis et al., 2008).

Wine productivity was calculated as grams of wine per liter to-

tal volume produced per day. Ethanol productivity was expressed

as g of ethanol produced per day per L liquid volume of bioreactor.

Conversion was calculated by the following equation:

ðInitial sugar concen:

Residual sugar concenÞ=Initial sugar concen: 100:

2.6.2. Determination of volatile by-products

Volatiles such as acetaldehyde, ethyl acetate, 1-propanol, isobu-

tanol and amyl alcohols were determined as described previously

(Kandylis et al., 2008).

2.6.3. Headspace solid phase micro-extraction (SPME) gas

chromatography–mass spectrometry (GC–MS) analysis

The volatile constituents of the wines produced by free and

immobilized cells on wheat were determined by means of gaschromatography–mass spectroscopy. More specifically, the vola-

tiles were isolated by the headspace solid phase micro-extraction

(SPME) method. The fibre used for the absorption of volatiles

was a 2-cm fibre coated with 50/30 mm divinylbenzene/carboxen

on poly-dimethyl-siloxane bonded to a flexible fused silica core,

(Supelco, Bellefonte, PA, USA). The conditions of headspace-SPME

sampling were as follows: 10 mL liquid sample, 3 g NaCl and inter-

nal standard (4-methyl-2-pentanol) were transferred into a 20-mL

headspace vial fitted with a teflon-lined septum sealed with an

aluminum crimp seal. The contents were magnetically stirred for

5 min at 60 C, and then the fibre was exposed to the headspace

for 45 min. The length of the fibre in the headspace was kept con-

stant. Desorption of volatiles took place in the injector of the gas

chromatograph in the splitless mode, at 240 C for 3 min. Beforeeach analysis, the fibre was exposed to the injection port for

5 min to remove any volatile contaminants. GC–MS analysis was

performed on a Shimadzu GC-17A gas chromatograph coupled to

a Shimadzu MS QP5050 mass spectrometer. Helium was used as

carrier gas (1.8 mL/min). Separation of compounds was performed

on a capillary column (Supelco CO Wax-10 60 m, 0.32 mm i.d.,

0.25 lm film thickness). Oven temperature was programmed at35 C for 6 min and then it was raised to 60 C with a rate of

2 C/min, held constant for 5 min, raised to 200 and 250 C with

a rate of 5 and 25 C/min respectively. Finally, it was held at

250 C for 6 min. The injector and interface temperatures were

set at 240 C. The mass spectrometer was operated in scan range

45–400 m/z. Identification of the compounds was effected by com-

paring (i) the Kováts’ retention indices based on the even n-alkanes(C10–C24) with those of standard compounds and by the literature

P. Kandylis et al./ Bioresource Technology 101 (2010) 7484–7491 7485


3/8

Kováts’ retention indices, and (ii) MS data with those of standard

compounds and by MS data obtained from NIST107, NIST21 and

SZTERP libraries. Semi-quantitative analysis was performed by

dividing the peak area of a compound with the peak area of the

internal standard and multiplying the result with the concentra-

tion of the internal standard (1.62 mg/L).

2.6.4. Electron microscopyPieces of the immobilized biocatalysts prepared with wheat

grains having immobilized S. cerevisiae AXAZ-1 cells were washed

with deionized water and dried overnight at 30 C. The samples

were coated with gold in a Balzers SCD 004 Sputter Coater for

3 min and examined in a JEOL model JSM-6300 scanning electron

microscope.

2.7. Experimental design and statistical analysis

All analyses were carried out in triplicate and the mean values

are presented (standard deviation for all values was about ±5% in

most cases). In the experiments conducted, the effect of immobili-

zation, fermentation temperature and scale-up on fermentation

parameters and formation of volatiles (ethanol, acetaldehyde, ethyl

acetate, isobutanol, 1-propanol and amyl alcohols) during glucose

synthetic medium and must fermentations were studied. The

experiments were designed and analyzed statistically by ANOVA.

Duncan’s multiple range test was used to determine significant dif-

ferences among results (coefficients, ANOVA tables and signifi-

cance ( p < 0.05) were computed using Statistica v.5.0 (StatSoft,

Inc., Tulsa, USA)).

3. Results and discussion

It is the first time that whole wheat grains were evaluated as

supports for yeast cell immobilization and their feasibility for re-

peated batch fermentation of glucose and grape must at various

temperatures was examined. Whole wheat grains were boiled for

15 min and after sterilization and cooling, immobilization took

place by mixing the wheat grains with a liquid culture of yeast cells

and the system left to ferment for 8 h. The immobilization was

confirmed by electron microscopy, showing yeast cells attached

on the surface of wheat grains and also mixed and entrapped in-

side them. Thus the available area of immobilization is increased

making possible the immobilization of a large number of cells

per grain. An enumeration of the immobilized cells by microbiolog-

ical analysis showed that more than 9.6 107 cells/g of wheat are

immobilized. In addition the immobilization was proved by using

the new biocatalyst for repeated batch fermentations of glucose

and grape must at various temperatures.

