WO2015101650A2

WO2015101650A2 - Food product and method for producing same

Info

Publication number: WO2015101650A2
Application number: PCT/EP2014/079495
Authority: WO
Inventors: France Thevenieau; Anne BOURDILLON; Alain Durand; Joëlle DE CONINCK; Stéphanie SUTTER
Original assignee: Fonds De Développement Des Filières Des Oléagineux Et Des Proteagineux Fidop
Priority date: 2013-12-31
Filing date: 2014-12-31
Publication date: 2015-07-09
Also published as: WO2015101650A3

Abstract

The invention relates to the use of at least one fungal strain for the production of a fermented solid edible food product intended for animal feed, from a byproduct originating from plants, in particular edible. The edible product comprises the fungus.

Description

Food product and its process for obtaining

The present invention relates to a food product and its method of production. By-products, residues or waste from the agriculture and agri-food industries account for 30% of world agricultural production. Of these wastes, lignocellulosic residues are the most important. The development of green technologies to reuse this energy source in animal feed is a very interesting valuation perspective. This strategy aims to reduce the pressure on agricultural productivity by efficiently using all the raw materials available, which considerably limits their accumulation in the form of waste. In addition to the environmental and health aspects, these by-products can represent a valuable biomass if protein enrichment technologies are implemented to address and address the real problems related to animal nutrition.

However, a very small amount of these agro-food by-products can be used in animal feed because of their low digestibility and nutrient content (essential protein and amino acid).

Also, there is a real need to provide agrifood by-products that are reusable for animal nutrition but have improved nutritional properties.

A solution to these problems has been proposed by the development of biotechnological processes, with the use of microorganisms in order to improve the nutritional quality of these residues represent a great opportunity for valorization in animal feed.

Microorganisms are capable of degrading the recalcitrant components of digestion, such as fibers and harmful compounds such as antinutrients while improving the nutritional quality of waste by enriching proteins and vitamins.

In particular, cakes have already been widely used in biotechnology for the industrial production of enzymes, antibiotics, biopesticides, vitamins and other biochemical compounds. Some of these products, mainly enzymes, are also commonly used as a feed additive in rations for livestock.

The carbohydrate and mineral content as well as the physical structure of the cake make it a good source of nutrients and / or support for the development of microorganisms. But these nutrients are not easily assimilated by the majority of industrial bacteria and yeasts (Wang et al., 2010, Enzyme and Microbial Technology, 47 (3), pp. 77-83). As a result, microbial production of enzymes in cakes is much more effective by molds than by bacteria because of their morphology and physiology (presence of many polymerases) allowing them to colonize this type of substrate more easily.

Strains of the genus Aspergillus, Penicillium and Rhizopus are the most famous for the production of enzymes on cakes [Sivaramakrishnan and Gangadharan, 2009 Edible OR Cakes (Biotechnology for Agro-Industrial Residues Use). Springer. 47 Op]

Although such processes make it possible to obtain a production of proteins, and in particular of enzymes, the digestibility of the cakes is not, however, increased.

Also, the need to improve the processes for the preparation of food products for animal feed remains, particularly with regard to animal nutrition.

One of the aims of the invention is to obviate the aforementioned drawbacks.

A second object of the invention is to provide a food product derived from vegetable by-products which is both easy to digest and which provides a sufficient nutritional intake.

Yet another object of the invention is to provide a food composition that provides nutritional benefit and other benefits to the animal that consumes it.

The invention relates to the use of at least one strain of micromycetes for the preparation of a fermented solid edible food product intended for animal or human nutrition from a by-product derived from plants, in particular edible, said edible product comprising said micromycetes.

The invention is based on the unexpected finding made by the inventors that the molds made it possible to obtain an edible food product from a by-product derived from plants, said edible product being enriched with available organic nutrients, in particular proteins, and having good digestibility for animals.

In the invention, mention is made of molds or micromycetes or even filamentous microscopic fungi in a uniform manner. These multicellular microorganisms have a vegetative apparatus called thallus consisting of filaments, the hyphae, which form the mycelium visible to the naked eye. Reproduction by asexual reproduction is most widespread with conidial production. In some fungi, the multiplication is also via a massive production of spores from sexual reproduction. These heterotrophic organisms are parasites (plants or animals) or live in symbiosis with plants, they are also saprophytes and participate in the decomposition of organic waste with significant lytic properties. Microscopic fungi are widespread, they play an important role because they are involved in major life cycles such as carbon and nitrogen and are present in many terrestrial biotopes. In particular, the invention relates to the use of at least one strain of lower fungus or filamentous for the preparation of a fermented solid food product for animal or human feed from a by-product derived from plants said edible product comprising said lower or filamentous fungus strain, said lower or filamentous fungus strain is a strain of Aspergillus soya, Rhizopus oligosporus, Rhizopus oryzae or Aspergillus oryzae, especially a strain selected from Aspergillus soya and Aspergillus oryzae.

In particular, the invention advantageously relates to the use of at least one filamentous mushroom strain for the fermentation preparation of a fermented solid edible product intended for animal or human food from a by-product derived from plants. ,

said plant derived by-product being derived from a strict high fat oilseed, said edible product comprising said micromycete strain, said strain of micromycetes is a strain of Aspergillus soya, or Aspergillus oryzae.

In this advantageous aspect of the invention, "strict fat-rich oleaginous" is understood to mean a plant capable of producing lipids, but which is generally not used for its production of protein. Also, in fact, the soybean which is a proteo-oleaginous plant is excluded from the plants used in the context of this embodiment. Similarly, wheat or beet are excluded from this embodiment.

In the invention, the micromycetes are as follows:

Aspergillus soya, for which the following strains can be mentioned:

ATCC® MYA4878 ™, ATCC® 9362 ™, ATCC® 20387 ™; an advantageous strain is the strain CBS 100934.

Rhizopus oligosporus, for which mention may be made in particular of the following strains: ATCC® 22959 ™, ATCC® 60826 ™, ATCC® 48010 ™, or

- Aspergillus oryzae, for which the following strains may notably be mentioned:

ATCC® 42149 ™, ATCC® 10124 ™, ATCC® 20719 ™, ATCC® 7252 ™; an advantageous strain is the strain Aspergillus oryzae UMIP 1042.72.

The inventors made the unexpected finding that it was possible to use the aforementioned mildew strains to prepare an edible solid fermented food composition from plant by-products, said nutritional composition having nutritional properties appropriate for the animal and good digestibility. In particular, the food composition according to the invention comprises immunomodulatory nutrients derived from the degradation of said by-products of fermented plants.

In the invention we will speak of nutrients, compounds or compositions immunomodulators uniformly.

In the invention, the term "edible solid fermented food composition" is defined as a substance which can be used for animal or human nutrition and which has the following characteristics:

it is fermented, that is to say that it has undergone degradation of its composition due to the fermentation of said aforementioned fungi (enzymatic degradation of proteins, sugars and lipids),

- it is solid, that is to say it has a relatively firm consistency as opposed to liquids, and despite the damage it has suffered or may suffer,

- it is edible, that is to say that it is suitable for use as food by humans or animals, and is not susceptible, provided that it is free from contamination by pathogens, to cause eating disorders or to cause pathologies.

In the invention, the composition is said to be solid, which means that it is firm and has a certain resistance, unlike liquids. Although the composition may contain more than 50% moisture before drying, the composition will be predominantly solid, have the above-mentioned properties, and will therefore be called "solid". After drying, the composition may comprise more than 5% moisture, and will still be considered "solid".

Solid-state fermentation (FMS) is characterized by microbial growth on moist solid particles in the absence of free water. It is particularly well adapted to fungal microorganisms. The latter develop in a system consisting of three phases: a solid matrix, a liquid phase linked to it and a gaseous phase trapped between and inside them (Rahardjo et al., 2006, Biotechnology Advances , 24 (2), pp. 161-179). The type of substrate generally used is an inexpensive natural raw material derived from agricultural residues that acts both as a carrier and source of carbon and energy for microorganisms (Rodriguez Couto and Sanroman, 2006, Journal of Food Engineering, 76). , pp. 291-302, Holer and Lenz, 2005, Current Opinion in Microbiology, 8, pp. 301-306).

Advantageously, the solid composition according to the invention comprises a moisture content of about 12%, which corresponds to the moisture content of the by-product derived from plants at the factory outlet.

In the invention, the term "by-product derived from plants" also defines the residues or wastes of the agricultural and agri-food industries.

