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Journal of Food Biochemistry ISSN 1745-4514
SCREENING OF ENZYMATIC ACTIVITIES IN
NON-SACCHAROMYCES CIDER YEASTS
jfbc_583
683..689
R. PANDO BEDRIÑANA1, A. LASTRA QUEIPO and B. SUÁREZ VALLES
Área de Tecnología de los Alimentos, Servicio Regional de Investigación y Desarrollo Agroalimentario (SERIDA), 33300 Villaviciosa, Asturias, Spain
1
Corresponding author. TEL: 34-985-890-066;
FAX: 34-985-891-854; Email:
[email protected]
Accepted for Publication May 2, 2011
doi:10.1111/j.1745-4514.2011.00583.x
ABSTRACT
The activities of polygalacturonase, pectin lyase, b-glucosidase, b-xylosidase and
protease were determined using solid media in 420 wild non-Saccharomyces cider
strains identified by internal transcribed spacer-restriction fragment length polymorphism. The identified species corresponded to Hanseniaspora valbyensis, Hanseniaspora uvarum, Hanseniaspora osmophila, Metschnikowia pulcherrima, Candida
parapsilosis and Pichia guilliermondii. The most common activity exhibited was that
of b-glucosidase (33%), with all the analyzed species having some strains able to
develop this activity. Strains of M. pulcherrima showed the greatest capacity to
produce b-glucosidase and protease. b-xylosidase was detected in 17 yeast strains
belonging to the genera Hanseniaspora, Pichia and Metschnikowia. All of the tested
species have some strains with the capacity to develop b-xylosidase activity, except
for C. parapsilosis. No strains were able to secrete pectin lyase, while polygalacturonase activity was observed in eight Hanseniaspora strains. Only two strains, belonging to the species H. uvarum and M. pulcherrima, developed three enzymatic
activities, namely b-glucosidase, b-xylosidase and protease.
PRACTICAL APPLICATIONS
The non-Saccharomyces yeasts are very interesting in cider making because of the
low level of ethanol in cider. In this paper, it is demonstrated that the yeast isolated
from Asturian cider represents a source of several enzymes, which have capacity to
release flavor compounds. In this sense non-Saccharomyces strains are being used as
inoculated cultures to ferment apple pomace and apple must, with the aim of
enhancing the aroma and flavor of the products.
INTRODUCTION
Most of the studies carried out with Asturian cider yeasts have
mainly focused on yeasts belonging to the Saccharomyces
genus, the main agents of alcoholic fermentation (Suárez
et al. 2005; Suárez et al. 2008). However, non-Saccharomyces
strains, present fundamentally during the early stages of cider
making, make an important contribution to flavor compounds (Suárez et al. 2007; Pando et al. 2010).
Yeast strains with enzymatic activity could be a significant
factor to improve the processing of beverages and a potential
source for the commercial production of enzymes. The genus
Saccharomyces is not recognized as being a good producer of
extracellular enzymes. In contrast, many of the nonSaccharomyces yeast species show natural enzymatic activiJournal of Food Biochemistry 36 (2012) 683–689 © 2011 Wiley Periodicals, Inc.
ties. Among the enzymes of special interest produced by these
yeasts are pectinases, proteases and glycosidases (CanalLlauberes 1993; Esteve-Zarzoso et al. 1998).
Pectic substances are structural heteropolysaccharides
and are the major constituents of the middle lamellae and
primary cell walls of higher plants (Talmadge et al. 1973).
Together with other polysaccharides (glucan and xylan),
pectic compounds influence clarification and are often
responsible for turbidity, viscosity and filter stoppages in
beverages (Meurens 1977; Dubourdieu et al. 1981).
Polygalacturonase and pectin lyase are depolymerising
pectinase enzymes able to cleave the glycosidic bonds
between two nonesterified galacturonic acid units and catalyze the b-elimination reaction between two methylated
galacturonic-acid residues, respectively. These activities
683
SCREENING OF ENZYMATIC ACTIVITIES
facilitate the pressing system and play an enhanced role in
the filtration of wines and ciders (Baron and Thibault 1985;
Canal-Llauberes 1989).
