METHOD OF SEQUENTIAL FUNGAL FERMENTATION OF LIGNEOUS RESOURCES

Abstract

The present disclosure relates to a method for transforming wood residues into an edible food product for a mammal, consisting of a sequence of fungal fermentations which make it possible to render ligneous resources edible; the invention also relates to the food product obtained by this method, and to the use thereof.

Claims

1. A method for transforming wood residues into edible food product for a mammal, the method comprising the steps of: 1) performing a first fermentation of a substrate composed of wood residues by a wood destroying edible fungus for a suitable duration corresponding to the maximum colonisation of the substrate before fructification by said fungus; 2) stopping the first fermentation by heat inactivation of said wood destroying edible fungus and grinding a product obtained from said first fermentation; 3) performing a second fermentation of the product obtained in step 2) by a fungus of the Aspergillus genus for a suitable duration corresponding to the maximum colonisation of the substrate before sporulation by said fungus of the Aspergillus genus.

2. The method for transforming wood residue into an edible food product for a mammal according to claim 11, wherein the pre-treatment step before step 1) comprises grinding of wood residues to obtain a size of wood residues less than or equal to 2 cm and/or heating at a temperature of at least 70 C. in a wet medium.

3. The method for transforming wood residues into an edible food product for a mammal according to claim 1, wherein said wood residues comprises a mixture of between 40 and 80% by weight of wood sawdust and between 20 and 60% by weight of wood meal.

4. The method for transforming wood residues into an edible food product for a mammal according to claim 12, wherein said alkalinizing mineral supplement represents an input of: 170 to 330 kg/t of calcium (expressed in the form of CaO), 20 to 60 kg/t of potassium (expressed in the form of K.sub.2O), 25 to 46 kg/t of magnesium (expressed in the form of MgO), 10 to 61 kg/t of phosphorus (expressed in the form of P.sub.2O.sub.5), metals, which are cofactors of digestive enzymes secreted by fungi, and has a pH, before mixing with wood residues, between 10 and 13.

5. The method for transforming wood residues into edible food product for a mammal according to claim 1, wherein the wood destroying edible fungus is selected from Pleurotus ostreatus Pleurotus pulmonarius, Hypsizygus ulmarius, Agaricus blasei and Agaricus braziliensis.

6. The method for transforming wood residues into edible food product for a mammal according to claim 1, wherein said fungus of the Aspergillus genus is selected from Aspergillus oryzae, Aspergillus niger, Aspergillus sojae, and Aspergillus awamori.

7. A fermented food product obtainable by the method according to claim 1.

8. The fermented food product according to claim 7, wherein the fermented food product comprises the following composition: between 5 and 10 U of xylanases/g of dry food product; between 5 and 10 U of amylases/g of dry food product; between 30 and 100 U of proteases/g of dry food product; between 20 and 40 mg of vitamin B3/g of dry food product; an amino acid profile comprising between 10 and 15 mg of histidine, between 30 and 45 mg of isoleucine, between 40 and 65 mg of leucine, between 20 and 30 mg of lysine, between 10 and 15 mg of methionine, between 25 and 40 mg of phenylalanine, between 12 and 19 mg of tyrosine, between 35 and 54 mg of threonine, between 25 and 40 mg of valine and between 8 and 12.5 mg of tryptophan/g of total proteins of said food product; a lignin content less than 18%.

9. A method of supplementing the diet of an animal, comprising incorporating the food product according to claim 7 in an animal food ration provided to the animal.

10. The method of claim 9, wherein the animal is a monogastric farm animal.

11. The method for transforming wood residue into an edible food product for a mammal according to claim 1, further comprising pre-treating the wood residues prior to the first fermentation of step 1).

12. The method for transforming wood residue into an edible food product for a mammal according to claim 1, wherein the substrate composed of wood residues in the first fermentation of step 1) further comprises 1 to 5% by dry weight of an alkalinizing mineral supplement.

13. The method for transforming wood residue into an edible food product for a mammal according to claim 1, further comprising stabilizing a product obtained from said second fermentation of step 3) by dehydration.

14. The method for transforming wood residue into an edible food product for a mammal according to claim 4, wherein the metals comprise one or more of Mn, Fe, Cu, and Zn in any proportion.

Description

FIGURES

[0100] FIG. 1: A. First fermentation: growth of Pleurotus ostreatus on oak sawdust after 40 days of incubation at 28 C. in the absence of or in the presence of mineral supplement (CM). B. Second fermentation: growth of Aspergillus oryzae after 3 days of incubation at 30 C. after different first fermentation conditions.