Livestock feeds include agricultural by-products, or vegetable by-products, to which ingredients must be added. to improve levels of essential amino acids, vitamins and minerals.

One of the advantages of the use of the aforementioned micromycetes is that it makes it possible to overcome this disadvantage of ingredient supply by allowing, by fermentation-type degradation, to enrich said by-product in elements such as proteins, vitamins, or sugar derivatives, and add value to the staple food.

By a calculation, the inventors know that a conversion of 50% of carbohydrates into biomass of the by-product derived from plants is obtained by the use of the mushrooms according to the invention. There is about 45% carbohydrate in the rapeseed meal, if we consider a consumption of only 20% of these carbohydrates by the fungi it means that for 100 g of cake 9 g of carbohydrates are converted into biomass, so 9% of biomass produced to the maximum.

Advantageously, the description relates to the aforementioned use, wherein said by-product derived from plants is chosen from: oilseed and proteaginous seeds, oilseed and proteinaceous hulls and shells, oilseed meal, cereals, cereal co-products (in particular starch, distillery, brewery products, etc.), sugar and fruit juice products (beet, citrus fruits, grapes, sugar cane), or maize.

Advantageously, the invention relates to the aforementioned use, wherein said by-product derived from plants is derived from plants belonging to the family Brassicaceae or Asteraceae.

More generally, all plant by-products comprising at least 1.5% fat relative to its total weight is advantageous. Examples of these by-products are given in Example 6.

Oilcakes are solid residues obtained after oil extraction from seeds or oleaginous fruits. These are the co-products of crushing, that is, the oil manufacturing industry. They usually represent 50 to 75% of the seed mass. The beneficial cakes of the invention are almond cake, peanut, safflower, hemp, rapeseed, copra (coconut), cotton, flax grown, maize (corn cakes) ), seaweed, walnuts, olives (olive cake), palm kernels (palm oil cake), poppy (poppy seed or poppy), sesame, soya or sunflower.

Beet pulp is a by-product of the sugar industry in temperate regions. During the extraction of sugar, the roots are roughly grated and the sweet juice is recovered by osmosis. The residue rich in digestible cellulose takes the name of beet pulp and can be used in animal feed in animals capable of digesting it (ruminants, horses, rabbits, ...) for ruminants (only able thanks to the bacteria of their rumen to digest the cellulose). It can be used in the form of fresh pulps rich in water (15 to 20% dry matter) and must generally be ensiled or in the form of dehydrated pulp packaged in "corks" which can thus be incorporated into compound feeds. In order to increase the digestibility of this pulp, it is advantageous to use the filamentous fungi as defined in the invention.

The sound, especially of wheat, is constituted by the envelope of caryopsis, separated from the almond cereals. When grinding cereals, bran is part of the issues, that is to say residues obtained after separation of the flour by sieving or blutage. The sound contains in particular cellulosic constituents (dietary fiber), proteins, mineral salts of phytic acid and vitamins. In order to increase the digestibility of the sound, it is advantageous to use the mushrooms as defined in the invention.

In an advantageous embodiment, the invention relates to the use defined above, wherein said by-product derived from plants is a rapeseed cake.

Advantageously, the invention relates to the use defined above, where said plants are rapeseed or sunflower, in particular rapeseed.

With more than 25 million tonnes per year, rapeseed meal production is second only to soybeans in world meal manufacturing. Rapeseed meal from the Brassica napus plant is the main by-product of biodiesel production in Europe.

Rapeseed meal has traditionally been used as an organic fertilizer. Its worldwide use as a feed for monogastric animals remains limited. Generally, oilcakes are a preferred protein source for livestock. Although new varieties low in glucosinolate and erucic acid have been developed, rapeseed meal as a food and protein source for animals remains unfavorable compared to soybean.

Indeed, rapeseed cake has a total protein content of 35% against 45% for soybean meal.

The total lysine concentration of rapeseed meal (1.8%) is 36% lower than that of soybean meal (2.8%) and the digestibility of lysine (75%) is also lower than that of soybean meal ( 91%) in swine. Rapeseed meal has more favorable total methionine values than soybean meal (0.70% vs. 0.64%) for a slightly lower digestibility (87% vs. 92%). The use of rapeseed cake therefore appears to be much less advantageous. that of soybean meal.

Thanks to the invention, the use of the aforementioned micromycetes makes it possible to valorize rapeseed cake and in particular to allow their enrichment in bio-available proteins or soluble proteins, amino acids (in particular lysine, methionine and arginine), and immunomodulatory substances.

In yet another advantageous embodiment, the invention relates to the above-mentioned use, wherein said by-product derived from plants is selected from: cakes, seeds, shells and pellicles of said strict oil-rich oleaginous plants. The by-product may be used alone, or optionally in admixture with at least one other plant by-product, or any other compound or edible substance.

In an advantageous embodiment, the invention relates to the use of at least one filamentous mushroom strain for the fermentative preparation of a fermented solid edible product intended for animal or human feed from a meal cake. rapeseed or sunflower,

said edible product comprising said strain of micromycetes, said strain of micromycetes is a strain of Aspergillus soya, or Aspergillus oryzae.

In another embodiment, the invention relates to the use according to the preceding definition, wherein the preparation of said edible solid fermented food product is obtained by fermentation in a solid medium.

The solid state fermentation process is defined as a culture of microorganisms (bacteria, fungi and the like) in a solid substrate without water flow. It involves exploiting the metabolism and the growth mechanism of these microorganisms and their metabolism on substrates to degrade the solid matter in order to produce substances with high added value. The microbial development is, on the surface and inside the solid matrix, in the absence of any liquid flow. The porous matrix may consist of a natural substrate or an inert carrier capable of absorbing nutrients that are dissolved in a solution.

An advantageous fermentation method in a solid medium is described below in the Examples section.

Advantageously, the invention relates to the aforementioned use, wherein said fermentation in solid medium is carried out under the conditions in which at least 1 .10 ⁷ lower fungus spores or micro fungi per gram of dry matter are inoculated with said by-product of plants, in particular about at least 1 .10 ⁸ lower mushroom spores or micromycetes per gram of dry matter are inoculated to said plant byproduct.

Advantageously, the invention relates to the above-mentioned use, wherein said solid-state fermentation is carried out under the conditions in which at least 1.10 ⁶ spores of micromycetes or filamentous fungi, in particular from 10 ⁶ to 10 ⁸ spores of micromycetes or filamentous fungus, especially about 10 ⁷ spores of micromycetes or filamentous fungi, per gram of dry matter are inoculated with the by-product of plants.

In one of its aspects, the invention relates to a method for producing a solid fermented product comprising a micromycete selected from Aspergillus oryzae and Aspergillus soya, said method comprising a fermentation step using said micromycete, a sub- plant product, as defined above, and in particular, a by-product derived from plants derived from a strict oleaginous fat-rich, advantageously a by-product from plants belonging to the family Brassicaceae or Asteraceae, in particularly a by-product of rapeseed or sunflower, and more particularly a rapeseed cake.

The invention also relates to a method for preparing a fermented solid food product for animal or human nutrition comprising:

a step of contacting at least one mushroom strain, said mushroom strain is a strain chosen from the following strains Aspergillus soya, Rhizopus oligosporus, Rhizopus oryzae and Aspergillus oryzae, in particular Aspergillus soya or Aspergillus oryzae, in particular Aspergillus spp. soybean, with a by-product derived from plants, and

a step of fermentation in a solid medium of said by-product derived from plants. Advantageously, the invention relates to the aforementioned method, said method comprising:

a step of contacting at least one strain of micromycetes, said strain of micromycetes, or filamentous fungus, is a strain chosen from the following strains Aspergillus soya, and Aspergillus oryzae, in particular Aspergillus soya, with a by-product. plant derived by-product derived from plants derived from a strict oilseed high in fat, and

- A fermentation step in a solid medium of said by-product from plants being derived from a strict oleaginous fat-rich.

Advantageously, the description relates to a process defined above, wherein said by-product derived from plants is chosen from: oilseed and proteinaceous seeds, oilseed and proteinaceous hulls and shells, oilseed meal, cereals, co-products of cereals (in particular starch products, distillery products, brewery products ...), products of the manufacture of sugar and fruit juices (beet, citrus fruits, grapes, sugar cane), or the But.

Advantageously, the invention relates to the aforementioned method, wherein said by-product derived from plants is derived from plants belonging to the family Brassicaceae or Asteraceae.

