Dopuri Vin 3

FOOD

MICROBIOLOGY

Food Microbiology 24 (2007) 271280

www.elsevier.com/locate/fm

Fungal strains isolated from cork stoppers and the formation of 2,4,6-trichloroanisole involved in the cork taint of wine

Sina Prak, Ziya Gunata, Joseph-Pierre Guiraud, Sabine Schorr-Galindo

UMR-IR2B cc023 Universite Montpellier II, Place Bataillon, 34095 Montpellier Cedex 5, France

Received 13 February 2006; received in revised form 24 April 2006; accepted 2 May 2006

Available online 13 May 2006

Abstract

Cork taint is mainly due to 2,4,6-trichloroanisole (TCA) produced through the activity of undesirable fungal strains. We observed that CFU mould number in TCA-containing stoppers was not quantitatively different to that of the stoppers not containing TCA (ca. 105 CFU/g). In contrast more fungi diversity was observed in TCA-containing stoppers. Penicillium spp (Penicillium chrysogenum,

Penicillium glabrum), Aspergillus spp (Aspergillus niger and Aspergillus oryzae), Chrysonilia sitophila, Mucor racemosus, Paecilomyces sp. and Trichoderma viride were found in TCA-containing stoppers, while C. sitophila and Penicillium sp. were the main fungi in the stoppers devoid of TCA. Conidia were numerous close to the lenticels and present from the lateral surface through to the centre of the stoppers. Strains of Aspergillus, Mucor, Paecilomyces, Penicillium and Trichoderma isolated from TCA-containing stoppers were able to convert 2,4,6-trichlorophenol (TCP) in TCA in resting cell or growing conditions. The best yields of conversion were obtained by green fungi

Paecilomyces sp. and P. chrysogenum, 17% and 20%, respectively. Chysonilia sitophila and Penicillium sp. did not produce TCA from TCP in our conditions.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Cork stoppers; Fungi; Trichlorophenol; Trichloroanisole

1. Introduction

Cork, the bark of the cork oak (Quercus suber) has been the traditional material in the manufacture stoppers since the 17th century for wine bottling, especially for high quality wines and for a proper maturation of the wine. The main components of cork are suberin, lignin, phenolic compounds and polysaccharides ( Pena-Neira et al., 2000; Alvarez-Rodriguez et al., 2002). Its particular features, impermeability to air and liquids, compressibility, ability to adhere to glass surface and chemical inertness makes it ideal for wine bottling. Nonetheless 27% of wines stoppered with corks developed a cork taint, a mouldy and musty off-odour which is considered to be unaccep-table by the consumer ( Butzke et al., 1999; Silva Pereira et al., 2000a). The incidence of cork taint has been considered to be the major problem associated with the use of these closures. Several volatile compounds, chlor-

Corresponding author. Tel.: +33 4 6714 4603; fax: +33 4 6714 4292. E-mail address: [email protected] (S. Schorr-Galindo).

0740-0020/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi: 10.1016/j.fm.2006.05.002

oanisoles, guaiacol, geosmine, 2-methylisoborneol, pyra-zines, 1-octen-3-ol and 1-octen-3-one were reported to contribute to cork taint attribute ( Buser et al., 1982; Amon et al., 1989; Simpson and Lee, 1990; Lee and Simpson, 1993; Sponholz and Muno, 1994; Pollnitz et al., 1996; Rocha et al., 1996a, b; Howland et al., 1997; Caldentey et al., 1998; Chatonnet et al., 2003; Ezquerro and Tena, 2005; Ezquerro et al., 2006). However, 2,4,6-trichloroanisol (TCA) is the compound most often associated with cork taint in wines possessing a very low sensory threshold (1.410.0 ng/l) ( Buser et al., 1982; Chatonnet, 1994; Evans et al., 1997; Butzke et al., 1999; Silva Pereira et al., 2000a; Alvarez-Rodriguez et al., 2002). In addition most of the tainted wines were found to contain TCA at or above its sensory threshold value ( Evans et al., 1997; Silva Pereira et al., 2000a).

The origin of chloroanisoles in cork stoppers is attributed to the microbial transformation of chlorophe-nols. This transformation is essentially a detoxification mechanism by the microbiological methylation of chlor-ophenols ( Zehnder et al., 1984; Silva Pereira et al., 2000a).