[0101] FIG. 2: A. Growth of Aspergillus oryzae on oak sawdust not fermented by Pleurotus ostreatus with and without combination with a mineral supplement and/or a nitrogenated supplement (in the form of protein, in the present case) (the insert has the growth of A. oryzae after sequential fermentation). B. Growth of Aspergillus oryzae on a culture medium containing 1.5% of glucose, 0.6% of NaNO.sub.3, 0.15% KH.sub.2PO.sub.4, 0.05% MgSO.sub.4, 0.05% KCl and minerals in trace form (Mn, Co, Zn and Fe) adjusted to different pHs. C. Growth of Aspergillus oryzae on a minimum culture medium (1.5% of glucose, 0.6% of NaNO.sub.3), complemented such as indicated in the figure and adjusted to pH 6.1.

[0102] FIG. 3: A. Growth of Pleurotus ostreatus on oak sawdust after 40 days of incubation at 28 C. after combination with different alkaline and/or mineral supplements. B. Development of Aspergillus oryzae following this first fermentation after 3 days of incubation at 30 C.

[0103] FIG. 4: Comparison of xylanase (A), amylase (B) and protease (C) activities secreted by Pleurotus ostreatus (from the first fermentation) (PO, clear grey) and by Aspergillus oryzae (from the second fermentation) (PO/AO, black), according to the percentage of mineral supplement added before the first fermentation. The activities are expressed in units (mol of product generated/min)/g of dry fermented product.

[0104] FIG. 5: Comparison of xylanase (A), amylase (B) and protease (C) activities secreted by Pleurotus ostreatus (PO, clear grey) and by Aspergillus oryzae (PO/AO, black), after different culture times of Pleurotus ostreatus on oak sawdust combined with 2.5% of ash. The activities are expressed in units (mol of product generated/min)/g of dry fermented product.

[0105] FIG. 6: Effect of the dehydration (A) and of the conservation (B and C) of the fermented product on xylanase, amylase and protease activities. (MC: moisture content).

[0106] FIG. 7: Enzymatic activities (Aamylase and xylanase and Bprotease) measured from the sequential fermentation method using Aspergillus oryzae or Aspergillus awamori during the second fermentation.

[0107] FIG. 8: Enzymatic activities (Aamylase and xylanase and Bprotease) measured from the sequential fermentation method using Aspergillus oryzae or Aspergillus awamori during the second fermentation, individually or in coculture.

[0108] FIG. 9: measurement of the laccase activity of the product from the first fermentation (histogram on the left) and that of the product from the second fermentation (histogram on the right).

EXAMPLES

Example 1Sequential Fermentation Method of Oak Wood with Pleurotus ostreatus then Aspergillus oryzae According to the Invention

1. EQUIPMENT AND METHODS

[0109] 1.1. Sequential Fermentation Method

[0110] Pre-Treatment of the Wood

[0111] The oakwood residues obtained in the form of sawdust have been coarsely ground using a blade grinder to obtain a minor fraction in the form of meal (about 20%). The aim is to reduce the size (between 50 m and 1 mm) and the crystallinity of a fraction of the lignocellulose of the wood with the aim of increasing its exchange surface and thus to facilitate enzymatic degradation (Saritha et al., 2012; Ravindran & Jaiswal, 2015).

[0112] They have been then subjected to a pre-treatment by heating to 90 C. in an aqueous medium in order to extract a portion of the extractables including water-soluble tannins.

[0113] After filtration on cellulose filter until obtaining a moisture content between 55 and 70% (Girmay et al., 2016) (Hoa & Wang, n.d.), an alkaline mineral supplement (ash) can be added then the substrate is autoclaved.

[0114] First Fermentation

[0115] The substrate is thus inoculated by Pleurotus ostreatus on rice. The inoculated substrate is then kept at 28 C., optimum growth temperature (Hoa et al., n.d.) for a period between 30 and 40 days until complete colonisation of the substrate by Pleurotus ostreatus (Hoa & Wang, n.d.).

[0116] Second Fermentation

[0117] From this first fermentation, the culture is ground using a blade grinder with the aim of making it homogenous and to serve as a base for the second fermentation. Oyster mushrooms are then inactivated by heating (between 70 C. and 120 C.), then 2.Math.10.sup.6 spores of Aspergillus oryzae are added per gram of dry fermented product with adjustment of moisture between 60 and 70%.