3.1. Wine making by wheat grains supported biocatalyst in laboratory

scale

The first four fermentations were carried out at 30 C using syn-

thetic glucose medium with glucose concentration from 109 up to

201 g/L for better adaptation of the biocatalyst. All the other fer-

mentations were carried out using grape must with initial sugar

concentration ranging between 190 and 211 g/L. After the comple-

tion of five repeated batch fermentations, the temperature was de-

creased for 5 C initially to 25 C and then to 20, 15, 10 and 5 C.

Fermentation temperature affected significantly fermentation

time, ethanol productivity and wine productivity ( p < 0.05), while

did not affect residual sugar, ethanol concentration and conversion

( p > 0.05). The immobilized biocatalyst retained its operational sta-

bility for a period longer than 5 months, even at low temperatures

and produced wines in 3 days at 20 C and 18 days at 5 C (Table 1).

The wines produced were of fine clarity and contained alcohol at

concentrations similar to those of dry table wines. The number of

free cells in the final product was low, especially at low tempera-

tures. This result proves that the fermentations were carried out

by immobilized cells. Ethanol and wine productivities were rela-

tively high while total acidity and volatile acidity were in the level

of commercial dry wines.

3.2. Wine making by wheat grains supported biocatalyst in 80 L

bioreactor

After the completion of fermentations in the laboratory scale

the biocatalyst was used for wine making in a scale-up system of

80 L. The bioreactor was cylindrical, made by stainless steal and

had 80 L capacity. The grape must was transferred to the bioreactor

using a pump through a second smaller tank. The growth of cells

took place inside the bioreactor, under aerobic fermentation and

the air was transferred through a bacteriostatic filter using a pump.

After the completion of each fermentation, the fermentation med-

ium was collected through spigots located at the bottom of biore-

actor. Inside the bioreactor, 5 cm higher than the exits, a stainless

steal grating was located having holes of 3 mmin diameter in order

to keep the biocatalyst (wheat) and prevent the closing of holes

and the same time to allow the must to pass through without

problems.

The system was used for eight repeated batch fermentations of

grape must, with initial sugar concentration ranging between 209

and 212 g/L, at various temperatures ranging from 20 to 2 C (Table

2). Fermentation temperature affected significantly fermentation

time, ethanol and wine productivity ( p < 0.05), while did not affect

residual sugar, ethanol concentration and conversion ( p > 0.05).

The immobilized biocatalyst retained its operational stability even

Table 1Kinetic parameters of low temperature repeated batch fermentations of grape must with immobilized S. cerevisiae AXAZ-1 yeast cells on wheat in laboratory scale.

Temperature

(C)

Batch Initial sugar

(g/L)

Fermentation

time (h)

Residual

sugar (g/L)

Ethanol

(% v/v)

Conversion

(%)

Ethanol productivity

(g/L/day)

Wine productivity

(g/L/day)

30 1glc 109a 52a Tr 5.9a 100a 22b,c –

2glc 143b 47a Tr 7.7b 100a 31a,b –

3glc 198c 44a 0.2a 9.7c 100a 42a –

4glc 201c 49a 19.9c 11.1d 90.0d,e 43a –

30 5gm 198c 54a 17.6b,c 11.7d 91.1c,e 41a –

25 6–10gm 202.6 ± 8.6c 67.2 ± 11.4a 9.5 ± 2.7a,b 11.5± 0.2d 95.3± 1.3a,c 33.6± 6.9a,b 253.8± 55.2a

20 11–15gm 201.6 ± 6.8c 72.8 ± 18.0a 14.9± 5.7a,c 11.5± 0.1d 92.6± 3.1b,c,d 31.2± 7.5a,b 238.3± 57.3a

15 16–20gm 197.8± 2.3c 81.8 ± 12.2a 9.9 ± 1.4a,b 11.4± 0.4d 95.0 ± 0.7b,c 27.0± 4.1b,c 205.9± 30.0a

10 21–25gm 194.5± 5.0c 131.0± 15.1a 13.2± 4.6a,c 11.6± 0.2d 93.2± 2.4b,c,d 17.0± 1.8c,d 127.6± 14.7a,b

5 26–30gm 202.0 ± 5.6c 414.8± 76.6b 6.8 ± 1.9a 11.5± 0.2d 96.7± 0.9a,b 5.4 ± 1.0d 40.9± 7.0b

Tr, traces; significant differences ( p < 0.05) in the same column are indicated by different letters in superscript.glc

Glucose medium.gm Grape must.

7486 P. Kandylis et al. / Bioresource Technology 101 (2010) 7484–7491


4/8

at low temperatures as in the fermentations of laboratory scale.

The wines produced were of fine clarity and contained alcohol at

concentrations similar to those of dry table wines. An increase in

the fermentation time was observed with the drop of temperature

from 20 to 15 C, however further decrease to 10 C led to lower

fermentation times. This trend was not observed in the experi-

ments in the laboratory scale and can be attributed to the higher

number of repeated batch fermentation in the same temperature

in the laboratory scale, which had as a result the better adaption

of cells in the drop of temperature. Ethanol and wine productivities

were relatively high while total acidity and volatile acidity were in

the level of commercial dry wines. Fermentation temperature af-

fected significantly ( p < 0.05) total and volatile acidity with lower

values observed at lower temperatures (Table 3).