272S. Prak et al. / Food Microbiology 24 (2007) 271280

The direct precursor of TCA is 2,4,6-trichlorophenol (TCP). Its presence in cork may have several origins: chlorine bleaching in the processing of corks which is nowadays abandoned ( Maujean et al., 1985; Sponholz and Muno, 1994), the use of polychlorophenol biocides in cork-oak forests ( Simpson and Lee, 1990) and dehalogenation of pentachlorophenol (PCP) and 2,3,4,6 tetrachlorophenol (TeCP) by micro-organisms ( Maarse et al., 1988; Sponholz and Muno, 1994).

Studies on the stoppers using electronic microscopy have shown the presence of numerous microflora (moulds, yeasts and bacteria) in cork stoppers ( Lee and Simpson, 1993; Jager, 1999). The most abundant micro-organisms were moulds. For instance 108 moulds, 104 yeasts and 104 bacteria per stopper ( Davis et al., 1981) and 5.7 104 moulds and 3 104 bacteria per g of cork ( Alvarez- Rodriguez et al., 2002) were reported. A drastic decrease in microbial population occurs during cork processing steps mainly by heat treatment. The cork flora can vary largely depending on the origin, processing, transportation and storage conditions ( Silva Pereira et al., 2000a). Among the microflora the moulds are usually the most resistant and some can survive in extreme conditions.

The moulds are considered to be mainly responsible for cork taint. Several authors screened fungal flora on cork stoppers and identified Penicillium sp., Trichoderma sp.,

Chrysonilia sp., Cladosporium sp., Fusarium sp., Acre-monium sp., Aspergillus sp., Monilia sp., Mucor sp., Paecilomyces sp., Rhizoctonia sp., Mortierella sp. and

Verticillium sp. ( Moreau, 1978; Davis et al., 1981; Castera-Rossignol, 1983; Lee and Simpson, 1993; Hill et al., 1995; Caldentey et al., 1998; Silva Pereira et al., 2000b; Alvarez-Rodriguez et al., 2002).

Eleven out of 14 fungal strains isolated from cork samples were able to produce TCA when they were cultured on cork supplemented with TCP ( Alvarez- Rodriguez et al., 2002). Among them Fusarium and Trichoderma strains were the most efficient ( Alvarez- Rodriguez et al., 2002). A S-adenosyl-L-methionine-depen-dant methyltransferase catalysing O-methylation of several chlorophenols including TCP was recently isolated from

Trichoderma longibrachiatum ( Coque et al., 2003).

With regard to the yeasts isolated from the bark of cork oak, Rhodosporidium sp. and Rhodotorula sp. ( Villa- Carvajal et al., 2004) were the most abundant. Among the bacteria Streptomyces sp, Streptococcus sp, Micrococ-cus sp., and Bacillus spp were observed in corks ( Fumi and Colagrande, 1988). Their ability to methylate chlorophe-nols has not been reported yet.

Mould flora composition of cork stoppers devoid of cork taint called here sound stoppers, and of those presenting cork taint were studied to improve our knowledge of the moulds which contribute to the off-odour of wine through the formation of TCA. The ability of isolated strains to transform TCP in TCA by resting and growing cells was examined both in solid culture medium consisting of cork stoppers and in liquid medium. The analysis of TCA was

performed through the headspace solid-phase micro-extraction (HS-SPME) technique. This technique has already been used for TCA analysis from cork and cork tainted wines ( Fischer and Fischer, 1997; Evans et al., 1997; Riu et al., 2002; Bianchi et al., 2003; Insa et al., 2004; Juanola et al., 2004; Ezquerro and Tena, 2005; Ezquerro et al., 2006).

2. Material and methods

2.1. Chemicals

TCP and TCA were obtained from Sigma (St. Louis, MO, USA).

2.2. Cork stoppers

Two batches of cork stoppers were supplied by Bouchons Abel Company (Le Boulou, France). There was no cork taint in the first batch whereas it was present in the second batch. In addition in the GC/MS analysis performed during the present work TCA was not detected in the first batch in contrast to the second one. The stoppers were stocked in a closed box at 2025 1C until analysis. The humidity level of the stoppers (length 4571 mm, diameter 2470.5 mm) was of 8%.