[0118] The Aspergillus oryzae spores have been collected beforehand after culture on PDA medium containing 0.6M of KCl in order to stimulate the sporulation (Song et al., 2001).

[0119] The culture is stopped after 3 days of incubation at 30 C., the fermented product is recovered.

[0120] Stabilisation

[0121] The fermented product is stabilised by dehydration: it is placed after homogenisation by mechanical stirring in a chamber at 24 C. until obtaining a moisture content of 11-12%, corresponding to an activity of water (aw) less than 0.6 and preventing the growth of microorganisms (Assamoi et al., 2009), then it is stored at 4 C. or ambient temperature.

[0122] 1.2. Characterisation of the Fermented Food Product Obtained

[0123] 1.2.1. Measurement of the Enzymatic Activities

[0124] Preparation of the Enzymatic Raw Extract

[0125] The enzymes secreted by fungi are isolated from the fermented product directly after the stopping the incubation period or after stabilisation. The equivalent of 0.1 g of dry fermented product is removed to an Eppendorf tube then placed in 2 ml of acetate buffer 50 mM pH 5.0 and stirred (incubator stirrer 150 rpm) for 30 minutes at 30 C. (Chancharoonpong et al., 2012). The supernatant containing the secreted enzymes is collected after centrifugation for 10 minutes at 10,000 g (4 C.).

[0126] Measurement of Xylanase, Amylase and Protease Activities

[0127] The xylanase activities are measured by using as a substrate, beech xylan at 1% in an acetate buffer 50 mM pH 5.0. Typically, 50 l of raw enzymatic extract are added to 150 l of substrate then incubated for 50 minutes at 50 C. (van den Brink et al., 2013). The appearance of reducing ends after enzymatic cutting is measured by colorimetric dosing at 405 nm after reaction with p-4-hydroxybenzhydrazide (Szilagyi et al., 2010).

[0128] The amylase activities are measured by using as a substrate, starch at 0.2% in an acetate buffer 50 mM pH 5.0. Typically, 50 l of raw enzymatic extract are added to 150 l of substrate then incubated for 50 minutes at 50 C. (van den Brink et al., 2013).

[0129] The protease activities are measured by using azocasein as a substrate as described (Janser et al., 2014) with a few modifications: 200 l of enzymatic extract are added to 200 l of azocasein at 0.5% in an acetate buffer 50 mM pH 5.0. The incubation is carried out for 1 hour at 55 C., then proteins are precipitated by adding 400 l of 10% trichloroacetic acid. After 10 minutes in ice, the tubes are centrifugated at 10,000 g for 10 minutes. 100 l of supernatant containing azopeptides and azo amino acids are transferred into a microplate containing 100 l of NaOH 5M. The absorbance is measured at 428 nm to determine the protease activity of the raw extract.

[0130] Measurement of the Laccase Activity

[0131] The laccase activity is measured by using ABTS 0.2 mM (2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) as a substrate (Valiskovi& Baldrian, 2006) in an acetate buffer 50 mM pH 5.0. Typically, 20 l of raw enzymatic extract are added to 140 l of acetate buffer pH 5.0 and 40 l of ABTS 1 mM. The enzymatic activity is then evaluated immediately by measuring absorbance at 420 nm, corresponding to the oxidation of ABTS by the laccase activity and, carrying out kinetics over 90 minutes.

[0132] 1.2.2. Determination of Lignin and Beta-Glucan Contents

[0133] The determination of the lignin content has been achieved by gravimetry after acid hydrolysis: the sample undergoes a succession of attacks by different solutions (neutral detergent, then acid detergent) in a Fibertec-type device (Boiling for 1 hour). At the end of each attack, the sample is carefully rinsed, dried and weighed. An attack with a highly concentrated acid is thus carried out, and the sample containing the lignin fraction is dried then weighed to determine the lignin content compared with the dry starting weight.

[0134] The determination of the beta-glucan content is achieved after specific enzymatic hydrolysis. The samples undergo successive enzymatic digestions. The glucose contained in beta-glucans 1.3-1.6, is thus released and determined by ion chromatography.

2. RESULTS

[0135] The Development of Aspergillus oryzae on Wood Residues is Dependent on the Prior Development of Pleurotus ostreatus.