Protein haze is considered the most common physical
instability in white wine and grape and apple juices (Hsu and
Heatherbell 1987; Hsu et al. 1989). Although the removal of
these haze-forming proteins by bentonite treatment is effective, this treatment results in a loss in beverage aroma (Miller
et al. 1985). The use of enzymatic preparations with proteolytic activity has been investigated as an alternative to
prevent protein haze formation in beverages, although the
search for fungal enzymes that may degrade wine proteins has
not been successful up to now (Van Resnburg and Pretorius
2000). Protease activity hydrolyzes proteins into smaller
soluble nitrogen molecules, thus favoring the clarification
and stabilization of beverages while helping to prevent
incomplete fermentations resulting from a deficiency of
assimilable nitrogen in the must. Extracellular protease production among various microorganisms is well known and
the applicability of yeast extracellular proteinases in brewing
and wine has been investigated in several studies (Feuillat
et al. 1980; Bilinski et al. 1987; Lagace and Bisson 1990; Dizy
and Bisson 2000).
The aroma and flavor properties of beverages can be
enhanced by glycosidase enzymes that hydrolyze nonvolatile
glycosidic precursors of the fruits (Gunata et al. 1985; Paillard
1990). Glycosidase activities include b-D-glucosidase, b-Dapiosidase, a-L-arabinofuranosidase, a-L-rhamnosidase and
b-D-xylosidase. Various authors have reported glycosidase
activities in studies in yeasts of enological origin (Delacroix
et al. 1994; Mcmahon et al. 1999; Lomolino et al. 2006).
In recent years, the potential of the industrial use of microorganisms as biotechnological sources has stimulated
renewed interest in the exploration of extracellular enzymatic
activity (Rosi et al. 1994; Charoenchai et al. 1997; Strauss
et al. 2001; Buzzini and Martini 2002). These types of screenings are usually done by rapid nonexpansive assays (plate
agar) that easily permit the analysis of large populations and
the observation of genetic variants.
The aim of this study is to report the enzymatic activities
(polygalacturonase, pectin lyase, b-glucosidase, b-xylosidase
and protease) of 420 wild non-Saccharomyces cider yeast
strains identified by internal transcribed spacer-restriction
fragment length polymorphism.
MATERIALS AND METHODS
Yeast Strains
Four hundred and twenty non-Saccharomyces cider strains
were analyzed, 14 of which belong to the SERIDA collection
of pure cultures while the rest were isolated from the two
cellars in Villaviciosa during three consecutive harvests
684
R. PANDO BEDRIÑANA, A. LASTRA QUEIPO and B. SUÁREZ VALLES
(2001–2003). The indigenous strains were grown at 28C in
10 mL glucose peptone yeast broth (5 g/L yeast extract, 5 g/L
peptone and 20 g/L glucose) twice and then 5 mL of the cultures was streaked onto the agar media. All trials were performed in duplicate.
Molecular Identification of Yeast
Yeast colonies were identified by amplification and restriction
of the rDNA 5.8S-ITS region using the primers ITS1 and
ITS4. Amplification, restriction and electrophoresis conditions, as well as the comparison of the DNA fragments against
reference strains and the database, were carried out following
Esteve-Zarzoso et al. (1999). Certified yeast strains of different species obtained from the Spanish Type Culture Collection (CECT) were used as reference strains in each
amplification and restriction reaction. The strains were Hanseniaspora uvarum (CECT 1444), Hanseniaspora valbyensis
(CECT 10122), Metschnikowia pulcherrima (CECT 10408)
and Candida parapsilosis (CECT 1449).