2.3. Scanning electron microscopy

Sound and TCA-containing cork stoppers were cut at different depths following the radius section (0, 2, 4, 6 and 8 mm) using a sterile blade to obtain squares chips (6 mm 6 mm). The cutting was parallel to the lateral surface and perpendicular to the lenticels. The samples were then desiccated for 24 h at 25 1C before microscopic observations were carried out (Cambridge Stereosca, JEOL JSM 6300F, Scanning Microscope).

2.4. Estimation of cultivable mould cells

Counts were performed on whole corks or on chips taken aseptically at different levels from the corks (from the surface to 2 mm radial depth; from 2 to 4 mm; from 4 to 6 mm; from 6 to 8 mm and from 8 mm to centre). For the isolation of the moulds, stoppers or chips were immersed in 200 ml (10 corks, ca. 35 g) or in 10 ml (ca. 1 g of chips) of previously sterilized tryptone-salt medium (tryptone 1 g/l and NaCl 8.5 g/l). After agitation (160 rpm, 45 min at 40 1C), 0.1 ml of the extraction medium was subjected to 10-fold serial dilutions and plated on Petri dishes contain-ing PDA-chloramphenicol (pH 3.5) medium ( Guiraud, 1998). After 35 days of incubation at 25 1C, the colonies were counted. The results are expressed as the number of viable colonies per g of cork (CFU/g cork) with IC 95.

S. Prak et al. / Food Microbiology 24 (2007) 271280

273

2.5. Estimation of mould population by epifluorescence microscopy

The direct epifluorescence filter technique (DEFT) ( Jaeggi et al., 1989) previously used for counting mould cells ( Kisko et al., 1997) was applied to our samples. For this purpose 10 ml of the extraction medium above were filtered on Millipore IsoporeTM 0.4 mm membrane (Milli-pore), followed by the addition of 1 ml of acridine orange solution (0.025 mg/ml). The membrane was then rinsed after 10 min with 10 ml of sterile water. The observation of the cells on the membrane was made through a drop of immersion oil under UV with an epifluorescence micro-scope (Olympus BX60). The counting is carried out on a known surface of microscopic field, distinguishing green coloured cells (predominance of DNA, theoretically in poor physiological state) and red coloured cells (predomi-nance of RNA, theoretically in good physiological state). The results reported are the mean of 3 counts.

2.6. Mould identification

Strains were isolated as single CFU by high dilutions in saline solution (NaCl 8.5 g/l) and the purity was controlled by subcloning. Minor strains observed in lower dilutions or contaminated by the predominant strains were isolated by picking and striating as long as necessary to obtain single colonies. Identifications were made by macroscopic ob-servations (shape, size, colour of colonies) and microscopic observations (slide stained by Cotton blue) through the comparison with data obtained in the same culture conditions ( Samson et al. 1995).

2.7. Mould cultures and TCA production

2.7.1. Inoculum preparation

All isolated fungal strains were grown on PDA medium (pH 3.5) at 25 1C for 5 days. Conidia were collected by scraping in a saline solution with 0.01% of Tween 80. After counting with a haemocytometer the suspension was standardized by dilution to the suitable concentration.

2.7.2. Bioconversion on solid cork stopper medium

Sound stoppers (15 g) were crushed in a waring blender (Waring blendor) with 75 ml of culture medium and autoclaved (20 min at 120 1C) in a Roux flask. The culture medium consisted of (per litre): 30 g glucose, 5 g caseine peptone, 5 g meat peptone, 5 g sodium chloride, 3.3 g sodium nitrate, 1 g calcium chloride, 1 g dipotassic phos-phate, 1 g ferric chloride, 1 g potassium chloride, 0.5 g magnesium sulfate (7H2O) and 0.1 g ferrous sulfate (7H2O). This medium was called solid cork stopper medium because it consisted in crushed cork stoppers impregnated with a liquid culture medium. The flasks were inoculated with 5 108 conidia of isolated and identified strains and then incubated during 3 weeks at 25 1C which was necessary to obtain a maximal biomass concentration.