[0136] The model selected for the development of the method is based on the use of oak sawdust which is ground coarsely and subjected to a hot aqueous extraction. After adjustment of the moisture by filtration and sterilisation, the substrate is inoculated by Pleurotus ostreatus and maintained for 40 days at 28 C. to allow the development of the fungus. The growth of Pleurotus ostreatus on oak sawdust can be optimised by adding a natural and easily available alkalinising mineral supplement (ash). FIG. 1A has different culture conditions achieved in the absence or in the presence of the mineral supplement.

[0137] After 40 days of culture, Pleurotus ostreatus is easily grown on oak sawdust in the absence of mineral supplement (CM 0%) while the growth, visible by the extension of white mycelium, increases with the mineral supplement percentage until being stabilised at about 5% of CM. The use of a grinder thus makes it possible to homogenise the fermented product while the inactivation by heating makes it possible to prevent a new growth of oyster mushrooms. After these treatment conditions, the fermented substrate regains an appearance, a brown colour characteristic of wood (mycelium is no longer visible to the naked eye) (see FIG. 1A, column 3 and FIG. 1B, column 1) and the growth is ineffective without any new inoculation (FIG. 1B, column 1).

[0138] Aspergillus oryzae spores are added to the fermented sawdust then the moisture level is brought to a value between 60 and 70% before initiating a second fermentation for 3 days at 30 C. in a wet chamber. FIG. 1B shows the growth of Aspergillus oryzae from this second fermentation. This is undetectable if the spores are added on sawdust not having been subjected to a first fermentation (FIG. 1B, column 2) and its level is correlated positively to the growth level of Pleurotus ostreatus obtained during the first fermentation (comparing the quantity of white mycelium in FIG. 1A, columns 1 to 4 and FIG. 1B, columns 3 to 6).

[0139] These results therefore show that the growth of Aspergillus oryzae on only oak sawdust is dependent on a first fermentation by Pleurotus ostreatus.

[0140] A certain number of factors could explain this dependence.

[0141] On the one hand, a decrease in the lignin content is expected given the lignivorous character of Pleurotus ostreatus, a decrease and partial degradation facilitating the access of holocellulose to the enzymes secreted by Aspergillus oryzae. To this is added a partial degradation of holocellulose by Pleurotus ostreatus itself leading to the release of reducing sugars easily assimilable by Aspergillus oryzae. Measuring reducing sugars after the first fermentation has been evaluated at 20 mg/g of dry fermented product against 2 mg/g of dry sawdust before fermentation. It has been estimated at 10 mg/g of dry fermented product from the second. These results therefore show that the first fermentation allows the release of reducing sugars which could be partially used during the second fermentation for the growth of Aspergillus oryzae.

[0142] On the other hand, it is possible that certain compounds secreted by the fungus serve as an additional carbon source (such as organic acids) and nitrogen source (proteins synthesised by Pleurotus ostreatus).

[0143] The minimum conditions for fermenting oak sawdust by Aspergillus oryzae are presented in FIG. 2 and make it possible to highlight the relevance of the sequential method on oak sawdust. Wood sawdust has been subjected to a pre-treatment similar to that performed before inoculation by Pleurotus ostreatus, namely a coarse grinding then an extraction in an aqueous medium at 90 C. After sterilisation (autoclaving), a new grinding is carried out before inoculation by the Aspergillus oryzae spores and incubation for 3 days at 30 C. As FIG. 2A shows, no growth is observable only on pre-treated wood (column 1), on pre-treated wood combined with 2.5% of alkalinising mineral supplement and 2.5% of neutral mineral supplement (here, ash, of which the pH has been adjusted to 7.5 by adding HCl) (columns 2 and 3), on pre-treated wood combined with 1.25% of alkalinising potash (column 4) and on pre-treated wood combined with 3% of nitrogenated supplement (column 5). However, growth is observed on pre-treated wood after addition of 2.5% of alkalinising or neutral mineral supplement and 3% of nitrogenated supplement (proteins) (columns 6 and 7). The growth level observed is greater when the mineral supplement is alkalinising compared with the neutral mineral supplement, but less than that observed after a first fermentation by Pleurotus ostreatus (comparing the inset and columns 6 and 7). These results suggest therefore that Aspergillus oryzae can be grown on oak sawdust in the presence of a mineral supplement and a nitrogenated supplement with a preferable development at alkaline pH. The combination of the nitrogenated supplement and alkalinising potash does not make it possible to see a development of Aspergillus oryzae on oak sawdust (column 8), confirming a dependence on the presence of mineral elements, absent in this experimental condition. The optimal growth conditions of A. oryzae combining the nitrogenated and alkalinising mineral supplements are not suitable with the published data having a growth optimum pH for this ascomycete between 6 and 7.5 (Krijgshield et al., 2013). FIG. 2B confirms a decrease of growth of A. oryzae at alkaline pH (the pH of the pre-treated wood combined with 2.5% of alkalinising mineral supplement is about 8.0) and therefore suggests that the positive effect of the alkalinisation observed on the growth Aspergillus would result in an effect on the wood (FIG. 2A, column 7) which weakens the lignocellulose of the wood and would facilitate its degradation by the enzymes secreted by Aspergillus (Rabemanolontsoa & Saka, 2015). The dependence of Aspergillus oryzae regarding mineral elements present in the mineral supplement added in the form of ash is highlighted in FIG. 2C. Compared with a complete medium and a minimal medium only containing a carbon and nitrogenated source and for which a very low growth is observed, the removal of phosphate limits any significant development of Aspergillus oryzae. The removal of magnesium and of sulphate is not limiting for the growth of the fungus but leads to a sporulation suggesting an ascomycete stress (result not presented).