Pectinase Activity
Pectinolytic activity was detected according to the method
described by Atlas (1993). Polygalacturonase activity was
studied using the MP5 medium (pH = 5.0) containing 10 g/L
glucose and 5 g/L polygalacturonic acid. Pectin lyase was
evaluated using the MP7 medium (pH = 7.2) containing
5 g/L glucose and 5 g/L apple pectin. Plates were incubated
for 72 h at 25C. Enzyme activity was indicated by the formation of a clear halo around the colony in an otherwise opaque
medium after flooding the plates with hexadecyltrimethylammonium bromide (10 g/L). In both assays, pure endopolygalacturonase (Megazyme) enzyme was used as positive
control to detect the activity.
Protease Activity
Gelatine was used as protein substrate to detect the production of proteolytic enzymes (Hankin and Anagnostakis
1975). The medium consisted of Difco Nutrient Agar
(Lawrence, KS) plus 4 g/L gelatine (pH = 6). Plates were incubated at room temperature for 10 days and then flooded with
an aqueous saturated solution of ammonium sulphate that
renders the agar opaque and enhances the clear zones around
colonies. Two M. pulcherrima strains obtained from the
Spanish Type Culture Collection (CECT 10408 and CECT
10546) were used as positive control.
b-Glucosidase Activity
This activity was evaluated in agar slant tubes using arbutin as
substrate. The basal medium composition was 5 g/L arbutin,
Journal of Food Biochemistry 36 (2012) 683–689 © 2011 Wiley Periodicals, Inc.
R. PANDO BEDRIÑANA, A. LASTRA QUEIPO and B. SUÁREZ VALLES
SCREENING OF ENZYMATIC ACTIVITIES
TABLE 1. IDENTIFICATION OF THE SPECIES STRAINS ANALYZED
Size of the PCR restriction fragments (bp)
Identification
AP (bp)
Cfo I
Hae III
Hinf I
Hanseniaspora valbyensis
Hanseniaspora uvarum
Hanseniaspora osmophila
Metschnikowia pulcherrima
Candida parapsilosis
Pichia guilliermondii
750
750
750
400
550
625
630,120
320,310,105
275,150,135,95,75
205,100,95
300,240
300,265,60
750
750
460,120,90,80
280,100
400,115
400,115,90
250,220,170,105
350,200,180
390,360
200,190
290,260
320,300
Number of isolates
Dde I
SERIDA
Cellar A
Cellar B
1
13
49
300,180,95,90,85
152
64
42
47
8
42
1
1
AP, 5.8S-ITS amplified product size; PCR, polymerase chain reaction.
1 g/L yeast extract and 20 g/L agar. Each tube contained 7 mL
of basal medium and 0.1 mL iron chloride (1%). Tubes were
incubated for 15 days at 28C. The yeast with this enzymatic
activity formed a dark halo around the slant. Pure
b-glucosidase enzyme (EC. 3.2.1.21, Sigma, St. Louis, MO)
was used as positive control.
b-Xylosidase Activity
This screening was carried out on agar plates (Manzanares
et al. 1999) containing 1.7 g/L yeast nitrogen base without
amino acids and ammonium sulphate (Difco), 5 g/L ammonium sulphate, 5 g/L D-xylose and 20 g/L agar (pH = 5.5).
Three hundred microliters of 4-methylumbelliferyl-b-Dxyloside (Sigma) was spread onto surface of the agar plates,
after which the yeasts were inoculated and the plates were
incubated at 25C for 24 h. Enzymatic activity was visualized
under UV illumination as fluorescent halos surrounding
yeast growth. Two H. uvarum strains obtained from the
Spanish Type Culture Collection (CECT 11105 and CECT
11106) were used as positive control.
in Hanseniaspora strains. The three Hanseniaspora species
isolated in ciders had strains that exhibited this activity; most
of these strains belonged to the species H. uvarum. Among the
321 Hanseniaspora strains, only eight yeasts were positive for
polygalacturonase activity. The production of extracellular
pectin lyase was tested and no yeast produced enzymes
capable of hydrolyzing bonds between methylated
galacturonic-acid residues.