Advantageously, the invention relates to the aforementioned method, wherein said by-product derived from plants is chosen from: oilcakes, seeds, shells and pellicles of said strict oleaginous fat-rich. In an advantageous embodiment, the invention relates to a process as defined above, wherein said by-product derived from plants is a rapeseed cake.

In yet another advantageous embodiment, the invention relates to a method as defined above, further comprising a step of inactivation of said molds. The inactivation step mentioned above is advantageous because it makes it possible to prevent the multiplication of molds and thus to stabilize the food composition. The inactivation of the fungi can be done by conventional techniques well known to those skilled in the art such as irradiation, especially with UV or ionizing β-rays or gamma rays (at doses up to 10 kilogray), or a heat treatment by heating, in particular type thermisation (treatment at a temperature from 60 to 62 ^<C), authorization pasteu (treatment at a temperature of 62 to 88Ό) at or sterilization (trait ly at a temperature of more than ^' Ι ΟΟ'Ό), or by cryogenization by spraying liquid nitrogen.

In yet another advantageous embodiment, the invention relates to the aforementioned method which further comprises a drying step. The drying has the effect of stabilizing the molecules of interest produced or released during the fermentation. This step is carried out by any techniques known to those skilled in the art, in particular by drying in hot or cold air, by vacuum spraying or by lyophilization.

The invention also relates to a solid food product intended for animal feed, and comprising at least one strain of micromycete, said strain of micromycete is a strain selected from the following strains Aspergillus soya, Rhizopus oligosporus, Rhizopus oryzae and Aspergillus oryzae, in particular Aspergillus soya and Aspergillus oryzae, in particular Aspergillus soya, and a by-product derived from plants, the food product being obtainable by the process as defined above.

Advantageously, the invention relates to a solid food product intended for animal feed, and comprising at least one strain of micromycetes, said strain of micromycetes, or filamentous fungus, is a strain selected from the following strains Aspergillus soya and Aspergillus oryzae, including Aspergillus soybean, and a by-product derived from plants, said by-product from plants derived from a strict oleaginous high fat, the food product being obtainable by the method defined above.

Advantageously, the invention relates to a solid food product intended for animal feed, and comprising at least one strain of fungus, said fungus strain is a strain of Aspergillus soya, Aspergillus oryzae, Rhizopus oligosporus or Rhizopus oryzae, and a by-product derived from plants, the food product being capable of being obtained by the process as defined previously.

The aforementioned composition is new. It is characterized in that it comprises a variable amount of available bio proteins, that is to say directly assimilable by the animal, including β-glucans also bio available. It also includes the remains of mushrooms that were used for fermentation.

The composition further comprises, for 100 g of its plant by-product mass, approximately 9 grams of carbohydrates converted into biomass, that is to say about 9% of biomass, at most.

In yet another advantageous embodiment, the aforementioned food composition comprises said fungi in an inactivated form, that is to say in a form that no longer allows their growth or multiplication. Said fungi are notably killed, so that only the macromolecules composing the microorganisms remain in the composition, the various mechanisms allowing the multiplication and reproduction of the micromycetes being inhibited or inactivated.

One way of inactivating the fungi of the food product is to subject it to a treatment known to those skilled in the art, and in particular a step of irradiation and / or heat treatment, or the aforementioned means.

A drying step may also be performed to stabilize the molecules of interest. This step is carried out by any techniques known to those skilled in the art, in particular by drying in hot or cold air, by vacuum spraying or by lyophilization.

In one advantageous embodiment, the invention relates to a fermented solid food product, in particular according to the preceding definition, intended for animal feed comprising rapeseed meal and at least one strain chosen from the following strains Aspergillus soya, Rhizopus oligosporus, Rhizopus oryzae and Aspergillus oryzae, in particular Aspergillus sojae and Aspergillus oryzae, in particular Aspergillus sojae, said fermented solid food product having an immunomodulatory activity at least three times higher than said unfermented food product (NBT test on blood neutrophils).

Advantageously, the invention relates to a fermented solid food product, in particular the preceding definition, intended for human or animal food comprising

- rapeseed cake and

at least one strain of micromycetes or filamentous fungi chosen from the following strains: Aspergillus sojae and Aspergillus oryzae, in particular Aspergillus soya, said fermented solid food product having an immunomodulatory activity at least twice greater than that of said non-food product fermented.

Other in vitro tests on peripheral blood monocytes (PBMC) or bone marrow differentiated macrophages (BMDM) also show higher immunomodulatory activity than the unfermented product. According to the literature, this immunomodulatory activity can be linked to the presence of β-glucans which are an integral part of the biomass of certain microorganisms such as fungi.

Β-Glucan (beta-glucan) is a polysaccharide consisting entirely of beta-linked D-glucose. The bonds can be β (1 -3), β (1 -4) or β (1-6), the numbers indicating the D-glucose atoms involved. Β-glucans form a diverse group of molecules. They are found mainly in the cellulose of plants, the sound of cereals, the cell walls of yeasts, especially baker's yeasts, and certain fungi and bacteria.

It has been shown that β-glucans are factors that improve the activity of macrophages and neutrophils. These compounds increase resistance to gram-negative bacterial infections, and are agents with anticancer, antibacterial and antioxidant properties. In addition, the effect of β-glucans can be enhanced by α-mannan also linked to the cell wall of microorganisms, including fungi. Β-glucans are considered of nutritional interest as soluble fibers or texturizing agents, especially those derived from oats or barley. The nutritional value of β-glucans is therefore major.

The fermentation of by-products derived from plants by the aforementioned fungi allows enrichment of β-glucans because they are an integral part of the fungal biomass, they are therefore present in a greater amount than said unfermented products. One way to evaluate the immunomodulatory activity of the products is to use the NBT assay described in the examples.

Advantageously, the invention relates to a fermented solid food product according to the preceding definition, wherein said immunomodulatory product comprises at least one β-glucan.

The metabolites stimulating the immune system are advantageous for the treatment and prevention of certain diseases in case of immunodeficiency (allergies, cancers, autoimmune diseases, and gastrointestinal diseases) or in combination with an antibiotic-based therapy. These bioactive molecules have essentially been isolated from medicinal mushrooms commonly used in Asia. Production in a liquid medium is more effective in the context of macromycetes or higher fungi because the culture in solid medium to obtain the carpophore is a long process that requires one to several months. Growth in a liquid medium accelerates growth and allows harvest mycelium with bioactive substances in a few days. These substances (polysaccharides including β-D-glucans and α-mannans, proteins or peptides, triterpenes, lipids and phenols) mainly induce mitogenicity and activation of immune cells such as hematopoietic stem cells, lymphocytes, macrophages, dendritic cells. and killer cells.

Furthermore, the invention also relates to a food product as defined above, for use as a medicament, especially as a veterinary medicinal product or for human use, or as a neutraceutical.

The invention further relates to a method for preparing an immunomodulatory product comprising:

a step of contacting at least one strain of filamentous fungus, said strain of filamentous fungus is a strain chosen from the following strains Aspergillus soya, Rhizopus oligosporus, Rhizopus oryzae and Aspergillus oryzae, in particular Aspergillus soya and Aspergillus oryzae, in Aspergillus soya, with a by-product derived from plants,

a step of fermentation in a solid medium of said by-product derived from plants, and

a step of extraction of the immunomodulatory products resulting from the fermentation in the preceding step.

Advantageously, the invention relates to a method for preparing immunomodulatory products comprising:

a step of contacting at least one strain of micromycetes or filamentous fungi, said strain of micromycetes being a strain of Aspergillus soya or Aspergillus oryzae, with a by-product derived from plants, said by-product of plants derived from a strict oilseed rich in fat

a step of fermentation in a solid medium of said by-product originating from plants originating from a strict oleaginous plant rich in fat, and

Advantageously, the immunomodulatory products obtained by the method described above may be β-glucans. .

The extraction and drying of the β-glucans of the fermentation product can be carried out by techniques known to those skilled in the art, in particular based on membrane technology, on liquid-liquid extraction, on absorption, under empty or secondary techniques.

So this step can be done by

(i) pressing of the liquid followed by spray drying (ii) in an aqueous medium in the presence or absence of a buffer where the water represents 1 to 5 times the weight of the substrate followed drying by lyophilization.

In order to improve extraction yields, to concentrate and purify β-glucans, the food product (cake + fungus) can be treated by techniques known to those skilled in the art, in particular it can be washed, in particular twice, with distilled water and then dried at about 50 ^ penda nt about 8 hours and then taken up in four volumes of ethanol at 95Ό and incubated for 8 hours at 4 ^<C.