[0144] These results therefore show that Aspergillus oryzae is capable of being grown on oak sawdust on the condition of adding to the substrate a mineral and nitrogenated supplement, growth is favoured if the mineral supplement is alkalinising with an effect which could be attributed to a weakening of lignocellulose (during the heating in the presence of the mineral supplement). However, the growth effectiveness is less than that observed during the sequential fermentation method.

[0145] In their entirety, these results show that the growth of Aspergillus oryzae on oak sawdust (without addition) is dependent on a first fermentation by Pleurotus ostreatus and suggest that this first fermentation, by weakening the wood and in particular, lignin, make available nutritional elements necessary for its development of which very probably, a nitrogen source which is accessible and essential for the growth of Aspergillus oryzae as well as the mineral elements essential to its development, in particular phosphate.

[0146] Effect of the Mineral Supplement on the Conduct of the Method According to the Invention

[0147] The relevance of the sequential fermentation method can also be highlighted through the analysis of the impact of the alkalinising mineral supplement on the fermentation by Pleurotus ostreatus. FIG. 3A presents the growth of Pleurotus ostreatus on oak sawdust combined with 2.5% of mineral supplement (column 1), at 2.5% of mineral supplement of which the pH has been adjusted to 7.5 (column 2), to 1.25% of potash (column 3) and to 1.25% of calcium carbonate (column 4) (1.25% of KOH have been added as ash contains about 50% of CaO mainly responsible for the alkalinity). The results obtained show that potash or calcium carbonate can be substituted for ash (comparing columns 1, 3 and 4). However, the adjustment of the mineral supplement at pH 7.5 leads to a notable decrease in the growth of Pleurotus on oak sawdust with however a greater growth of oyster mushrooms under these conditions to that observed without adding any mineral supplement (comparing with FIG. 1, column 1).

[0148] Thus, these results show that the alkalinity of the ash has a more determining effect on the growth of oyster mushrooms than the addition of minerals.

[0149] It is probable that the alkalinising effect here also works on wood and not on the growth of the fungus, itself. Indeed, different studies have shown an optimum pH for the growth of oyster mushrooms between 5 and 7 during the culture on a synthetic medium or straw (Romero-Arenas et al., 2012, Tripathi and Yadav, 1992, Belletini et al., 2016).

[0150] Finally, FIG. 3B presents the growth of Aspergillus oryzae in secondary fermentation under these experimental conditions. More surprisingly, these results show that the presence of additional mineral elements is not essential to the growth of Aspergillus oryzae during the second fermentation since the ash can be substituted by potash or calcium carbonate (comparing FIG. 3B, columns 1, 3 and 4) and reinforce the importance of the quality of the first fermentation on the second by making available nitrogenated nutritional elements and minerals taken from wood for Aspergillus oryzae.

[0151] The Method According to the Invention can be Applied to Different Wood Species

[0152] Complementary experiments have been carried out on different wood species. The species models have been selected according to their level of harvesting, their classification as species of value and their immediate availability: spruce, beech and nannyberry have thus served as a study model. Beech is the third most harvested hardwood after oak and poplar, spruce forms part of the most harvested conifers, and mountain ash is a valuable species.

