Expression of Multidisciplinary Flavour Science
β-GLUCOSIDASE PRODUCTION BY
YEASTS ISOLATED FROM VINEYARD
NON-SACCHAROMYCES
T. ARROYO1, G. Cordero1, A. Serrano1, and E. Valero2
1
2
Agroalimentación, IMIDRA, El Encín. A-2 Km 38,200. Alcalá de Henares,
28800Madrid, Spain
Biología Molecular e Ingeniería Bioquímica. UPO, Sevilla, Spain
Abstract
In order to select yeasts with biotechnological properties, 156 strains of nonSaccharomyces yeasts have been analysed. 83 were isolated in the vineyard under
two forms of biological defence, organic culture and conventional culture. The others
73 strains came from the IMIDRA (Spain) collection. The strains have been studied
under the point of view of their β-glycosidase activity. The yeasts that present this
activity were genetically identified by PCR-RFLP of ITS region of chromosomic DNA.
29% of vineyard strains and 33% of the IMIDRA collection showed β-glucosidase
activity under the assay conditions. The yeasts with high β-glucosidase activity were
isolated from the vineyard under organic culture conditions.
Introduction
β-Glucosidase commercial enzyme is one of the most interesting glycosidases
especially used for hydrolysis of glycoconjugated aroma precursors, in musts and
wines (1). An alternative to the commercial enzymes could be the use of specific
enzymes contained in yeasts forming part of the wine ecosystem. NonSaccharomyces yeasts produce endogenous and exogenous enzymatic activities
into microbial cells able to develop odorous compounds. Recent investigations about
biodiversity of yeasts strains in French and Portugal vineyards showed a large
proportion of non-Saccharomyces strains (2). In relation to these results the aim of
this study was to investigate the β-glucosidase enzymatic activities of nonSaccharomyces strains isolated under different conditions of biological defence of the
vineyard.
Experimental
Non-Saccharomyces strains and molecular identification. 96 grape samples were
harvested in the Origin Apellation “Vinos de Madrid” during 2006 and 2007
campaigns, by two systems of biological defence, organic culture and conventional
culture.
83 non-Saccharomyces strains yeasts of vineyard selected according to its ability
to grow in a medium containing L-lysine and 73 non-Saccharomyces strains from
IMIDRA yeasts collection (CLI): Candida (38), Metschnicowia (11), Hanseniaspora
(1), Hansenula (2), Kloeckera (10), Pichia (7), Rhodotorula (4) was used in this study.
The molecular identification of yeast non-Saccharomyces was carried out with
PCR amplification of the ITS ribosomal region. The PCR products were treated with
the restriction endonucleases CfoI, Hae III and HinfI (3).
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Expression of Multidisciplinary Flavour Science
β-Glucosidase enzymatic activity. Test to study β-glucosidase enzymatic activity
were carried out using plates containing medium with arbutin (Sigma) as substrate
(yeast extract 0.3% w/v, malt extract 0.3 % w/v, meat-peptone 0.5% w/v, arbutin
0.5% w/v and YNB lys+ 0,67% w/v). After sterilization (121ºC, 20 min) a sterile
solution of ferric ammonium citrate (1% w/v) was added to the medium. The plates
inoculated with 0.1 mL of the yeast to be tested, were incubated at 30º C for 5 days.
Hydrolysis of arbutin by yeasts appeared as a dark brown colour in agar. 1mg/mL of
an enzyme extracted from almonds (EC.3.2.1.21, Sigma, 5.2 U/mg solid) was used
as a positive control. An activity scale from 0 (null) to 7 was elaborated in order the
semiquantitative activity yeasts assessment.
Results
A vineyard of Arganda region (Origin Apellation “Vinos de Madrid”) was sampling in
two harvest seasons (2006 and 2007). In total, 96 grape samples were collected, and
900 colonies was isolated from the initial population and spontaneous fermentation.