After 3 weeks of incubation, 5 g of solid state culture on cork medium were transferred to 20-ml vials and spiked with 150 ml of a TCP solution (1 g/l). Bioconversion was carried out for 2 and 7 days at 25 1C. Samples were then subjected to headspace solid phase micro-extraction (HS-SPME) and gas chromatography/mass spectrometry (GC/ MS) analysis. To estimate biomass from the solid cork stopper medium after 3 weeks of culture or 2 and 7 days of bioconversion, the cork rubble was taken into a saline solution with 0.01% Tween 80 and conidia were counted with a haemocytometer.

2.7.3. Bioconversion in liquid medium

Czapeck medium (per litre: 30 g sucrose, 2 g sodium nitrate, 1 g potassic phosphate, 0.5 g potassium chloride, 0.5 g magnesium sulfate (7H2O), 0.1 g ferrous sulfate (7H2O)) was dispensed in 250 ml Erlenmeyer flask (100 ml medium). The flasks were inoculated with 5 104 conidia of identified strains and incubation was carried out at 25 1C for 1 week. This incubation time was necessary to obtain a maximal biomass concentration. Thereafter 15 ml of medium containing resting cells were poured into 20-ml vials and 150 ml of a TCP solution (1 g/l) was added to the medium. Bioconversion was carried out for 2 and 7 days at 25 1C. Samples were then subjected to HS-SPME and GC/ MS analysis. To evaluate biomass after 1 week of culture, or after 2 and 7 days of bioconversion, the culture medium was filtered onto a membrane and the resulting biomass was evaluated following drying at 105 1C for 24 H.

2.7.4. Production of TCA during culture in liquid medium

The culture medium used for bioconversion experiments (100 ml) was spiked with 1 ml of TCP solution (1 g/l), inoculated (5 104 conidia) and cultured for 1 week at 25 1C. Thereafter 15 ml of medium were poured into 20 ml vials for HS-SPME and GC/MS analysis. Biomass evalua-tion was performed as in the case of bioconversion in liquid medium.

2.8. TCA analysis from corks and mould cultures

2.8.1. TCA extraction from corks

For TCA extraction 10 cork stoppers were immersed in 150 ml of water-ethanol solution (88/12; v/v) and subjected to agitation (160 rpm) during 24 h at 25 1C.

2.8.2. HS-SPME

Twelve milliliters of cork macerates were placed in a 20-ml vial and 2 g/l NaCl were added with. The vial was capped with a Teflon septum and placed for equilibrium for 24 h at 20 1C. The culture media above were also transferred in a 20-ml vial and were subjected to the same procedure. Polydimethylsiloxane (PDMS, 100 mm film thickness, Supelco, USA) fibre from SPME device was exposed to the headspace over the sample of 20-ml vial for 40 min at 20 1C ( Evans et al, 1997; Fischer and Fischer,


1997). Thermal desorption of volatiles from the SPME fibre occurred in the GC injection port at 250 1C for 4 min.

2.8.3. GC analysis

GC analysis was performed with a Varians 3300 chromatograph (Walnut Creek, CA, USA) equipped with a fused silica DB-WAX capillary column (30 m 0.32 mm i.d., 0.25 mm film tickness, J&W scientific). Injection was in splitless mode and injector temperature was set at 250 1C. The vector gas (hydrogen) flow rate was 1.5 ml/min. The column temperature was programmed from 40 to 250 1C at 10 1C/min and maintained at 250 1C for 10 min. Detection was by a flame ionization detector (FID) set at 300 1C.

Quantification was carried out using the external standard method with a TCA calibration curve established with TCA solution at 20, 50, 100, 200, 500 and 1000 ng/l.

2.8.4. GCMS

TCA recovered by SPME fibre was identified with a GCMS apparatus (HP-6890A GC connected up to an HP-5973N MS) mounted with a capillary column identical to that aforementioned. Temperature programming for oven and injection port were as above. The mass range scanned was from 40 to 350 amu at a scanning rate of 2.89 scans/s. The transfer line temperature was 260 1C. The vector gas (Helium) flow rate was 1.5 ml/min. The ionization method used was electronic impact with ioniza-tion energy of 70 eV. TCA was analysed by the selective ion monitoring (SIM) analysis mode with following target ions: m/z 167, 195, 197 and 210.