[0153] The experimental conditions are based on the reference protocol developed on oak.

[0154] The pre-treatment conditions of wood, the conditions of first fermentation (with and without alkaline) and of second fermentation are similar to those used for oak.

[0155] The effectiveness of the method has been evaluated from the second fermentation by measuring xylanase, protease and amylase activities and presented in table 1. To facilitate the comparison of the results, these have been standardised to the values obtained during the use of oak as a substrate (1 arbitrary unit).

TABLE-US-00001 TABLE 1 Enzymatic activities measured from the sequential fermentation method using sawdust coming from different wood species. Enzymatic activity (arbitrary unit) Amylase Xylanase Protease 0% 2.5% 0% 2.5% 0% 2.5% alkaline alkaline alkaline alkaline alkaline alkaline supplement supplement supplement supplement supplement supplement Oak 0 1 0.05 1 0 1 Beech 0.57 0.91 0.194 0.62 0.18 1.4 Nannyberry 0.54 0.91 0.25 0.84 0.21 1.12 Spruce 0.44 0.8 0.18 0.58 0.05 0.62

[0156] The results obtained show that i) the method developed on oak sawdust can be applied to other species with ii) a significant improvement in the production of amylase, xylanase and protease activities during the second fermentation if the method is conducted in the presence of alkaline supplement during the step of pre-treating sawdust before the first fermentation, that iii) the production of these activities is less dependent on the addition of the alkaline supplement for oak, mountain ash, and spruce species compared with oak, iv) the production of amylase, xylanase and protease is generally less when spruce is used as a substrate.

[0157] The Pre-Treatment of Water has No Damaging Effect on the Xylanase and Amylase Content of the Product Obtained by the Method According to the Invention

[0158] Complementary experiments have been carried out in order to evaluate the possible effect of different pre-treatment conditions.

[0159] The effect of the extraction temperature has been evaluated first. For this, wood (oak) residues mixed with water have been heated to 50 C., 90 C. and 120 C. or have been mixed with water without heating before proceeding with the filtration step, then adding alkaline supplement before sterilisation before inoculation by Pleurotus ostreatus for the first fermentation. The effectiveness of the method has been evaluated from the second fermentation by measuring xylanase and amylase activities. The two fermentation steps have been conducted as described above for oak residues.

[0160] No significant difference has been observed on the level of secretion of the amylase and xylanase activities from the second fermentation, confirming the possibility of using a quite wide temperature range during the extraction step.

[0161] Impact of Moisture on the Substrate on the Conduct of the Method According to the Invention

[0162] Complementary experiments have been carried out in order to confirm the impact of the moisture content of the substrate on the sequential fermentation method. For this, the moisture level of the substrate (oak residues such as used in the reference method) from the filtration has been adjusted to 40%, 50%, 60%, 70% and 80% before adding the alkaline supplement, sterilisation and inoculation. The effectiveness of the method such as described above has been evaluated from the second fermentation by measuring the xylanase and amylase activities. No significant difference has been observed on the level of secretion of the amylase and xylanase activities from the second fermentation, confirming the possibility of using the moisture levels between 40 and 80%.

[0163] Implementation of the Method According to the Invention with Other Basidiomycete Strains

[0164] The sequential fermentation method has been implemented according to the protocol described above with Pleurotus pulmonarius and Hypsizygus ulmarius substituting for Pleurotus ostreatus. As above, the effectiveness of the method has been evaluated from the second fermentation by measuring the xylanase and amylase activities secreted by Aspergillus oryzae. The table below presents the results obtained from these activities. To facilitate the comparison of the results, these have been standardised to the values obtained during the use of Pleurotus ostreatus for the first fermentation (1 arbitrary unit).

TABLE-US-00002 TABLE 2 Measurement of the xylanase and amylase activities from the fermentation by Aspergillus oryzae according to the basidiomycete used for the first fermentation. Enzymatic activity (arbitrary unit) Amylase Xylanase Pleurotus ostreatus 1 1 Pleurotus pulmonarius 1 1.25 Hypsizygus ulmarius 0.92 0.56

[0165] The results obtained show that the sequential fermentation method according to the invention can be applied to Pleurotus pulmonarius and Hypsizygus ulmarius, two wood destroying fungi not leading to a significant difference on the production of amylase by Aspergillus oryzae.