3.3. Major volatile by-products

Most of the compounds that contribute to the aroma of wines

are produced during must fermentation and very few derived from

grapes. The most abundant compounds in the wine aroma are acet-

aldehyde, ethyl acetate, 1-propanol, isobutanol and amyl alcohols,

accounting for more than half of them (Regodón Mateos et al.,2006). Their concentrations are responsible in great matter for

the formation of characteristic flavor of wines, however extremely

high concentrations have been correlated with spoilage in wines

and cider (Polychroniadou et al., 2003). The concentrations of these

compounds in the wines produced in 80 L bioreactor are summa-

rized in Table 3. Fermentation temperature affected significantly

( p < 0.05) all the major volatiles by-products. Acetaldehyde ranged

in concentrations from 11 to 35 mg/L. The lowest concentration

was observed at 20 C, while reduction in fermentation tempera-

ture led to higher concentrations, but still inside the usual levels

produced by S. cerevisiae strains (Fleet and Heard, 1993; Antonelli

et al., 1999). Ethyl acetate, the most important and abundant ester

in wines (Plata et al., 2003; Ribérau-Gayon et al., 2000; Rapp and

Mandery, 1986), ranged in concentrations from 57 mg/L at 20 C

to 86 mg/L at 2 C and a significant increase ( p < 0.05) with the

drop of fermentation temperature was observed. It is considered

that such low concentrations of ethyl acetate (50–80 mg/L) con-

tribute to wine olfactory complexity having a positive impact on

wine quality and only at concentrations higher than 120 mg/L

may spoil the bouquet with an unpleasant, pungent tang (Ribé-

rau-Gayon et al., 2000). Higher alcohols are considered as the larg-

est group of flavor compounds in wines and among them amyl

alcohols, 1-propanol and isobutanol have high importance (Longo

et al., 1992). They were detected at concentrations that contribute

to the pleasant flavors of the wines produced (Rapp and Mandery,

1986; Ribérau-Gayon et al., 2000) and a significant drop ( p < 0.05)

in their concentrations with the drop of fermentation temperature

was observed, as in many previous studies (Etievant, 1991; Vidrih

and Hribar, 1999; Kandylis and Koutinas, 2008; Kandylis et al.,

2008).

3.4. SPME-GC–MS analysis of wines

For the evaluation of the aromatic profile, wine samples, pro-

duced by free and immobilized cells were analyzed using aSPME-GC–MS technique and the results are presented in Table 4.

In the case of immobilized cells the results from the wines pro-

duced in laboratory scale and in 80 L bioreactor are presented. In

total, 88 compounds were detected, of which 47 in wines produced

at 15 C by free cells and 54 by immobilized cells in laboratory

scale and 80 L bioreactor. Further reduction of fermentation tem-

perature did not affect significantly the qualitative aroma profile.

Semi-quantitative analysis showed that immobilized cells pro-

duced significantly higher concentrations of esters and other com-

pounds that provide improved characteristic flavor. The reduction

of fermentation temperature from25 to 15 C led to higher concen-

trations of all compounds, while further reduction of fermentation

temperature led to lower concentrations of all compounds

Table 3

Volatiles and acidity of wines produced by low temperature repeated batch fermentations of grape must using immobilized S. cerevisiae AXAZ-1 yeast cells on wheat in 80 L

bioreactor.

Temperature

(C)

Batch Volatile acidity

(g of acetic acid/L)

Total acidity

(g of tartaric acid/L)

Acetaldehyde

(mg/L)

Ethyl acetate

(mg/L)

1-Propanol

(mg/L)

Isobutyl alcohol

(mg/L)

Amyl alcohols

(mg/L)

Total

volatiles (mg/L)

20 1 0.5a 5.6a 11a 57a 13a 23a 226a 330a

20 2 0.4a,b 5.7a 14a 59a 10a,b 20a 226a 329a

15 3 0.4a,b 5.7a 14a 62a 9b,c 20a 183b 288b

15 4 0.5a 5.7a 32c 63a 7b,d 16b 122c 240c

12 5 0.4a,b 5.7a 23b 76b 6c,d,e 12c,d 89d 206d

10 6 0.3b,c 5.4b 27b 74b 4d,e 13b,c 96d 214d

5 7 0.3b,c 5.2c 33c 80b,c 5d,e 11c,d 87d 216d

2 8 0.2c 5.2c 35c 86c 3e 9d 80d 213d

Significant differences ( p

< 0.05) in the same column are indicated by different letters in superscript.

Table 2

Kinetic parameters of low temperature repeated batch fermentations of grape must with immobilized S. cerevisiae AXAZ-1 yeast cells on wheat in 80 L bioreactor.

Temperature

(C)

Batch Initial sugar

(g/L)

Fermentation time

(h)

Residual sugar

(g/L)

Ethanol

(% v/v)

Conversion

(%)

Ethanol productivity

(g/L/day)

Wine productivity

(g/L/day)

20 1 210.6 184a 14.2 11.1 93.3 11.4a 90.0a,b

20 2 212 173a 14.9 11.0 93.0 12.1a 95.7a

15 3 209 305b,c 13.8 11.3 93.4 7.0b,c,d 54.3c

15 4 209.3 375c 10.7 11.4 94.9 5.8c,d,e 44.1c

12 5 208.9 216a,b 13.8 11.3 93.4 9.9a,c,d 76.6b

10 6 211 192a 13.3 11.2 93.7 11.1a,b,d 86.2a,b

5 7 210.3 624d 14.2 11.3 93.2 3.4d,e 26.5d

2 8 209.5 1152e 12.7 11.4 93.9 1.9e 14.4d

Significant differences ( p < 0.05) in the same column are indicated by different letters in superscript.