3. Results and discussion

3.1. TCA levels of cork stoppers

TCA levels in two batches of cork stoppers sorted according to the presence or absence of off-odour by the supplier was measured by the combination of SPME and GC/MS techniques after the extraction of 10 stoppers using a water/ethanol mixture. TCA was not detected in the so-called sound corks while 10.5 ng/g. of cork was found in the cork-taint attributed batch, an amount capable to exert cork taint in wine. This value was in the range of the levels encountered in TCA-contaminated corks ( Juanola et al., 2004). Note that a high variability in TCA concentration of cork stoppers may occur due to the influence of several factors: the origin of oak slabs, the processing and conservation of the material, the winery environment, microflora, etc.

3.2. Fungal flora of stoppers

3.2.1. Scanning electronic microscopy

The examination of the microphotographs from sound and TCA-containing stoppers shows that the conidia are numerous close to the lenticels ( Fig. 1), rare outwards but are present through to the centre of the stopper. This is in accordance with the observations of Jager (1999) and Jager et al. (1996). For both types of stoppers contamination occurs at the different positions observed ( Figs. 1 and 2). There is no obvious difference under electronic microscopy

Fig. 1. Scanning microscopy of TCA containing stopper at cut level 2 mm. General view ( 30) (a) and zoom ( 1000) of compact zone (b) and lenticel (c).


275

Fig. 2. Scanning microscopy ( 1000) of sound stopper (a, c) and TCA containing stopper (b, d) at cut level 4 mm near lenticel (a, b) and in compact zone (c, d). Arrows indicate Penicillium-like chains.

observation between sound and TCA-containing stoppers, except for Penicillium-like chains of conidia which are clearly distinguishable from TCA-containing stoppers ( Fig. 2).

3.2.2. Estimation of mould population from cork stoppers and identification

The colonies of moulds from cork stoppers were isolated and identified ( Table 1). They belong to genus Aspergillus, Penicillium, Mucor, Trichoderma and Chrysonilia. Many of them have been already detected on corks showing that the cork flora characterized here could be considered repre-sentative of cork flora composition ( Hill et al., 1995; Alvarez-Rodriguez et al., 2002).

The levels of some moulds were significantly higher in TCA-containing cork stoppers than in sound stoppers except for Penicillium sp. The latter and Chrysonilia sitophila were among the dominant ones in both stoppers. C. sitophila is often the dominant fungus during the traditional production of cork stoppers where the cork slabs are subjected to the so-called maturation stage after boiling. The presence of C. sitophila was considered to be favourable as it inhibits the development of other fungi which contribute to the formation of cork taint. Interest-ingly this fungus does not have the ability or has a very low ability to produce TCA ( Silva Pereira et al., 2000b). Several mould species were present at high levels in TCA-contain-ing stoppers: Penicillium spp (Penicillium chrysogenum, Penicillium glabrum), Aspergillus spp (Aspergillus niger and Aspergillus oryzae), C. sitophila, Mucor racemosus, Paeci-lomyces sp. and Trichoderma viride. The cork flora is considerably influenced by the processing, the surrounding

environment and storage conditions but C. sitophila, Penicillium spp and Trichoderma sp. were often among predominant moulds in cork slabs or cork stoppers ( Fumi and Colagrande, 1988; Lee and Simpson, 1993; Silva Pereira et al., 2000a, b; Alvarez-Rodriguez et al., 2002) which is in agreement with the present study. Aspergillus sp. was found in our study at quite high levels in TCA tainted corks. However, this genus has not often been reported in previous studies or reported only at low levels in cork stoppers ( Silva Pereira et al., 2000a, b; Alvarez- Rodriguez et al., 2002). Some strains were also detected in this work but only at a low level: Acremonium sp.,

Cladosporium sp. and Fusarium oxysporum.