After centrifugation at 11 000 g at 4 ^<e C for bout 40 minutes, the precipitate is suspended in an equivalent volume of 75% ethanol and centrifuged again. The precipitate is then dried at 40 ° to remove residual ethanol (Shenbhagaraman et al., 2012, International Journal of Biological Macromolecules, 50, pp. 957-964).

Alternatively, the product can also be taken up in three volumes of 95% ethanol. After centrifugation at 11 000 g at 4 ^<C dur ing 10 minutes, the precipitate is dialyzed through cellulose membrane against distilled water for 48 h. The resulting fraction is lyophilized and the proteins of the fraction are removed by the Sevag method (Matthaei et al., 1962, PNAS, 48, pp. 666-677). The β-glucans are then fractionated by Q Sepharose Fast Flow column (Sigma Aldricht) and eluted with NaCl. The last purification step is carried out on Sepharyl S-400 column with NH ₄ CO ₃ as eluent before final lyophilization.

Another aspect of the invention also relates to food rations consisting essentially, or from 95% to 100%, of the food product as defined above, or to a food composition comprising from 0.1% to 95% of the food product such as as defined previously added to a conventional food according to the animal, and in particular minerals, nutritional supplements, dietetic foods, complementary foods, production concentrates, or nitrogen correctors.

In other words, the aforementioned composition can be used:

- either directly as an edible product for animals,

- as a supplement or food additive to be added to the conventional diets of the animal concerned.

In the context of human nutrition, it is possible to use the food composition according to the invention in the form of an additive. This additive can be in the form of oral or injectable solution, tablets, tablets, granules, capsules, or in any other galenic forms that the skilled person is capable of producing in this field. This additive may also be provided in combination with other compounds or additives, such as vitamins, sugars, amino acids, or any other additive well known to those skilled in the art.

As a food additive for human or animal nutrition, it is recommended to provide from 0.1% to 5% of the composition according to the invention in relation to the mass total of the food ration.

In another aspect, the invention relates to a method for feeding animals comprising providing in their diet a solid fermented food product as defined above.

This method consists in providing animals with food rations consisting essentially of 95% to 100% of the food product as defined above, or a food composition comprising from 0.1% to 95% of the food product. as defined above added to a conventional food according to the animal, and in particular minerals, nutritional supplements, dietetic foods, complementary foods, production concentrates, or nitrogen correctors.

For example, for a ruminant or a horse, the method will consist in providing the animal in addition to forage, either the food product as defined above, or in a mineral food, or in a bolus, or in a nutritional supplement, distributed in the state, either in a production like, or in a nitrogen corrector supplemented with 0.1% to 95% of the food product as defined above. For example, the production feed for dairy cows may consist of cereals, cereals, protein crops, seeds and oil cake, beet pulp, molasses, urea, minerals and vitamins. .

For monogastrics, dogs and cats, the method will consist in providing the animal with either the food product as defined above, or a complementary or complete food comprising from 0.1% to 95% of the food product as defined above. . For example the product may be incorporated into a piglet food consisting of cereals, from cereals, seeds and oil cake, dairy products, synthetic amino acids, minerals and vitamins.

These examples are purely illustrative and should not be interpreted as limiting the method of the invention.

In yet another aspect, the invention relates to a solid fermented product comprising

a filamentous fungus, said filamentous fungus is a strain chosen from the following strains Aspergillus soya, Rhizopus oligosporus, Rhizopus oryzae and Aspergillus oryzae, in particular Aspergillus soya, and

- a by-product of plants,

for its use in the prevention or treatment of disorders related to a decrease in the activity of the immune system, including disorders related to animal or human digestion. In an advantageous embodiment, the invention relates to a solid fermented product comprising

a micromycete, said micromycetes being Aspergillus soya or Aspergillus oryzae, and

a by-product derived from plants originating from a strict oleaginous plant rich in fat,

for its use for its use as a medicament or a neutraceutical, in particular in the context of the prevention or treatment of disorders related to a decrease in the activity of the immune system, in particular for the treatment and the prevention of certain diseases in the event of immunodeficiency such as allergies, cancers, autoimmune diseases, and gastrointestinal or digestive diseases.

As mentioned above, metabolites stimulating the immune system, and especially β-glucans, are advantageous for the treatment and prevention of certain diseases in the case of immunodeficiency, such as allergies, cancers, autoimmune diseases, immune, and gastrointestinal diseases.

A food composition comprising a solid fermented product as defined above, because of the presence, inter alia, of bio available β-glucans therefore makes it possible to treat and especially to prevent the gastrointestinal problems encountered by the animals during treatment. provision of food rations from by-products of plants.

Also, in another embodiment, the invention relates to a solid fermented product comprising

- a by-product of plants,

for its use in order to modulate the immune status of animals, thereby increasing the resistance of animals to diseases, including bacterial or viral infectious diseases.

In yet another embodiment, the invention relates to a solid fermented product comprising

- a by-product of plants,

for its use in stimulating the immunity of young animals, particularly through the nutrition of breeders. In this aspect of the invention, the breeding adults consuming the composition according to the invention, defined above, are capable of transmitting, in particular via the mother's milk, the compounds resulting from the activation of the immune system by the compounds immunomodulators, particularly by β-glucans, resulting from the fermentation according to the aforementioned method, and thus stimulate the immune system of young animals.

- a by-product of plants,

for its use in stimulating the viability, health status and growth performance of young animals by stimulating immunity.

- a by-product of plants,

for use in modulating human immune status, thereby increasing disease resistance or treating immune disorders.

- a by-product of plants,

for its use in order to modulate the human digestive status, thereby reducing digestive disorders.

Advantageously, the invention relates to a solid fermented product mentioned above for its aforementioned use, wherein said by-product derived from plants is derived from plants belonging to the family Brassicaceae or Asteraceae.

Advantageously, the invention relates to a fermented product for its aforementioned use, wherein said by-product derived from plants is chosen from: the cakes, seeds, shells and films of said strict oil-rich oleaginous plants.

Advantageously, the invention relates to a solid fermented product for its use the preceding definition, wherein said by-product from plants is selected from: oilseeds and protein crops, oilseed and proteinaceous hulls and shells oilseed cakes, cereals, cereal co-products ( especially products of starch, distillery, brewery ...), the products of the manufacture of sugar and fruit juices (beet, citrus, grapes, sugar cane), or corn.

In another advantageous embodiment, the invention relates to a solid fermented product for its above-mentioned use, wherein said by-product derived from plants is a rapeseed meal.

In the invention which is described above, the man is included in the concept of "animals".

The invention thus relates to compositions and uses that are transposable to humans. The invention will be better understood with the aid of the following figures and examples:

Legend of figures:

FIG. 1 represents a graph showing the evolution of the percentage of CO ₂ of mushroom cultures on rapeseed meal over time. The x-axis represents the time in days and the y-axis represents the percentage of C0 ₂ produced. The strains tested are as follows: 1 = Aspergillus niger; 2 to 7 = Aspergillus sp. ; 8 = Aspergillus oryzae; Aspergillus soyae; Rhizopus oryzae; 11 = Rhizopus oligosporus; 12 = Penicillium brasilianum; 28 = Trichoderma harzianum; 42 = Rhizopus sp. ; T = cake alone (control).

FIG. 2 represents a graph showing the evolution of the loss of dry mass of mushroom crops on rapeseed cake. The x-axis represents the time in days and the y-axis represents the percentage of dry weight loss of the rapeseed cake. The strains tested are as follows: 1 = Aspergillus niger; 2 to 7 = Aspergillus sp. ; 8 = Aspergillus oryzae; Aspergillus soyae; Rhizopus oryzae; 11 = Rhizopus oligosporus; 12 = Penicillium brasilianum; 28 = Trichoderma harzianum; 42 = Rhizopus sp.

Figure 3 is a histogram showing the percentage of activated neutrophils in A = blood alone, B = blood with bacteria from the kit, C = blood with cake alone, D = blood with a sample of cake fermented with Aspergillus oryzae, E = blood with a sample of cake fermented with Aspergillus soya, F = blood with a sample of meal fermented with Rhizopus oryzae and G = blood with a sample of meal fermented with Rhizopus oligosporus,

FIG. 4 represents a graph showing the percentage of β-glucan in rapeseed meal alone (A), or Rhizopus oryzae mycelium (B), mycelium Aspergillus soya (C), Aspergillus oryzae mycelium (D) or Rhizopus oligosporus mycelium (E). In control is shown the use of β-glucan yeast (F).