[0166] The regulation of the secretion of the xylanase activities has been widely studied and this is controlled by the respective levels of inducers (xylan, xylose with low concentration, nitrogen, etc.) and repressors (xylose with high concentration, glucose, etc.) potentially present in the medium. It is possible that the first fermentation conducted at the production/release of inducing compounds and/or repressors varying according to the fungus species used, which would explain the difference of production of xylanase when the first fermentation is carried out in the presence of Hypsizygus ulmarius.

[0167] In conclusion, the sequential fermentation method can be applied to different wood destroying fungi species by ensuring the level of secretion of the enzymatic activities sought.

[0168] Implementation of the Method According to the Invention with Aspergillus awamori and in Coculture of Aspergillus oryzae and Aspergillus awamori

[0169] The sequential fermentation method has been applied to Aspergillus awamori, a mould of the Aspergillus genus used in traditional Japanese food.

[0170] The steps of pre-treating oak sawdust then of inoculation by Pleurotus ostreatus have been carried out according to the reference protocol. The substrate from the first fermentation has been adjusted to 65% moisture level before being inoculated by 2.Math.10.sup.6 Aspergillus awamori or Aspergillus oryzae spores per gram of dry fermented product then incubated for 3 days at 30 C. (reference protocol). The effectiveness of the method has been evaluated by measuring xylanase, amylase and protease activities from the second fermentation. The results obtained are presented in FIG. 7. They are expressed in U/g of dry fermented product.

[0171] These show that the secretion of amylase by Aspergillus oryzae and awamori is comparable (panel A). However, the xylanase and protease activities are significantly different between the two species. The xylanase activity is greater for Aspergillus awamori (about 2.5 times) (panel A) while the protease activity is less for A. awamori (about 3.5 times) (panel B).

[0172] Taken together, these results show i) that another species of the Aspergillus genus can be grown on fermented wood residues by following the steps established for Aspergillus oryzae, making it possible to propose that the method is applicable to other species of the Aspergillus genus, ii) that the enzymatic activities sought initially, namely the xylanase, amylase and protease activities are present from the fermentation for the two species used, awamori and oryzae and iii) that the level of secretion of the xylanase and protease activities is variable according to the species considered.

[0173] According to this last observation, modulating the level of the xylanase and protease activities by combining the species can be contemplated. FIG. 8 shows the results of the measurements of the amylase, xylanase and protease activities obtained by producing Aspergillus awamori and Aspergillus oryzae cocultures during the second fermentation, the percentage of each mould varying between 100, 75, 50 and 25% of the coculture. As expected, the level of secretion of the amylase activities is similar whatever the respective percentage of A. oryzae or awamori (panel A). The level of secretion of xylanase activity increases when the percentage of Aspergillus awamori increases to be stabilised when the A. oryzae/A. awamori ratio is identical (about 12 U/g of fermented product) (panel A).

[0174] Correlating to the results presented in FIG. 7, the level of protease activity decreases when the percentage of A. awamori increases, this decrease being significant from an identical A. awamori/A. oryzae ratio (50/50). In their entirety, these results show that i) the coculture of moulds of the Aspergillus genus on the fermented wood residues can be considered and ii) a controlled coculture at the moment of the inoculation makes it possible to modulate the respective level of secreted enzymes. For example, A. oryzae can be used by itself, if the protease activities are sought and combined with A. awamori in order to increase the level of secretion of the xylanase activities.

[0175] The First Fermentation of the Method Leads to the Production of Laccase by P. ostreatus

[0176] The presence of laccase activity is detected from the first fermentation; however, this enzyme is hardly or not detected from the second fermentation (see FIG. 9).

[0177] Knowing that this activity is responsible for the degradation of lignin and that the lignin content of the wood from the method is 12% (against a theoretical content between 17 and 25% before fermentation), the decrease of the lignin content can be associated with this first fermentation.

[0178] The Sequential Fermentation Allows the Production of Xylanases, Amylases and Proteases by Aspergillus oryzae

[0179] The enzymes secreted by filamentous fungi are widely used in animal food to improve the digestibility of food and to increase the growth performances of farm animals (Asmare, 2014). Aspergillus oryzae has been used in human food for several millennia and described for its capacity to synthesise and to secrete enzymes involved in the degradation of starch-rich and also lignocellulosic substrates (Brink & Vries, 2011; Kobayashi et al., 2007; Vries & Visser, 2001).