5/8

Table 4

Volatile compounds (mg/L) identified in wines produced by free and immobilized S. cerevisiae AXAZ-1 yeast cells on wheat at laboratory scale and 80 L bioreactor using SPME-GC-

MS technique.

KI Compound Free cells Immobilized cells of S. cerevisiae-AXAZ-1 on wheat

Laboratory scale 80 L bioreactor

25 C 15 C 5 C 25 C 20 C 15 C 10 C 5 C 20 C 15 C 10 C 5 C

Esters

925 Ethyl formateb 0.013 0.009 0.0301040 Ethyl butanoatea 3.246 0.175 1.011 1.153 2.093 1.025 1.006 0.489 1.185 1.024 1.000

1123 3-Methylbutyl acetatec 5.218 7.976 9.051 8.618 6.642 4.987 7.852 5.769 6.995

1258 Ethyl hexanoatea 0.797 0.606 0.612 3.412 4.015 6.034 5.033 3.804 3.175 5.761 5.688 4.520

1307 Hexyl acetatec 0.008 0.016

1361 Ethyl heptanoatec 0.012 0.008 0.019 1.130 0.968 1.123

1386 Ethyl 2-hydroxypropanoatec 1.591 3.464 2.121 2.332 3.321 5.505 3.993 3.344 6.245 6.096 9.280 6.288

1396 Heptyl acetatec 0.034 0.039

1401 Methyl octanoatea 1.089 1.870 2.032 1.877

1451 Ethyl octanoateb 2.495 4.505 1.658 4.217 6.785 9.154 8.564 7.459 1.885 6.309 4.849 3.046

1477 3-Methylbutyl hexanoateb 0.015 0.005 0.010 0.014

1497 Octyl acetateb 0.012 0.030

1506 Ethyl 7-octenoateb 0.117 0.022 1.047 2.019 3.043 2.013 1.644 0.987 1.847 2.036 1.955

1553 Ethyl 3-hydroxybutanoatec 0.057 0.068 0.043

1564 2-Furanmethanol acetatec 1.123 0.024 0.021 0.356 0.030 0.031 1.153 0.964 1.045

1652 Ethyl decanoateb 1.772 1.297 1.176 3.916 5.188 7.426 5.851 4.368 6.523 8.631 7.668 8.045

1676 3-Methylbutyl octanoateb 0.038 0.225 0.150 0.084 0.025 0.054 0.025

1700 Diethyl butanedioate

b

0.900 2.382 1.981 1.327 2.197 3.374 2.614 2.208 3.265 3.895 2.773 3.5701709 Ethyl 9-decenoateb 1.312 2.166 3.623 3.384 5.101 7.168 5.658 4.830 0.126 2.336 1.505 1.346

1736 Phenylmethyl acetateb 0.039

1808 Methyl salicylatec 0.018 0.048 0.070 0.018 0.033 0.025

1809 Ethyl benzeneacetateb 0.133 0.038 0.148 0.030 0.092 0.098 0.037 0.019 0.021 0.019 0.020

1847 2-Phenylethyl acetateb 0.893 2.585 1.817 5.027 7.208 8.398 6.991 4.085 0.694 2.721 1.761 2.286

1850 Ethyl dodecanoateb 0.734 0.184 0.267 2.356 4.658 5.348 3.915 2.967 0.156 4.719 4.195 4.456

1889 3-Methylbutyl decanoatec 0.117 0.097 0.061

2041 Isopropyl myristatec 0.030 0.083 0.053 0.117

2094 Ethyl tetradecanoatec 0.238 0.154 0.341 2.061 3.011 3.501 2.243 2.135 1.786 2.186 2.000 2.214

2185 Dibutyl phthalatec 0.269 0.814 1.267

2271 Ethyl hexadecanoatec 0.845 0.590 0.955 0.465 2.020 2.369 2.413 1.934 0.600 1.434 1.506 1.931

2292 Ethyl 9-hexadecenoateb 2.330 0.443 2.364 2.481 1.275 6.626 4.441 3.930 0.473 3.185 3.280 3.864

2365 Diethyl phthalatec 8.231 10.121 5.002 1.216 3.505 4.243 2.247 1.509

2416 Ethyl octadecanoatec 0.024 0.100 0.330 0.158 1.145 1.160 1.025 0.124 2.197 2.139 2.316

2435 Ethyl 9-octadecenoatec 0.231 0.326 1.289 0.798 1.099 0.263 1.296 1.308 1.331

Sum of esters 25.902 28.944 24.040 39.769 59.646 86.628 68.032 54.353 33.808 65.878 62.186 53.395