Figures 5A to 5L show photographs of cell pellets of blood cells (PBMC).

Figure 5A shows a cell pellet of cells treated with the medium alone.

FIG. 5B represents a cell pellet of cells treated with medium supplemented with 10 ng / ml of LPS.

FIG. 5C represents a cell pellet of cells treated with the medium supplemented with 5 μl of ZVAD.

FIG. 5D represents a cell pellet of cells treated with medium supplemented with 100 μl of prednisolone.

FIG. 5E represents a cell pellet of cells treated with sample A1 (soluble fraction of Aspergillus soya mycelium) at a concentration of 0.125 mg / ml. FIG. 5F represents a cell pellet of cells treated with sample A1 (soluble fraction of Aspergillus soya mycelium) at a concentration of 0.25 mg / ml. Figure 5G shows a cell pellet of cells treated with sample A1 (Aspergillus soyae mycelium soluble fraction) at a concentration of 0.5 mg / ml.

FIG. 5H represents a cell pellet of cells treated with sample A1 (soluble fraction of Aspergillus soyae mycelium) at a concentration of 1 mg / ml. Figure 51 represents a cell pellet of cells treated with sample A2 (soluble fraction of a cake culture with Aspergillus soya) at a concentration of 0.125 mg / mL.

FIG. 5J represents a cell pellet of cells treated with sample A2 (soluble fraction of a cake culture with Aspergillus soya) at a concentration of 0.25 mg / ml.

FIG. 5K represents a cell pellet of cells treated with sample A2 (soluble fraction of a cake culture with Aspergillus soya) at a concentration of 0.5 mg / ml.

FIG. 5L represents a cell pellet of cells treated with sample A2 (soluble fraction of a cake culture with Aspergillus soya) at a concentration of 1 mg / ml.

FIG. 6 represents a histogram showing the percentage of cell viability of blood cells (PBMC) treated with A = the culture medium, B = the culture medium supplemented with 10 ng / ml of LPS, C = the culture medium supplemented with 10 ng / ml of LPS and 5 μl of ZVAD, and D = the culture medium supplemented with 10 ng / ml of LPS and 100 μl of prednisolone.

FIG. 7 is a curve representing the cell viability of the blood cells as a function of the doses of A1 or A2 sample supplemented with 10 ng / ml of LPS. The axis abscissa represents the concentration of extract expressed in mg / ml. The curves with white or black triangles respectively represent the viability results obtained with the samples A2 and A1. The curve with a white circle represents the viability of the blood cells treated with an extract of cake having fermented with a macromycete (Trametes versicolor).

FIG. 8 is a histogram showing the concentration of interleukin IL-1 β secreted in the cell medium by A = the culture medium alone, B = the culture medium supplemented with 10 ng / ml of LPS, C = the medium culture medium supplemented with 10 ng / ml of LPS and 5 μl of ZVAD, and D = the culture medium supplemented with 10 ng / ml of LPS and 100 μl of prednisolone. The ordinate axis represents the IL-1 β concentration expressed in pg / mL. ^*** : significant difference determined by the statistical analysis of variance analysis (ANOVA)

FIG. 9 is a histogram showing the concentration of interleukin IL-1β secreted in the cell medium by PBMCs in the culture medium alone and the culture medium supplemented with 10 μg / ml of LPS (A) or supplemented with 10 ng / mL of LPS and different concentrations of the cake sample alone (T): (B): 7 μg / mL, (C): 15 μg / mL, (D): 31, 2 μg / mL, (E) ): 62.5 μg / mL, (F): 125 μg / mL and (G): 250 μg / mL The y-axis represents the concentration of IL-1β expressed in μg / mL.

FIGS. 10A and B are histograms showing the concentration of interleukin I L-1β secreted into the cell medium by PBMCs in the culture medium alone and by the culture medium supplemented with 10 ng / mL of LPS and different concentrations of A1 and A2 products.

FIG. 10A is a histogram showing the concentration of interleukin IL-1β secreted in the cell medium by PBMCs in the culture medium alone (A) and by the culture medium supplemented with 10 ng / mL of LPS and different concentrations ((B): 0.125 mg / mL, (C): 0.25 mg / mL, (D): 0.5 mg / mL and (E): 1 mg / mL) of A1 product. The ordinate axis represents the IL-1 β concentration expressed in mg / mL.

FIG. 10B is a histogram showing the concentration of interleukin I L-1β secreted in the cell medium by PBMCs in the culture medium alone (A) and by the culture medium supplemented with 10 ng / ml of LPS and different concentrations ((B): 0.125 mg / mL, (C): 0.25 mg / mL, (D): 0.5 mg / mL and (E): 1 mg / mL) of A2 product. The ordinate axis represents the IL-1 β concentration expressed in mg / mL.

FIG. 11 is a histogram showing the concentration of interleukin IL-1β secreted in the cell medium by BMDMs in:

A = the culture medium alone,

B = the culture medium supplemented with 1 μg / ml of LPS,

C = the culture medium supplemented with 1 μg / ml of LPS and 5 μl of ZVAD,

D = the culture medium supplemented with 1 μg / ml of LPS and 70 μl of prednisolone and E = the culture medium supplemented with 1 LPS and 1 μl of DEXA.

The ordinate axis represents the IL-1 β concentration expressed in pg / mL.

FIG. 12 is a histogram showing the concentration of interleukin I L-1β secreted in the cell medium by BMDMs in:

A = the culture medium supplemented with LPS,

B = the culture medium supplemented with LPS and 1 μg / ml of PC product (commercial product rich in β-glucan yeast),

C = the culture medium supplemented with LPS and 10 μg / ml of PC product,

D = the culture medium supplemented with LPS and 100 μg / ml of PC product.

The ordinate axis represents the IL-1 β concentration expressed in pg / mL.

FIG. 13 is a histogram showing the concentration of interleukin I L-1β secreted in the cell medium by BMDMs in:

A = the culture medium supplemented with LPS,

B = the culture medium supplemented with LPS and 1 μg / mL of cake alone,

C = the culture medium supplemented with LPS and 10 μg / mL of cake alone,

D = the culture medium supplemented with LPS and 100 μg / mL of cake alone.

The ordinate axis represents the IL-1 β concentration expressed in pg / mL.

FIG. 14 is a histogram showing the concentration of interleukin I L-1β secreted in the cell medium by BMDMs in:

A = the culture medium supplemented with LPS,

B = the culture medium supplemented with LPS and 1 μg / mL of culture cake (48h) with Aspergillus soyae produced in vats,

C = the culture medium supplemented with LPS and 10 μg / mL of cake culture (48h) with Aspergillus soyae produced in vats,

D = the culture medium supplemented with LPS and 100 μg / mL of culture on cake (48h) with Aspergillus soyae produced in vats.

The ordinate axis represents the IL-1 β concentration expressed in pg / mL.

EXAMPLES

Example 1 - Culture Processes in Solid Medium

I. Establishment of a Micrometek Work Bank

Slant agar plates (including PDA medium, potato dextrose agar) are used to maintain pure strains and are regularly maintained by transplanting.

For sporulating strains, the fungus spores are harvested by scraping the surface of the agar with sterile water. The harvested spores are then stored in -80% glycerol.

The PDA medium is prepared as follows:

The potato infusion is prepared by boiling 300 g of sliced potatoes (washed but not peeled) for 30 minutes and then decanting the resulting broth or filtering through a cheesecloth. It is then diluted by adding distilled water for a final volume of one liter. Then 20 g of dextrose and as many agar-agar powder are added before sterilization by autoclave at 100 kPa for 15 minutes.

II. Culture in non-aerated conditions: Erlenmever vial

Inoculation and substrate preparation

In order to be able to have enough spores for the experiments, the strains are subcultured in a Roux vial also containing agar medium. These are then incubated for 4 days at 30 ° C.

The cultures are performed in 500 mL wide-mouth Erlenmeyer flasks. Before inoculation with the fungus, the solid medium is autoclaved 20 min at 121 ^<C. The medium is adjusted to between 40 and 50% dry matter with sterile water containing sporespour obtain an inoculation rate of 1 x 10 ⁷ spores / g sec. Air diffusion is very limited through the cotton stopper

Culture monitoring

Throughout the duration of the cultures, measurements of C0 ₂ are made in order to follow the mycelial growth. Macroscopic and microscopic observations are made regularly to visualize the appearance of biomass. At the end of growth, the entire contents of a flask are homogenized and recovered for drying in an oven and for measuring the percentage of dry matter and pH. The product, once dried, is stored at room temperature awaiting tests for determining the immunomodulatory activity.

