FR2705607A1 - Discovery relating to the involvement and role of moulds in the process for naturally drying oak wood, in particular the moulds: Aureobasidium pullulans, Trichoderma harzianum, Trichoderma koningii - Google Patents

Abstract

- The invention relates to a process for naturally drying oak wood by innoculation of moulds. - To this end, the invention proposes a process for drying oak wood characterised in that moulds are innoculated onto the wood to be treated in order to obtain a viable fungal flora in place which constitutes means for naturally drying and for controlling the composition of oak wood. - Application to the drying of oak wood used for the manufacture of barrels.

Description

TECHNICAL FIELD OF THE INVENTION

  THE DIFFERENT TECHNIQUES OF DRYING WOOD.

  Interests and limits The drying of wood is an essential prerequisite for its use, be it for joinery, carpentry or cooperage. Fresh wood does not have a constant hygrometry and during its dehydration, its mechanical properties vary. The wood loses its water spontaneously (from 40% maximum to 12% minimum); during this stage its density is modified and its mechanical properties gradually stabilize. The wood is dry when its relative humidity is in equilibrium with the hygrometry of the environment

STATE OF THE PRIOR ART

  The drying is obtained naturally, in the open air, artificially, in an oven or by semi-accelerated processes, combining the first two principles.20 Artificial drying Artificial drying is widely used in industry, it has the advantage of be fast and produce a very homogeneous wood in terms of hygrometry. This practice also makes it possible to limit the financial fixed assets of a timber yard. But it is a heavy consumer of energy and requires investment

  important in specific equipment and little versatile. In cooperage, artificial drying is a complementary method, allowing, in particular, to complete the natural drying and homogenize the hygrometry of a batch of wood.

  The wood mounted in batteries is deposited in a closed enclosure, subjected to a powerful ventilation to accelerate the drying, the temperature is adjustable (30-40 C). The drying of the green wood must be gradual to avoid the appearance of cracks. To limit the loss of wood during drying, the moisture is increased and the drying is divided into sequences (GIORDANO, 1971): the wood undergoes periods of steaming more or less long, interspersed with period of stabulation under ventilated shelter . The drying is then progressive and the appearance of slits is limited. The duration of

drying is several weeks.

  Natural drying Natural drying is generally used for timber intended for cooperage. It is a long operation of several tens of months, which requires a wood park ensuring a regular supply of quality raw material for the manufacture of barrels or barrels. The wood used for assembly

  Thunders are most often artificially dried.

  The seriousness of a cooper is directly related to the volume of wood available, because only natural drying can provide suitable wood for breeding and for the improvement of wines (TARANSAUD,

  1976). No description and investigation work on the mode

  has not been realized, although its interest has recently been

reaffirmed (CHATONNET, 1989).

  Natural drying is carried out on large flat and unobstructed surfaces. The soil is generally concreted and water flows are arranged, to avoid, on the one hand, the dirt of the bottom of the piles of wood by the ground, and on the other hand the stagnation of waters constituting foci of bad odors and micro-organisms responsible for putrid alterations. Drying is traditionally 24 months (21 mm staves) or 36 months (28 mm staves). We

  estimates that drying is increasing by 10 mm per year.

  In fact, our work has shown that the drying of wood is much faster, since the first 10 months are the period of intense dehydration. In addition, the succession of rains and dry periods causes only small fluctuations in

wood moisture (2-8%).

  This second period, however, is essential for the ripening of wood, because it is the seat of a set of reactions that improve the qualities of the wood (mechanical properties, aromatic and taste qualities). One can estimate at first approach that a drying of 12 to 8 months is sufficient, beyond

drying is no longer justified.

  Studies carried out with specimens placed in piles of wood, within a park, show that an average rainy period does not allow to wet the wood located in the pile. In addition, a high-speed 12-hour sprinkler system does not allow

3 2705607

  to wash the inner part of a pile. The liquid movements throughout the drying remain essentially localized on the external part of the piles. The hygrometry at the center of the stack is greater than that of the ambient environment, which explains why the innermost staves of the stack have the least number of withdrawal slots. Beyond these technical conditions, natural drying leads to wood whose drying level is very heterogeneous, their color, from light gray to dark brown, as a control. Drying after immersion Artificial or natural drying is sometimes practiced after

  immersion of the wood for a few days in renewed water.

  This practice makes it possible to wash the wood thoroughly and ensures effective removal of bitter and astringent substances. But it has the disadvantage of hardening the wood and losing some mechanical properties. In addition, the more or less complete elimination of polyphenols from wood is not always desirable. These elements actively participate in the breeding of wines (GLORIES,

1 992).

  This technique although anecdotal remains used by some

  coopers to shorten the drying period.

  Mixed drying process Mixed drying consists of using both natural drying and artificial drying, benefiting from the main advantages of

each technique.

  Natural drying is used for deep changes

  that it induces in the wood. It is a stage of wood refining.

4 2705607

  Artificial drying ensures homogeneous and rapid dehydration. It allows a more flexible use of the wood yard, for example following a heavy rain rehumid the layer

superficial staves.

  This technique, more and more frequently used, allows, without excluding natural drying, to limit the volume of stocks of

  and the immobilization of sometimes important financial sources.

Description of the figures.

  The invention will be described in more detail in the description which will

  to follow, description given by way of example only with regard to

  attached drawings in which: FIG. 1 is a diagram illustrating the influence of the density of the

  grain on the extractibility of wood polyphenols.

  -Figure 2: Influence of the Botanical Origin of Oak on the Extractibility of Polyphenols -Figure 3: Drying oak staves during natural drying. Year 992

  Relative relative humidity (Hr,%) constant after 250 days of drying.

  -figure 4: rehumectation of the staves after a rain. Influence of the position of the wood in the pile -Figure 5: Optical microscopy observation of microorganism populations distributed on oak staves (x 1000) -figure 6: Morphological structure of Trichoderma harzianium, (1) of Trichoderma koningil (2) and Aureobasiduim pullulans (3) -Figure 7: Evolution of the number of spores and the surface of the stems colonized by fungi during natural drying - Figure 8: Distribution of mushroom species during natural drying - Figure 9: Autolysis of mold strains isolated from oak wood depending on the time of culture. The autolysis is followed by the measurement of the D0210 nm -figurelO: Influence of the carbon substrate on the production of exocellular polysaccharides by Aureob. sweater. after 1 month of

culture.

  11: Estimation of galactosidase activities, glucosidases,

  manosidase, rhamnosidase and arabinosidase from Aureob. sweater.

  FIG. 12: Variation of the amylase activity during incubation

  from Aureob. sweater. in the presence of various carbon substrates.

  FIG. 13: Variation of p-glucosidase activity during

  incubation of Aureob. sweater. in the presence of various carbon substrates.

  14: Influence of the drying mode and the geographical origin of the wood on the water-soluble extract and ashes FIG. 15 Influence of the drying mode on the scopoletin content of the wood FIG. 16 Enzymatic hydrolysis of some phenolic compounds wood with a suspension of Asp. non-proliferative oryzae -Figure 17: Fractionation of the crude exocellular enzymes of some fungi encountered on staves by high performance chromatography of molecular sieving, on TSK G600-WP column -figure 18: Release of gallate units of pentagalloyl-glucose during its hydrolysis by Asp's tannase. Figure 19: Degradation of castalagine (A) and digallic acid (B) by the exocellular crude enzymes of Aureob. pull-figure 20: Release of glucose from a solution

  of ellagitannins (10%) in the presence of Aureob's mycelium. pullulans.

  21: Demonstration of the enzymatic hydrolysis of

  aesculin by coupling HPLC / Fluorimetry.

  22: Formation of aldehyde phenols during development

from Trich. harzianum.

  23: the enzymatic pathways of degradation of ellagitannins and gallotannins. Figure 24: Enzymatic degradation of monoglucoside coumarins (CET: coumarin etherase) -Figure 25: Assumption of Aldehyde Phenols Production

  enzymatic (aldehyde-phenols synthetase) from lignin.

  FIG. 26: representation of LINEWEAVER and BURK of the hydrolysis of

  ellagitannins of the oak by Auresob's heterosidase phenols activity.

sweater. and Trich. Harz.

  Determination of kinetic constants.

  -figure 27 Influence of Ph on Aureob's PeH activity. pull and

Trich. Harz.

  -figure 27a Influence of the nature of tannins (condensed tannins or hydrolysable tannins) and their concentrations on growth

of Aureob. sweater.

6 2705607

  -Figure 28: Influence of the concentration of free ellagitannins on

  Aureob's PeH activity. sweater and Trich. Harz.

  29: Extract with ethyl acetate of the TP / TS fraction of a red wine. (1): Control; (2) enzyme 2% crude enzymes; 7.5 mg: 1 TP / TS. Incubation 20 C, 16 H in 0.2M NaAc buffer, pH = 5 Identification of peaks: 1: ac gallic; 2 / (+) catechin We will now develop more precisely the technique of

NATURAL DRYING

  Natural drying encompasses a set of phenomena subordinate to climatic conditions. This is the leaching of wood,

  progressive dehydration and the development of micro-

  organisms on its surface. The latter has so far only been suspected (PONTALLIER et al., 1982). A fourth parameter to consider is the botanical and geographical origin of the

  wood. All of these factors will be addressed.

  Mechanisms for drying and leaching wood The wood stored in the open air undergoes over time a significant loss of water constitution. If on arrival on the timber yard it contains an average of 60% water, the nutter considers that it is dry when the residual moisture reaches a minimum of 12%. But the first millimeters of wood exposed to bad weather have a very heterogeneous hygrometry and subject to daily weather conditions (transient rains can at the end of drying rehumid 5 to 10 mm of wood). For this reason, it is necessary, before use, to provide a period of stabling in a

  local highly ventilated to restore a satisfactory moisture threshold.

  In the park, rainwater ensures intensive leaching of the wood. But this operation depends on its natural hydration rate; the wood fibers must be sufficiently inflated to ensure, on the one hand, the penetration of the water to the tissues, on the other hand the flow of the charged liquid. The lower the density of the wood, the more intense the leaching. The following experiment (Table 1) illustrates these phenomena: the tests were carried out with 2 categories of fresh, fine grain and loose grain wood, superficially dried for 3 weeks at 20 ° C. in the presence of Na 2 SO 4 to absorb the excess of 'humidity. The

7 2705607

  samples are then placed in 2 enclosures, for 1 to 2 days; One at 95% hygrometry and the other is an oven at 50 C. Then macerated for 5 days in a methanol-water-acetone mixture (1/1/2). The phenolic content of the extracts is estimated by the FOLIN-CIOCALTEU (IFC) reagent. After 5 days, the IFC are approximately identical in the 8 samples studied, however

  slight difference between fine and loose grains.

  The extractability index (E) expresses as a percentage the ratio between the IFC obtained after 1 day of maceration and that obtained

after 5 days.

  E = FClj / FC 5j x 100 In optical microscopy, there is a tightening of the fibers and therefore a lower ability to yield the constituents trapped in the tissues. The cross-texture of the wood "plywood effect" (ROLAND, 1980) accentuates the phenomenon. This explains why a rich wood

  polyphenols may have a low extractability index.

8 2705607

table I

  influence of the mode of drying of the wood on the extractability of the total phenols Extractability index E = IFC1j / IFCSj fine grains loose grains characteristics of the local time (G)

1 72% 98%

% hygro.

2 60% 98%

1 5_

  1 5 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

1 25% 34%

Steaming

2 26% 42%

  The density of the grain can be classified, for the samples considered, in descending order of porosity determined visually: Limousin; Vosges; Nevers; Allier; Tronçais. In this case, these are types of wood with the characteristics of

majority of the woods in these regions.

  This classification is well correlated with the wood extractibility index (fig.1). These are different samples that are naturally dried identically, sorted only by type, regardless of geographical origin. The results presented correspond to the average of about fifty samples in each case. In each of the regions there is a mixture of wood of different grain character, but

  whose average remains the reflection of this visual ranking.

  Over a long drying period, leaching loss is important in the first few months and then when the moisture content of the wood goes down. low enough, the phenolic depletion is considerably slower, as in the model experiment in Table I. In fact, the more we advance in drying and the less the constituents

9 2705607

  parietals are likely to be driven by rainwater.

  Throughout the drying, the oxidative mechanisms, of enzymatic or chemical origin, lead on the one hand to a decrease in the polyphenol content, and on the other hand to different structural modifications characterized by polymerizations and condensations with the polymers. of the wall (cellulose, lignin ...) (SHUYUN PENG et al., 1991). Recently we have been able to confirm these results for

  French oak species in natural drying situation.

  Analytically, natural drying leads to a depletion of wood (Table 11) compared to artificial drying. But the leaching and the lowering of the moisture content are not enough to explain the profound changes undergone by the wood. table It influences the drying mode on the phenol acid content of oak (Allier) g sawdust (60 mesh) for 20 ml of maceration (MeOH / H20 / HCOOH 50/40/10) 96h under nitrogen with magnetic stirring Natural drying Artificial drying ac. gallic (mg / l) 64.0 98.0 ac. ellagic (mg / l) 58.0 70.0

IFC (1) 49 67

  I ellagitannins (2) 0.08 0.12 I gallotannins (3) 0.26 0.68 1: folin index ciocalteu 2: reaction with nitrous acid (Bate-Smith, 1972) expressed as an index ( A DO) 3: reaction with potassium iodide (Haslam, 1965) expressed as an index (A DO)

2705607

  ww Influence of Botanical Origin: Oaks belong to the genus Quercus; but the timber destined for cooperage is attached to the smaller section of Lepidobalanus, in which we find the species of economic interest of the first order: sessile oak (Quercus sessilis, Quercus petraea or oak) and pedunculate oak ( Quercus pedunculata or Quercus robur). These species have been chosen for their ability to act favorably on

wines (GLORIES, 1987).