Protease Activity
Fifteen percent of the isolates were able to liquefy gelatine and
produced clear zones on agar plates (Table 2). All the species
analyzed had some strains that exhibited this activity, except
for the Pichia guilliermondii species. The percentages of
strains exhibiting protease activity were approximately 5%
for H. valbyensis and C. parapsilosis species and 17% for H.
uvarum and H. osmophila species. M. pulcherrima was the
species that showed the greatest capacity to produce this
activity (49%).
RESULTS
b-Glucosidase Activity
The approximate length of the amplified products and the
restriction fragments observed after digestion for all the
tested strains are summarized in Table 1. The 420 isolates
identified by polymerase chain reaction-restriction fragment
length polymorphism analysis of the 5.8S-rDNA gene region
belonged to six different yeasts species and were obtained as
representatives of non-Saccharomyces cider strains in previous studies (Suárez et al. 2007; Pando et al. 2010). The investigated isolates were mostly strains of the genus
Hanseniaspora (76%), with H. valbyensis being found to be
predominant in both cider cellars.
The ability of several yeast species to produce enzymes on
solid media is shown in Table 2.
b-Glucosidase activity was the one most commonly exhibited
by cider strains. One hundred forty-one isolates cleaved
arbutin; that is to say, 33% of strains was positive for
b-glucosidase activity (Table 2). All the analyzed species had
some strains able to develop this activity. The H. valbyensis
species had the lowest number of strains exhibiting this activity (16%), while M. pulcherrima was the species with the
greatest percentage of activity (73%).
Pectinase Activity
Pectinase activity was not found to be common in wild cider
yeasts (Table 2). Polygalacturonase activity was only observed
Journal of Food Biochemistry 36 (2012) 683–689 © 2011 Wiley Periodicals, Inc.
b-Xylosidase Activity
The production of b-D-xylosidase activity using D-xylose as
inducer was detected in 17 strains. All the species tested have
some strains with the capacity to develop this activity, except
for C. parapsilosis. The H. uvarum and M. pulcherrima
species, with seven and five positive strains for b-xylosidase
activity, respectively, were the species with the greatest percentages of activity.
685
1
6
2
4
–
–
1
–
–
1
–
1
–
1
–
–
–
–
28
35
15
32
–
–
Cellar A
SERIDA
Cellar B
Cellar A
4
–
–
8
14
1
1
3
–
–
–
–
SERIDA
Cellar B
12
12
7
19
–
–
–
–
–
8
2
–
Cellar A
SERIDA
–
1
–
–
–
–
1
2
2
–
–
–
Cellar B
Cellar A
No. of strains
202
77
42
55
42
2
Species
Hanseniaspora valbyensis
Hanseniaspora uvarum
Hanseniaspora osmophila
Metschnikowia pulcherrima
Candida parapsilosis
Pichia guilliermondii
SERIDA
–
–
–
–
–
–
Protease
Polygalacturonase
TABLE 2. EXTRACELLULAR ENZYMATIC ACTIVITY OF YEASTS ISOLATED FROM CIDER FERMENTATIONS
686
R. PANDO BEDRIÑANA, A. LASTRA QUEIPO and B. SUÁREZ VALLES
Table 3 shows the 50 strains that exhibited more than one
enzymatic activity. Most of the strains (46 yeasts) developed
b-glucosidase and protease activities, the majority of which
belonged to the M. pulcherrima species (43.5%). In this
respect, the eight M. pulcherrima strains isolated in cellar A
were positive for b-glucosidase and protease activities
(Table 2). One H. uvarum strain developed b-glucosidase and
polygalacturonase activities, and one P. guilliermondii strain
exhibited b-glucosidase and b-xylosidase activities. Only two
strains, belonging to the H. uvarum and M. pulcherrima
species, developed three enzymatic activities, namely
b-glucosidase, b-xylosidase and protease.
DISCUSSION
–
3
–
–
–
–
b-Glucosidase
b-Xylosidase
Cellar B
SCREENING OF ENZYMATIC ACTIVITIES
In this study, the tested yeasts were grown in defined media
under conditions that optimized the expression of extracellular enzymatic activity. Among the 420 yeast strains analyzed,
175 strains exhibited some enzymatic activity. Within the
same species, strains differed in producing enzymatic activities. Most developed one enzymatic activity (71.4%), while
only two strains were positive for three enzymatic activities.