III. Culture in aerated condition: Vats

Inoculation and substrate preparation

The cultures are carried out in tanks of maximum capacity 50L. They are fed in parallel with air which can be adjusted temperature, relative humidity and flow. This air crosses from bottom to top the layer of culture. The reactors are sterilized with live steam for 3 hours. Oilseed substrate is autoclaved (1 h at 121 ^<C). The oleaginous substrate is then inoculated with a spore suspension to obtain a seeding rate of 1 x 10 ⁷ spores / g dry and its final dry matter percentage is adjusted between 40 and 50%. The starting instructions are: temperature 28 °, humidity 95% and air flow between 7 and 12m / s.

Culture monitoring

At regular time intervals temperature measurements are made in the outlet air and at different points in the culture mass. Regular samples are taken for the drying of culture samples and the dosage of immunomodulatory activity.

Example 2 - Cultivation of mushrooms on rapeseed cake

Fourteen mold strains were grown on rapeseed cake. The evolution of the percentage of C0 ₂ and of the loss of dry mass was determined for all the strains in order to be able to compare their growth.

strains

The 14 strains are Aspergillus niger (isolated from cereals), Aspergillus sp. (6 strains isolated from soil), Aspergillus soya CBS 100934 and Aspergillus oryzae UMIP 1042.72, Rhizopus oryzae CBS 131512, Rhizopus oligosporus CBS 338.62, Penicillium brasilianum (isolated from soil), Trichoderma harzianum (isolated from soil) and Rhizopus sp. (isolated from a food). The strains are stored in the working bank in the form of an agar plate inclined with PDA medium at 25 ° C. and in a frozen bank in the form of a suspension of spores in glycerol at -80 °.

Inoculation procedure

The inoculum, in the form of a spore suspension, was obtained from Roux vine precultures containing PDA agar medium. These are incubated for 4 days at 30%. A suspension of the order of 1 ^* 10 ⁸ spores / ml according to the strain is obtained following scratching of the surface of the agar.

Culture conditions

The strains are grown on rapeseed meal in an unventilated condition (Erlenmeyer flask). The amount of rapeseed meal is adapted to obtain a wet layer height of about 2 cm. The percentage of dry matter is adjusted between 40% and 50% with sterile water containing the spores. Four vials per strain are thus produced in order to perform kinetic measurements requiring the entire sacrifice of the culture (1 sample per day). All the flasks are incubated at 25 ° C.

Analytical methods

The evolution of the percentage of C0 ₂ is measured by means of a gas analyzer and the percentage of dry matter using the drying method in an oven for 24 hours at the ^" OS". mass loss over time taking into account the dry weight of the culture at the initial time Once dried these culture samples are used for the determination of immunomodulatory activity with the NBT kit (Sigma). Both culture samples and the control cake alone are crushed to a fine powder (<0.5 mm) using a mortar and stored at room temperature in the dark, which are then incorporated into human blood to obtain a final concentration of about 8 mg / mL A total of 100 neutrophils are counted three times by blood smear for more precision.

Results Determination of the parameters related to growth: CO?

The percentage of C0 ₂ increases after seeding for all the strains studied. There is thus germination of spores, growth of microorganisms and consumption of the substrate (Figure 1).

Strain number 8 (Aspergillus sp.) With a peak at 9% CO ₂ after one day of culture is ahead of all other strains. The evolution is globally comparable between the other strains of fungi. On the third day of cultivation, a slowing down of growth linked to the depletion of available nutrients occurs. The absence of C0 ₂ production for the "meal-only" control (T) confirms the absence of contaminants.

Determination of growth-related parameters: loss of dry mass

The loss of dry mass represents the loss of mass of the substrate in the form of CO ₂ and / or other volatile compounds. The loss of mass of the meal grown with the 14 strains is on average between 15 and 25% at the end of culture (Figure 2). This is in agreement with the evolution of the percentage of C0 ₂ , in fact the lowest performing strain (n ° 11) has the lowest dry mass loss over time. The other strains remain quite close as for the evolution of C0 ₂ .

All of these results show that the 14 strains of mold are able to grow on the substrate rapeseed meal.

Example 3 In Vitro Study of the Immunomodulatory Effect of Solid Fermented Products According to the Invention (NBT Test)

To study the immune response with rapeseed cake cultures, the inventors selected, from among the remaining strains, only those that are used in human nutrition, namely fungi used for the production of Asian fermented products. All other strains of fungi are thus removed from the screen because they could present a potential risk for animal health (presence of possible mycotoxins). We therefore have a total of 4 strains of fungi namely Aspergillus oryzae, Aspergillus soya, Rhizopus oryzae and Rhizopus oligosporus.

Immunomodulatory activity is evaluated with a NBT kit (NitroBlue Tetrazolium; Sigma). The method has been diverted from its primary use, ie, in vitro diagnosis for the identification of neutrophil dysfunction or pyrogenic infection in humans. NBT is a salt of tetrazolium, a small molecule that easily penetrates inside white blood cells. In the presence of a stimulating agent (bacteria, toxins, latex particles and glass, yeast membrane protein etc.) the white blood cells are activated and generate reactive oxygen derivatives leading to the reduction of NBT which precipitates then in the cell in the form of formazan (blue-black grains). A microscopic examination then allows determine the percentage of activated white blood cells, ie the cells presenting the insoluble intracytoplasmic deposits of formazan.

A heparinized blood sample is incubated with a NBT buffered solution in the presence of culture samples, cake alone or stimulants (deactivated bacteria) provided in the NBT Sigma Kit.

Preparation of a culture extract for incorporation into the blood

Only 5 μl of culture extract can be incorporated into the blood for the stimulated condition of the NBT test. In order to get stimulation of the white blood cells, the extract must be quite concentrated as the bacterial stimulant provided with the kit. To do this, 4 g wet of a sample from a culture on rapeseed cake are milled with ultraturax with a minimum of osmosis water (15 ml). A volume of 5 μΙ, of pasty consistency, is then taken using a cone whose end has been cut to allow the passage of a few culture fragments in the blood. In order to have a maximum of biomass and thus of immunomodulatory molecules, the choice of sampling times for the analysis is established taking into account the evolution of the percentage of C0 ₂ over time as well as the sporulation, for strains producing spores. Regarding micromycetes the 2nd or 3rd day of culture is chosen according to the strain. During these kinetic times, the 4 strains have not yet begun to sporulate but have however produced a lot of biomass since the C0 ₂ peak has passed.

Results

The evolution of the percentage of C0 ₂ and the loss of dry mass makes it possible to discriminate these four strains in terms of growth. A. soya and A. oryzae are the most successful strains on rapeseed meal. The percentage of C0 ₂ is higher and is maintained over a longer period compared to the two strains of Rhizopus. This shows a metabolism better adapted to the culture medium including the synthesis of enzymes to further degrade the substrate to maintain growth after depletion of available nutrients.

Neutrophils are counted under the microscope.

The choice of the kinetic time for the immune response is the 3rd day because the microorganisms have not yet begun to sporulate but they have produced a maximum of biomass since the C0 ₂ peaks are passed. The results in Figure 3 show that only cultures with A. oryzae and A. sojae have immunostimulatory potential for activation of blood neutrophils. The response with the two strains of Rhizopus is equivalent to the meal alone which induces a stimulation of 5,67%.

Of these 4 strains, A. sojae and A. oryzae have a higher immunomodulatory potential important since the β-glucan content of their biomass or mycelium alone is higher compared to Rhizopus. The immune response assayed by the NBT test (Figure 3) is correlated with the amount of β-glucan present in the biomass or mycelium of the filamentous fungi (Figure 4).

EXAMPLE 4 In Vitro Studies of Several Products on Human Blood Mononuclear Cells (PBMC): Investigation of Cytotoxic and Immunomodulatory Effects

The effect of three products Aspergillus (T: cake alone, A1: mycelium alone of A sojae and A2: cake culture with A. sojae was evaluated on cell toxicity and production of IL-1β by cells blood-borne mononuclear cells (PBMCs) stimulated by endotoxin (lipopolysaccharide; LPS).

LPS, used at a non-cytotoxic concentration of 10 ng / mL overnight, induces IL-1 β production by PBMCs.