  The soil influences the structural characteristics of wood and their phenolic content. The results in Table III represent the average of 20 samples per soil studied. These results can be explained by the dominant characteristics of each of the regions responsible for types 15.

table III

  influence of geographical origin on the phenolic richness of Quercus sp. Q. pedunculata Q. sessilisQ. sessilisQ. sessilis terroirs Limousin Center Vosges Nevers

IFC (1) 46 28 26 63

  catechins (μg / g) 872 440 512 586 I ellagitannins (2) 0.21 0.08 0.12 0.28 1: FOLIN-CIOCATEU 2 index: reaction with nitrous acid expressed as an index (A DO) Some biometric analyzes of wood samples indicate that spring wood is less abundant for Q. sessilis (16%) compared to Q. pedunculata (38%). This wood rich in large diameter bundles promotes the extraction of phenolic compounds from tissues (Figure 2). The results obtained are

  comparable to those of FLETCHER (1,978).

  Influence of the local climate The climatic conditions of each region significantly orient the drying phenomena. Thus, the succession of rainy and dry period, hot and cold, determines both the

  speed and intensity of natural drying.

  Climatic parameters useful to the reasoning of drying: The reasoning of the drying and the choice of the date of beginning of the operation passes by a perfect knowledge of the local climate (Macro-climate). Thus, some areas with little rain and too dry are not recommended, because even if the drying is coupled with a system of frequent spraying of the wood, the too fast dehydration will induce a loss of wood little compatible with the profitability of the technique (cracks withdrawal, incomplete leaching). French climates (oceanic, transitional oceanic, continental,

  Mediterranean) seem, on the other hand, to be appropriate.

  Three parameters are taken into account to choose the beginning of the drying: First, the drying must be preceded by a phase of leaching abundant wood, essential to its quality; - Then comes the establishment of a flora, which requires sufficient moisture and rainfall allowing the

development of thalli.

  - Finally, we end with a sufficiently dry period to induce progressive dehydration and

continuous wood.

  Climatic conditions: example of the Bordeaux climate On an average of 50 years of meteorological records, the Bordeaux climate is characterized by (VIVAS, 1 987) * A moderate thermal amplitude (ATha) (ATha = Tmin Tmax = 15 C); including a minimum in January (+5 C) and a maximum offset in August (+20 C). The role of thermal flying played by the ocean

  close explains regular results and without extremes.

12 2705607

  * Precipitation regularly distributed throughout the year, without a period of true drought. Some years undergo a more or less accentuated subsistence. The degressive precipitation classification, for the determination of the climatic type gives: Winter (229 mm), Autumn (222mm), Spring (212mm), Summer (154mm). The formula H.A.P.E is characteristic of

oceanic climates.

  Although the last decade (80-90) was marked by record rainfall deficits and abnormally high temperatures, the average ocean climate is perfectly

climatic profile presented.

  Impact of the average regional climate on the choice of the beginning of the drying: example of the Bordeaux climate Over a period of 12 months, and to meet the presented requirements (cf 3.1), the natural drying in the Bordelais must begin in November, a months before maximum rainfall (December precipitation: 100 mm). The summer period

  relatively little rain allows a homogeneous drying of the wood.

  The use of wood in cooperage may be preceded by a changeover to

the oven a few days.

  The drying operation is spread over 12 to 24 months, taking care to use the wood at the end of the summer period, or after a few weeks of stabling in a ventilated room. The drying period is more efficient, adapted to climatic conditions, it can be shortened. The decision to interrupt the drying is related to the appearance of the wood (visual observations) and its hygrometry

(measured by probes).

  The natural dehydration of oak wood during a dry year followed by a period of heavy rainfall. Year 1992 Regardless of the geographical origin of the wood (fig.3), the dewatering profiles of the staves are comparable. Surface wood quickly loses its water, but Hr remains dependent on periods of rain. A passing rain can cause an increase in Hr of 10%. But the loss of this water is fast, in 10 days the surface returns to its initial hygrometry. The inner layers of the wood dehydrate quickly during the first 100 days and then

13 2705607

  rate of drying is noticeable. In 12 months the wood

  reaches a Hr of 18% compatible with the manufacturing constraints.

  Comparing the 1992 results with those of 1987, we observe the same type of evolution with however a significant drying period spread over 200 days instead of 100 days. Additional analyzes show that a rain of 80mm (over 72 H) does not cause a significant variation in the Hr of a dry wood (fig.4). On the other hand, on green wood the wetting is more important, this is to relate with the previous results

  concerning the accessibility of the solvent to the tissues.

TECHNICAL PROBLEM POSE

  The study of the different methods of drying oak wood has made it possible to draw up a summary of the advantages and

disadvantages of these techniques.

  The porosity problems associated with this operation should be defined and specified, as well as the organoleptic properties of the phenolic compounds of the wood after natural drying. Some

  Answers will be made in the second part of the work.

  Natural drying is traditionally an operation that occurs without the intervention of man. Molds grow when they are sufficient and when the environment is favorable. It is an operation that is not mastered and gives

  wood for cooperage of very heterogeneous quality.

  After a program of scientific research on the subject we have highlighted the existence of molds perfectly adapted to wood and likely to cause modifications compatible with a better quality of this wood

14 2705607

SUMMARY OF THE INVENTION

MATERIALS AND METHODS

  1 - HPLC Analysis of the Phenolic Compounds of Oak Wood

  1. - Determination of gallic acid and these galloyl derivatives

  glucose The oak is devoid of digallic acid; on the other hand, the chestnut contains a few hundred pg / g of wood 10 (SALAGOITY et al., 1986, VIVAS et al., 1992). This molecule is particularly bitter (JOSEPH and MARCHE, 1975) and its hydrolysis, leading to the release of gallic acid, causes the complete disappearance of this sensation. This type of observation is also valid for galloyls-glucose. Its study is therefore important because it illustrates the impact of fungal esterases on the flavor of

wood extracts.

  The extraction and assay technique is derived from the protocol of SALAGOITY et al. (1986). It comprises a purification of the aqueous medium by extraction with ethyl acetate at pH = 7 (elimination of neutral compounds) and a second extraction at pH = 2 to collect and concentrate gallic acid, ellagic acid, acid

  digallic and m-digallic, trigalloyl-glucose and pentagalloyl-

  glucose. The identification of the peaks is carried out by injection of the reference products and by comparison of the UV spectra between 240

  and 400nm. The results are expressed in mg / g of wood.

  1.2- Separation and identification of oak ellagitannins The phenolic composition of unburned wood is relatively simple. Essentially, we find in water extracts vescalagin, catalagine, gallic acid, ellagic acid and some dimeric forms, roburines and grandinin, in small quantities. The separation is carried out on column C18 (ODSTM ultrasound, dp:, p: di: 4, 6mm, long: 25 cm). The solvents of elutions A and B have the following composition: A = H20

  ultra pure + 5% HCOOH R.P; B = MeOH bidistilled + 5% HCOOH R.P.

2705607

  * Elution program:% B: O for 10 Omn; % B: 2% in 10 minutes; % B:

% in 20 minutes; % B: 100 for 1 Omn.

  The purity of the peaks is controlled by the UV spectrum measurement between 240 and 400nm. The molecules are identified by comparing the retention times obtained from the pure products injected under the same chromatographic conditions, which has the 1H / 13C NMR spectrum, and by measurement of k. max in the UV. Products

  we were provided by A.SCALBERT.

  1.3- Dosage of coumarins The coumarins contained in 20ml of water extract with water are extracted with 3 x 20ml of diethyl ether. The organic phase is evaporated to dryness, taken up in MeOH and chromatographed in

  the conditions described by CHAUVET et al (1992) in fluorimetry.

  Calibration is performed by injection of coumarin solutions into

known quantities.

  1.4- Research of the transformation products The products resulting from the enzymatic degradation of the phenolic compounds of the wood are identified: On the one hand, thanks to the TR obtained in HPTLC under standard conditions (cf 2./2.2) and compared to those of pure products. Then by recovery of the different spots separated on TLC plate (C18 silica scraped) and solubilized in ultrapure water, centrifuged (4500 rpm, 20 minutes), concentrated under partial vacuum to 100upl at C and injected in HPLC; On the other hand, by direct injection of the reaction media

  after centrifugation (4500 rpm, 20 min) in HPLC.

16 2705607

  2. Use of high performance thin layer chromatography 2.7 - Purification of certain phenolic compounds * The phenolic compounds of grapes and wine are isolated according to the techniques developed by GLORIES (1978). * Phenolic compounds of wood: The main ellagic tannins (vescalagin, castalagine) are isolated from a green chestnut extract depleted with water. The separation of the two isomers is carried out by HPTLC. 10 μl over 5 mm of a 1 g / l solution are deposited on RP1 8 plates (silica grafted with octadecyl groups); the migration is carried out in a linear growth chamber with a mixture of H 2 O / CH 3 COOH / MeOH (89/10/1, v / v / v); reading is done at 280

nm with a CAMAG densitometer.

  Gallotannins and especially pentagalloyl-glucose was isolated from commercial tannic acid or Hamamelis tannins

according to the same protocol.

  2.2- Follow-up of the enzymatic transformation of certain phenolic compounds The enzymatic hydrolysis of the wood tannins is monitored by HPTLC under the previously described conditions (see 2./2.1), with a dilution factor of 10 or 100. The results are expressed as the decimal logarithm of the surface of the

corresponding peak (log Sp).

  The degradation is measured over time by taking 100 μl of supernatant from the centrifuged reaction medium (4500 rpm, min). The heterosidase activity is stopped by 10 μl of N.HCl.

17 2705607

  3- Microbiological analyzes 3.1- Isolation of micro-organisms The molds are isolated: a) from sawdust (60 mesh) representing the first 5 millimeters of the surface wood (sawdust is stored at -20 C) of a set of staves dried naturally from 6 to 18 months and spread over the entire wood yard. The fungal suspensions are obtained by sonication (2 x 5s) of 1 g of surface sawdust

staves in 10 ml of sterile water.

  b) from smear of the surface of the woods being dried.

  A sterile hydrophilic cotton sweeping the surface of the staves is

stirred in 10 ml of sterile water.

  Solid media are inoculated with 1 ml of the sawdust maceration water. The incubation is 24 or 72H at 25 C. Morphologically different strains are isolated by fragmentation of mycellium portion. Beyond 5 days of culture the box is completely covered by molds and the selection of pure strains becomes impossible. Only individuals encountered in all isolates are kept for purity and identification; because many fungal isolates are present only on certain samples. The purpose of the work being to define the specific flora of natural drying we have kept only a few

  specimens found consistently throughout the drying process.

  Successive subcultures, necessary to purify each identified strain, are carried out on MAG solid medium: - malt agar extract: 45g - agar 1 5g - glucose: 25g - distilled water: 1000ml

autoclaved 15 minutes at 1 15 C.

18 2705607

  3.2- Culture and constitution of collection * culture media - Medium CZAPEK-DOX: (NH4) H2PO4 5.75 g / I K2HPO4 1.0 g / I KCl 0.5 g / I MgSO4 0.5 g / I FeSO4 0.005 g / I Glucose 25 g / I o0 Distilled water qs 1 I pH = 5; sterilized 1 5min 120 C - Non-Proliferative Medium (NP) (NH4) H2PO4 5.75 g / I K2HPO4 1.0 g / l KCl 0.5 g / l MgSO4 0.5 g / I FeSO4 0.005 g / I Distilled water qsp 1 I pH = 5; sterilized 1 Smin 120 C -milieu MAG: malt agar extract 45 g / I Agar 15 g / I Glucose 25 g / I Distilled water qsp 11

sterilized 15 minutes at 115 C.

  - Wood medium Oak extract with water 1 g / I Malt agar extract 30 g / l Agar 20 g / I

  Distilled water qs 1 I pH = 5; 0.22p sterile filtration.

  * collection The molds in culture are preserved either on wood medium on inclined agar, or in petri dish after sporulation on

  MAG medium, in NP liquid medium supplemented with 20% glycerol and stored at -20 ° C.

19 2705607

  3; 3- Identification of the strains The identification is carried out in collaboration with A. LABARERE (INRA-University of Bordeaux II) and E. GUEHO (PASTEUR Institute,

Paris, mycology laboratory).

  APITM tests are performed on liquid medium. The liquid culture (CZAPEK-DOX) is inoculated with a suspension of spores obtained from culture on solid MAG media. After 15 days of culture at 20 ° C. (+ 2 ° C.), on a stirring table, the mycelium is separated from the culture medium and homogenized with the ultraturrax (30s); 10 ml of homogenate are removed and centrifuged (20 min, 4500 rpm), rinsed with sterile water and recentrifuged. The pellet is suspended in a non-proliferating medium. After 48 hours at 30 ° C., the mixture is used to

  assimilation and fermentation tests.