Total esters 18 17 19 20 22 25 23 27 21 21 25 23

Organic acids

1534 Acetic acidc 0.322 6.689 11.314 0.083 1.330

1633 Butanoic acidb 0.007 0.022

1962 Hexanoic acidc 0.726 0.850 0.626 0.115 0.135 0.151 4.120 1.890 0.150

2156 Octanoic acidc 9.880 11.145 8.730 7.224 8.941 10.369 11.588 10.442 2.526 3.350 1.762 3.072

2213 Nonanoic acidc 0.354 0.790 0.073 0.119 0.126 0.757

2336 Decanoic acidc 1.723 2.113 0.580 3.483 4.503 5.038 5.404 4.362 0.924 2.356 1.172 1.608

2390 Undecylenic acidc 0.814 1.772 0.506 0.247

2425 Dodecanoic acidc 0.366 0.011

Sum of acids 13.771 21.587 21.257 10.780 13.570 15.407 16.992 15.753 3.720 11.703 6.786 5.834

Total acids 6 5 5 3 4 2 2 4 4 6 6 5

Alcohols

1179 1-Butanolb 0.038 0.114 0.117 0.015 0.300 0.063 0.014 0.043

1305 3-Penten-1-olc 0.086 0.323 0.150 0.293 0.130 0.136 0.284 0.111 0.038 0.019 0.021 0.038

1311 3-Hexen-1-olb

0.043 0.044 0.0561329 4-Methyl-1-pentanolb 0.004 0.029 0.011 0.017 0.007 0.013 0.023 0.011

1350 3-Methyl-1-pentanolb 0.009 0.014 0.049 0.077 0.044 0.069 0.026 0.358 0.053 0.130 0.062

1370 1-Hexanola 0.079 0.090 1.694 0.967 2.275

1419 3-Ethoxy-1-propanolc 0.035 0.008 0.026 0.020 0.032 0.045 0.063 0.099 0.062

1433 2-Butoxy-ethanola 0.026 0.012

1470 1-Heptanolb 0.031 0.079 0.183 0.211 0.120 0.076 0.208 0.123 0.042 0.055 0.051 0.092

1520 2-Ethyl-1-hexanolc 0.007 0.013 0.042 0.023 0.025 0.027 0.004 0.030

1559 2.3-Butanediolb 3.143 3.883 2.791 8.947 8.738 7.366 6.308 5.873 2.256 4.377 2.370 1.402

1570 1-Octanola 0.601 0.488 0.321 0.708 0.757 0.321 0.325 0.016 0.193

1590 1.3-Butanediolb 2.673 2.343 1.765 4.540 4.918 4.615 3.870 3.592 0.628 2.948 1.362 0.703

1605 Inositolc 0.028 0.167 0.126 0.224

1628 2-(2-Hydroxyethoxy) ethanola 0.094

1680 2-Furanmethanola 0.367 0.261 0.029

1730 3-(Methylthio) 1-propanolb 0.385 0.244 0.530 0.751 1.422 0.791 0.538 0.696 0.139 0.248 0.195 0.102

1783 Decanolc 0.150 0.241 0.085 0.086 0.097 0.024

1916 Benzyl alcohola 0.045 0.049 0.016 0.034 0.015 0.033 0.143 0.116



6/8

(Mallouchos et al., 2007; Kandylis et al., 2008; Kandylis and

Koutinas, 2008). However, an increase in the percentage of total es-ters and a decrease in the percentage of higher alcohols with the

drop of temperature from 15 to 5 C was also observed (Fig. 1),

which is in agreement to other recent studies and is considered

to have a positive impact to wine aroma (Mallouchos et al.,

2002; Kandylis et al., 2008). No significant differences were

observed in qualitative and quantitative characteristics of wines

produced in laboratory scale and in 80 L bioreactor ( p > 0.05),

showing that the scale-up of the systemdid not affect the aromatic

characteristics of the produced wines.

A total of 36 esters detected in the present study in wines pro-

duced by immobilized cells while only 18 by free cells. In both free

and immobilized biocatalysts, an increase in the concentrations of

esters was observed with the decrease of temperature to 15 C,

while further reduction of temperature led to a decrease. However,in all temperatures wines produced by immobilized cells pre-

sented higher numbers and concentrations of esters than those

produced by free cells, especially at 15 C. Very few esters are pres-ent in grapes and the majority of them were produced during fer-

mentation as a result of yeast metabolism (Fraile et al., 2000). The

main ester in our samples and wines in general was ethyl acetate.

Other esters present in all wines samples were those of fusel alco-

hols and sort chain fatty acids, the so-called ‘‘fruit esters”. In the

case of immobilized cells their content was higher compared to

free cells. Some examples are ethyl hexanoate, contributing fruity

notes to wine aroma, ethyl octanoate, having a floral fruity, musty

impact and ethyl dodecanoate which is known for its smokey,

earthy, dried fruit, spicy and toasty aroma (Miranda-Lopez et al.,

1992). In addition high concentration of esters of aliphatic acids

like decanoic, dodecanoic, tetradecanoic etc. are responsible for a

yeast tone (Molnar et al., 1981), which is characteristic in wines

with small aging (Pisarnitskii, 2001). Furthermore, many studiessupport that any factor that decreases the speed of fermentation

Table 4 (continued)

KI Compound Free cells Immobilized cells of S. cerevisiae-AXAZ-1 on wheat

Laboratory scale 80 L bioreactor

25 C 15 C 5 C 25 C 20 C 15 C 10 C 5 C 20 C 15 C 10 C 5 C

1933 2-Phenylethanola 9.596 6.086 4.923 17.236 19.776 18.571 14.346 10.307 12.428 10.225 7.290 4.280