The IL-1β production inhibition tests carried out in this study were validated by the use of two internal anti-inflammatory references: the caspase inhibitor and the NLRP3 inflammasome inhibitor (ZVAD). and a corticosteroid (PRED, prednisolone). Added to cell cultures at the same time as LPS, these 2 molecules significantly inhibit the production of IL-1β by more than 50%.

Protocol:

From a pocket of human buffy coat (provided by the French Blood Establishment), the separation of blood mononuclear cells (lymphocytes + monocytes or PBMC) from healthy donors is obtained by Ficoll centrifugation with a density of 1.077 g / mL. The opalescent ring containing the mononuclear cells is recovered carefully. The cells are washed three times with phosphate buffered saline (PBS) by centrifugation at 4 ^<C. After counting of viable cells are, 1 × 10 ⁵ cells are seeded in each well of 96-well round bottom in RPMI + 10% fetal calf serum (FCS) + 1% glutamine + 1% PSA.

Treatment of the cells with T, A1 and A2 products or internal anti-inflammatory references was performed at seeding as well as stimulation with endotoxin (Lipopolysaccharides extracted from Escherichia coli, LPS 0128: B12). The soluble fraction of the products supplied (A1, A2) was tested at 4 concentrations 1 - 0.5 - 0.25 - 0.125 mg / mL (stock solutions prepared extemporaneously in culture medium at the initial concentration of 17 mg / mL). The product T has been tested at the following concentrations: 7 - 15 - 31 .2 - 62.5 - 125 - 250 μg / mL.

Two internal anti-inflammatory references were tested: ZVAD (caspase inhibitor and inhibitor of the inflammasome NLRP3) at the concentration of 5μΜ and PRED (Prednisolone, corticosteroid) at the concentration of 100μΜ. LPS was used at a concentration of 10 ng / mL. The cell supernatants were recovered the next day (17:30 post-LPS) and then frozen at -20 ° in order to subsequently perform the I L-1 β assay. A cell viability test using XTT was performed on the cell mat. A photographic report was made after 17:30 of treatment using an inverted phase contrast optical microscope equipped with a camera (Nikon TMS).

Principle of the cell viability test: The XTT, derivative of tetrazolium, makes it possible to measure the metabolic activity of living cells in culture. XTT is reduced by the mitochondrial or extra-mitochondrial dehydrogenases of active living cells to an orange compound highly soluble in water. The amount of reduced compound formed is proportional to the amount of metabolically active cells and can be quantified by measuring the absorbance at a wavelength between 450-500 nm (490nm) and a counter reading between 630 - 690 nm (660 nm). nm). The equipment used for this measurement is the Infinity 200M® Pro (Tecan company). The percentage of cell viability is determined by the ratio to the solvent control considered to be 100% cell viability. The results are expressed in% of cell viability with respect to the corresponding control (culture medium) and expressed as an average ± standard deviation from the mean.

Principle of the IL-W assay: IL-Ι β is assayed by ELISA sandwich immunoassay. The wells of a 96-well plate are coated with a capture antibody. After addition of the cell culture supernatant, the antigen (IL-1β) will bind to the capture antibody. A detection antibody is added and binds to the antigen. A secondary antibody bound to an avidin-HRP enzyme is added and binds to the detection antibody. The substrate is added and converted by the enzyme into a quantifiable form by measuring the absorbance at 450 nm. The equipment used for this measurement is the Infinity 200M® Pro (Tecan company).

Results

1 - Aspects of cell pellets after 17:30 of contact of PBMCs stimulated with LPS with A1 and A2 products:

PBMCs, composed of monocytes and lymphocytes, are cells in suspension. The cells are visualized in the form of a cell pellet since round-bottomed plates have been used in order to facilitate the grouping of the cells and thus preserve their proliferative capacities. Treatment of the cells may change the appearance of the cell pellet (Figures 5). The increase or decrease in the size of the pellet indicates proliferation-inducing or cytotoxic effects, the complete dissociation of the cell pellet can also be observed, as is the case in particular with the product A1, indicating a modification of the cellular interactions.

2- Cell viability: After 17:30 of incubation, LPS 0128: B12, used at the concentration of 10 ng / mL induces no cytotoxicity compared to the control (culture medium). The addition of 5μΜ of ZVAD or 100 μΜ of PRED does not statistically modify the viability of PBMCs. The results are shown in Figure 6.

As shown in FIG. 7, the A1 product tested at a maximum concentration of 1 mg / ml, shows no major cytotoxicity, just like a negative control. The product A2 induces a cytotoxicity of about 40% from the lowest concentration tested, ie 0.125 mg / ml. However, it is noted that this cytotoxicity does not increase with concentration. An interaction with the XTT test may be considered.

3- IL-1 beta assay:

After 17h30 of incubation, the stimulation with LPS, used at the concentration of 10 ng / mL, induced a production of IL-1 β by the PBMCs (1357 ± 152 pg / mL) in comparison with the basal level in the presence Control Culture medium (4 ± 2 μg / mL).

In the presence of LPS, ZVAD (5 μΜ) or PRED (100 μΜ) induces a significant 56% decrease in IL-1β production by stimulated PBMCs.

These results are shown in Figure 8.

In the presence of LPS, the two products A1 and A2 are capable of significantly reducing the IL-1 β production by stimulated PBMCs compared to the meal alone. These results are shown in Figures 9 and 10.

These results demonstrate that in the presence of LPS, the two products used at different concentrations are capable of significantly inhibiting IL-1β production by LPS-stimulated PBMCs and in a dose-dependent manner. For the meal alone the values remain at the same level as the cell medium alone, so there is no immunomodulatory effect with the unfermented product.

Example 5 - In vitro studies of several products on mouse bone marrow cells differentiated into mature macrophages (BMDM): Search for cytotoxic and immunomodulatory effects

The effect of three products (PC: commercial product rich in yeast β-glucan (40%) used in animal feed, T: cake alone and C48: culture produced in vats on cake with A. soya) was evaluated on the cellular toxicity and IL-1β production by mature mouse macrophages stimulated by endotoxin (lipopolysaccharide; LPS). The initial approach was aimed at verifying the presence of a pharmacological activity in the soluble part of the products tested without toxicity for the most suitable cell culture model: PBMCs. However, since the active principle is considered insoluble (β-glucan), the presence of a pharmacological activity has been carried out on the complete product including the insoluble particles. To do this another cellular model has been tested, namely macrophages, since it is more suitable for the presence of insoluble particles, the cells in particular have the ability to phagocyte the particles.

LPS, used at a non-cytotoxic concentration of 1 μg mL for 24 hours, induces macrophage proliferation and IL-1β production.

Protocol:

The bone marrow cells of mice are isolated and removed from healthy mice before being cultured for 5 days for macrophage differentiation in the presence of a cytokine mMCSF at 50 ng / mL. One night after inoculation of BMDM in 96-well plate, the cells are treated with complete products or internal anti-inflammatory references. BMDM stimulation by endotoxin (Lipopolysaccharides extracted from Escherichia coli, LPS 0128: B12, μg / mL) was performed 24h after treatment. After one night of stimulation, the cell supernatants were recovered and then frozen at -20 ° in order to subsequently carry out the IL-Ι β assay. A cell viability test using XTT was performed on the cell mat. The principles of the XTT test and the IL-1β assay are identical to those presented in Example 4 for PBMCs.

The complete products were tested at different concentrations: 1 - 10 - 100 μg / mL. The stock solutions of the products were prepared extemporaneously in culture medium. Three internal anti-inflammatory references were tested: ZVAD (caspase inhibitor and inflammasome inhibitor NLRP3) at the concentration of 5 μΜ; PRED (Prednisolone, corticosteroid) at a concentration of 7 ΟμΜ and DEXA (Dexamethasone, corticosteroid) at a concentration of 1 μΜ. LPS was used at a concentration of 1 μg / mL.

Results

1 - Cell viability:

The complete products, used alone, show no toxicity on BMDM at 1000 μg / mL, except PC (responsible for a decrease in cell viability of 60%). In the presence of LPS, the 3 products induce a cell proliferation of about 70% as for LPS alone compared to the control medium.

The addition of 5 μl of ZVAD, 70 μl of PRED or 1 μl of DEXA in the presence of LPS does not statistically modify the viability of BMDMs and indeed induce cell proliferation.

2 - IL-1 beta assay:

LPS, used at a non-cytotoxic concentration of 1 μg / mL overnight is inducing the production of IL-1β by BMDM. In the presence of LPS, the anti-inflammatory internal reference products (ZVAD, PRED and DEXA) reduce the IL-1β production induced by the stimulated BMDMs. A decrease of more than 75% is also obtained with PRED and DEXA. All of these data validate the experiment. These results are presented in Figure 11.