  E. GUEHO, (PASTEUR Institute, Paris) from solid cultures

  on MAG, performs the taxonomic description after culture on

  several specific media, allowing in particular the development of fructifications 4. Measurement of enzymatic activities 4. 1 Precipitation of total exocellular proteins The precipitation of ammonium sulphate proteins [(NH4) 2SO4] at 80% saturation (66g / 100ml) is a quick method of separating exocellular enzymes from a culture medium. 100 g of a centrifuged culture medium of a mushroom strain, in a CZAPEK-DOX liquid medium, 66 g of (NH 4) 2 SO 4 are added in 2 steps at 4 ° C. The medium is supplemented a first time with 33 g of salt, and stirred 3H at 4C (350 rpm). Then saturated with a second add of 33g and stirred 3H at 4 C (350 rpm). The medium is centrifuged (4500 rpm, 30 min). The pellet is solubilized in 2 ml of NaAc buffer (0.2M, pH = 5) and supplemented with (NH4) 2SO4 until its solubility limit at 4 C, it is stirred again (2H, 4C, 300rpm)

and centrifuged (4500 rpm, 30 min).

2705607

  The precipitated fraction containing a strong activity 1-

  glucosidase (assayed according to the method of DARRIET et al., 1988) is taken up with 2 ml of NaAC buffer and used directly, or

  freeze-dried and stored at -20 C.

  4: 2- Coumarin Etherase Activity * The demonstration of the activity is made on a 1 mg / 10 ml solution of aesculin in NaAc buffer (0.2M, pH = 4.5) incubated at 25 ° C. for 12 hours. in the presence of 10% of the (NH4) 2SO4 precipitate (80% saturation). The aglycone formed (aesculetin) is dosed

directly by HPLC.

  The activity is measured by the release of native glucose from solution containing about 1 mg of coumarin / 10 ml of NaAC buffer. High concentrations are obtained by extraction at pH = 7 of an oak extract with water and dry evaporation of the extract. The method of glucose determination uses a kit suitable for the determination of

  glucose in blood serum (GLUCOSE [HK] TM; SIGMA diagnoses).

  The results are expressed in mM glucose / ml / h.

  4.3- Measurement and monitoring of the phenol heterosidase activity: * The demonstration of the activity is carried out by the incubation (25 C, 12H) of pentagalloyl-glucose (1 mg / 1 Oml) in the presence of 10% total protein precipitate, and assay gallic acid released by HPLC. * The measurement of the enzymatic activity is based on the release of carboxylic functions lowering the pH of a medium of

reaction.

  In a screw tube is placed 9 mg of an aqueous extract of green oak in 9 ml of a NaCl solution (0.1M, pH = 4.5) and 1 ml of the precipitate with (NH4) 2SO4 at a rate of 1%. A control tube is produced under the same conditions, without enzymes. The pH is measured in both tubes at zero time (pHto: for the control, pHeo: for the enzyme tube) and after 4H incubation at 25 C (pHt4: for the control; pHe4: for the

enzyme tube).

21 2705607

  Optimum conditions were established beforehand, the correlation between the release of glucose (enzymatic method) and the drop in pH are perfectly similar after the first hour of incubation (r = 0.98). The Phenol heterosidase activity is calculated from the relation: (pHto - pHt4) - (pHeo - pHe4) x 100

  PeH in 10 u pH / ml / h = -----------------------------

  4.4- characterization of an aldehyde-phenol synthetase activity The mode of action of this activity not being known to us

  simply highlight the transformations.

  The synthesis of the aldehyde-phenols is studied from humidified oak sawdust (5g; 60 mesh), distributed in a petri dish and seeded with fresh mycelium, in the form of the pellet of a culture of 15j (1 mg mycelium / 100ml of CZAPEK-DOX culture medium). The dishes are incubated at 30 ° C. for 15 days. The sawdust is then extracted by means of a hydroalcoholic solution (50% v / v + 1% N HCI) for 24h at 25 ° C. 10 ml of centrifuged liquid (4500 rpm, 20 min) is adjusted to pH = 7 (NaOH N) and exhausted with 10.5 and then 5 ml of diethyl ether. The organic phases are combined in a separating funnel and then evaporated to dryness; the residue is taken up in 1 ml of MeOH. The extract is chromatographed under the conditions

  described by SALAGOITY et al. (1 987).

  Vanillin production is syringaldehyde is quantified

  from standard solutions injected under the same conditions.

  - Determination of fungal macromolecules 5.1- Precipitation and determination of polysaccharides a) Precipitation: The polysaccharides are precipitated (24H at 4 C) from 0.5ml of centrifuged culture medium (4500rpm, 20mn) for 2.5ml

  ethanol 95% v / v (1/5, v / v). After washing with 95% ethanol

   C and centrifugation (4500 rpm, 20 minutes), the precipitate is dissolved in 10 ml of water

22 2705607

  b) Neutral polysaccharide assay: The neutral polysaccharides are determined by the

sulfuric phenol.

  1 ml of phenol (5% aqueous solution) and 5 ml of sulfuric acid RP are added to 1 ml of the solution to be determined. After immediate stirring, the tubes are placed in a water bath at 100 ° C. for 1 min and then cooled to 0 ° C. The optical density is read at 490 nm under 1 cm. The values come from an average of three assays, the

  results are expressed in mg / l of glucose equivalent.

  c) Assay for acid polysaccharides: The acid polysaccharides are assayed by the meta-phenylphenol method. To 1 ml of the solution to be dosed is added 5 ml of sulfuric acid RP, operating in an ice-water bath. After stirring, the tubes are placed in a water bath at 100 ° C. for 3 minutes, cooled to 0 ° C. and supplemented with 100 μl of meta-diphenylphenol (0.15% in NaOH).

  0.5%). After 15 Smn the optical density is measured at 520 nm under 1 cm.

  The values come from an average of three assays, the

* results are expressed in mg / I ac equivalent. galacturonic.

5.2- Determination of total protein

  The proteins are assayed by the Coomassie blue method.

  To 100 pl of centrifuged culture medium (4500 rpm, 20 min) was added 1 ml of Coomassie blue G250 [10 mg bl. Coom. G250 + 5ml EtOH 95% (v / v) + 10ml 85% phosphoric acid (w / v) + distilled water (qs 100ml)]. After 5 minutes the optical density is measured at

595 nm under 1 cm.

  The results are expressed in mg / l of BSA equivalent (bovine serum albumin)

23 2705607

  HIGHLIGHTS OF THE FUNGAL FLORA OF

OAK WOOD

  1- Observation in situ In optical microscopy, fragments of surface wood appear covered by places of black agglomerations and

  brilliant (x 400). Each grouping seems to consist of sub-groups

  more or less spherical units. The highest concentration of population is located in the defecated areas and in the slits of withdrawal, very frequent at the end of drying. At a higher magnification (x 1000), the flora is more easily distinguished and can be classified according to color and shape: - clusters of more or less elongated black spheres cover the wood (Figure 5a). In the absence of these molds, the fibers are "opaque brown" and leave little light; on the other hand, after the implantation of this type of micro-organisms, the fibers become white, translucent and not very thick. It can be assumed that these are cellulolytic or lignolytic microorganisms; the mycelium is distributed among the wood fibers and remains difficult to observe. It can be demonstrated by culturing some blackish masses in a glucose solution (fig.5Sb); - Microorganisms forming small green spots (fig.5c). They accumulate and develop by covering the previous organisms (fig.5d). The mycelium is distributed on the surface

wood.

  In scanning electron microscopy: [cf. annex sheet]

  2- isolation and identification of strains.

  Location on the timber yard The micro-organisms are isolated either from sawdust (60 mesh) representing the first 5 mm of the surface wood (sawdust is stored at -20 C), or from sterile cotton rubbed on the surface of the wood. wood and shaken in 10 ml of sterile water to collect the

spores.

  On the one hand, the liquid inoculum is immediately seeded on MAG solid medium at a rate of 1 ml per 10 ml of agar; else

24 2705607

  24 = / w part, the sawdust containing the inoculum is added to sterile water (

  1/10, p / v) and sonicated 10 sec before being seeded.

  Fifty successive subcultures will be necessary to isolate mushrooms, of which 3 represent 99% of the biomass and 1 more than 80%. For the latter, we have 4 strains isolated at different

  drying times and at different locations on the lumberyard.

  A sample of each species is deposited in the collection of the Institute of oenology; PASTEUR Institute (Paris) owns

also some copies.

  The identification results are collated in Tables IV and V and the board We identified: - Aureobasidium pullulans (de Bary) Arnaud; -Trichoderma harzianum Rifai -Trichoderma koningii Oud - Rhizopus stolonifer (Ehrenb ex Lindner) and isolated an undetermined strain coded "ECLR2-91" The sources of inoculation are diverse (table vi) since with the exception of ECLR l all species meet with more or less importance throughout the environment. Thus, during drying, the spores and conidia will settle on the surface of the wood and only the best adapted can be established and become the majority. The ECLR fungus can be considered as a contamination because it comes exclusively from the bark of trees. R. Stolonifer is also a strain whose presence is accidental insofar as it has been isolated from a large number of moldy substrates (BOTTON et al., 1985) and is not specific to the

  fungal flora related to natural drying.

2705607

table Vl

  localization of the different sources of contamination on the timber yard isolated from: runoff water (a), wood surface swabbing (b), fine soil maceration (c) agar slab stall air (d) water bark wood logs soil atmosphere isolation abc - d genera / species Rhizopus stolonifer - + +++ Aureobasidium pullulans ++ + Trichoderma harzianum + + + - + + Trichoderma koningii ++ + +

"ECLR2-92" 0 +++ 0 0

  0: absent; -: rare (<isouche / 10); +: present (1/10); ++: frequent (2 to 8/10); +++: dominant species (> 8/1 O)

26 2705607

  3- Ecology of molds during natural drying 3.1 - succession of molds during drying Over the entire wood yard, staves are taken at different stages of drying; wood samples (5x5 cm) are cut and distributed into sterile bags before analysis. Each sample (10 test pieces) represents a different drying time (0, 4, 6, 12, 18, 20 months). In the laboratory the test pieces are washed in 100 ml

  sterile water for 2 hours on a stirring table; then sonicated (5x2s).

  Wash water is used for spore counting on MAG; the rinsed specimens are placed in petri dishes and covered with cooled MAG agar (40 C), they will be used for the study

  of the distribution of mycelias on wood.

  After 4 days of incubation (25 C) for spores and 6 days for mycelium, we count: - the number of visible colonies (SFT = spores forming thalli). This criterion should be used with caution. It can give an indication of the pressure of the inoculum on the wood surface and also expresses the ability to sporulate or the ability of the mycelium to

  fragment. Each SFT can encompass one or more spores.

  -% of surface mycelium (% mycelium). This method makes it possible to better express the importance of fungal biomass by

relative to the surface of the wood.

  Throughout natural drying, the number of spores increases (Fig. 7a); beyond 12 months the rate of accumulation of the inoculum (spores, conidia, chlamydospores) is significantly reduced. At the same time (fig.7b) the colonization of wood by mold is

fast and maximum in 12 months.

  a) evolution of inoculum quantity and rate of implantation of mycelias (fig.7a and b) Conidia, spores and chlamydospores do not participate in the drying process, but they will be deposited by wind or by the leaching water on the green stave. The mycelia implanted on wood (Trichoderma sp.) Or in surface fibers (A. pullulans) are derived from this native inoculum which germinates and forms a thallus. Plus the inoculation pressure is great and

  the faster the wood is covered with mushrooms.

27 2705607

  The increase in the number of spores in the first months is mainly due to a contribution of the surrounding environment (wind, water, dust ...), but as soon as the first thalli have reached their phenological maturity, the wood cover is realized of close by. When colonization becomes saturating (12 months: 87%), the sporogenous inoculum remains constant. The whole surface is occupied by thalli more or less overlapping and the expansion of the populations in place is null. Spores will disperse

stumps on green wood.

  b) distribution of species according to the duration of drying (Fig. 8) According to the classification of JARWIS (1978) Aureob. sweater. is a constant species (frequency> 80%), Trich sp. is occasional (frequency 1020%) and the other species represent less than 10%

  of the population are accidental (R. stolonifer., ECLR).

  Population dynamics remain relatively stable. At the beginning of drying, the presence of 3 to 4 spores of different species may be noted, but as early as the 6th month only A. pullulans is present. At the end of drying the part of Trichoderma sp. increases. Observations are renewed for implanted mycelia, but with a faster and more extensive exclusive presence of A. pullulans. This suggests that it is a species well adapted to the development of

  wood and has a source of inoculation into the environment.

  The study of the ecology of the species, implanted on the wood, reveals that A. pullulans is a specific mold of the natural drying of oak wood; at least in Bordeaux. Other mushrooms

  can be considered as a species of contamination.

  3.2- The inhibition phenomena The relative homogeneity of the fungal flora and the late implantation of Trichoderma sp., On the wood already colonized by A. pullulans in stationary phase, suggest that a certain number of

  Inhibition mechanisms can act.

  On the one hand, we evaluate the possible inhibitory effect of Trichoderma sp. and A. pullulans on several microorganisms frequently encountered in the environment. The strains tested are taken from solid media and deposited by successive streaks in the center of the. box (MAG solid medium). After incubation for 96 hours at 25 ° C., 1 ml of a spore suspension is added to the medium.