[0180] The xylanase, protease, amylase, glucanase and phytase activities are the activities the most sought for animal food (Shallom & Shoham, 2003; Kuhad et al., 2011; Asmare, 2014). Assays for xylanase, protease and amylase activities have been developed in order to evaluate the level of secretion of these enzymes by Aspergillus oryzae while comparing it to that of Pleurotus ostreatus.

[0181] FIG. 4 presents the xylanase, amylase and protease activities secreted by Pleurotus ostreatus from the first fermentation and Aspergillus oryzae from the second. Three culture conditions have been compared, one carried out without adding any mineral supplement and two carried out with the addition of 1 and 5% of mineral supplement, two conditions stimulating the development of Pleurotus ostreatus and consequently that of Aspergillus oryzae.

[0182] Thus, if the fermentation carried out by Pleurotus ostreatus is a condition sine qua non to the growth of Aspergillus oryzae, the second fermentation gives the fermented product, added value in particular through the presence of digestive enzymes. It is important to note that the level of secretion of these enzymes by Pleurotus ostreatus remains much less than that of Aspergillus oryzae whatever the duration of the first fermentation. FIGS. 5A and B shows almost zero secretion of xylanases and amylases after 20, 30, 40, 50 and 60 days of fermentation by Pleurotus ostreatus in the presence of 2.5% of ash. The xylanase and amylase activities secreted by Aspergillus oryzae increase between 20 and 30 days of first fermentation (by Pleurotus ostreatus) to be stabilised then (the incubation duration of Aspergillus oryzae remained constant for 3 days). The differences in the levels of secretion of the protease activities are less pronounced between Pleurotus ostreatus and Aspergillus oryzae whatever the duration of the first fermentation, but are always in favour of Aspergillus oryzae under the conditions where the mineral supplement has been added at a level of 2.5% (see FIG. 5C) and 5% (see FIG. 4C).

[0183] Taken together, these results show that the sequential fermentation method on wood residues using Pleurotus ostreatus then Aspergillus oryzae allows the production of enzymes, such as xylanases, amylases and proteases.

[0184] Sequential Fermentation Allows the Degradation of Lignin.

[0185] One of the obstacles to using lignocellulosic compounds and particularly wood for animal food is its rigidity due to its lignin content of about 25% (Guerriero et al., 2016).

[0186] The lignin content of the fermented product from the sequential fermentation has been evaluated and represents 11% of lignin, value a lot less than the lignin content of the wood residues (starting product).

[0187] The Enzymatic Activities of the Fermented Product can be Stabilised on the Substrate.

[0188] The secretion of digestive enzymes during the second fermentation opening up prospects of enhancing fermented wood sawdust, it appears essential to develop a method for stabilising and for conserving these simple and inexpensive enzymes.

[0189] In the method implemented, from the second fermentation, the fermented product of which the moisture level is close to 60% is made homogenous by mechanical stirring then placed in a chamber at 24 C. until obtaining a moisture content of 12%, the moisture level stabilising the product from a microbiological standpoint and preventing an increase in growth or the development of other types of microorganisms (Assamoi et al., 2009).

[0190] FIG. 6A presents the residual enzymatic activities after dehydration to 12% and shows that dehydration carried out under these conditions does not lead to any loss of xylanase, amylase and protease activities.

[0191] FIG. 6B presents the same residual activities after conservation at 4 C. of the fermented and dehydrated product for one, two, three or four weeks. No significant loss of activity is observable whatever the activities measured.

[0192] FIG. 6C presents the residual xylanase, amylase and protease activities after conservation at ambient temperature of the fermented and dehydrated product for one, two, three and four weeks. As above, no significant loss of activity is observable whatever the activities measured.

[0193] These results therefore confirm the possibility of using the fermented substrate as a support for stabilisation/immobilisation of the secreted enzymes.

[0194] The beta-glucan content (main compounds of the wall of the filamentous fungi) present in the final fermented product is measured at about 8%.

3. CONCLUSION

[0195] The experimental data highlight the production of a fermented product of food quality from wood; this product is complex and composed of residual digested lignocellulose (in a proportion similar to that of straw), mycelium and compounds secreted by fungi comprising enzymes. These enzymes of interest are usually added to the animal food ration (Asmare, 2014). In current methods, the purified enzymes must be diluted by mixing with mineral meals or matrices before being incorporated in the food. The enzymes secreted and stabilised on the final fermented product according to the invention are already diluted by the presence of the residual substrate and of mycelium, a premixing with a meal or a matrix can be avoided, facilitating the production and limiting the production cost of the enriched food.

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