The total fungal flora was estimated by counting the number of viable colonies of identified mould from cork stoppers ( Table 1). 2.970.3 105 CFU/g of cork was observed in TCA-containing stoppers. This value was quite similar to that obtained in the sound stoppers (2.670.8 105 CFU/g). These levels are close to those reported in cork stoppers by Alvarez-Rodriguez et al. (2002). Furthermore, the physiological state of fungal cells was estimated by epifluorescence microscopy after having recuperated the moulds by membrane filtration ( Table 2). The level of moulds detected was lower than those obtained by colony counting. This may be due to the retention of many cells by crushed stopper rubble during filtration. The level of conidia was lower in sound stoppers (9.071.1 103 per g) than in TCA-containing stoppers (12.471.1 103 per g). Green coloured conidia were predominant both in sound and TCA-containing stoppers representing 61% and 63% of the conidia population, respectively. This indicates a relatively bad physiological state of fungi in the two


stopper types. TCA content had no influence on the physiological state of fungal cells. However, this bad physiological state resulted in a poor incidence on cultivability of micro-organisms since the level of CFU was significantly higher than that obtained by epifluores-cence microscopy.

The fungal flora population was also evaluated accord-ing to the radial depth in TCA-contaminated stoppers from the surface to the centre by counting the viable conidia ( Table 3). The various moulds were present from the lateral surface to the centre as it was observed by scanning electron microscopy. The level of C. sitophila and M. racemosus decreased from the surface to the centre. There was a similar tendency for T. viride whereas Penicillium sp. levels increased from the surface up to the centre of the cork. All the moulds detected here in cork are aerobic species. Therefore, oxygen limitation inside the stopper

Table 1

Levels of predominant fungal flora in sound and TCA containing cork stoppers

Flora

Sound stoppers

TCA-containing stoppers

Acremonium sp.

+

10

3

Aspergillus niger

1.570.5

3

Aspergillus oryzae

3

1.270.5

10

3

Chrysonilia sitophila

1.770.3

10

5.370.4

10

Cladosporium sp.

+

Fusarium oxysporum

+

10

4

Mucor racemosus

+

1.170.3

2

Paecilomyces sp.

9.270.6

10

5

Penicillium chrysogenum

1.070.2

10

5

Penicillium glabrum

5

1.670.3

10

Penicillium sp.

2.670.8

10

+

10

4

Trichoderma viride

+

0.870.2

: Not detected. Detection limit of the method: 60 UFC/g. +: Only rare colonies were found (o1.2 102 UFC/g).

becomes a very unfavourable condition for their growth that explains their presence preferably in the lenticels ( Moreau, 1978; Lefebvre et al., 1983; Silva Pereira et al., 2000a). Their position in lenticels could be related to the humidity level which regulates fungi growth ( Moreau, 1978; Lefebvre et al., 1983). The progressive reduction of oxygen level and aw in the heart of the stopper could explain the results obtained for C. sitophila and M. racemosus. Both strains especially C. sitophila are known to be oxygen-demanding. Indeed C. sitophila when cultured in Petri-dish develops colonies with irregular tufts espe-cially at the margin of the Petri-dish ( Samson et al., 1995). Moreover, aw of 0.87 was found to be more favourable for the growth of Mucor and Trichoderma spp isolated from cork while Penicillium spp could grow at aw of 0.8 ( Castera- Rossignol, 1983).

3.3. TCA production from TCP by the isolated moulds

This work was carried out using either resting cells (bioconversion) or growing cells. Resting cells were recovered from the fungi culture either on solid cork medium or liquid medium.

3.3.1. Bioconversion of TCP in TCA by fungi grown on solid or liquid medium

Nine fungi strains isolated from cork stoppers were assayed for their ability to transform TCP in TCA after growth on a solid cork medium or liquid medium. Seven strains produced TCA ( Tables 4 and 5) except for C. sitophila and Penicillium sp. This result supports the absence of TCA in the sound cork stoppers since both fungal strains were the dominant fungi in these stoppers especially Penicillium sp. which was detected at trace level in TCA-containing stoppers. Besides C. sitophila was found not to produce TCA from TCP or to produce it at very low

Table 2

Physiological state of fungal conidia by acridine orange epifluorescence technique

Cells/g stoppers (%)

Green conidia

Red conidia

Total

3

3

3

Sound stoppers

5.571.1

103

(61)

3.570.9 103

(39)

9.071.1 103

(100)

TCA stoppers

7.872.3

10

(63)