28 2705607

  (Trichoderma sp.) Or chiamydospores (A. Pullulans); after 8 days of incubation the results are collected - the inhibition is positive (+) when the previously implanted strain is covered by the species tested and the box is practically occupied by a single type of microorganism; - the inhibition is negative (-) when the test species has not developed; - the inhibition is weak (+/-) when the two mycelia continued their development each in a part

free from the culture medium.

  On the other hand, we study more particularly the competitions between Trichoderma sp. and A. Pullulans. The same protocol is used and inhibition is followed through snapshots

  performed at different incubation times.

  Table VII Inhibition of several fungi by Trichoderma sp. and Aureobasidium pullulans +: +/- inhibition: weak inhibition -: no effect Species tested Inhibitory species

Trich. sp. Aureob. sweater.

S. cerevisiae (EG8C) + +

Trichoderma sp. / + / -

A. pullullans + / B. cinerea + +

A. niger + +/-

Penicillium sp. + +/-

R. stolonifer +/-

ECLR2-91 + ±

  It is found (Table VII) that both species (Trichoderma sp, A. pullulans.) Inhibit all microorganisms tested. It is therefore understandable that when A. pullulans, after 18 to 24 months of development, leaves an ecological gap on the wood, T. harzianum and koningii

can take root.

29 -2705607

  Due to their adaptation to woody environments and their virulence, these fungi have the potential to colonize the wood surface and constitute a limited and specific fungal flora. Antagonism of Trichoderma sp. with regard to Botrytis cinerea has already been reported by WOOD (1951). ARTIGUES (1981) highlights the existence of a positive correlation between the level of the & (1-3) glucanase activity of

  Trichoderma sp. and the destruction of the mushroom.

  Inhibition may also be related to certain antibiotic, fungicidal or fungistatic substances (Wylie and Morenzoa, 1977-1978). To illustrate this phenomenon we have completed our observations with a study concerning competition and inhibition.

of A. pullulans by Trichoderma sp.

  Trichoderma sp. establishes then colonizes all the surface of the culture in 15j. We can also note on the line delimiting the two species the appearance of a yellow-orange color that is not present in pure culture. On the other hand, this zone contains a water-soluble substance which causes growth arrest of A. pullulans (orange agar dissolved in distilled water, sterile filtered 0.22p and added to a MAG solid medium inoculated with A.

pullulans).

  4- Discussion and conclusion 1- The microscopic observation of the oak wood surface during natural drying reveals the presence of a certain number of microorganisms. Some developing their mycelium between

  wood fibers, the others remaining on the surface.

  2- This fungal flora is represented for more than 80% by Aureobasidium puflulans. There are also some isolates of Trichoderma harzianum and koningii. The presence of Rhizopus stolonifer and an unidentified strain coded ECLR2-91 remains limited.

2705607

  3- During a drying period of 18 to 24 months, the flora is exclusively constituted by Aureobasidium pullulans; Trichoderma sp. appears at the end of the period and then represents 10% of the flora. The implantation and the colonization of the wood by Aureobasidium pullulans are linked first to a very important inoculation pressure by the whole environment,

  followed by inhibition of a number of micro-

  organisms, finally by a remarkable adaptation in the middle.

  Nevertheless, when this species colonized the entire surface of the wood (stationary phase), Trichoderma sp. can be implanted, thanks to a suitable enzymatic and antibiotic material. The wood progresses gradually from the natural drying stage to that of decomposing substrate; the flora is then deeply modified: apparition

  large colonies of R. stolonifer and Trichoderma sp.

  sometimes accompanied by Geomyces sp., Geotrichum sp.

Trichothechom ... (MOREAU, 1952).

  DEVELOPMENT AND GROWTH OF MOLD

  All laboratory tests mainly concern A. pullulans (PNDS4-91, SNDS1-91), T. harzianum (VFFS1-91) and

  ECLR2-91. The cultures are practiced on liquid medium CZAPEK-

  DOX or in a non-proliferating medium. Growth is evaluated from

dry weight of mycelium produced.

  1- Mold growth To measure the ability of fungi to grow, we test several species, isolated from wood, in liquid medium (1mg of dry mycelium / 100ml of medium). After 1 or 2 months on a stirring table at 200 ° C. (+2 ° C.), the cultures are centrifuged (4500 rpm, min). The pellet is rinsed 3 times with sterile water and 3 times with 95% v / v ethanol; each operation is followed by centrifugation (4000 rpm, 20 min). The mycelia are then dehydrated in an oven (60 C), to constant weight. By difference in weighing between the empty calibrated tube and containing the dry centrifugation pellet, the amount of mycelium produced is evaluated in mg / 100 ml of medium or in mg / g of

carbon substrate consumes.

31 2705607

  The growth of microorganisms in glucose medium is very different depending on the species studied (Table VIIi): - ECLR2-91 develops little; moreover, on wood or on agarwood extracts, its growth is also very slow; - R. stolonifer (SB-92) has a very limited growth whereas in solid medium, on wood or on agarwood extracts its growth is very fast; it seems, therefore, that the liquid medium is not suitable for its culture; - T. koningii (VCFS1-91) and A. pullu! Ans (SNDS1-91) have a low capacity to develop on a liquid medium agar media give the same type of results; T. harzianum (VFFS2-91) and A. pullulans (PNDS4-91) are best adapted to the liquid medium, their growth is rapid; the use of sugar is complete. In addition these features

Table VIII

  [2 months culture; carbon substrate: glucose 25g / i; inoculumr: tmg / 100mlj stem species weight rmyceliumrn residual sugar mg mycellium c / l per g substrate mg / 100ml mg / g substrate medium Trichoderma: - T. harzianumVFFS2-91 100 40 0.5 41 - T. koningiiVCFS1- 91 7.5 3 9.0 4.7 Aureobasidium: - A. pullulansPNDS4-91 127.5 51.2 53.5 - A. pullulansSNDS1-91 7.1 3.1 9.5 4.5 Rhizopus: -R . stolonifer SB-92 6.2 2.5 8.4 3.7 unknown

ECLR2-91 2 0.8 12 1.5

32 2705607

  also express themselves on wood and in agar media

of wood extract.

  Carbohydrate metabolism (with directly assimilable glucose) is deficient for most species tested except for T. koningii (VCFS1-91) and A. pullulans (SNDS1-91).

Table IX

  Incidence of the carbon substrate on the growth of Trichoderma harzianum and Aureobasidium pullulans [culture: lmois; inoculum: 1 mg / 100ml] mg of mycelium mycelium mg / g / 100 mi of substrate

  substrate dose g / I T. harz. A. pull. T.harz A. pull.

  glucose 25 24 34 9.6 16.3 polysaccharides 25 10 8 4 3.2 wood oak tannins 25 33 46 13.2 18.4 with water 2 5 cellulose 5 14 9 28 18 To test the influence of the energy source on the growth of T. harzianum and A. pullulans, we carried out cultures under the same conditions using a range of substrates that can be found in oak - glucose wood: D (+) - glucose - polysaccharides: extracts from oak sawdust (60 mesh) in 0.1% HCl (1 kg of sawdust per 5 I, stirred for 72 hours at room temperature). The bluish-colored liquid is then added with 95% EtOH at the rate of 1 vol to 9 vol EtOH. After 3 days at -4 ° C., the precipitate formed is centrifuged (5000 rpm, 30 min), rinsed with 3 times with 95% EtOH at -15 ° C. and centrifuged (5000 rpm, min). The pellet is taken up by the minimum of water and freeze-dried. The resulting powder contains 20% neutral polysaccharides. Most

33 2705607

  wood polysaccharides being in combined form, we had to abandon purification on pvpp because it

profitable.

  - cellulose: cellulose DIFCOTM - oak tannins: extracts by water exhaustion of 1 kg of sawdust (1/5, w / v) for 96 hours at room temperature, on stirring table. The crude solutions are collected, the volume reduced to 1/2 by evaporation and the concentrated aqueous extract is then purified.

  by demixion according to the technique of GLORIES (1978).

  The two species studied (Table IX) can be grown on all the substrates used, it can be assumed that they have enzymatic activities specific to the metabolism of the different sources of osides (glycosides, holosides). The efficiency of use of these compounds can be presented in descending order of the weight of mycelium produced per g of substrate: A. pullulans: tannins> cellulose> glucose >> polysaccharides T. harzianum: cellulose >> tannin> glucose> polysaccharides Ce ranking points out a higher affinity for cellulose for T. harzianum (cellulolytic fungus) and a cellulose / oak tannin affinity for A. pullulans (saprophytic, lignolytic fungus). The cultivation of these fungi on EGGINS and PUGH medium confirms the established classification, as well as the cellulolytic character of Trichoderma sp. in general (BOTTON et al., 1985). This medium consists of (NH4) 2SO4 0.5g; L-asparagine: 0.5 g; KH2PO4: 1.0g; KCI 0.5g; MgSO4 0.2g; CaCl2 0.1g; DIFCO

  Yeast Extract: 0.5g; Agar: 20g; cellulose: 10g; water qs 1000ml.

  This is why A. pullulans develops first and transforms the wood surface into a fibrous, white mass. The

  Cellulosic substrate can then be attacked by T. harzianum.

  These fungi do not seem suitable for the hydrolysis and assimilation of wood polysaccharides, but growth is improved by using yeast polysaccharides or even Botrytis cinerea glucan. This is related to the existence of glucanase that is suitable for the release of the saccharide monosaccharides (DUBOURDIEU, 1982, LLAUBERES, 1988).

34 2705607

  2- Release of macromolecules a) autolysis of T. harzianum and A. pullulans After a more or less long stationary phase, the fungi enter a phase of decline. A progressive endocellular autolysis then begins under the action of various enzymatic activities studied in Saccharomyces sp. (protease: SILVA et al., 1987, FEUILLAT, 1985, esterase: CHEN et al., 1980, glucanase: LLAUBERES, 1987); in Pediococcus (LLAUBERES, 1987) and in

  Botrytis cinerea (MARTINEZ et al., 1983).

  The study concerns the turbidity of cultures during mold growth (SIGRIST PHOTOMETER KTL65 Turbidimeter) in CZAPEK-DOX liquid medium (1mg of dry mycelium / 100ml) on a stirring table; as well as on the lysis phenomena in the same medium followed by measurement of the OD 210nm according to the protocol of

GARBAY and LONVAUD (1 991).

Paintings

  Evolution of the turbidity of culture media during the development of Trichoderma harzianum, Aureobasidium pullulans and ECLR2 [culture: liquid medium on stirring table, inoculum: 1 mg / 100ml] unit: NTU species Trichoderma harzianum Aureobasidium pullulans Unknown strains VFFS2- 91 SNDS1-91 PNDS4-91 ECLR2-91 time in weeks:

1 10 22 8 2

2 10 36 10 6

4 11 42 26 9

6 28 72 28 15

8 32 110 89 18

36 112 92 21

2705607

  The turbidity of the medium increases very rapidly for A pullulans; on the other hand, T. harzianum and ECLR2 do not opacify the medium. Turbidity is proportional to the weight of mycelium produced and beyond 10 weeks all crops lose a bit of trouble. This observation indicates the end of the growth of mushrooms and the beginning of their autolysis (GARBAY and LONVAUD,

1991).

  The measurement of the OD 210nm shows (fig._f) that the autolysis starts from the first days of culture. The release of UV absorbing macromolecules 10 is limited during the first 4 weeks (D0210 <10) and takes an exponential rate beyond 10 weeks of culture. It appears that the lysis of the coded strain ECLR2 is earlier; which is to relate with its weak growth capacity in

liquid medium.

  During the development of molds, there is a balance between lytic enzymes and building enzymes: - in the growth phase: the cell mass increases and the lytic enzymes play a minor role; - in phase of decline: the cellular mass is constant and the lysis of the tissues becomes predominant. The fungi then use the parietal macromolecules as a nutrient (DUBOURDIEU, 1982). The lysis is triggered either by the disappearance of the carbon or nitrogenous substrate, or by the absence of adequate substrate, or by the

environmental toxification.

  b) production of polysaccharides and exocellular proteins during growth and lysis Media and culture conditions are identical to those used for previous experiments (2 / a)). The cultures are carried out for up to 3 months and the following analyzes are carried out on the supernatants obtained after centrifugation (4500 rpm; 30 minutes) -metaphenylphenol acidic polysaccharides (DURBOURDIEU,

1982);

  neutral polysaccharides with sulfuric phenol (DUBOURDIEU,

1982);

  -the total proteins with COOMASSIE blue (BRADFORD, 1976).

  At the end of active growth, A. pullulans released in the medium between 150 and 350 mg / l of neutral polysaccharides (FIG. 10). On a

36 2705607

  unstirred medium it can be assumed that the production of

  polysaccharides is more limited (LLAUBERES, 1988).

  The nature of the energy source strongly influences the production of polysaccharides. It is observed that the more glucose is difficult to invest in the metabolism (cellulose, ellagitannins), and the more the fungus forms exogenous polysaccharides. In addition, the more the substrate is poor in ose (ellagitannins), the more the release is intense. Thus the production of polysaccharides seems to be linked to

  growth difficulties in the environment.