When the L-lysine method was applied, 700 isolates were grouped under nonSaccharomyces yeasts. The large proportion of non-Saccharomyces strains founded
in the vineyard probe the importance of this source like a non-Saccharomyces
biodiversity reserve. In order to study the distribution of β-glucosidase activity in
yeasts from vineyard and yeasts isolated in wines, 156 strains were taken, 83 were
isolated in the vineyard under different conditions of defence biological, organic crop
and conventional crop and 73 strains belonging to the IMIDRA yeasts collection.
These strains were tested to obtain the restriction patterns of ITS ribosomal region.
The ITS and the restriction fragments size of this studied strains are summarized in
(Table 1). In base to this restriction fragments, the 83 isolates from vineyard were
grouped under 13 different species of yeasts, types 1-13 in the Table. Although in the
majority of cases, the PCR products from strains of the same species had identical
molecular sizes, and species of the same genus had similar sizes for the amplified
fragment, these PCR products showed a high degree of length variation, between
500 and 900 bp. The comparative study of this profiles which the profiles compiles in
bibliography o database of reference collections has not been always possible. For
this reason will be useful the use of classical method and other molecular techniques
to recognize the genus and species of this patterns. The 56% of isolates investigated
were strains type 1. In this case the PCR products and the amplified fragment of this
type had showed the same size that the strain 1962 CECT (Spanish collection of
type cultures), identified as Kluyveromyces thermotolerans. Types 2 and 6 are the
second most abundant and each one represents no more than 8%. The 73 yeasts
strains of IMIDRA collection previously identified to level of genus, corresponding to
genus Candida (38), Hanseniaspora (1), Hansenula (2), Kloeckera (10),
Mestchnicowia (11), Pichia (7), and Rhodotorula (4).
Table 1 shows also the results of β-glucosidase activity measured by using plates
with arbutin. When the activity is present, the splitting of arbutin is observed by the
dark brown colour by reaction of hydroxyquinone and ferric ammonium citrate. In
order to establish a rapid semiquantitative method to measure the yeasts activity, an
scale of colour (cream until dark brown) have been made increasing until seven
times the enzyme extracted from almonds (EC.3.2.1.21, Sigma). The results showed
that 29 % of vineyard strains present activity β-glucosidase in the assay conditions.
All types of non-Saccharomyces species, except the type 8, were β-glucosidase
positive. The types 3 and 13 were only isolates in vineyard in organic crop and the
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Expression of Multidisciplinary Flavour Science
Table 1. Origin, type/genus, ITS and RFLPs and activity control of the positive βglucosidase strains. CLI= IMIDRA yeasts collection. A= Arganda, SM= San
Martín, N= Navalcarnero. CV= Conventional culture. OC= Organic culture.
IMIDRA
β-glucosidase
Collection
Activity
PCR-AP
Origin
Type/Genus sp
RESTRICTION FRAGMENTS (RFLPs)
ITS
HaeIII
CfoI
HinfI
23A-9C
+++++++
CV
5
650
600
550
300
23A-3A
+++++++
CV
3
650
600
550
350
23A-6C
+++++++
CV
5
650
600
550
300
6 - 5A
++++++
OV
13
500
475
200+100
250+250
19A-1A
++++++
CV
6
450
400+150
300+250
300X2
19A-2A
++++++
CV
6
450
400+151
300+251
300X3
21A-5C
++++++
CV
5
650
600
550
300
6-2A
+++++
OV
13
500
475
200+100
250+250
6 - 10A
+++++
OV
13
500
475
200+100
250+250
23-2A
+++++
CV
9
650
575
300
300X2
23-2C
+++++
CV
12
800
800
300
350+200+175
24A-1A
+++++
CV
7
375
375+300+100
200+100
200X2
23-3A
++++
CV
9
650
575
300
300X3
24A-2A
++++
CV
7
375
375+300+100
200+100
200X2
12A-1A
+++
CV
11
750
750
300+100
350+100
2 - 6C