4.670.8 10

(37)

12.472.0 10

(100)

Table 3

Levels (UFC/g) of predominant fungal flora in TCA containing cork stoppers according to the depth

Flora

0 to 2 mm

2 to 4 mm

4 to 6 mm

6 to 8 mm

8 mm to centre

Average

4.971.5

3

3.171.2

3

2.370.9

3

1.270.4

3

0.970.2 103

3

Chrysonilia sitophila

104

104

104

104

2.5

104

Mucor racemosus

6.971.7

105

5.770.9

105

5.070.8

105

4.871.1

105

3.370.6 104

5.2

105

Penicillium sp.

1.670.8

104

3.871.2

104

3.970.7

104

3.171.7

104

1.570.4 105

2.8

104

Trichoderma viride

2.171.1

10

2.970.8

10

1.770.6

10

0.670.1

10

o 60

1.5

10

Total

2.5 105

4.7 105

4.6 105

3.7 105

1.8 105

3.4

105

Detection limit of the method: 102 UFC/g.


277

Table 4

Bioconversion of TCP to TCA by filamentous fungi growing on solid cork medium

Time (day)

TCA (ng/g)

Bioconversion of TCP to TCA (%)

Residual TCP (%)

Biomassa (107 UFC/g)

Paecilomyces sp.

0

nd

27.1

2

36.2

0.55

37.5

7

290.0

3.65

23.08

P. glabrum

0

nd

28.8

2

10.0

0.13

24.04

7

174.0

2.18

23.08

P. chrysogenum

0

nd

15.5

2

24.0

0.32

26.92

7

273.0

3.29

20.19

Mucor racemosus

0

nd

6.96

2

3.4

0.11

63.46

7

29.4

5.21

24.04

Trichoderma viride

0

nd

65.5

2

35.2

4.89

70.19

7

190.5

4.86

21.15

A. oryzae

0

nd

7.26

2

5.7

0.06

15.38

7

18.8

0.21

13.46

A. niger

0

nd

7.34

2

5.1

0.06

15.38

7

14.4

0.16

14.42

nd: not detected.

aBiomass concentration obtained after 7 days of culture before use as resting cells in bioconversion experiments. This concentration remained constant during bioconversion.

levels ( Silva Pereira et al., 2000b). Although Penicillium-related species isolated from cork stoppers flora were among the most efficient moulds in TCA production from TCP, a significant difference between strains in the yield of TCA production was observed ( Silva Pereira et al., 2000a; Alvarez-Rodriguez et al., 2002). Some strains were very weak producers which could explain why the Penicillium sp. that we have isolated did not produce TCA at measurable levels.

On solid cork medium, the TCP level was decreased by 3085% according to the fungal strain used after 7 days of incubation ( Table 4). Only a small part of TCP was converted to TCA by fungi on cork medium. Indeed the bioconversion yield of TCP to TCA was from 0.06% to 5.21% depending on the fungal strain. This phenomenon has already been reported in the literature and it has been suggested that TCP metabolites could have been incorpo-rated into cellular material ( Silva Pereira et al., 2000a; Alvarez-Rodriguez et al., 2002). Further studies should be carried out to clarify this point.

The highest yield was obtained with Paecilomyces sp., followed by P. chrysogenum and T. viride, respectively. Except for M. racemosus which was identified for the first time in our study in cork flora, the other strains have already been known to be producers of TCA ( Silva Pereira et al., 2000a; Alvarez-Rodriguez et al., 2002). A. niger and

A. oryzae converted also TCP to TCA but at a lesser extent than other fungal strains.

Among fungi T. viride, P. chrysogenum, Paecilomyces sp, and P. glabrum showed good growth ability on cork medium. Similar results were obtained in a previous study with T. viride and P. chrysogenum ( Alvarez-Rodriguez et al., 2002). We observed the highest yields of TCA production by biomass with P. chrysogenum, Paecilomyces sp, and P. glabrum.

In liquid cultures ( Table 5), bioconversion yield of TCP to TCA was higher for all strains than in cork media except for A. niger. However, TCP degradation by fungal strains was significantly higher in cork medium than in liquid cultures. This may be related to the immobilization of fungi on cork stoppers. In fact Pallerla and Chambers (1998) reported that fungal biomass immobilization can improve both the chlorophenolic compound degradation capacity of fungi and tolerance to the toxicity of compounds.