Table Xl

  Release of protelins and exocellular polysaccharides by different fungi isolated from oak during their lysis [culture: 4 months in liquid medium, on stirring table; inoculum: 1 mg / 1 00ml Carbon source: glucose] Neutral polysaccharide acid polysaccharides Total protein mg / l eq. ac. galacturonic mg / I eq. glucose mg / I eq. BSA Trichoderma koningii

(VCFS1-91) 0 198 1.5

  Trichoderma harzianum (VFFS1-91) 0 495 tr Aureobasidium pullulans (PNDS4-91) 0 806 tr

(SNDS1-91) 0 1375 12

  Rhizopus stolonifer (SB1-92) 0 1053 tr Undiagnosed strain

(ECLR2-91) 0 341 0, 25

  tr: trace; BSA: bovine albumin serum

37 2705607

  Table Xl shows, on the one hand, the absence of acidic polysaccharides in the strains studied, on the other hand, the low proportion of proteins, except for A. pullulans (SNDS1-91), and finally the high production of polysaccharides. neutrals by Aureobasidium sp. (1 μg / l) and R. stolonifer (lg / l). The polysaccharidogenic properties of A. Pullulans have been established for a long time (BOUVENG et al., 1963) and are likely to allow the inactivation of the astringent fraction of wood tannins. In addition, there was likely protein hydrolysis during the 4 months of incubation by

fungal proteases.

  3-Characterization of enzymatic activities The ability of certain species of fungi to develop on particular media (cellulose, polysaccharides, oak tannins) suggests the existence of suitable enzymatic material ensuring

  the use of the saccharide fraction of the different substrates.

  3.1- main exocellular enzymatic activities. -Evolution during development The studies focus on Aureob. pull. because it is the species most frequently encountered throughout the

natural drying.

  A preliminary test on API LRA, OSIDASETM reveals a large number of exocellular activities osidic in the first 10 hours of culture, non-proliferating medium or oak wood extract. On the other hand, the medium supplemented with glucose has an enzymatic profile (OSIDASES) which is too weak to read. The main activities of A. pullulans are evaluated using pNP (p-nitrophenol) substrates according to the technique of DARRIET et al. (1988) and expressed in ADO 400nm / ml / 12H incubation at 25 C. The amylase activity is measured with the AMYLASE 3TM kit (SIGMA) on 20jl of test portion (protocol adapted from the measurement of amylase activity in the urine) and is expressed in

103 units / ml / min.

  a) demonstration of some osidic activities at Aureobasidium pullulans:

  Aureob. pullulans has essentially an activity I-

  glucosidase (Fig. $ 1), which can be inhibited by glucose (DUBOURDIEU et al., 1988). Trichoderma sp. presents an enzymatic profile

38 2705607

  comparable. The non-proliferating pellet or the culture supernatant also has a strong Rglucosidase activity. This activity is thermodegradable (1H at 65 ° C), strongly repressed by condensed grape tannins (100 mg / l). On the other hand, ellagitannins have no effect on the enzyme activity even at higher concentrations (1 μg / l). Finally, the activity does not seem to be inhibited by NaCl contents of the order of 0. 5 to 1

Mr. I-1.

  b) evolution of f3-glucosidase and amylase activities over time During A growth. pullulans in liquid medium (CZAPEK-DOX, 1 mg mycelium / 100ml) on different substrates (100mg / 1 OOml) two types of activities are followed (fig.2 and 13): - an amylase activity (EC: 3.2 .1.2) the maximum of which is reached in less than 10 OH of culture and remains insignificant whatever the medium. Only oak polysaccharides appear to stimulate the production of this enzyme. Its role in the metabolism of

substrates is only very weak.

  - a -glucosidase activity (EC: 3.2.1.21), the maximum of which is 48H of culture. This activity is intense during

  the entire phase of use of the carbon substrate by the mycelium.

  The glucosidase activity profiles show two remarkable facts: on the one hand, a very high r-glucosidase activity is noted in the non-proliferating medium; on the other hand, an increase of this same activity in the medium supplemented with glucose beyond 60H of culture. These two observations show the adaptability of the fungus in the medium: production of exocellular β -glucosidase in the non-proliferating medium, to obtain an energy substrate essential for its growth. Beyond several hours, the activity

  drops rapidly, in the absence of a source of glycosides.

  production of R-glucosidase in the medium containing 1 g / l of glucose after 60H of culture. This increase corresponds to

  the virtual disappearance of native glucose from the medium.

  3.2- Total enzymatic profile. Monograph of activities

esterases and osidases.

  The tests are performed on API galleries. ZYM. LRATM, following the protocol established by the manufacturer. The results are grouped in

Table XII.

39 2705607

  The average enzymatic profiles (n = 5) presented will allow easier identification of these two fungi. It is interesting to note that A. pullulans has a broader spectrum of osidic and esterase activity than T. harzianum. The maltosidase and lactosidase activity can be

  frequent) and the butyrate and valerate esterase activities of A.

  pullulans. Table XII: Osidase and esterase profiles of Trichoderma harzianum and Auresobasidium pullulans (see Appendix) 4- Discussion and conclusion 1-Study of the growth, development and certain enzymatic activities of fungi isolated from oak during its drying natural allows to better understand the ecology of this specific flora 2-Rapid colonization of the medium by A. pullulans (fertility, growth rate, use of low PM easily assimilable substances). It is a lignicolous fungus, which degrades lignin and ellagitannins. The result is a better accessibility of cellulosic fibers (appearance of wood) for other microorganisms. 3-Possibility for Trichoderma sp. to degrade after the passage of A. pullulans, cellulose by feeding on the autolytic debris of A. pullulans. Many species of Trichoderma are fungal hyperparasites. So we have during drying two categories of different mushrooms with two metabolisms

  distinct; we therefore have two phases of evolution of the wood.

  4-All the fungi found on the wood release into the medium a large number of enzymes, including R-glucosidase

  (estimated by pNP-E-D-glucose) is the main one.

  INFLUENCE OF THE SURFACE FUNGAL FLORA OF

  OAK WOOD ON PHENOLIC COMPOSITION

  1. Observation on the influence of the drying method on the composition of the wood In general, the natural drying causes a decrease in the water-soluble fraction of the wood (table Xlil fig.14). The leaching of wood exposed to the air for several months causes

2705607

  simultaneously elimination of phenolic compounds (80%

  the water-soluble extract) and salts or free mineral elements.

  It should be noted that the elimination of phenolic compounds is greater than for the mineral matter; the ratio of ash (C) / water-soluble extract (H) increases by 5 to 10% between

  natural drying and artificial drying.

  If the wood undergoes during natural drying a simple leaching, the

  The C / H ratio must remain constant between the two drying modes.

  On the other hand, if an enzymatic action ensures the destruction of the heterosidic molecules and allows the accumulation of simple phenols, easily leachable and less bulky sterically, then the C / H ratio increases. In addition, the insolubilization of a fraction of ellagitannins may accentuate the phenomenon (VIVAS and GLORIES, 1992). It is this second mechanism that we observe during the

natural drying.

  In agreement with the results of CHATONNET (1992), natural drying depletes the wood of flavonols, ellagitannins and phenolic acids. The density of the grain (tight grains or loose grains) can

  accelerate or slow down these phenomena.

  The disappearance of compounds embedded in the wood matrix (cellulose, hemicellulose, lignin ...) facilitates the accessibility of solvents to the fibers (ROSEN, 1978). CHARUK and RAZUMOVA (1974) established that pinewood extraction increases its gas permeability. Finally, the importance of shrinkage slits during drying seems to depend on the extractable wood content; the more the wood is poor in water-soluble extract and the more the withdrawal slots will be abundant or deep (SALOMON and KOZA, 1968). By its action on the ultrastructure of the oak, the drying mode can therefore modify the intensity of the

  oxidative phenomena during the aging of wines in barrels.

41 2705607

Table XIII

  Effect of the drying method on the phenolic composition of the wood oak of the central region Natural drying Artificial drying (24 months) water-soluble extracts (mg / g) 97 +10 (1) 134 +5

IFC (2) 19.6 +1 22.5 +1.5

  d440nm 0.03 +0.001 0.03 0.001 Flavanols (mg / g eq. (+) catechin) 0.21 + 0.01 0.46 +0.05 I ell (3) 0.12 + 0.01 0, 18 +0.05 gallic gall (mg / g) 26 + 4 39 6 ac ellagic (mg / g) 17 +3 24 +3, 5: deviations from the mean: Folin Ciocalteu Index: Ellagitannins Index (ADO600nm; Bates- SMITH, 1972) 2. Observations on the transformation of certain wood compounds during natural drying During natural drying, the water-soluble phenolic constituents are removed by leaching during rainy periods. But, there is also a set of mechanisms causing a change in the composition of oak wood. We study here certain phenolic molecules characteristic of oak and likely to affect the taste qualities of wines

(astringency, bitterness).

42 2705607

  2. 1- evolution of ellagitannins Green oak or naturally dried oak contains 10% of ellagitannins (MOUTOUNNET et al., 1992, MOUTOUNNET, 1992), most of which is represented by castalagine (50%) and vescalagin (30%). ). The high performance liquid chromatography study during drying shows, on the one hand, that the contents of castalagine and vescalagin slightly decrease in favor of the

  castaline and vescaline - after losing a Hexa group

  hydroxy-diphénique-; on the other hand, that insoluble forms related to the wall of the wood appear, as well as oxidized molecules

(increase of d 440 nm).

  Nevertheless, these variations are of low intensity and do not allow to differentiate clearly the mode of drying. Two interpretations can be advanced to explain these conclusions - First, during drying, the degradation of

  ellagitannins leads to simple phenols (gallic ac, especially ac.

  ellagic) easily removed by leaching. The increase in the C / H ratio completes this interpretation; - Lastly, since sampling variability is often greater than the variability associated with the drying mode,

  results become difficult to exploit.

  According to MOUTOUNNET (personal communication), vescalagin and castalagine are only slightly involved in the taste properties of wines; on the other hand, the pedunculagin and certain gallotannins isolated from Quercus sp. by ISHIMARU (1987) can actively participate in the bitter and astringent sensations of wood. In all objectivity the sapid substances responsible for the impressions of green, sap, astringency are still unknown today. Their enzymatic destruction releases into the

  medium of simple phenols relatively neutral in terms of taste.

  2.2- Evolution of coumarines Coumarins have been studied in wines and spirits for a very long time. JOSEPH and MARCHE (1972) were the first

  to isolate scopoletin, aesculectin, umbelliferone and methyl-

  umbelliferone, in extracts of Cognac. They also highlighted some of the heterosidic forms, particularly the

scopoline and aesculine.

43 2705607

  If in the state of aglycone (aesculetin, scopoletin) coumarins are relatively neutral in terms of taste, in contrast, in heterosidic form (aesculine, scopoline) they are strongly bitter and give wines feelings of greenness and bitterness pronounced. The blind tasting of a series of wines supplemented with aesculetin (lmg / l) and aesculin (lmg / l) clearly underlines the role of coumarines heterosidiques in the properties

organoleptic wines.

  As assumed by JOSEPH and MARCHE (1972), during natural drying, the disappearance of coumarins linked to glucose under the action of microorganisms, allows to attenuate strongly

the bitterness of the wood extracts.

  On a sample of wood from Allier, Limousin and Vosges, meeting the characteristic criteria of these different types of wood (VIVAS and GLORIES, 1992), we practice either natural drying or artificial drying; then the extracts with ether

  are analyzed. Fig.15 groups together all the determinations.

  Whatever the geographical origin of wood, natural drying allows a significant increase in scopoletin levels. The most likely precursor is scopolin. The disappearance of the glycosidic fraction of wood coumarines makes it possible to limit the feelings of bitterness of wines aged in casks. This type of result is related to tastings made on wines aged in France.

  barrel, whose wood is either artificially dried or naturally dried.

  In general, artificial drying produces wines that are more bitter, with impressions of sap and greenness (PONTALLIER, 1981, PONTALLIER

et al., 1998).

  2.3- Evolution of the phenol aldehydes The phenol aldehydes come from the degradation of the lignins. These polymers are constituted by condensation of phenyl-propenoic alcohol: 4-hydroxy-cinnamic alcohol (structure

  hydroxy-benzoyl; coumarilic alcohol), hydroxy-4-methoxy-3- alcohol

  cinnamic (guaiacyl structure, coniferilic alcohol), hydroxy alcohol

  4-dimethoxy-3,5-cinnamic (syringyl structure, sinapyl alcohol).

  After chemical or enzymatic oxidation the corresponding aldehydes accumulate in the wood (SEIKEL et al., 1971): hydroxy-4-methoxy-3-cinnamic aldehydes (coniferaldhehyde), 4-hydroxy-3,5-dimethoxycinnamic (sinapaldehyde) .L'apparition

  hydroxy-4-methoxy-3-benzoic acid (vanillin) and hydroxyl

44 2705607

  4-dimethoxy-3,5-benzoic acid (syringaldehyde) is thought to be due to the oxidation of ac-propanones (CHO-CH = CH), according to the mechanism of ROBERT and

  CHEN (1987), reprinted by MONTIES (1992).