+
OV
2
900
300+200+100
300+300
350
7-6B
+
OV
10
800
800
325+225+150
400+300
7-8B
+
OV
4
750
750
325
350+175+150
350+200+175
23-9C
+
CV
12
800
800
300
12A-7C
+
CV
2
900
300+200+100
300+300
350
3 - 4A
+/-
OV
1/ Kluyveromyces thermotolerans
700
310+215+90+90
315+285+95
355+345
10 - 10C
+/-
CV
1/ Kluyveromyces thermotolerans
700
310+215+90+90
315+285+95
355+435
CLI 1
+++++++
A
Hansenula sp
610
600
490
300
CLI 70
++++++
N
Rhodotorula sp
600
375+200
275+225+75
300+200
CLI 457
++++++
A
Metschnikowia pulcherrima
375
250+100
200+75
190
CLI 219
+++++
A
Metschnikowia pulcherrima
400
375+250
190+75
190
CLI 50
+++++
N
Rhodotorula sp
600
400+200
275+225+75
350+200
CLI 68
+++++
N
Metschnikowia pulcherrima
400
250+75+50
190+75
190
CLI 460
+++++
A
Metschnikowia pulcherrima
400
250+100
200+75
190
CLI 461
+++++
A
Metschnikowia pulcherrima
375
250+100
200+75
190
CLI 463
+++++
A
Metschnikowia pulcherrima
375
250+100
200+75
190
CLI 49
+++
N
Rhodotorula sp
600
400+200
275+225+75
350+200
CLI 560
++++
SM
Metschnikowia pulcherrima
375
275+100
190+75
200
CLI 72
+++
N
Kloeckera sp
750
700
300+100
300+190+175
CLI 903
++
EN
Kloeckera apiculata
750
700
300+100
300+200+190
CLI 458
+++
A
Metschnikowia pulcherrima
375
250+100
200+75
190
CLI 225
+++
A
Kloeckera sp
750
700
300+100
300+200+190
CLI 417
+++
A
Hanseniospora sp
-
-
-
-
CLI 3
++
A
Kloeckera sp
750
750
300
350
CLI 29
++
A
Kloeckera sp
-
-
-
-
CLI 31
++
A
Kloeckera sp
-
-
-
-
CLI 187
++
SM
Kloeckera sp
-
-
-
-
CLI 190
++
SM
Kloeckera sp
-
-
-
-
CLI 194
++
SM
Kloeckera sp
750
700
650+100
225+200+175
CLI 512
++
N
Kloeckera
750
300+100
300+100
300+200+190
types 5 and 11 were founded in conventional crop. The profile type 1 has presented
a very weak activity and is present in both culture conditions. The 33% of the strains
of IMIDRA collection have presented also β-glucosidase activity. Similar results were
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Expression of Multidisciplinary Flavour Science
founded in other studies about Spanish wines. In La Mancha region, the 25% of the
non-Saccharomyces strains presented β-glucosidase activity (4). In sequence
ascending the genus with the higher activity were: Hansenula, Metschnicowia,
Hansesniaspora, Kloeckera and Rhodotorula. (Table 1) also shows the intensity of
enzymatic activity. Hansenula and Metschnicowia have been the most actives with
values between 4 and 6 times the value of the control activity. Metschnicowia
pulcherrima was linked to the enzyme β-glucosidase in the studies carried out in La
Mancha (4). Strains belonging to genus Candida and Pichia have not shown βglucosidase activity in this study. Strauss et al. (5) detected null o some weak
activity β-glucosidase activity in genus Candida when arbutin was used as substrate
of enzyme. Kloeckera has a lower activity, 2 or 3 times the control value,
Hanseniaspora and Kloeckera (anamorphic form of Hanseniaspora) are highly
present in the first stages of the vinification, its can support at least 5 alcoholic
degrees. The proportion of yeasts isolates in vineyard with enzymatic activity is
similar in contrast with yeasts of wine. The results have shown that the types 3, 5 and
13 are 6 to 7 times more active than the control. This result to indicate that of
vineyard present a high biodiversity of non-Saccharomyces yeasts with an important
proportion of strains with β-glucosidase production. The distribution of this kind of
yeast in the vineyard seems to be dependent of the sanitary treatments applied. The
most active yeasts, types 3 and 13, have been isolates in organic conditions. We can
conclude saying that the vineyard is an important source of non-Saccharomyces
yeasts with a high β-glucosidase activity.
Acknowledgements.
This work was supported by INIA project RM2006-00012-00-00.
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