Surprisingly A. niger grew well in liquid medium, but did not methylate TCP. Furthermore, the liquid culture was more convenient for M. racemosus to pro-duce TCA than the cork medium. All these results show the importance of the culture medium when fungal strains were assayed for their ability to synthesize TCA.

278


Table 5

Bioconversion of TCP to TCA by filamentous fungi growing on liquid medium

Time (day)

TCA (ng/ml)

Bioconversion of TCP to TCA (%)

Residual TCP (%)

Biomassa (mg/ml)

Paecilomyces sp.

0

nd

0.78

2

288.4

13.11

83.33

7

298.3

4.45

49.24

P. glabrum

0

nd

1.23

2

3.16

0.11

82

7

390.2

5.74

54.66

P. chrysogenum

0

nd

3.24

2

59.9

5.44

90

7

287.7

5.23

50

Mucor racemosus

0

nd

3.93

2

2.7

0.11

83.33

7

220.9

5.21

71.73

Trichoderma viride

0

nd

1.68

2

63.6

4.89

92.89

7

252.9

4.86

71.04

A. oryzae

0

nd

3.48

2

86.6

2.79

82.78

7

101.7

1.17

51.67

A. niger

0

nd

6.85

2

nd

0

100

7

nd

0

98.67

nd: not detected.

aBiomass concentration obtained after 7 days of culture before use as resting cells in bioconversion experiments. This concentration remained constant during bioconversion.

Table 6

Formation of TCA by filamentous fungi after 7 days of culture on liquid medium supplemented with TCP

TCA (ng/ml)

Conversion of TCP to TCA (%)

Residual TCP (%)

Growtha (mg/ml)

Paecilomyces sp.

222.12

17.07

61.54

0.68

P. glabrum

737.45

20.43

16.63

1.88

P. chrysogenum

587.78

7.87

4.18

2.99

Mucor racemosus

467.18

5.21

1.97

4.12

Trichoderma viride

156

3.37

31.71

1.58

A. oryzae

680.33

14.26

9.98

6.66

A. niger

87.92

0.65

8.31

5.95

aFinal biomass concentration after 7 days of culture.

3.3.2. Formation of TCA from TCP during culture in liquid medium

When TCP was added to the liquid medium at the beginning of the culture at the same level used for bioconversion ( Table 6), fungi growth was not influenced significantly compared to the culture conducted in the same medium in the absence of TCP ( Table 5). This indicates that TCP did not have a toxic effect on fungi growth. The transformation yield of TCP to TCA during culture with TCP ( Table 6) was higher than that observed in bioconver-sion conditions in liquid medium ( Table 5). This was the case mainly for the cultures with Paecilomyces sp, P. glabrum, P. chrysogenum, A. oryzae and A. niger. A. niger was not able to produce TCA from TCP in bioconversion

conditions while the addition of TCP in the beginning of the culture enabled it. This may be attributed to the induction of the synthesis of the TCP methylating enzyme in this case.

4. Conclusion

Stoppers with cork taint showed a fungal population quantitatively little different to that in sound stoppers, but showed an increased diversity of species. Strains often observed in sound or tainted corks such as Chysonilia sitophila, Penicillium spp, Trichoderma spp, Mucor spp, etc. were identified. However, the isolation of strains of

Aspergillus niger, A. oryzae and Mucor racemosus is new


279

in cork tainted stoppers. From the 9 predominant strains tested only 7 were able to produce TCA from its putative precursor TCP, both as resting and growing cells. C. sitophila and Penicillium sp. did not produce TCA in our culture and bioconversion conditions. In all the experi-ments carried out in this study the fungi which produced the most TCA were P. chrysogenum, Paecilomyces sp, and

P. glabrum. It is noteworthy that in the cork industry the mouldy taint is traditionally linked to the occurrence of black fungi but our study shows that green fungi were a very potent TCA producer from TCP.

Acknowledgements

The authors are grateful to the Bouchons Abel Company (Le Boulou, France) for providing them with the cork stoppers used in this study.

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