  In Table XIV are grouped the results of a test carried out in 1991 on woods of different geographical origin (Allier, Limousin, Vosges) of which each lot was divided in two; one artificially dried, the other naturally dried. The analysis of the phenol aldehydes is carried out on the ethereal extract of the neutral fraction of the

wood (extraction at pH = 7).

table XIV

  Determination of certain phenol aldehydes on artificially dried oak wood and naturally A: wood of the ally; L: limousin wood; V: Vosges SN wood: natural drying; SA: artificial drying results in mg / l origin / drying mode vanillin syringaldehyde coniferaldehyde sinapaldehyde

ASA 0.28 0.92 0.09 0.12

ASN 0.58 1.26 0.28 0.12

LSA 0.35 0.56 0.17 0.07

LSN 0.41 0.55 0.10 0.10

VSA 0.32 0.61 0.10 0.10

VSN 0.46 1.08 0.15 0.15

average aldehyde-phenols

SA 0.31 0.69 0.12 0.09

SN 0.48 0.96 0.17 0.12

  30 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

  percentage increase55 40 40 35

(SN / SA)

  The variability of the sampling is greater than the variability related to the geographical origin. On the other hand, the differences related to the drying modes are significant. The percentage increase between SA (artificial drying) and SN (natural drying)

  represents 30 to 50% of the total phenol aldehydes.

2705607

  The phenol aldehydes of oak wood are mainly represented by vanillin and syringaldehyde; these molecules increase strongly during natural drying. Coniferaldehyde and sinapaldehyde are weakly present and relatively unaffected by the drying mode. These results suggest that the mechanisms that have the effect of increasing the content of phenol aldehydes, essentially of the benzoic series, originate from: either the rupture of the terminal cinnamic alcohols of the lignins, followed on the one hand by their oxidation to aldehydes and on the other hand, the oxidation of the a-propanones giving rise to the benzoic aldehydes (see IV / 4 / 4.1); - either the aldehydes linked to the parietal compounds, according to the MONTIES (1992) hypothesis, releasable chemically or

enzyme.

  We prefer to refer to the first hypothesis, in so far as MONTIES (1992) itself expresses reservations about the existence of covalently bonded aldehydes. Finally, because these bound aldehydes can include the cinnamic alcohols constituents lignin patterns and liberable by hydrolysis

alkaline or acidic.

  3- Microbiological origins of the transformations In order to confirm the action of microorganisms isolated from the oak on wood glycosides and on lignin, it is advisable to reproduce the phenomena under controlled conditions, in the laboratory. 3.1- enzymatic degradation of wood tannins. identification of the hydrolysis products a) enzymatic degradation of some phenolic heteroside compounds of the wood in a non-proliferating medium A non-proliferating suspension of Aspergillus oryzae or Aureob. pulL or Trich. sp., bringing together tannins

  hydrolyzable purified (vescalagin, pentagalloyl-glucose and ac.

  digallic), can hydrolyze them in less than 80 hours. The ranking in descending order of half-degradation (50% of the molecule-hydrolyzed) gives the following order:

46 2705607

  - ac. digallic (6h), - vescalagin (8h),

pentagalloyl glucose (30h).

  We present in Fig.11 an example of enzymatic hydrolysis profiles obtained. The genera Trichoderma, Aspergillus and Aureobasidium have the same type of curve. The precipitate (1%) with (NH4) 2SO4 (80% saturation) of the total proteins of a culture medium of these molds, contain a β-glucosidase activity.

  Majority (R-glucosidase: 4.2 u OD 440nm / ml / 12h).

  The kinetic study of the degradation curves of digallic acid and pentagalloyl-glucose show: on the one hand, the existence of active depsidase activity in the first 10 hours of incubation (Vmax = 5h), causing early and rapid hydrolysis of digallic acid; on the other hand, the existence of an esterase activity, the maximum activity of which follows the depsidase activity (Vmax = 20h). These results are confirmed by the work of BEVERINI and

METCHE (1990).

  These authors purified a commercial tannase, in two tannases denoted I and 11, on Sephadex G50, Boigel P-300 and CON A-Utragel. Tannase I has a greater esterase activity (substrate: methyl gallate), whereas tannase II has a predominant depsidase activity (substrate: digallic ac). Specific activities vary depending on the substrate used. In collaboration with LAGUNE (Laffort) we confirmed the existence of two tannases from the exocellular enzymes of a pure culture of Aureob. sweater. by high performance molecular sieving chromatography, TSK G6000 PW column (figl7). Figure 6 shows clearly the difference between the degradation of digallic acid and pentagalloyl-glucose. These observations are consistent with the work

cited above.

47 2705607

  It is therefore possible to envisage the separation of tannase activity into true tannase activity: depsidase (EC: 3.1.1.200); - Esterase activity: phenol heterosidase (EC: 3.1.1.201) b) Identification of the hydrolysis products FIG. 18 shows the enzymatic degradation of pentagalloyl-glucose. At the same time, a disappearance of gallotannins and the appearance of the corresponding monomer, gallic acid, are observed. It should be pointed out that in this example only the increase of gallic acid is followed, nevertheless pentagalloyl-glucose gives intermediate products: trigalloyl-glucose, digalloyl-glucose and monogalloyl which, in turn, release gallic acid, under

the action of tannase.

  The chromatographic profiles of Fig.19 show the degradation of castalagine on castalagine and ellagic acid (Figure 19a) and of acid digallic acid on the other.

gallic (fig.19b).

  Degradation of oak extract with water, purified by demixion

  (GLORIES, 1978), in the presence of non-proliferative suspension of A.

  pullulans, allows the release of native glucose dosed by method

enzymatic (fig.20).

  3.2- degradation of the heterosidic coumarins: a) incidence of the exocellular enzymes of A. Aesculin pullulans From aesculin purified on a cellulose plate from Aesculus hypocastanum, a solution of 10 μg / ml of a

  precipitated with (NH4) 2SO4 (80% saturation) of a pure culture of A.

  pullulans, a solution without enzyme constitutes the control. The two tubes are kept in the dark for 12h at 25 C. The contents of each tube are extracted with 2xlml of diethyl ether (300rpm, 5mn), the organic phases are combined in a separating funnel and then evaporated to dryness and taken up by 1 ml of MeOH. The extracts are

  then analyzed by HPLC (SALAGOITY et al., 1998).

  The coumaric glycoside is not fluorescent, but its aglycone is (FIG. 21), which makes it possible to demonstrate by HPLC coupled with a fluorimetric detector the appearance of aesculetin (dihydroxy-6,7coumarin). ) and the disappearance of aesculin ([6,7-dihydroxy-7-coumarin] -7-1-D-monoglucoside). A. pullulans therefore has an exocellular

48 2705607

  to explain in part the increase of stopoletin and aesculetin during natural drying. The hydrolysis of coumaric glycosides is confirmed by the enzymatic determination of glucose

in the reaction media.

  "The nomenclature enzyme" does not present in its last version of hydrolase capable of acting on the ether linkages of coumarin-7-monoglucoside. "It therefore seems advisable to place in hydrolase (EC: 3), a category numbered EC: 3.1.8, entitled "carboxylic ether hydrolase" for classifying coumarin etherase, for

  which we propose the following code: (EC: 3.1.8.10).

  b) use of the exocellular enzymes of A. pullulans precipitated from a culture medium, to reveal the coumarines

heterosides from oak wood.

  Extracts with water (20%, w / v) of sawdust (60 mesh) of green oak, dried artificially and naturally, are concentrated and lyophylized. The powders obtained serve to constitute 1 g / l solutions in a 0.2 M NaAc buffer, pH = 5. 1 ml of each extract are incubated at 25 ° C. for 12 hours in the presence of 10% of the (NH4) 2SO4 (80% saturation) precipitate, of a culture of A. pullulans at maximum Iglucosidase activity; the control is supplemented with 1 ml of water. The reaction media are depleted with 10.5.5 ml of diethyl ether (300 rpm, 5 min); the organic phases are combined in a separating funnel and evaporated to dryness, then taken up in 1 ml of MeOH and chromatographed (SALAGOITY et al., 1987). The results of the assay are summarized in Table XV. The non-enzymed control contains the coumarins in aglycone form and the enzymed tubes represent the total coumarins (aglycones) + heterosidics). The results do not allow comparison of the different oak samples, since they each represent a sample of sawdust from a working day at the cooperage. On the other hand, the profiles in glycosides and in aglycones can be compared. Our protocol allows to dose simultaneously, on the same

  sample aesculin, scopolin, aesculetin and scopoletin.

  We can also verify the impact of natural drying on the content of coumarins and glycosides aglycones. The latter makes it possible to transform, under the effect of the exocellular enzymes of

49 2705607

  molds, almost all of aesculin and scopoline in aglycone corespondant. Artificial drying, without causing such complete transformations eliminates% glucosidic coumarins; suggesting the existence of coumarin-etherase activity in wood or, more likely, the possibility of chemical hydrolysis under

artificial drying.

Table XV

  Assay of aesculetin of scopoletin and their glycosides in the

  oak wood dried artificially or naturally.

  [Water oak extract incubated in 0.2M NaAc buffer, pH = 5 in the presence of exocellular A enzymes. pullulans (10%)] results in g / li oak green oak drying oak artificial natural drying Aesculine 6 4 0.2 Scopoline 36 26 4.1 j heterosides 42 30 4.3 Aesculetin 0.1 0.8 4.8 Scopoletin 2 2.6.2 Synthesis of aldehyde phenols The synthesis of phenol aldehydes is studied from moistened sawdust (5g; 60 mesh), distributed in a box. Petri and seeded with Trich fresh mycelium. harz., in the form of the centrifugation supernatant of a culture of 125 I (1 mg mycelium / 100 ml of CZAPEK-DOX culture medium). The dishes are incubated at 30 ° C. for 15 days. The sawdust is then extracted with a hydroalcoholic solution (50% v / v + 1% HCl N) for 24 hours at C. 10 ml of centrifuged liquid (4500 rpm, 20 minutes) is adjusted to pH = 7 (NaOH N ) and used up with 10.5.5 ml of diethyl ether. The phases

2705607

  The organic compounds are combined in a separating funnel and then evaporated to dryness and taken up with 1 ml of MeOH. The extract is chromatographed under the usual conditions. An experiment is conducted under the same conditions with, on the one hand, the mycelium of A. pullulans and on the other hand, with sawdust which has been removed by hot water the compounds

extractables water-soluble.

  Fig.22 shows a significant increase in the phenol aldehydes, especially vanillin and syringaldehyde, in the presence of T. harzianum. The microbiological origin of the phenol aldehydes.

  natural drying is therefore very likely.

Table XVI

  Production of phenol aldehydes during the incubation of mycelium, in the presence of oak sawdust [inoculum: fresh mycelium of a liquid culture of 15 days, incubation: 15 days at 30 ° C] results in mg / l sawdust spent sawdust - ------------------ --- with water + T. harzianum + A. pullulans + A. vanillin pullulans +0.3 +0.15 +0.4 syringaldehyde +1.1 +0.6 +0.7 T. harzianum and A. pullulans are capable of releasing significant amounts of vanillin and syringaldehyde (Table Xvl ). The removal of a large amount of water-soluble extract by hot water (90 ° C) does not limit the production of aldehydes microbially.

  The precursor of these phenol aldehydes may be lignin.

  But this origin will have to be checked from purified lignin from

  oak wood (lignin-dioxane, lignin-ethanol).

  This enzymatic activity of fungal origin is called "aldehyde phenol synthetase". A detailed study should allow

to specify its mode of action.

51 2705607

  4- Heterosidase activities of Aureobasidium pull. and Trichoderma sp. Study of phenol heterosidase and coumarin etherase activities 4.1Research for substrates suitable for phenol heterosidase and coumarin etherase activities The search for suitable substrates for phenol heterosidase (PeH) and coumarin ether ether (CET) activities is carried out in non-proliferating liquid medium: - NaCl buffer 0 1M pH = 4.5; for the activity PeH and depsidase;

  - 0.2M NaAc buffer pH = 5; for the CET activity.

  The substrates are used at the dose of 1 mg / l Oml and the exocellular enzymes from the (NH4) 2SO4 precipitate (80% of

  saturation) of a maximum culture of R-glucosidase activity.

  The CET activity is measured by the release of native glucose (enzymatic method) and the PeH and depsidase activity by the "ApH" method. The tests are positive when, on the one hand, the difference in pH is greater than 10% of that of the non-enzyme control, and on the other hand when the release of native glucose is greater than 20% of the non-enzymed control. The results are collated in Table XVII

52 2705607

Table XVII

  Heterosidic substrate capable of being hydrolysed by the exocellular enzymes of Aureobasidium pullulans and Trichoderma harzia n um [measured activities: PeH by ApH; CET by glucose release]

  activity / Aureob substrate. sweater. Trich. Harz.

  -PeH: * ellagitannins: chestnut extract + + oak extract + + vescalagin + + * gallotannins: gall nut extract + + tara tannin + + myrobolan tannin + + tannic acid +/- + trigalloyl- glucose + + pentagalloyl-glucose + + * condensed tannins: grape seed tannin fraction TP of a red wine +/- + * anthocyanins: malvidin monoglucoside + + * flavonol: kaempferol monoglucoside + + -CET: aesculin + + -Depsidase: digallic acid + + In general, all the heterosidic substrates are used by the enzymatic activities of fungi isolated from oakwood.

53 2705607

  Condensed tannins are inhibitors of PeH activity; although the TP fraction (Tannins-polysaccharides and Tannins-Proteins, GLORIES, 1978) is modestly used. The inhibition appears partially lifted when the astringency sites are partially blocked. pathways of transformation of certain phenolic compounds of oak wood The enzymatic activities studied are PeH, CET and aldehyde phenol synthetase; they concern three types of substrate: hydrolysable tannins (gallotanins, ellagitannins) and their

  depsides, glycosilated coumarins and lignin.