M. pulcherrima and H. uvarum species have the greatest
number of strains with a capacity to produce extracellular
enzymes. The results from the screening of enzymatic activities in non-Saccharomyces cider yeasts suggest their potential
to improve the aroma and flavor of cider used as natural
inocula (Cabranes et al. 1997; Xu et al. 2006).
The activities that non-Saccharomyces cider yeasts were
found to have the least capacity to develop were pectinase
activities. These results were in agreement with those
obtained in previous work. Panon et al. (1995) evaluated the
production of polygalacturonase enzyme in H. valbyensis and
M. pulcherrima strains isolated in French cider, observing that
no strain exhibited this activity. In another screening carried
out with 24 wine yeasts, other researchers observed that none
of the analyzed H. uvarum and M. pulcherrima strains exhibited polygalacturonase activity (Charoenchai et al. 1997).
However, a screening of 110 Kloeckera apiculata strains, an
imperfect state of H. uvarum, and 54 Candida pulcherrima, an
imperfect state of M. pulcherrima, showed four and one
strains with this activity, respectively (Strauss et al. 2001). In
our study, no strain exhibited pectin lyase activity; in fact, this
activity is scarce in enological yeast (Gainvors et al. 1994).
Proteolytic activity has been reported in C. parapsilosis, M.
pulcherrima and H. uvarum wine strains (Charoenchai et al.
1997; Braga et al. 1998; Dizy and Bisson 2000; Fernández et al.
2000; Strauss et al. 2001). Our study has confirmed that
several species of indigenous cider yeast can produce proteolytic activity, the species M. pulcherrima being the one that
had the greatest number of strains exhibiting this activity, in
agreement with that reported elsewhere (Fernández et al.
2000).
Journal of Food Biochemistry 36 (2012) 683–689 © 2011 Wiley Periodicals, Inc.
0
1
0
0
0
0
0
0
0
1
2
0
0
0
20
0
5
0
6
0
1
0
13
1
No. of strains Hanseniaspora uvarum Hanseniaspora osmophila Hanseniaspora valbyensis Metschnikowia pulcherrima Pichia guilliermondii Candida parapsilosis
SCREENING OF ENZYMATIC ACTIVITIES
The significance of b-glucosidase lies in its capacity to
release flavor compounds from glycosidically bound nonvolatile precursors. It should be borne in mind that
b-glucosidase activity varies appreciably according to the base
medium and the substrate employed (Hernández et al. 2002).
Arbutin was chosen as substrate and the medium was not
supplemented with glucose, as the addition of glucose
decreases the levels of hydrolytic activity in most nonSaccharomyces yeast strains (Fia et al. 2005). The percentage
of strains exhibiting b-glucosidase capacity was similar to
that observed in wine yeasts (Rosi et al. 1994; Arévalo et al.
2005). The high percentage of M. pulcherrima strains with the
capacity to synthesize the b-glucosidase enzyme has already
been reported (Fernández et al. 2000). The capacity to
produce b-xylosidase had been previously reported as quite
rare in wine yeasts and was associated with the genera Hanseniaspora and Pichia (Manzanares et al. 1999). However, five
M. pulcherrima strains exhibited this activity in our study.
These observations disclose interesting new perspectives,
demonstrating that yeast isolated from Asturian cider actually represents a source of several enzymes that are potentially
exploitable for biotechnological purposes. However, more
detailed studies on the secretion of enzymes from yeasts are
needed to determine whether these enzymes would be produced and secreted by the yeast during cider fermentation.
ACKNOWLEDGMENTS
The authors are indebted to Dr. Briones Pérez (Department
of Food Technology, University of Castilla La Mancha) for his
professional advice, and also to the Government of the Principado de Asturias (Spain) and to the National Institute of
Research and Agro-Food Technology (INIA), Project No.
RM06-00008.
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