1998 Dodecanola 0.150 0.105 0.176 0.086 0.040 0.055 0.109 0.007 0.057

2022 Phenolc 0.038 0.031 0.023 0.021

2063 3-Phenoxy-1-propanolc 0.157 0.059 0.154 0.045 0.0632312 2.4-bis-(1.1-Dimethylethyl) phenolc 0.516 0.537 0.587 0.232 0.185 0.079 0.218 0.111 2.931 0.363 0.268

2359 Hexadecanolc 0.395 0.249 0.171 0.337 0.285 0.138 0.291 0.139

Sum of alcohols 17.682 1 4.286 1 1.883 3 3.015 36.428 33.264 27.133 2 1.985 1 8.517 22.224 1 2.341 9.783

Total alcohols 15 13 11 18 18 20 19 18 14 19 13 16

Carbonyl compounds

1390 Nonanalb 0.006 0.011 0.010 0.016 0.011

1486 Furfurala 0.352 0.135 0.042 0.051 0.034 0.062 0.012 0.010

1533 Benzaldehydeb 0.293 0.934 0.043 0.080 0.063 0.061 0.142 0.093 0.277 0.295

1578 5-Methyl-furfuralc 0.498 0.732 0.065 0.284 0.130 0.315

1834 b-Damascenoneb 0.024 0.007 0.056

2005 c-Dodecalactoneb 0.028 0.024 0.025

Sum of carbonyl compounds 1.143 0.159 1.708 0.166 0.398 0.063 0.236 0.443 0.158 0.105 0.301 0.341

Total carbonyl compounds 3 2 3 4 4 1 4 4 2 2 2 4

Terpene c ompounds1189 Limonenea 0.081 0.201 0.021 0.576 0.018

1454 cis-Linalool oxideb 0.022 0.034 0.017 0.030 0.019 0.029 0.019 0.014

1556 b-Linaloolb 0.032 0.159 0.262 0.116

1679 Citronellol acetatec 0.205 0.177 0.017

1715 a-Terpineolb 0.267 0.645 0.169 0.309 0.094 0.271 0.2891738 a-Fernesenec 0.259 0.0901741 Geranialc 0.056 0.035 0.189 0.200 0.091

1790 b-Citronellolc 0.101 0.088 0.188 0.141 0.221 0.053 0.076 0.095

1843 Anetholec 1.105

1928 Isophytolc 0.148 0.025 0.045 0.108 0.109 0.146 0.460 0.444

2090 Nerolidolc 0.058 0.081 0.117 0.036 0.065 0.116 0.033 0.063 0.081

2243 Farnesyl acetatec 0.248 0.155 0.141 0.387 0.341

2343 Farnesolc 0.136 0.181 0.520 0.146 0.382 0.387 0.100 0.205 0.057 0.067

Sum of terpene compounds 0.551 1.629 1.199 0.916 1.526 1.066 1.358 1.675 0.318 0.712 0.600 1.119

Total terpene compounds 3 8 3 9 10 5 8 10 4 4 5 6

Miscellaneous compounds930 1.1-Diethoxy ethanec 0.429 1.322 1.115

1063 3-Fluoro-1-propenec 0.141 0.388 0.047 0.015 0.059 0.022

1079 2-Fluoro-1-propenec 0.938 0.137 0.820 0.841 0.355 0.227 0.723 0.335 0.684

Sum of miscellaneous compounds 0.141 0.817 0.047 0.953 0.196 0.820 0.863 0.355 0.227 2.045 0.335 1.799

Total miscellaneous compounds 1 2 1 2 2 1 2 1 1 2 1 2

Sum of compounds detected 59.190 67.422 60.134 85.599 111.764 137.248 114.614 94.564 56.748 102.667 82.549 72.271

Total compounds detected 46 47 42 56 60 54 58 64 46 54 52 56

KI, Kováts’ retention indices.a MS data and Kováts’ index in agreement with those of authentic compound.b MS data and Kováts’ index in agreement with those in literature.c MS data in agreement with those in NIST107, NIST21 and SZTERP libraries.



7/8

like temperature, pH and low oxygen conditions simultaneously

increase the amount of ethyl esters (Etievant, 1991). 2-Phenylethyl

acetate, detected in all wine samples, contribute to the aromatic

complexity of wines giving a banana–apple aroma (Ribérau-Gayon

et al., 2000). Acetates other than ethyl acetate like 3-methylbutyl

acetate, hexyl, heptyl and octyl acetate were detected in our sam-

ples and are responsible for a pleasant fruit-like aroma (Rapp and

Mandery, 1986). Their number and concentration were relatively

high in the immobilized cells compared to free cells. Another ester

with higher concentration in immobilized cells was ethyl-9-

decenoate, which described as exhibiting a very pleasant odor (Eti-

evant, 1991).

Fatty acids due to their low odor threshold values and rather

high concentrations in wines are considered to have flavor impactin wines (Etievant, 1991). This impact is positive, contributing to

the complexity of the aroma bouquet, at concentrations up to their

threshold values, while is negative at higher concentrations ( Jack-

son, 1994). A positive correlation between concentrations of hexa-

noic, octanoic and decanoic acid and the quality of the produced

wines has been reported (Etievant, 1991). In the present study

these acids detected in all wines but in higher concentrations in

those produced by immobilized cells. The same trend was ob-

served for all volatile fatty acids.