  4.2- The proposed transformation routes are from

  the analysis of reaction products between substrates and enzymes.

  a) degradation of ellagitannins and gallotannins The acidolysis of wood tannins gives the same type of residues as those obtained enzymatically: - The mild acidolysis of gallotannins (HCI N, 100 C, 30 min) gives native glucose and gallic acid and sometimes, depending on the structures, quinic acid; - The acidolysis of ellagitannins (6N HCl, 100 C, 60mn) gives ellagic acid and bound glucose, hardly released by

  chemical. This property is used for their specific dosage.

  The enzymatic degradation of the wood tannins (fig.23) leads in all cases, if the reaction is conducted until its teme, to simple phenols (often gallic or ellagic acid) and to glucose (more rarely pentoses) . The activity PeH acts on the ester bonds between the hydroxyls of the glucose and the carboxyls of the acid

  for galloyl derivatives and hexa-hydroxy-

diphenic for ellagitannins.

  The degradation is carried out in several stages - For the gallotannins, the progressive loss of the gallate units is observed and one can follow by HPLC the appearance of a

  tetragalloyl-glucose as well as tri-, di- and monogalloyl-

  glucose. The end of the reaction corresponds to the release of all gallate units of glucose, which can be used for

  energy metabolism of the fungus.

  - The ellagitannins give at first hexa-hydroxydiphenic acid (HHDP) and vescaline or castaline

54 2705607

  (for this example, Fig. 22). HHDP acid is stable only in glycosilated form, so it evolves rapidly by intramolecular esterification into ellagic acid (Scalbert, personal communication). In a second step, vescaline or castaline are degraded to give a small amount of gallic acid, glucose

  and HHPD acid that instantly produces ellagic acid.

  b) degradation of monoglucoside coumarins The acid hydrolysis of coumarin-monoglucosides generally leads to a transformation of the molecule, which then loses its

  fluorescence properties, in the aglycone state.

  Enzymatically, the breakdown of the ether bond between

  OH of coumarin and glucose has been known for a long time (JOSPEH and MARCHE, 1972). The term of the reaction can be easily followed by the appearance of a characteristic fluorescence

aglycone (fig.24).

  During the enzymatic hydrolysis of a green oak coumarin concentrate, extracted with diethyl ether, from a neutralized aqueous solution (pH = 7), we observed the decrease of umbeliferone and the appearance of a peak at the end of

  chromatogram (C18 grafted silica column) also fluorescent.

  This peak revealed after enzymatic treatment has the same UV spectrum and the same retention time as sporalen o o oo Sporalen MURRAY et al. (1982) have particularly studied this route, which uses as precursor 7-dimethylalyl-pyrophosphate and umbelliferone. Although we have not definitively identified furanocoumarin such as sporalen, this reaction needs to be confirmed.

2705607

  c) hypothesis of enzymatic formation of phenol aldehydes from lignin Although accepted by CHEN and CHANG (1985), the degradation of lignin by fungal populations has not yet been formally demonstrated. MONTIES (1992) still speaks of hypothesis

for this way of transformation.

  The production of phenol aldehydes from lignin concerns phenylglycerol terminal units whose benzyl function "a" is free or etherified (fig.25). Two mechanisms are envisaged elimination "y-D" which allows on the one hand, the rupture of the arylether bond and the release of the cinnamoyl radical, on the other hand, the production of cinnamaldehyde. In our example (fig.25),

  we observe the production of coniferaldehyde (hydroxy-4-

  methoxy-3-cinnamaldehyde). The benzoic aldehydes are derived from the oxidation of α-propanones; he then accumulates in the middle of the

  vanillin (4-hydroxy-methoxy-3-benzaldehyde).

  - the elimination "c-e" which leads after oxidation of the

  benzoic aldehydes propanones directly.

  4.3- characteristics of the phenols heterosidases of A. pullulans.

and Trichoderma sp.

  Since we did not purify the different enzymes, we estimated enzymatic kinetics, substrate affinities and pH-dependent activities, from supernatant of culture to

maximum activity [3-glucosidase.

  Fig. 26 is the representation of LINEWEAUER and BURK, in the form of: 1 / V = f (1 / S) [S: concentration in mg / II; V: reaction rate 10-3 u pH / ml / h], enzymatic hydrolysis of green oak ellagitannins 3o allows the calculation of Km and Vm. The kinetic constants are estimated at: km = 14.5 and Vm 0.14 for T. harzianum; Km = 70 and Vm = 0.45 for A. pullulans. A. pullulanshave a better affinity

  that Trichoderma sp. for the studied substrate.

  The PeH activity is maximal for both genera at pH = 5 (fig.27). at pH = 2 the activity is practically non-existent, beyond

pH = 6 it decreases rapidly.

56 2705607

  4.4- inhibition of phenol heterosidase activity The condensed tannins of grapes and wine have the property of combining proteins, they are therefore likely to limit

  mold growth and inactivate the released enzymes.

  Although oak ellagitannins do little to affect mold growth, condensed grape seed tannins significantly inhibit their growth (fig.27a), even at

low concentration (500 mg / l).

  The PeH activity is inhibited by the free glucose of the culture media as early as 10 g / l, whereas the disappearance of the glucose makes it possible to lift the inhibition. The condensed tannins repress the PeH activity, since the mycelia washed and resuspended in a suitable medium do not recover their initial osidase activity. Finally, for high concentrations of free ellagitannins (> 1 g / l) the PeH activity is inhibited (FIG. 28). In the wood, the ellagitannins impregnate the fibers and are released gradually. Such concentrations are not found in the free state in the wood; Moreover, the leaching of wood, preceding the implantation of the fungal populations, depletes

  the layers of surface fibers.

  Inhibition of activity by grape and wine tannins is greatly diminished by the inactivation of astringency sites by proteins or polysaccharides. We see in fig.29 the effect of

  the PeH of A. pullulans on the TP-TS fraction of a wine (GLORIES, 1978).

  The chromatogram shows, on the one hand, a marked increase in the peak of gallic acid and a small increase in (+) catechin,

  on the other hand, the appearance of two peaks at the end of the chromatogram.

  4.5- research of a performing strain The previous epidemiological study has shown that natural drying induces a rather limited fungal flora. A. pullulans is the most commonly encountered strain on the

  wood and this throughout drying.

  The search for a strain of this mushroom, particularly adapted to the conditions of natural drying can find an application in cooperage in the same way as in others

  biotechnologies (dairy products, beers, wines, pantries ...).

  The use of a suitable fungal leaven could provide a

  "drying" of wood more complete and more homogeneous.

  Screening screens are split over two levels

57 2705607

  - selection according to the criteria of classical microbiology.

  A mold has a number of phenological, physiological and ecological characteristics, most of which are found in the many fungal monographs. - Selection according to criteria specific to the technology implemented. These criteria are directly related to the constraints of

the production.

  a) selection according to the criteria of the classical microbiology To develop on the wood, during drying, the mushroom must find in the medium a minimum hydric potential of growth (awo), ranging between 0.75 and 0.60 (JAY, 1978). The water potential of the wood (aw) varies during drying from 0.45 to 0.12. But A. pullulans develops on the surface of the wood and

  can thus benefit from the ambient hygrometry and the rains.

  The relative atmospheric humidity in oceanic climate varies from 55 to% (aw = 0.55 -0.95), most of the time the values are greater than 0.70; which allows an almost continuous activity of the mushroom. A. pullulans can grow on dry substrates with aw less than 0.70. Trichoderma require aw

  higher (0.80) as well as Rhyzopus sp. (0.93), according to JAY (1978).

  According to CORLETT and BROWN (1980) the acceptable pH range for the growth of many fungal organisms ranges from 1.5 to

  10. On oak wood pH is not a limiting criterion.

  Temperature is also an important factor. On the park, the summer sun can cause a rise of the surface temperature up to 45-50 c in the most extreme cases. A. pullulans is a heat resistant mushroom; it can withstand conventional sterilization treatment of foods (JENSEN, 1960; KING et al., 1969). In addition the thermal amplitudes

  and thermal shocks stimulate germination of spores.

  b) selection on criteria related to production constraints Fungi must not form odorous compounds that may affect the quality of the wood. Few works have been done on this subject. It is known that Trichoderma sp. can produce from methylacetate, -formate, -propionate and -myristate of

menthol and iso-menthol.

58 2705607

  The mushroom must quickly become established on the wood and represent a majority flora. A. pullulans has many sources of inoculation in the environment. Its adaptation to the environment also allows it to establish itself quickly to represent 70 to 80% of the fungal flora of drying. So, the

  selection pressure seems to be in favor of A. pullulans.

  The fungus must possess an exocellular β-glucosidase activity that is sufficiently intense to modify the heterosidic phenolic profile of the wood. The production of polysaccharides capable of inactivating certain astringent molecules completes the

chosen spectrum of action.

  A. pullulans has a satisfactory response to all selection screens presented for the natural drying of oak in

the Bordelais.

  5. Discussion and conclusion The superficial fungal flora of oak wood is likely to cause changes in the structure of phenolic compounds. Natural drying represents a set of physicochemical, microbiological and enzymatic reactions capable of

to affect the quality of the wood.

  1- The artificial drying causes a simple dehydration of the

  wood and partial destruction of certain phenolic compounds.

  In comparison, natural drying profoundly changes the composition of oak wood. This method of preparation induces a greater elimination of the water-soluble phenolic compounds, with respect to the mineral fraction of the wood. The ash / water soluble extract ratio increases due to natural drying. At this stage, we may suspect either a change in the accessibility of the solvent-impregnated fabrics or a decrease in the steric hindrance of the water-soluble phenolic molecules. Both hypotheses are likely and only a set of reactions of hydrolysis of wood and tannins impregnating the matrix

  seem possible (MONTIES, 1992).

  2- Under the conditions of natural drying, we note both the hydrolysis of ellagitannins and glycosidic coumarins,

  the appearance of characteristic reaction products (gallic ac, ac.

  ellagic, vescaline, castaline, coumaric aglycones and native glucose) and modification of wall composition

59 2705607

  (insolubilization of ellagitannins by combination with constituents of the wall and alteration of lignins giving rise to phenol aldehydes). All these mechanisms have already been observed (JOSEPH and MARCHE, 1972, SEIKEL et al., 1971, ROBERT and CHEN, 1987, SHUYUN PENG et al., 1991, VIVAS and GLORIES, 1992), but their fungal origin has never been formally established

  (MONTIES, 1992, VIVAS et al., 1991).

  3- During the growth of the fungal flora characteristic of natural drying in Bordeaux (A. pullulans, Trichoderma sp.) We were able to reproduce in the laboratory the mechanisms observed in the field. But this fungal biomass can also act in a non-proliferating condition. Thus, when the wood is completely covered by fungi, the exocellular activities continue to be secreted and act after infiltration into the wood by capillarity. Three enzymatic activities have been identified: - The phenol heterosidase (PeH) activity, which acts on the ellagitannins and wood gallotannins; it corresponds to two enzymes: a true tannase, the depsidase for which we propose the code (E.C .: 3.1.1.200), and an esterase for which we propose the code (E.C: 3.1.1.201). The tannase activity (E.C .: 3.1.1.20) is therefore divided into two subclasses (200 and 201). The existence of these two distinct activities has recently been confirmed by

BEVERINI and METCHE (1990).

  The coumarin etheretherase (CET) activity has been particularly studied on scopolin, aesculin and their respective aglycones: scopoletin and aesculetin. This activity is of carboxylic nature ether hydrolase, so we propose the code (E.C:

3.1.8.10).

  - Finally, an aldehyde phenol synthetase activity of which we do not know exactly the mode of operation. Lignin

  is most likely the specific substrate of this activity.

  4-Phenol heterosidase activity acts on a wide variety of substrates. The heterosidic nature of the molecules seems to be the only criterion determining the transformation of the substrate. Condensed tannins strongly inhibit activity; but the inactivation of certain sites of astringency by oxidation or by combination with

  Proteins limit this phenomenon.

2705607

  Enzymology work must be undertaken to purify these

  different activities and to determine their specific characteristics.