In the presence of LPS, unlike the inactive product T, the PC and C48 products significantly increase IL-1β production by the stimulated BMDMs and in a dose-dependent manner. The immunomodulatory activity with the culture sample (C48) appears to be greater than that observed with the commercial product (PC), with a difference of 10 μg / mL for the production of IL-1β at the same tested concentration of 100 μg / mL in the presence of BMDM. The immune response is more than doubled with this cell model for the fermented product (C48) compared to the unfermented product (cake alone: T) or cell medium alone with only LPS.

These results are shown in Figures 12, 13 and 14.

Example 6 - Plant by-products comprising at least 1.5% fat.

The following table gives examples of plants, and their by-products, usable in the context of the invention.

Raw materials MG rate

WHEATFEED 2

SOLUBLES OF STARCHING 3,5

NON-DETOXIED PEANUT OIL MELT 2

PEANUT SEED 48.5

OATS NOW 8.3

CORNFEED BNA MP COEF 3.1

OAT PICK 2.1

COOK OF COCOA 5

LINK COVER 5

TOURTEAUX COLZA DESH. HP 2.05

COAT OF COPRAH DESHUILE 2

SUNFLOWER HULL 3,6

PAT 50-55% MAT DEGRAISSE (MG <15) 6.8

DRECHE DE BLE 3,7

DRECHE of MAIS 9.5

DRECHE D ORGE 7

BONE AND MEAT POWDER 10,81

FLOUR OF TENDERED WHEAT 1, 5

LOW HARD FLOUR 2.7

BULGOUR of DURUM WHEAT 4

FEVEROLE TANNINS + (> 0.3%) 1, 6

FIRST FLOUR OF MAIZE 2

FLOUR SECOND OF MAIZE 5.5

SHRIMP FLOUR 4,96

TUNA FLOUR 9

FISH FLOUR 18

POULTRY PAT 13.5

GRAIN OF GRANULATED COLZA 43.5

FLOUR COLZA SEED 43.5

COTTON SEED 20 Raw materials MG rate

BROWNED RAW LINED SEED 30.9

GLUTEN OF WHEAT 1, 6

GLUTEN OF MAIZE 60 2.5

GERME MAIS SEMOULERIE 20% MF 22

EXTRUDED SOUS EXAMPLE 19.7

SUNFLOWER SEED 40.4

FISH OIL 99

ACID PALM OIL 97

RICE SOUND OIL 97.5

SUNFLOWER OIL ACID 98

LINEN OIL 98.4

PALM OIL 99

OIL OF COPRAH 99

FREE FATTY ACIDS 99

SUNFLOWER OIL 99.1

PEANUT OIL 99.1

SOY OIL 99.5

POND OIL 99.5

MAIZE OIL 99.5

FROM CEREALS CELULLOSE 1, 5

LACTO SWEET 1, 5

ACUTE LACTOSERUM 2.5

CASEIN 1, 5

PROTEIN CONCENTRATE OF LACTOSERUM 2

WHOLE MILK - DAIRY PRODUCTS 26

YEAST TORULA 2

SWEETS LINES DESHUILE 5

LUPINE BLUE TOASTE 5.3

LUPINE BLUE 5.3

WHITE LUPINE 7,5

LUZERNE RABBIT HORSE MATT 17% 1, 9

MAIS 3,5

MARC OF PEAR WITH PECTIN 1, 5

APPLE MARC DEPECTINIZED 4

SOY MIX 3.2

MILLET 4.3

MUSTARD SOUND 19.4

BARLEY 1, 7

STRAW 1, 71

PALMISTE DESHUILE TOURTEAU 2

FAT FLOUR DEGREASES 7

GRAPE PULP 5

COCENTRE PROTEIC DE LUZERNE (PX) 9.3

WHITE TURN WHITE REPLACEMENT 2.8

REMOULAGE HUE BLUE 3,4

2.5 PORK PLASMA

HARD WHEAT Granulated 2,555

SOYA SWEETS 48 BRAZIL 1, 7

TURKEY FLOUR SOUND 3.1

SORGHOS TANNINS = 0.4 2.8 Raw materials MG rate

SOUND OF RICE DESHUILE 3.1

SOUND OF RICE GRAS 19

COTTON BATH 7

GERMY BUTTERS BUT DESHUILE 5

OLEIC LP SUNFLOWER (MG <5 MAT <29) 1, 5

PEPINS OF GRAPE 12

ARGAN EXPELLER 10.9

EXTRUDED FLAXSEED 38.5: fat

Claims

1. Use of at least one strain of filamentous fungus for the fermentative preparation of a fermented solid edible product for animal or human feed from a by-product of plants,

said by-product derived from plants being derived from a strict oil-rich oleaginous,

said edible product comprising said strain of micromycetes,

said strain of micromycetes is a strain of Aspergillus soya, or Aspergillus oryzae.

2. Use according to claim 1, wherein said by-product derived from plants is derived from plants belonging to the family Brassicaceae or Asteraceae.

3. Use according to claim 1 or claim 2, wherein said plants are rapeseed or sunflower, in particular rapeseed.

4. Use according to any one of claims 1 to 3, wherein said by-product derived from plants is selected from: cakes, seeds, shells and pellicles of said strict oil-rich oleaginous.

5. Use according to any one of claims 1 to 4, wherein the preparation of said edible solid fermented food product is obtained by fermentation in solid medium.

6. Use according to claim 5, wherein said fermentation in solid medium is carried out under conditions where at least 1 .10 ⁶ spores of filamentous fungus including 10 ⁶ to 10 ⁸ spores of filamentous mushroom per gram of dry matter are inoculated to said sub -product of plants.

A process for preparing a fermented solid food product for animal nutrition comprising:

a step of contacting at least one strain of filamentous fungus, said strain of micromycetes is a strain chosen from the following strains Aspergillus sojae, and Aspergillus oryzae, in particular Aspergillus sojae, with a by-product derived from plants under -produced plant derived from a strict oilseed rich in fat, and

a step of fermentation in a solid medium of said by-product derived from plants by-product derived from plants derived from a strict oilseed rich in fat.

8. The method of claim 6, wherein said by-product derived from plants is derived from plants belonging to the family Brassicaceae or Asteraceae.

9. The method of claim 7 or claim 8, wherein said by-product derived from plants is selected from: cakes, seeds, shells and pellicles of said strict oil-rich oleaginous plants.

A solid food product for animal feed, comprising at least one strain of micromycetes, said strain of filamentous fungus is a strain selected from the following strains Aspergillus sojae and Aspergillus oryzae, in particular Aspergillus sojae, and a by-product derived from of plants, said by-product from plants being derived from a strict oleaginous fat-rich, the food product being obtainable by the method defined in any one of claims 7 to 9.

11. Fermented solid food product, especially according to claim 10, intended for human or animal food comprising:

- rapeseed cake and

at least one strain of filamentous fungi chosen from the following strains Aspergillus soya, and Aspergillus oryzae, in particular Aspergillus soya, said fermented solid food product having an immunomodulatory activity at least twice greater than that of said unfermented food product.

The fermented solid food product of claim 11, wherein said immunostimulatory products comprise at least one β-glucan.

A process for preparing immunomodulatory products comprising:

a step of contacting at least one strain of filamentous fungi, said strain of micromycetes being a strain of Aspergillus soya or Aspergillus oryzae, with a by-product derived from plants, said by-product derived from plants being from a strict oilseed high in fat

14. Solid fermented product comprising

a micromycete, said micromycete being Aspergillus soya or Aspergillus oryzae, and

for use as a drug or neutraceutical.

15. Solid fermented product comprising

a micromycete, said filamentous fungus being Aspergillus soya or Aspergillus oryzae, and

for use in the prevention or treatment of disorders related to a decrease in the activity of the immune system, particularly for the treatment and prevention of certain diseases in case of immunodeficiency such as allergies, cancers, cancers, autoimmune diseases, and gastrointestinal diseases or related to digestion.

16. A solid fermented product for use according to claim 14 or 15, wherein said by-product derived from plants is derived from plants belonging to the family Brassicaceae or Asteraceae.

A solid fermented product for use according to claim 14 or 15 or claim 16, wherein said vegetable derived by-product is selected from: oilcakes, seeds, shells and pellets of said high fat oilseeds.