Fusel alcohols are generally considered to have rather unpleas-

ant odors therefore it is believed that they contribute more to the

intensity of the odor of the wine that to its quality (Etievant, 1991).

2-Phenylethanol is one of the few fusel alcohol described with

pleasant odor as old rose (Tsakiris et al., 2004; Etievant, 1991). Itwas detected in all wines but in higher concentrations in wines

produced by immobilized cells than those produced by free cells.

A reduction in its content at low temperatures (10 and 5 C) was

observed, which is in accordance to other studies (Mallouchos

et al., 2002; Kandylis et al., 2008; Kandylis and Koutinas, 2008 ).

Benzaldehyde detected in almost all wines has a bitter almond

odor. b-damascenone has a complex smell of flowers, tropical fruit

and stewed apple (Ribérau-Gayon et al., 2000; Pisarnitskii, 2001).

Furfural and 5-methyl furfural are characterized by a toasted al-

mond odor, however in our samples were detected in very low lev-

els probably because is formed mainly during ageing of wine in

barrel (Rapp and Mandery, 1986).

Terpenes, which are mainly derived from the grape, are princi-

pal components that contribute to the characteristic aroma of awine (Vilanova and Sieiro, 2006). Linalool gives a flowery odor

(Vilanova and Sieiro, 2006) and was detected only in wines by

immobilized cells at levels little higher than its perception thresh-

old (0.05 mg/L, (Ribérau-Gayon et al., 2000)). a-Terpineol, farnesoland cis-linalool oxide were detected in almost all wines samples

produced by free and immobilized cells, however due to their high

perception thresholds, varying from 0.4 mg/L (for a-terpineol) upto 1–5 mg/L (for cis-linalool oxide) , have very little olfactory im-

pact on wines (Ribérau-Gayon et al., 2000). b-Citronellol was de-

tected at levels much higher than its perception threshold

(0.018 mg/L, (Ribérau-Gayon et al., 2000)) in wines produced by

immobilized cells at low temperatures (10 and 5 C) and is consid-

ered that gives an aroma of citrus, sweet, floral note ( Vilanova and

Sieiro, 2006; Peña-Alvarez et al., 2006).

4. Conclusions

In the present study wheat grains supported biocatalyst proved

capable for wine making in a wide range of temperatures in labo-

ratory scale and in a scale-up system of 80 L, producing quality

wines with great aroma. The growth and immobilization of cells

and fermentations were carried out at the same bioreactor leading

to a reduction of investment and operational cost. Thus, industrial-ization of the propose system has a great potential, considering the

high fermentation efficiency and high quality wines produced,

especially in low temperatures.

Acknowledgement

This work is part of the 03ED657 research project, implemented

within the framework of the ‘‘Reinforcement Programme of Hu-

man Research Manpower” (PENED) and co-financed by National

and Community Funds (20% from the Greek Ministry of Develop-

ment-General Secretariat of Research and Technology and 80%

from EU-European Social Fund).

References

Antonelli, A., Castellari, L., Zambonelli, C., Carnacini, A., 1999. Yeast influence on

volatile composition of wines. J. Agric. Food Chem. 47, 1139–1144.

Chandel, A.K., Narasu, M.L., Chandrasekhar, G., Manikyam, A., Rao, L.V., 2009. Use of

Saccharumspontaneum (wild sugarcane) as biomaterial for cell immobilization

and modulated ethanol production by thermotolerant Saccharomyces cerevisiaeVS3. Bioresour. Technol. 100, 2404–2410.

Colagrande, O., Silva, A., Fumi, M.D., 1994. Recent applications of biotechnology in

wine production (review). Biotechnol. Prog. 10, 2–18.

Etievant, X.P., 1991. Wine. In: Maarse, H. (Ed.), Volatile Compounds in Foods and

Beverages. Marcel Dekker, New York, pp. 483–533.

Fleet, G.H., Heard, G.M., 1993. Yeasts-growth during fermentation. In: Fleet, G.H.

(Ed.), Wine Microbiology and Biotechnology. Harwood Academic Publishers,

Chur, Switzerland, pp. 27–55.

Fraile, P., Garrido, J., Ancin, C., 2000. Influence of a Saccharomyces cerevisiae selectedstrain in the volatile composition of rose wines. Evolution during fermentation.

J. Agric. Food Chem. 48, 1789–1798.Fumi, M.D., Trioli, G., Colombi, M.G., Colagrande, O., 1988. Immobilization of

Saccharomyces cerevisiae in calcium alginate gel and its application to bottle-fermented sparkling wine production. Am. J. Enol. Viticult. 39, 267–272.

Jackson, R.S., 1994. Wine Science: Principles and Applications. Academic Press Inc.,

San Diego, California, USA. pp. 184–185, 187.

Iconomopoulou, M., Psarianos, K., Kanellaki, M., Koutinas, A.A., 2002. Low

temperature and ambient temperature wine making using freeze dried

immobilized cells on gluten pellets. Process Biochem. 37, 707–717.

Kandylis, P., Goula, A., Koutinas, A.A., 2008. Corn starch gel for yeast cell

entrapment. A view for catalysis of wine fermentation. J. Agric. Food Chem.