  One of the applications of the work is the selection of a mushroom adapted to natural drying. Its cultivation and use in the form of fungal leavens is envisaged. A. Pullulans seems to be the most suitable species; some particularly efficient strains have been isolated and constitute the basis for the selection and industrial production of the fungus

table IV

  identification of mushroom strains isolated from oak wood. Morphological tests (in collaboration with E. GUEHO, Institut PASTEUR, PARIS) [rganes described in: Genres and species colonies -92 9 S 1 FFS1-91 -91

1 SB PNDoS- 1 IvCFS-91Iv -

  color: white-gray black olive green dark green shape: no chars irregular round round aspect: filamentous icy dusty powdery growth of thalli: very fast medium fast fast

slolons: + -

  Sporocytospore reproductive tract: + high (mm) 2.5 spores: + angular diameter (μm) 350 long (um) 10-15 chlamydospores: + + conidia: + + + f o r m elliptic subglobular elliptic elliptic. or oblong forresbl uolngue diamr (um) 7-16 2,8-3,2 3,5 long (μm) 3,5-7 2,5-2,8 2-2,8 division classification Amastigomicota Amastigomicota Amastigomicota Amastigomicota groups Zygomycetes Deuteromycetes Deuteromycetes Deuteromycetes Mucorales orders Hypomycetes Hypomycetes Hypomycetes identification genera Rhizopus Aureobasidium Trichoderma Trichoderma species stolonifer pullulans harzianum koninii stolonlfer pullulans harzianum koningii

Table V

  identification of Aureobasidium pullulans and Trichoderma harzianum by physiological assimilation and fermentation tests

  PHYSIOLOGICAL TESTS FERMENTATION ASSIMILATION

  CARBON SUBSTRATES Aureobasidfum Trichoderma Auraobasidlum Trichoderma pullulans harzianum pullulans harzlanum glycerol - + erythritol + + D-arabinose - + L-arabinose - + + ribose ± + + D-xylose ± + L-xylose + + adonitol + + B methyl-xylose galactose + + + + D-glucose + + + + D-fructose + - + + + D-mannose + + + + L-sorbose + + +

rhamnose + + + -

  dulcitol + + inositol mannitol + + +

sorbitol + + -

methyl-D-mannoside + -

  at! methyl-D-glucoside + + N- acethyl glucosaminidase - + + + amygdalin + + + arbutin + + + + esculin + + + + salicin. + + + cellobiose + + maltose + + lactose + + + melibiose + + sucrose + + trehalose + + + inulin + + melezitose + D-raffinose - + starch + + + glycogen - + + + xylitol B gentobiose - + + D- turanose + + + + D-Iyxose + + + +

D-tagatose + -

  D-fucose + - + D-arabitol L-arabitol + + + _gluconate - - + - + 2-keto-gluconate + + - '+ + -cetogluconate + - +

Table Xii

  Osidase and esterase profiles of Trichoderma harzianum and Aureobasidium pullulans.

  +; +; ++; +++ increasing intensity of the color reaction.

  A ctivities osidases A ctivities esterases Act lvi tés Trichoderma Aureobasidium Activities Trichoderma Aureobasidium harzianum pullulans harzianum pullulans α-D-galactosidase +++ +++ C4-esterase +++ b-Dgalactosidase - +++ C5-esterase.- ++ aL- rhamnosidase + +++ C6-esterase +

  α-L-arabinosidase +++ C8-esterase ++ ...

  α-D-glucosidase ++ ++ C9-esterase +++ β-D-glucosidase. +++ CoOlO-esterase + ++ b-D-galacturonase + O12-esterase, I- ± + b-D-glucuronosidase + C14-esterase +

  a-D-matosidase + C16-esterase ++ - + ±

  bD-maltosidase + + C18-esterase + + N-acetyl-α-D-glucosaminidase +++ = +++ N-acetyl-bD-glucosaminidase +++ ++ aL-fucosidase +++ +++ bD-fucosidase ++ +

b-L-fucosidase +++ t-

  b-D-Lactosidase ++ ++ α-D-manosidase + β-D-manosidase + α-D-xylosidase. b-D-xylosidase: b: J3

64 2705607

BIBLIOGRAPHY

  ARTIGUES.M -1981- Research criteria for selection of Trichoderma clones active against Sclerotinia minor and Sclerotium rolfsii. Thesis doct-ing, Montpellier

  BATE-SMITH.E.C -1972- detection and determination of ellagitannins. Phytochem.

11, 1153-1156

  BEVERINI.M and METCH.M -1990- Identification, purification and physicochemical

  properties of tannase of Aspergillus orizae. Sc. Foods.

  BOTTON.B; BRETON.M; LEVRE.M; GUY.Ph; LARPENT.P and VEAU.P -1988-

  Useful and harmful molds. Industrial importance. ed. Masson. Paris BOUVENG.H.O; KIESSLING.H; LINDBERG.B and Mc KAY.J -1963Polysaccharides elaborated by Pullularia pullulans (Aureobasiduim pullulans). Acta Chem. Scand. 17

- 1351-

  BRADFORD.H -1976- A rapid and sensitive method for the quantification of

* microorganisms quantities of proteins using the principles of protein binding.

  Ann. Biochem. 72, 248-254 CHARUK.E.V and RAZUMOVA.A.F -1974- Der e influss von holzextrakstoffin auf permeabilitât von. Holz. Holztecnol. 15, 36-37 CHATONNET.P -1989- Selection and preparation of wood for the breeding of quality wines. Rev. OEnol. 52, 21-25 CHATONNET.P - 1991- Incidence of oak wood on the chemical composition and organoleptic qualities of wines - Technological applications. Thesis DER, University

Bordeaux Il.

  CHATONNET.P -1992- Origin and processing of timber in cooperage. in "the wood and the

  quality of wines and eaux-de-vie "ed.Vine and wine publ.

  CHAUVET.S; VIVAS.N; GLORIES.Y and SUDRAUD.P -1992- Oenological tannins: Characterization of the nature of commercial products. Rev. OEnol. 18, 64, 8-10

2705607

  CHEN.C.H; JAMIESON.A.M; VAN GHELUW -1980- The release of fatty acids as a consequence of yeast aulpJysis. AMRN. Soc. Brewing. Chemist., J. 38, 1, 13-18 CHEN.C.L and CHANG.H.M -1985- Chemistry of lignin biodegradation. in "Biosynthesis and Biodegradation of wood conponents" ed. Higuchi DARRIET.Ph; BOIDRON.J.N and DUBOURDIEU.D -1988- The hydrolysis of terpene glycosides of small-bodied Muscat by the periplasmic enzymes of Saccharomyces cerevisiae. Wine Vine Knowledge. 22, 3, 189-195 DUBOURDIEU.D -1982- Research on polysaccharides secreted by Botrytis

  cinerea in the grape berry. State Thesis Science. University of Bordeaux II.

  DUBOURDIEU.D; DARRIET.Ph; OLLIVIER.C; BOIDRON.J.N and RIBEREAU-GAYON.P -

  1988- Role of the yeast Sacchamomycès cerevisiae in the hydrolysis of terpene heterosides of grape juice. C.R. Acad. Sc. Paris. Ill. 306, 489-493 FEUILLAT.M -1985- Aluine caratteristiche del metodo classico di elaborazione degli spumante. Enotecnico. 21, 5, 475-484 GARBAY.S and LONVAUD-FUNEL.A -1990- Study of the lysis of Leuconostoc oenos. J. Int. Sc. Wine Vine. 24, 4, 157-167 GIORDANO.G -1971- Characteristiche fisiche and meccaniche del legno. in "'Technologia

del legno ", vol 1. Utet ed.

  GLORIES.Y -1978- Research on the coloring matter of red wines. Thesis doct. science. Bordeaux II University GLORIES.Y -1992- Oxidative mechanisms linked to wood preservation. in "the

  wood and the quality of wines and eaux-de-vie. "Vigne et vin intern.

  GREAVES.H -1970- The effect of some wood inhabiting bacteria on the permeability of

  Eucalyptus regnans and Pinus radiata sapwood and heartwood. HFA. 24, 6-

  14 HIGUCHI.T; FUKAZAWA.K and SHIMADA.M -1967- Res. Bull. College Exp. For. 25, 167-

66 2705607

  ISKIMARU.K -1987- (in Japanese) Chemical study of the tannins of the family of

Quercus. Thesis doct ..

  JARVIS.B -1978- Methods for detection of fungi in foods and beverages. in "food and beverage mycology". ed. Beuchat. Wesport Conn. Avi. Pub. Inc. JOSEPH.E; MARCH.M; GOIZET.A and AUDEBERT.J -1975b- Theoretical study on cognac and its aging in oak barrels. Rev. Bro. OEnol. 57, 1-17 LLAUBERES.R.M -1988- Polysaccharides secreted in wines by Saccharomyces cerevisiae and Pediococcus sp. Thesis doctor of the University of Bordeaux Il MARCHE.M and JOSEPH.E -1975a- Theoretical study on the cognac, its chemical composition and its aging in oak barrels. Rev. Bro. OEnol. 57, 1-106 MONTIES.B -1992- Chemical composition of oak wood: phenolic compounds, relationship with some physical and chemical properties likely to influence the

  quality of wines and eaux-de-vie. in "wood and the quality of wines and eaux-de

  Life "ed., vine and wine, internat.

  MOREAU.F -1952- Mycological Encyclopedia. Volume I and II. ed. Paul Lechenvallier.

  Paris. MOUTOUNNET -1992- The tannins of the oak wood. in C.R. Colloque "impact of oak on the quality of wines" under direction Y.GLORIES (to appear) MOUTOUNNET.M; RABIER.P; SARNI.F and SCALBERT.A -1992- The tannins of the oak: The conditions of their presence in the wines. in "the wood and the quality of the wines and

  eaux-de-vie ", ed., wine and wine, internat.

  MURRAY.R.D.H; MENDEZ.J and BROWN.S.A -1982- The coumarin naturals: Occurence, chemistry and biochemistry. Wiley ed. Chichester PENG.S; SCALBERT.A and MONTIES.B -1991- Insoluble ellagitannins in Castanea sativa and Quercus petraea woods. Phytochem. 30, 3, 775-778

67 2705607

  PENG.S; SCALBERT.A and MONTIES.B -1991- Insoluble ellagitannins in Castanea sativa and Quercus petreae woods. Phytochem. 30, 3, 775-778 PONTALLIER.P -1981- Research on the breeding conditions of red wines. Role of oxidative phenomena. Doct-lng thesis, University Bordeaux Il PONTALLIER.P; SALAGOITY.M.H and RIBERAU-GAYON.P -1982- Intervention of oak wood in the evolution of red wines aged in barrels. Wine vine knowledge. 16

1, 45-61

  ROBERT.D and CHEN.C.L -1987- Structural Analysis of Biodegraded Lignins from Peacock Wood by Phanerochaetes chrysosporum by Quantitative 13C NMR spectroscopy. IV [nt. Wood and Pulping Chem. ISNPC. Paris. 139145

  ROSEN.H.N -1978- Interaction of alcohols and organics acids with wood. Wood Science.

151-157

  SALAGOITY.M.H; TRICARD.C; MARSAL.F; SUDRAUD.P -1986- Preliminary Investigation for the Differentiation of Cen- tative Determination of Gallic Acid and Its Derivatives. Am. J. Eno. Vitic. 37, 4, 301-303 SALAGOITY.M.H; TRICARD.C and SUDRAUD.P - 1987Dosing simultaneous aldehydes

  aromatics and coumarins by high performance liquid chromatography.

  Application to wines and eaux-de-vie aged in oak barrels. J. Chromatogr. 392,

379-387

  SALOMON.M and KOZA.K -1968- Shrinkage of sapwood and heartwood of young douglas fir trees. Forest Prod. J. 18, 90-94 SEIKEL.M.K; HOSTETTLER. F.D and NIEMANN.G.L -1971- Phenolics of Quercus rubra

  SILVA.A; FUMI.M.D; MONTESISSA.G; COLOMBI.M.G and COLANGRANDE -1987-

  Impact of preservation in the presence of yeasts on the composition of sparkling wines. Wine vine knowledge. 21, 3, 141-162 TARANSAUD. J -1976The book of cooperage. ed. The diffusion book wheel

68 2705607

  VIVAS.N -1987- Experimentation of several lyre driving systems in the Bordeaux vineyard. Mem .. [9TS viti-oeno. ed. INRA-Bordeaux. 110p VIVAS.N; CHAUVET.S; GLORIES, Y and SUDRAUD.P -1992- Definition and characterization of commercial preparations of oenological tannins. J. tnt. Sc. Wine Vine. (subject to acceptance). VIVAS.N; GLORIES.Y and FRANCOIS.J -1991- Focus on the breeding of red wines in oak barrels. Rev. OEnol. 17, 62, 17-21 VIVAS.N and GLORIES.Y -1992- Function e importancia del sistema de secado in the production of madera of roble destinada to toneleria. Vib'Expo 92, 19-21 June 1992. Madrid VIVAS.N and GLORIES.Y -1992- Role and importance of the drying mode of wood for the manufacture of barrels: Impact on the porosity and flavor of phenolic compounds. (in preparation) WOOD.R -1951- The control of lettuce diseases by the use of antagonistic

  organisms. 1- The control of Botritis cinerea. Ann. Appl Biol. 38, 203-

  WYLLIE.T.D and MORENHOUSE.L.G -1977- Micotoxic fugi, mycotoxin and

  micotoxicoses. Vol I; l; lil. ed. Dekker. New York.

Claims (10)

R E V E N D I C A T ION S   1. A process for drying oak wood, characterized in that mold is molded on the wood to be treated in order to   to obtain a viable fungal flora in place, constituting means of natural drying and control of the composition of the oak wood.   2. Method according to claim 1, characterized in that the molds are previously isolated from the natural flora of the oak wood being dried. 3. Method according to claim 1 or 2, characterized   what molds are used in the form of fungal leaven.   4. Method according to one of claims 1 to 3, characterized in that the molds belong to the genus   AUREOBASIDIUM PULLULANS.15 5. Method according to one of claims 1 to 3, characterized in that the molds belong to the genus   TRICHODERMA HARZIANUM. 6. Method according to one of claims 1 to 3, characterized in that the molds belong to the genus TRICHODERMA KONINGII.   7. Drying method according to claim 1, characterized in that the composition of the wood is controlled   in order to act on the aromatic and gustatory quality of a wine or a brandy of wine, or an alcohol in contact with the oak wood.   Oak wood obtained by the process according to any one of claims 1 to 7.   9. Oak wood according to claim 8, characterized in that it is intended for cooperage.   10. Oak wood according to claim 8, characterized in that it is intended for the aging of wines, eaux-de-vie of wine or spirits.