56, 12037–12045.

Kandylis, P., Koutinas, A.A., 2008. Extremely low temperature fermentations of

grape must by potatoes supported yeast-strain AXAZ-1. A contribution is

performed to catalysis of alcoholic fermentation. J. Agric. Food Chem. 56, 3317–

3327.

Kopsahelis, N., Agouridis, N., Bekatorou, A., Kanellaki, M., 2007. Comparative study

of spent grains and delignified spent grains as yeast supports for alcohol

production from molasses. Bioresour. Technol. 98, 1440–1447.

Kopsahelis, N., Nisiotou, A., Kourkoutas, Y., Panas, P., Nychas, J.-E.G., Kanellaki, M.,2009. Molecular characterization and molasses fermentation performance of a

5 10 15 20

30

35

40

45

50

55

60

alcohols

esters

% t

o t a l v o l a t i l e s

fermentation temperature ºC

Fig. 1. Effect of temperature on percentages of total esters and total alcohols on

total volatiles of wines produced by immobilized cells on wheat in the 80 L

bioreactor.



8/8

wild yeast strain operating in an extremely wide temperature range. Bioresour.

Technol. 100, 4854–4862.

Kourkoutas, Y., Dimitropoulos, S., Kanellaki, M., Marchant, R., Nigam, P., Banat, I.M.,

Koutinas, A.A., 2002. High temperature alcoholic fermentation of whey using

Kluyveromyces marxianus IMB3 yeast immobilized on delignified cellulosicmaterial. Bioresour. Technol. 82, 177–181.

Kourkoutas, Y., Bekatorou, A., Banat, I.M., Marchant, R., Koutinas, A.A., 2004.

Immobilization technologies and support materials suitable in alcohol

beverages production: a review. Food Microbiol. 21, 377–397.

Longo, E., Velázquez, J.B., Sieiro, C., Cansado, J., Calo, P., Villa, T.G., 1992. Production

of higher alcohols, ethyl acetate, acetaldehyde and other compounds by 14Saccharomyces cerevisiae wine strains isolated from thesame region(Salnés NWSpain). World J. Microbiol. Biotechnol. 8, 539–541.

Mallouchos, A., Komaitis, M., Koutinas, A.A., Kanellaki, M., 2002. Investigation of

volatiles evolution during the alcoholic fermentation of grape must using free

and immobilized cells with the help of solid phase microextraction (SPME)

headspace sampling. J. Agric. Food Chem. 50, 3840–3848.

Mallouchos, A., Loukatos, P., Bekatorou, A., Koutinas, A.A., Komaitis, M., 2007.

Ambient and low temperature winemaking by immobilized cells on brewer’s

spent grains: effect on volatile composition. Food Chem. 104, 918–927.

Miranda-Lopez, R., Libbey, L.M., Watson, B.T., McDaniel, M.R., 1992. Odor analysis of

Pinot Noir wines from grapes of different maturities by gas chromatography–

olfactrometry technique (Osme). J. Food Sci. 57, 985–993.

Molnar, I., Oura, E., Suomalainen, H., 1981. Study of volatile substance produced

during the autolysis of champagne yeast. Acta Alim. 10, 27–36.

Peña-Alvarez, A., Capella, S., Juárez, R., Labastida, C., 2006. Determination of

terpenes in tequila by solid phase microextraction–gas chromatography–mass

spectrometry. J. Chrom. A. 1134, 291–297.

Pisarnitskii, A.F., 2001. Formation of wine aroma: tones and imperfections caused

by minor components. Appl. Biochem. Microbiol. 37, 552–560.

Plata, C., Millán, C., Mauricio, J.C., Ortega, J.M., 2003. Formation of ethyl acetate and

isoamyl acetate by various species of wine yeasts. Food Microbiol. 20, 217–224.

Polychroniadou, E., Kanellaki, M., Iconomopoulou, M., Koutinas, A.A., Marchant, R.,

Banat, I.M., 2003. Grape and apple wines volatile fermentation products and

possible relation to spoilage. Bioresour. Technol. 87, 337–339.

Rapp, A., Mandery, H., 1986. Wine aroma. Experentia 42, 873–884.Regodón Mateos, J.A., Pérez-Nevado, F., Ramírez Fernández, M., 2006. Influence of

Saccharomyces cerevisiae yeast strain on the major volatile compounds of wine.Enzyme Microb. Technol. 40, 151–157.

Ribérau-Gayon, P., Glories, Y., Maujean, A., Dubourdieu, D., 2000. The Chemistry of

Wine Stabilization and Treatments. Hanbook of Enology, vol. 2. John Wiley and

Sons Ltd., Chichester, UK (pp. 41–54, 193–196).

Tsakiris, A., Bekatorou, A., Psarianos, C., Koutinas, A.A., Marchant, R., Banat, I.M.,

2004. Immobilization of yeast on dried raisin berries for use in dry white wine-

making. Food Chem. 87, 11–15.

Vidrih, R.,Hribar, J., 1999. Synthesis of higheralcoholsduring cider processing. Food

Chem. 67, 287–294.

Vilanova, M., Sieiro, C., 2006. Determination of free and bound terpene compounds

in Albariño wine. J. Food Comp. Anal. 19, 694–697.


salto de escala1.pdf

Documents